Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry

Opportunity, Challenge and Scope of
Natural Products in Medicinal

Chemistry
Editor
Vinod K. Tiwari
Department of Chemistry, Faculty of Science, Banaras Hindu University
Varanasi-221005, India
Co-editor
Bhuwan B. Mishra
Research Signpost, T.C. 37/661 (2), Fort P.O., Trivandrum-695 023

Kerala, India
Published by Research Signpost

2011; Rights Reserved
Research Signpost
T.C. 37/661(2), Fort P.O.,
Trivandrum-695 023, Kerala, India
Editor
Vinod K. Tiwari
Co-editor
Bhuwan B. Mishra
Managing Editor
S.G. Pandalai
Publication Manager
A. Gayathri
Research Signpost and the Editors assume no responsibility
for the opinions and statements advanced by contributors
ISBN: 978-81-308-0448-4
Preface
Natural products extracted from tissues of terrestrial plants, marine
organisms or microorganism fermentation broths are the evolutionary shaped
molecules with a profound impact on human health. Nature's biosynthetic
engine produces innumerate metabolites with distinct biological properties
that make them valuable as health products or as structural templates for drug
discovery. In the early 1900s, before the Synthetic Era, 80% of all
medicines were obtained from roots, barks and leaves with a belief that for
every ill there exists a cure in the plants of field and forest. However, with the
advent of robotics, bioinformatics, high throughput screening, molecular
biology-biotechnology, combinatorial chemistry, in silico (molecular
modelling) and other methodologies, the pharmaceutical industry largely
moved away from plant derived natural products as a source for leads and
prospective drug candidates. There are also several misconceptions that
constrained the utilization of plant products for discovery and development of
pharmaceuticals. Among some of practical aspects while trying to explain the
difficulties associated with natural product research are: low yield, one-sampleone-source problem; high structural complexity and occurrence of multiple
stereoisomer; lacking of follow-up studies, since most efforts (e.g. in academic
environments) are not the part of focused drug development programs.
Pharmaceutical discovery is a numbers game in which thousands of
chemicals must be evaluated to find a hit. The interesting chemicals
identified as natural products are derived from the phenomenon of
biodiversity in which the interaction of organisms among each other and their
environment formulate the evolution of diverse complex natural entities in
the organisms that enhance their survival by protecting them against a wide
variety of microorganisms, arthropods and vertebrates and maintain
competitiveness in the ecosystem. Importantly, nature has been doing
combinational chemistry for eons and supplying almost unimaginable
chemical diversity, which yields stereochemically complex structures with
diverse functional groups and molecules ideal for interacting specifically with
biological targets. As Aristotle said, Nature does nothing without purpose
or uselessly, the world of plants, and indeed all natural sources, represents a
virtually untapped pool of novel drugs awaiting imaginative and progressive
organizations.
This book covers almost all natural product drug discoveries that have
been made in past few decades. The book editorial (Chapter 1) sumarises the
natural products, semi-synthetic natural products and natural product derived
compounds that have been registered, undergoing registration or in clinical
development, while chapters 2-12 are focused on natural product drug

discoveries by disease area i.e. infectious (bacterial, fungal and parasitic etc.)
diseases and Oncology.
The chapter 13 is focused on mutasynthesis that couples the power of
chemical synthesis with molecular biology to generate derivatives of
medicinally important natural products while the last chapter of the book
highlights significance of carbohydrate containing natural products in
medicinal chemistry.
I am thankful to all the authors and reviewers who helped me in
compiling this book. Lastly, I want to draw the attention of readers about the
increasing loss of much of the worlds forests, particularly in the tropics,
where the potentially remarkable properties of plant constituents not yet
discovered are threatened. Several plant species are on the brink of extinction
and in need of urgent conservation measures, otherwise, many future drugs
and other useful plant products would remain undiscovered and the often
surprising chemical structures produced by the genetic diversity of plants
might not be envisioned by future chemists.
Vinod K. Tiwari
Editorial Advisory Board

Prof. A. D. Kinghorn, USA
Prof. A. W. Lipkowski, Poland
Prof. Atta-ur-Rahman, Pakistan
Prof. B. Pirotte, Belgium
Prof. D. L. Boger, USA
Prof. D. S. Bhakuni, India
Prof. D. Strack, Germany
Prof. De-Yun Wang, Singapore
Prof. G. A. Cordell, USA
Prof. G. Appendino, Itly
Prof. G. Bringmann, Germany
Prof. G. H. Veeneman, Netherland
Prof. G. P. Bolwell, UK
Prof. G. S. Singh, Botswana
Prof. G. Vo-Thanh, France
Prof. Ganesh Pandey, India
Prof. Guisen Zhao, China
Prof. H. Ila, India
Prof. J. A. R Rodrigues, Brazil
Prof. J. C. Stockert, Spain
Prof. J. D. Connolly, UK
Prof. J. S. Yadav, India
Prof. Jamie Simpson, Australia
Prof. K. C. Nicolau, USA
Prof. K. D. Janda, USA
Prof. M. Garson, Austria
Prof. M. I. Choudhary, Pakistan
Prof. M. J. Chan-Bacab, Mexico
Prof. M. P. Kaushik, India
Prof. M. Salzet, France
Prof. M. Shibasaki, Japan

Prof. N. Tagmatarchis, Greece
Prof. Norbert Haider, Austria
Prof. P. G. Wang, USA
Prof. P. S. Portoghese, USA
Prof. Prabhat Arya, Canada
Prof. Pradeep Kumar, India
Dr. Prabhu P Mohapatra, USA
Prof. R. A. Lewis, Switzerland
Prof. R. M. Singh, India
Prof. R. P. Tripathi, India
Prof. R. R. Schmidt, Germany
Prof. S. Neidle, UK
Prof. R. Robins, France
Prof. S. Chandrashekhar, India
Prof. S. Komatsu, Japan
Prof. Seokjoon Lee, South Korea
Prof. Shang-Cheng Hung, Taiwan
Prof. Thomas Kurz, Germany
Prof. V. K. Singh, India
Prof. W. Boland, Germany
Prof. Xi Chen, USA
Prof. Y. Asakawa, Japan
Prof. Y. B. Tripathi, India
Prof. Y. H. Wong, Hong Kong
Prof. Y. Hashimoto, Japan
Prof. Y. Hu, China
Prof. Y. Yamamoto, Japan
Prof. Yogendra Singh, India
Prof. Nity anand, India
Contents
Chapter 1
Natural products in drug discovery: Clinical evaluations and investigations
Bhuwan B. Mishra and Vinod K. Tiwari
Chapter 2
Natural products in discovery of potential and safer antibacterial agents
Girija S. Singh and Surendra N. Pandeya
63
Chapter 3
Anti-tubercular activity of natural products: Recent developments
L. N. Rogoza, N. F. Salakhutdinov and G. A. Tolstikov
103
Chapter 4
Scope of natural products in fighting against leishmaniasis
B. B. Mishra, R. R. Kale, V. Prasad, V. K. Tiwari and R. K. Singh
121
Chapter 5
Naturally occurring antihyperglycemic and antidyslipidemic agents
T. Narender, T. Khaliq and G. Madhur
155
Chapter 6
Bio-flavonoids with promising anti-diabetic potentials: A critical survey
Goutam Brahmachari
187
Chapter 7
Marine natural alkaloids as anticancer agents
Deepak Kumar and Diwan S. Rawat
213
Chapter 8
Microtubule binding natural substances in cancer chemotherapy
Ram C. Mishra
269
Chapter 9
Natural products: Anti-fungal agents derived from plants
Tasleem Arif, T. K. Mandal and Rajesh Dabur
283
Chapter 10
Sesquiterpene lactones: Structural diversity and their biological activities
Devdutt Chaturvedi
313
Chapter 11
A review on natural products with mosquitosidal potentials
Navneet Kishore, Bhuwan B. Mishra, Vinod K. Tiwari
and Vyasji Tripathi
Chapter 12
Soybean constituents and their functional benefits
Ajay K. Dixit, J. I. X. Antony, Navin K. Sharma
and Rakesh K. Tiwari
335
367
Chapter 13
Mutasynthesis of medicinally important natural products through
manipulation of gene governing starter unit
Deepak Sharma, Syed Khalid Yousuf
and Debaraj Mukherjee
385
Chapter 14
Carbohydrate-containing natural products in medicinal chemistry
Hongzhi Cao, Joel Hwang and Xi Chen
411
Research Signpost
37/661 (2), Fort P.O.
Trivandrum-695 023
Kerala, India
Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 2011: 1-62
ISBN: 978-81-308-0448-4
1. Natural products in drug discovery:

Clinical evaluations and investigations
Bhuwan B. Mishra and Vinod K. Tiwari
Abstract. Natural products (NPs) have provided the source for the
majority of FDA-approved agents and continue to be one of the
major sources of inspiration for future drug discovery. The R&D
thrust in the pharmaceutical sector today is focused on
development of new drugs, innovative/indigenous processes
for known drugs, development of NP-based drugs through
investigation of leads obtained from the traditional systems of
medicine as well as other resources. Present review describes
natural products (NPs), semi-synthetic NPs and NP-derived
compounds that have been registered, undergoing registration or in
clinical development since 1998 till June 2010 by disease area i.e.
infectious (bacterial, fungal, parasitic and viral), immunological,
cardiovascular, neurological, inflammatory and related diseases
and Oncology. This review also highlights the recently launched
natural product-derived drugs, new natural product templates and
late-stage development candidates.
1. Introduction
The interesting chemicals identified as NPs are derived from the
phenomenon of biodiversity in which the interactions among organisms and
their environment formulate the diverse complex chemical entities within the
Correspondence/Reprint request: Dr. Vinod K. Tiwari, Department of Chemistry, Faculty of Science, Banaras Hindu
University, Varanasi-221005, India. E-mail: tiwari_chem@yahoo.co.in
Bhuwan B. Mishra & Vinod K. Tiwari
organisms that enhance their survival and competitiveness. Today, R&D

thrust in the pharmaceutical sector is focused on development of new drugs,
innovative/indigenous processes for known drugs and development of
plant-based drugs through investigation of leads obtained from the traditional
systems of medicine as well other resources [1,2].
History of medicine dates back practically to the existence of human
civilization and use of NPs by human have been traced from ancient records
such as the use of Artemisia annua in China, opium poppy (active principle
morphine) in Egypt, snakeroot plant (active principle reserpine) in India,
willow tree (salicin) & foxglove (active principle digitalis - a mixture of
compounds such as digitoxin, digitonin, digitalin) in England and
ipecacuanha root (active principle emetine), coca bush (active principle
cocaine) and cinchona bark (active principle quinine) in Mesoamerica. The
current accepted modern medicine or allopathy has gradually developed over
the years by scientific and observational efforts of scientists. However, the
basis of its development remains rooted in traditional medicine and therapies.
Plants have always been a rich source of NP leads e.g. morphine,
cocaine, digitalis, quinine, tubocurarine, nicotine, muscarine, paclitaxel
(TaxolTM) and artemisinin. The success of penicillin encouraged the
discovery of new antibiotics from microorganisms. Mining of the bacterial
genome and identification of crucial targets followed by study of new
bacterial or fungal strains have resulted in discovery of significant
antibacterial agents such as the cephalosporins, tetracyclines,
aminoglycosides, rifamycins and chloramphenicol. Since past five decades,
marine sources e.g. coral, sponges, fish and marine microorganisms have
attracted scientists from different disciplines leding to the discovery of
several marine NPs with promising biological activity such as curacin A,
eleutherobin, discodermolide, bryostatins, dolostatins, and cephalostatins.
Venoms and toxins (peptides and non-peptides) occurring in snakes,
spiders, scorpions, insects, and other microorganisms are also significant in
drug discovery due to their specific interactions with macromolecular
targets in the body, and have been proved crucial while studying receptors,
ion channels, and enzymes. Toxins like -bungarotoxin (from cobras),
tetrodotoxin (from puffer fish) and teprotide (from Brazilian viper) etc. are
in clinical trials for drug development. Similarly, the neurotoxins obtained
from Clostridium botulinum (responsible for botulism, a serious food
poisoning), has been found significant to prevent muscle spasm.
The review summarizes the 3 groups of compounds classified as NPs,
semi-synthetic NPs and NP-derived compounds that have been registred,
undergoing registration or in clinical development since 1998 to June 2010
by disease area i.e. infectious (bacterial, fungal, parasitic and viral),
Natural products in drug discovery
immunological, cardiovascular, neurological, inflammatory and related

diseases and oncology. The compounds which have biological activities
and are derived from natural sources, e.g., plants, animals and
microorganisms have been grouped as NPs. The compounds that
are derived from a NP template using semi-synthesis have been
grouped in semi-synthetic NPs while the compounds that were synthetically
derived or in some cases inspired from a NP template have been
classified as NP-derived compounds [3-5]. The review also presents
an update of previous reviews published in relevance to present context
[6-10].
Table 1. NP-deived drugs launched during 1998-2004; lead compounds and
therapeutic area.
Year
1998
1998
1999
1999
1999
2000
2001
2001
2001
2001
2002
2002
2002
2002
2002
2003
2003
2003
2003
2003
2004
Trade name
orlistat (Xenical)
cefoselis (Wincef)
dalfopristin and
quinupristin (70 :30
mixture) (Synercid)
valrubicin (Valstar)
colforsin daropate (Adele,
Adehl)
arteether (Artemotil)
ertapenem (InvanzTM)
caspofungin (Cancidas)
telithromycin (Ketek) 55
pimecrolimus (Elidel)
galantamine (Reminyl)
micafungin (Funguard)
amrubicin hydrochloride
(Calsed)
biapenem (Omegacin)
nitisinone (Orfadin)
miglustat (Zavesca)
Lead compound
lipstatin
cephalosporin
streptogramin B 44 &
streptogramin A 45
Disease area
Antiobesity
Antibacterial
Antibacterial
doxorubicin 164
forskolin
Oncology
Cardiotonic
artemisinin 65
thienamycin 5
pneumocandin B
erythromycin 51
ascomycin
galantamine
FR901379
doxorubicin 164
Antimalarial
Antibacterial
Antifungal
Antibacterial
Atopic dermatitis
Alzheimers disease
Antifungal
Oncology
thienamycin 5
leptospermone
1-deoxynojirimycin
mycophenolate sodium
(Myfortic)
rosuvastatin (Crestor)
pitavastatin (Livalo)
daptomycin (CubicinTM)
everolimus
(CerticanTM) 24
mycophenolic acid
Antibacterial
Antityrosinaemia
Type 1 Gaucher
disease
Immunosuppression
mevastatin
mevastatin
daptomycin
sirolimus 10
Dyslipidemia
Dyslipidemia
Antibacterial
Immunosuppression
2. Drug approval processes

The Investigational New Drug (IND) application is submitted to the FDA or
EMA before commencement of clinical trials. Once clinical trials are successfully
completed, the applicant files New Drug Application (NDA) in the US or
Marketing Authorization Application (MAA) in Europe seeking drugs approval
for marketing, to which the agency replys in the form of approval letter, nonapproval letter or approvable letter. An approval letter allows the applicant
to begin marketing of product, while a non-approval letter rejects the
application. An approvable letter informs the applicants that the agency have
completed their scientific review and determined that the application can be
approved pending resolution of minor deficiencies identified in the letter or
during an inspection of the manufacturing facilities.
Table 2. NP-deived drugs launched during 2005-2010; lead compounds, and
therapeutic area.
Year
2005
Lead compound
Dronabinol
1/cannabidol 2
Fumagillin 3
Thienamycin 5
Disease area
Pain
2005
2005
Trade name
Dronabinol 1/Cannabidol 2
(Sativex)
Fumagillin 3 (Flisint)
Doripenem 4 (Finibax/DoribaxTM)
2005
2005
2005
Tigecycline 6 (Tygacil)
Ziconotide 8 (Prialt)
Zotarolimus 9 (EndeavorTM stent)
Tetracycline 7
Ziconotide 8
Sirolimus 10
2006
Anidulafungin 11
(EraxisTM/EcaltaTM)
Exenatide 13 (Byetta)
Lisdexamfetamine 14 (VyvanseTM)
Retapamulin 16
(AltabaxTM/AltargoTM)
Temsirolimus 18 (ToriselTM)
Trabectedin 19 (YondelisTM)
Ixabepilone 20 (IxempraTM)
Methylnaltrexone 22 (Relistor)
Everolimus 24 (Afinitor)
Telavancin 25 (VibativTM)
Romidepsin 27 (Istodax)
Capsaicin 28 (Qutenza)
Monobactam aztreonam 29
(CaystonTM)
Echinocandin B 12
Antibacterial
Pain
Cardiovascul
ar surgery
Antifungal
Exenatide-4 13
Amphetamine 15
Pleuromutilin 17
Diabetes
ADHD
Antibacterial
Sirolimus 10
Trabectedin 19
Epothilone B 21
Naltrexone 23
Sirolimus 10
Vancomycin 26
Romidepsin 27
Capsaicin 28
Aztreonam 29
Oncology
Oncology
Oncology
Pain
Oncology
Antibacterial
Oncology
Pain
Antibacterial
2006
2007
2007
2007
2007
2007
2008
2009
2009
2009
2009
2010
Antiparasitic
Antibacterial
3. NP based drugs approved during 1998-2004

A total of 21 NP and NP-derived drugs were launched in the United
States, Europe or Japan during 1998-2004 that can be classified as 3 NPs,
10 semi-synthetic NPs and 8 NP-derived drugs (Table 1).
3.1. NP based drugs approved during 2005-2010

A total of 19 NP based drugs were approved for marketing worldwide in
between the year 2005 to April 2010, among which 7 being classified as NPs,
10 semi-synthetic NPs and 2 NP-derived drugs (Table 2).
VeregenTM (Polyphenon E ointment), a defined mixture of catechins
obtained from green tea, is the first ever herbal medicine to receive FDA
approval in 2006. VeregenTM was developed by MediGene AG and launched
in the US by Bradley Pharmaceuticals in December 2007 for topical use
against genital warts. In March 2010, Solvay launched Veregen (10 %
ointment) in Germany.
Sativex, a mixture of dronabinol 1 and cannabidol 2 obtained from the
cannabis plant, is the world's first pharmaceutical prescription medicine that
was launched in Canada (April 2005) and was later approved by Health
Canada (August 2007) as adjunctive analgesic for severe pain in advanced
cancer patients [11]. Sativex has been recommended by FDA to enter
directly in Phase III trials and as of November 2009, GW Pharmaceuticals
have completed the recruitment for Phase II/III trial against cancer pain. In
March 2010, GW Pharmaceuticals provided an update on the progress of
regulatory submission for Sativex oromucosal spray for the treatment of the
symptoms of spasticity due to multiple sclerosis.
Fumagillin (Flisint, Sanofi-Aventis) 3, an endothelial cell proliferation
inhibitor isolated from Aspergillus fumigatus [12], was approved in France in
September 2005 for the treatment of intestinal microsporidiosis. Fumagillin 3
can also block the blood vessel formation through binding to methionine
aminopeptidase II and is under clinical investigtions as an angiogenesis
inhibitor for the treatment of cancer.
Among carbapenem-type -lactams, doripenem (Finibax, DoribaxTM) 4
is an ultra-broad spectrum injectable antibiotic launched in Japan (2005) by
Shionogi & Co. while ertapenem, a NP derived compound based on structure
of thienamycin 5 is being marketed by Merck as InvanzTM. In October 2007,
Johnson & Johnson (J&J) obtained formal FDA approval for use of 4 in intraabdominal and urinary tract infections.
Tigecycline (Tygacil) 6, a glycylcycline antibiotic structurally similar to
teracycline 7, was approved by FDA in June 2005 against intra-abdominal
and complicated skin and skin structure infections (SSSIs). Tigecycline 6 was
developed by Francis Tally and contains a centralised four-ring carbocyclic
skeleton substituted at the D-9 position confering broad spectrum activity.
Tigecycline 6 inhibits protein translation by connecting with 30S ribosome
and hinders amino-acyl tRNA molecules coming to A site ribosomal subunit
[13]. As of May 2006, the 6 has been approved in Europe and later a
supplementary NDA for community-acquired pneumonia (CAP) was
submitted to the FDA in October 2007.
Ziconotide (Prialt) 8, a synthetic-conotoxin and calcium channel
blocker, isolated from Conus magus [14], causes pain relief by inhibiting
pro-nociceptive neurochemical releases in the brain and spinal cord [15].
In December 2004, the FDA approved 8 when delivered as infusions into the
cerebrospinal fluid using intrathecal pump system. In 2005, Elan launched
8 in US and Europe while rights for marketing 8 in Europe were obtained by
Eisai in March 2006.
CH3
CH3
OH
OH
H2C
H3C
O
H3C
CH3 HO
CH3
CH3
CH3
1
CH3
HO
H
CH3
CH3
H3C
S
N
OCH3
H
N
O
O
HO
O
N
H
NH2
S
O
CO2H
3
HO
H
4
H3C
CH3
CH3
H3C
NH2
CH3
N
H
OH
H3C
S
N
H
N
H3C
N
H
H3C
HO
CH3
HO
OH
CONH2
O
6
H3C
HO
OH
CH3
N
CH3
OH
H2N-CKGKGAKCSRLMYDCCTGSCRSGKC-CONH2
CONH2
OH
OH
OH
Zotarolimus 9, a derivative of sirolimus 10, is an active principle of

EndeavorTM stent that is being used as anti-proliferative agent by Medtronic
[16,17]. In July 2005 EndeavorTM was approved by European comission for
the sale while FDA approved it in February 2008 for the treatment of
coronary artery disease.
Anidulafungin 11 (EraxisTM in US, EcaltaTM in Europe), a semi-synthetic
derivative of echinocandin B 12, was originally developed by Eli Lilly
against invasive and oesophageal candidiasis and candidemia. Anidulafungin
11 was later licensed to Vicuron Pharmaceuticals, which was purchased by
Pfizer in June 2005. Pfizer gained FDA approval for EraxisTM in the US
(February 2006) and EcaltaTM in Europe (July 2007).
Exenatide 13 (Byetta), a 39 amino acid peptide isolated from the oral
secretions of Heloderma suspectum (Gila monster) [18], mimics the
antidiabetic or glucose-lowering properties of incretins. In April 2005, Eli
Lilly obtained FDA approval for 13 while EMEA in November 2006
approved it to Amylin Pharmaceuticals for use in type 2 diabetes mellitus
[19]. Amylin Pharmaceuticals, Eli Lilly and Alkermes submitted a NDA in
May 2009 for subcutaneous dosing of 13 once weekly that was accepted in
July 2009 by the FDA.
Attention-Deficit Hyperactivity Disorder (ADHD), a neurodevelopmental
disorder in which dopaminergic and noradrenergic neurotransmission are
supposed to be dysregulated, is primarily characterized by the co-existence of
attentional problems and hyperactivity. Despite abuse potentials
methylphenidate and amphetamines were used for Attention-Deficit
Hyperactivity Disorder (ADHD) management since many years.
14
Lisdexamfetamine
(VyvanseTM,
NRP104)
consisting
of
dextroamphetamine coupled with L-lysine was designed by New River
Pharmaceuticals produces effects similar to placebo on intravenous
administration, however on oral administration it converts into
D-amphetamine 15 in the gastrointestinal (GI) tract [20]. In February 2007,
FDA approved 14 to treat ADHD.
Pleuromutilin 16, a fungal metabolite inhibiting protein synthesis
in bacteria [21], is the lead compound of retapamulin (SB-275833) 17
developed by GlaxoSmithKline. In June 2007, EMEA approved an ointment
containing 1% retapamulin 17 called AltabaxTM in the US and AltargoTM in
Europe for topical treatment of impetigo caused by Staphylococcus aureus or
Streptococcus pyogenes.
Temsirolimus (Torisel, CCI-779) 18, a derivative of 10 and mTOR
inhibitor [22] developed by Wyeth in various Phase III trials was approved in
May 2007 by the FDA and November 2007 by the EMEA for the treatment
of renal cell carcinoma (RCC) [23].
Trabectedin (Yondelis, ecteinascidin-743, ET-743) 19, an alkaloid

obtained from Ecteinascidia turbinate [24], is a DNA minor groove binder
that inhibits cell proliferation by disrupting the cell cycle. Trabectedin 19
is sold by Zeltia and J&J against advanced soft tissue sarcoma (STS).
In September 2007, the EMEA has approved 19 for use against ovarian
cancer and STS. In November 2009, Yondelis received its second marketing
authorisation from the European Commission against relapsed platinumsensitive ovarian cancer when administered in combination with DOXIL/
Caelyx.
R
O
CH3
CH3
O
OH
O
CH3
O
CH3
O
N
CH3
O
CH3
CH3
CH3
9R=
N
N
O
HO
10 R =
O
H3C
OH
CH3
HO
OH
O
HO
11 R =
NH
NH
H3C
N
HN
H3C
CH3
H3C
H
N
HO
NH
HO
OH
O
OH
O
OH
12 R =
H3C
HO
13 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS
NH2
H
N
NH2
NH2
CH3
CH3
15
14
O
OH
CH2
CH3
CH3 OH
OH
O
CH3
O
R
CH3
H
O
CH3
O
H3C
H3C
O
CH3
CH3
O
N
16 R = OH
17 R =
OH
CH3
O
CH3
CH3
O
CH3
NCH3
O
O
O
CH3 H3C
HO
H3C
HO
OCH3
CH3
18
H3C
S
CH3
CH3
O
O
OH
O
H3CO
NH
H3C
OH
H3C
CH3
H3C
N
X
CH3
O
HO
19
CH3
OH
20 X = NH
21 X = O
Ixabepilone (IxempraTM, BMS-247550) 20, a semi-synthetic derivative of

epothilone B 21 produced by Sorangium cellulosum [25], was developed by
Bristol-Myers Squibb (BMS) as an anticancer drug that binds to -tubulin
and suppresses the dynamics of microtubule. In October 2007, BMS gained
FDA approval for 20 as a monotherapy and in combination with Xeloda for
the treatment of breast cancer, resisting standard therapy [26].
Methylnaltrexone (MOA-728, Relistor by Wyeth) 22, a derivative of
naltrexone 23 that blocks peripheral opioid receptors activated by opioids and
10
thus is significant in management of alcohol and opioid dependence [27].

Wyeth and Progenics in May 2007 filed an NDA for subcutaneous doses of
22 against opioid induced constipation (OIC) and other pain indications that
was approved in April 2008 by Health Canada and the FDA. As of May 2009
an oral formulation of 22 is under Phase II trials against OIC in chronic pain.
HO
CH3
O
H3C
O
N
HO
O
CH3
HO
OH
HO
CH3
22
CH3
O
CH3
CH3
O
CH3
O
HO
N Br
CH3
HO
O
H3C
CH3
24
23
Everolimus (LuveniqTM or LX211) 24, an mTOR inhibiting derivative of

10 is marketed as immunosuppressant by Novartis under ZortressTM (USA)
and CerticanTM (Europe and other countries) in transplantation medicine, and
Afinitor for use in advanced renal cell carcinoma (RCC). CerticanTM was
approved in 2004 as immunosuppressant while in March 2009 the FDA has
approved 24 against advanced RCC after failure of Sutent (sunitinib) or
Nexavar (sorafenib).
OH R1
HN
H3C
O
CH3
OH
O
OH
OH
Cl
O
OH
HO
Cl
O
H
N
H
N
H
N
N
H
N
H
N
H
HN
CH3
CH3
O
O
HO
CH3
NH2
O
OH
H
N
OH
CH3
R1 =
HO
R2
25
R2 =
N
H
26 R1 = H, R2 = H
PO3H2
11
Telavancin (VibativTM, TD-6424) 25, a semisynthetic derivative of

vancomycin 26 that inhibits bacterial growth through binding to D-Ala-D-Ala
[28], was developed by Theravance in partnership with Astellas for use
against Gram-positive cSSSIs and MRSA that was approved in September
2009 by the FDA. Theravance has also submitted telavancin 25 to the FDA in
a second indication against nosocomial pneumonia or hospital aquired
pneumonia (HAP). In November 2009, the FDA released a complete
response letter to Theravance for telavancin 25 NDA against nosocomial
pneumonia.
Romidepsin (depsipeptide, FK228, FR901228, Istodax) 27 extracted
from the bacteria Chromobacterium violaceum, is a histone deacetylase
(HDAC) inhibitor [29] developed by Gloucester Pharmaceuticals under
National Cancer Institute (NCI) sponsorship for treatment of cutaneous and
peripheral T-cell lymphoma (TCL). In November 2009, the FDA approved
27 to use in the treatment of selective cutaneous TCL patients previously
treted with minimum of one prior systemic therapy. In January 2010, Celgene
completed the acquisition of Gloucester Pharmaceuticals.
Capsaicin (Qutenza) 28, isolated from chili peppers of genus Capsicum
[30], produces burning sensation on contact to tissues though binding to
vanilloid receptor subtype 1 (VR 1) [31]. In November 2009, the FDA
approved Qutenza (a transdermal 8% patch of 28) to use in treatment of
neuropathic pain combined with postherpetic neuralgia. In April 2010,
NeurogesX launched Qutenza in US. Aztreonam lysine (CaystonTM) 29 is an
inhaled lysine salt formulation [32] that was evaluated by Gilead in various
Phase III trials against cystic fibrosis (CF) patients infected with the Gramnegative bacteria Pseudomonas aeruginosa. In February 2010, the FDA
approved 29 against CF patients.
O
H3CO
CH3
H
N
H3C
NH
CH3
S
O
S
O
O
H3C
CH3
CO2H
O
NH
H
N
N
O
CH3
H2N
H3C
27
CH3
28
HO
O
HN
CH3
N
H
CH3
29
O
S
OH
12
4. Infectious diseases
4.1. Antibacterial
NP-derived drugs have played their crucial role in anti-infective drug
discovery and the majorities of antibacterial drugs currently in clinical use are
NPs or were designed using NP templates. Despite having complex structure
the development of a NP to an antibacterial drug entirely depends on its
ability to penetrate bacterial cell membranes. The success of penicillin
encouraged the discovery of other compounds from natural sources against
bacterial infections and as a result nearly all novel classes of antibiotics
belong to NP sourced scaffolds [33].
Ceftobiprole medocaril (BAL-5788) 30, a cephalosporin antibiotic
with excellent activity against methicillin-resistant Staphylococcus aureus,
penicillin-resistant Streptococcus pneumoniae, Pseudomonas aeruginosa, and
Enterococci [34], was filed for regulatory approval in the US and Europe
in July 2007 by Basilea Pharmaceutica and J&J affiliated Cilag GmbH
International to use in the treatment of cSSSIs. In November 2008, the
approval of 30 was declined by the FDA with recommendation of two new
studies to access safety and efficacy in treatment of cSSSIs. Additionly,
various Phase III trials are underway for HAP/CAP. Ceftaroline acetate (PPI0903, TAK-599) 31, discovered by Takeda and licensed to Cerexa, shows
efficacy against the penicillin-resistant S. pneumoniae and is under Phase II
development by Forest Laboratories to use in the treatment of cSSSIs and
CAP [35].
Tebipenem pivoxil (ME-1211, L-084) 32, an oral carbapenem antibiotic
is under Phase III clinical development by Meiji Seika in Japan for
treatment of otolaryngological/respiratory infections. Tomopenem (CS-023,
RO4908463, R1558) 33 [36], by Daiichi Sankyo for treatment of common
nosocomial infections and PZ601 (SM-216601, Protez) 34 [37], against
MRSA and Pseudomonas aeruginosa, are currently in Phase II trials.
ME1036 (CP5609) 35, a DHP-1-stable parenteral carbapenem having
excellent in vitro activity against multidrug-resistant (MDR) staphylococci
and Enterococcus faecalis was licensed by Cerexa and Forest Laboratories
from Meiji Seika Kaisha. ME1036 35 is currently under Phase I evaluation.
Likewise, sulopenem (CP-70429) 36, is being evaluated by Pfizer in various
Phase I trials [38].
Faropenem daloxate (SUN-208, BAY-56-6824) 37 is a penem-type
-lactam licensed to Replidyne by Daiichi Suntory Pharma for marketing in
conjunction with Forest Pharmaceuticals [39]. In December 2005, Replidyne
submitted an NDA to the FDA for use of 37 in the treatment of bacterial
13
sinusitis (BS), chronic bronchitis (CB), CAP and uncomplicated (SSSIs).

In response to Replidynes NDA, the FDA in March 2007 agreed for Phase
III placebo-controlled trials of 37, one each in BS and CB along with two
non-inferiority CAP trials. However, these additional trials have certainly
delayed the launch of drug.
OH
N
H2N
H
N
N
S N
N
O
HO
CH3
H3C
N
O
O
OH
H H
CH3
H3C
H3C CH3
30
S
N
32
CH3
CH3
N
OH
HO
OH
H H
O
H
H
N
H
N
OH
H H
H3C
H
N
N
H
N
H
CH3
O
O
HN
36
S
Cl
H
N
H
N
NH
HO
O
OH
OH
HO
OH OH
O
H
N
38 R =
39 R = OH
Cl
HO
OH
O
37
N
H
N
O
N
H
HH
H3C
OH
CO2H
HO
CH3
OH
H3C
CH3
NH2
35
HO
H3C
OH
HO
NH2 O
NH
N
CH3
HH
O
N
33
OH
CH3
H3C
NH
34
OH
H H
HO
CH3
H3C
CH3
N
HO
31
CH3
CH3
N
CH3
N
H
CH3
14
Dalbavancin (Zeven, BI-397) 38, a semi-synthetic derivative of the

teicoplanin analogue A40926 39, was discovered by Biosearch Italia and
being developed by Pfizer for the treatment of cSSSIs [40]. In February 2005,
Vicuron Pharamaceutical (now a part of Pfizer) filed an NDA for 38 to use
in the treatment of patients suffering from cSSSIs. In response, the FDA
released an approval letter in December 2007, however, as of September
2008 Pfizer have withdrawn all the marketing applications of 38 for running
another Phase III trial.
Oritavancin (NuvocidTM, LY-333328) 40, a chloroeremomycin 41
derivative inhibiting cell-wall synthesis, was discovered and developed by Eli
Lilly and acquired by InterMune in 2001 and later tansferred to Targanta
Therapeutics in 2005. In February 2008, Targanta submitted an NDA for 40
to the FDA that was not approved due to insufficient data. Aditionally, a
MAA was submitted by Targanta for 40 to EMEA that was accepted for
review in June 2008. TD-1792 42, a vancomycin-cephalosporin heterodimer
successfully evaluated by Theravance in Phase II trials against cSSSIs
including MRSA, has been designed to target 2 key targets in bacterial cell
wall synthesis. In July 2007, Theravance disclosed to meet primary and
secondary endpoints of non-inferiority trial compared to vancomycin 26.
Ramoplanin factor A2 (known as ramoplanin) 43, the major
component of the lipopeptide antibiotic drugs obtained from Actinoplanes
ATCC 33076 [41], inhibits cell wall synthesis in bacteria by forming
U-shaped structures that are able to bind and capture Lipid II (C35-MurNAcpeptide-GlcNAc), a specific intermediate in membrane formation [42].
Oscient Pharmaceuticals hold the North American right and are evaluating
orally active doses of 43 in Phase II trials against Clostridium difficile
associated GI tract infections [43].
NXL-103 (XRP2868), an orally available mixture (70:30) of flopristin
(RPR132552A, streptogramin A-type) 44 and linopristin (RPR202698,
streptogramin B-type) 45 that inhibit bacterial protein synthesis through the
synergistic binding to different sites on the peptidyltransferase domain of the
50S ribosomal subunit [44], was discovered by Sanofi-Aventis [45]. Novexel
in October 2008 announced for positive Phase II trials of NXL-103 against
CAP and cSSSIs including MRSA.
Friulimicin B 46, a lipopeptide antibiotic produced by Actinoplanes
friuliensis HAG 010964 [46], exerts activity through complex formation with
bactoprenol-phosphate, resulting in inhibition of peptidoglycan and teichoic
acid biosynthesis in bacteria [47], is under Phase I clinical development
(July 2007) by MerLion Pharmaceuticals. Structure of friulimicin B 46 was
confirmed after the crystal structure of amphomycin tsushimycin (A-1437 B)
47, an aspartic acid analogue of 46 was published in late 2005.
15
R
HN
HO
H3C
O
CH3
H2N
HO
H3C
OH
O
O
CH3
Cl
Cl
H
N
OH
OH
OH
O
O
N
H
HN
O
H
N
N
H
HO
H
N
N
H
CH3
NH2
CH3
CH3
OH
OH
HO
40 R =
Cl
41 R = H
OH
H3C
NH2
O
CH3 O
OH
OH
OH
Cl
O
OH
HO
Cl
O
O
H
N
N
H
HN
H
N
N
H
H
N
N
H
CH3
NH2
NH
42
H
N
N
H2N
S
OH
OH
HO
Cl
CH3
CH3
O
CO2H
OH
OH
H2N
OH
H3C
O
H
N
H3C
HN
OH
H
N
N
H
O
H
N
N
H
O
N
H
NH
N
H
O
NH2
O
O
H2N
OH
H3C
O
HN
H
N
OH
O
H
N
N
H
O
H
N
N
H
NH2
NH
N
H
CH3
OH
OH
O
O
O
OH
OH
OH
43
CH3
OH
CH3
CH3
HO
HO
HO
H3C
Cl
16

CH3
H3C
N
O
N
O
F
CH3
CH3
N
O
O
O
CH3
H
N
HO
H3C
CH3
H
N
N
H
N
NH
OH
N
H
45
COOH
CH3
O
O
O
CH3
44
HN
HN
H3C
O
H3C
NH
H3C
N
O
N
H
COOH
HN
HOOC
CH3 O
N
H
H3C
H
N
HN
O
O
O
NH
NH2
46 R = NH2
47 R = OH
H3C
CH3
Moli1901 (duramycin, 2262U90) 48, obtained from Streptomyces

cinnamoneum [48], inhances the chloride transport and increases fluid
secretion in vitro, thus finds significance for the treatment of CF [49].
Moli1901 48 is currently under clinical development by AOP Orphan in
colaboration with Lantibio in Europe. In March 2007, Lantibio announced
the positive results of Phase II trial of aerosolized 48 in adolescents and
adults suffering from CF. An ophthalmic solution of 48 for treatment of dry
eye syndrome is also under Phase II trials by Lantibio.
Omiganan 49, originally purified from neutrophils of bovine, is an
indolicidin 50 derivative that can interact with the bacterial cytoplasmic
membrane and has been found significant against antibiotic-resistant
and sensitive bacterial infections [50]. Omiganan 49 was developed by
MIGENIX and later licensed to Cadence Pharmaceuticals and Cutanea
Life Sciences for catheter-related infections (coded OmigardTM, CPI-226,
MBI-226) and dermatological diseases (coded as CLS001, MX-594AN),
respectively. Cadence Pharmaceuticals are currently evaluating a gel-based
formulation of 49 in Phase III trials while another phase III trials for
treatment of rosacea, a chronic inflammatory skin disorder are underway.
17
NH
H-Ala-Lys-Gln-Ala-Ala-Ala-Phe-Gly-Pro-Phe-Abu-Phe-Val-Ala-HOAsp-Gly-Asn-Abu-LysOH
S
S
S
48
ILRWPWWPWRRK-NH2
49
ILPWKWPWWPWRR-NH2
50
Erythromycin 51, macrolide antibiotic produced by actinomycetes, exerts

antibacterial activity through inhibition of protein synthesis by binding
to peptidyltransferase site of 50S subunit [51]. Among other derivatives,
cethromycin 52, EP-420 53 and BAL-19403 54 are currently in clinical
development. Cethromycin (ABT-773) 52 was discovered by Abbott
Laboratories and later acquired by Advanced Life Sciences to use in the
treatment of CAP and anthrax [52]. Advanced Life Sciences in October 2008
submitted a NDA to use 52 in the treatment of mild-to-moderate CAP which
was accepted by FDA in December 2008. The cethromycin 52 (RestanzaTM)
has demostrated clinically and statistically significant survival rate in
placebo-controlled non-human primate studies with anthrax, plague and
tularemia. In September 2009, the FDA has given orphan drug designation to
52 for the treatment of plague and tularemia. Likewise, EP-420 (EP-013420)
53, a bridged bicyclic derivative of 51 is currently under Phase II clinical
development by Enanta and Shionogi for treatment of CAP [53].
BAL19403 54, a macrolide antibiotic significant against clinical isolates
of Propionibacterium acnes with mutations in the 2057 to 2059 region of
23S rRNA conferring resistance to 51, is under clinical development by
Basilea for the treatment of acne [54]. Telithromycin (Ketek) 55 is the first
approved ketolide developed by Sanofi-Aventis that received approval from
the European Commission (July 2001) and the FDA (in 2004) for treatment
of respiratory infections. Telithromycin 55 displays bactericidal activity by
blocking the progression of the growing polypeptide chain through binding
with the 50S subunit of ribosome. Tiacumicin B (OPT-80, PAR-101) 56,
a macrolactone isolated by Abbott [55] from actinomyces, inhibits
RNA synthesis and is under phase III clinical development by Optimer
Pharmaceuticals for the treatment of Clostridium difficile-associated diarrhea
(CDAD) [56].
PTK-0796 (MK-2764) 57 is an aminomethylcycline inhibiting protein
synthesis in bacteria, was discovered and evaluated by Paratek in Phase II trials
for the treatment of common hospital infections. PTK-0796 57 was in-licensed
18

O
H3C
CH3
H3C
H3C
OH
OH
HO
CH3
HO
O
H3C
O
O
O
H3C
N
O
CH3
H3C
N
CH3
HO
O
CH3
H3C
O
H3C
CH3
OH
CH3
CH3
O
CH3
OCH3
H3C
H
N
CH3
CH3
CH3
CH3
51
52
N
HO
CH3
H3C
H3C
H3C
H3C
CH3
HO
O
CH3
OCH3
H3C
N CH3
CH3
HO
CH3
O
O
H3C
O
H C
S 3
CH3
N
O
N
CH3
H3C
O
H3C
CH3
CH3
OCH3
O
CH3
CH3
54
53
H3C
H3C
HO
O
O
H3C
CH3
N
H
CH3
H3C
O
O
H3C
O
O
O
OCH3
H3C
H3C
CH3
OHO
N CH3
CH3
OH
H3C
OH
O
N
N
CH3
OH
CH3
H3C
CH3
H3C
CH3
OCH3
O
OH
O
O
O
OH
Cl
O
H3C
OH
CH3
OH
CH3 Cl
CH3
55
56
by Novartis form Paratek for collaborative Phase III clinical development.

In October 2009, Novartis gained exclusive marketing rights of 57 to use in
the treatment of MRSA, MDR Streptococcus pneumoniae and vancomycinresistant enterococci.
Eritoran (E5564) 58, a second-generation lipid A antagonist [57]
designed by Eisai from Rs-DPLA 59 isolated from Rhodopseudomonas
sphaeroides [58], inhibits endotoxin response through antagonism of the
Toll-like receptor 4 (TLR4) [59,60].
19
CBR-2092 60, a hybrid antibiotic inhibiting RNA and DNA synthesis is

being developed by Cumbre Pharmaceuticals for treatment of gram-positive
cocci infections. CBR-2092 60 is supposed to exert antimicrobial activity
through combined effects on RNA polymerase, DNA topoisomerase IV and
DNA gyrase. Currently, CBR-2092 60 is in Phase IIa trial by Cumbre for
treatment of infections caused by gram-positive cocci [61].
H3C
CH3
H3C
CH3
H3C
H
CH3
OH
H
N
H3C
OH
CONH2
OH
OH O
57
OCH3
H2O3PO
OH
O
O
HO
HN
O
H2O3PO
O
O
HN OPO3H2
O
O
HO
H3CO
CH3
O
HN
HO
HO
O
CH3
CH3
O
O
HN OPO3H2
O
O
CH3
H3C
H3C
H3C
CH3
CH3
58
H3C
59
O
CH3 CH3 CH3
O
H3CO
OH
CH3
OH
OH
O
OH
H3C
CH3
NH
O
OH
O
CH3
N
N
CH3
CO2H
N
CH3
60
4.2. Antifungal
Invasive fungal infections infections of the bloodstream and organs
within the body (e.g. meningitis, pneumonia, peritonitis) are important
causes of morbidity and mortality in liver, pancreas, heart, kidney and lung
(i.e. solid organ) transplant recipients [62]. Fungi are eukaryotes and, despite
20
the presence of a cell wall, fungi are more similar to mammalian cells on a
cellular level than to bacteria, making the treatment of mycotic infections
difficult [63]. Only 2 NP-derived compounds, aminocandin 61 and SPK-843
62 are undergoing clinical evaluation. Due to lack of biological target, 1,3-D-glucan synthesis in human, echinocandin derivatives have been considered
significant against refractory aspergillosis and invasive infections by Candida
species [64]. Among other semi-synthetic echinocandins, caspofungin
(launch 2001, Cancidas, Merck), micafungin (launch 2002,
Mycamine/Funguard, Astellas) and anidulafungin 11 (launch 2006,
EraxisTM/EcaltaTM, Pfizer) have been approved. Deoxymulundocandin 63,
isolated from Aspergillus sydowii [65], is the lead compound of aminocandin
(NXL-201, IP960, HMR-3270) 61 and exhibit excellent activity against
Candida albicans and C. tropicalis by destabilizing the fungal cell
membrane. SPK-843 62, a semi-synthetic derivative of patrician-A 64, is
under clinical development by Dutch company APARTS BV [66] that has
acquired world wide rights for the development of 62.
4.3. Antiparasitic
The use of medicinal plants against parasitic diseases has been
traced to ancient times i.e. bark of Cinchona calisaya and Strychnos
pseudoquina, root and leaves of Deianira erubescens, bark of Remijia
ferruginea [67].
Artemisinin (Artemotil) 65, obtained from traditional Chinese medicine
Artemisia annua, was approved in the year 2000 for the treatment of
chloroquine-resistant Plasmodium falciparum malaria and cerebral malaria.
The World Health Organization has strongly discouraged the use of 65 as a
monotherapy since malarial parasites are developing resistance to the drug.
However, combination therapies that include 65 are the preferred treatment
for malaria and are both effective and well tolerated in patients. Artemotil is
currently used only as a second line drug in severe cases of malaria and is
also increasingly being used against vivax malaria. As of May 2009,
arterolane (RBx11160, OZ-277) 66, a trioxolane modelled on artemisinin 65
pharmacophore, is under Phase III clinical development for the treatment of
malaria by Ranbaxy in combination with piperaquine [68].
Paromomycin 67 (HumatinTM, King Pharmaceuticals), an orphan drug
extracted from Streptomyces krestomuceticus [69], was approved in
September 2006 by Drug-Controller General of India for the treatment of
patients suffering from visceral leishmaniasis (VL). Paromomycin 67 was
developed by the Institute for OneWorld Health [70] and is an off-patent
antibiotic marketed in the US to treat intestinal parasites.
H3C
HO
NH2
H
N
HO
21
OH
O
O
HO
NH
NH
N
H
NH
H3C
HN
O
OH
HN
OH
O
HO
O
CH3
NH
HO
H
N
NH
O
CH3
N
H
N
OH
H3C
OH
OH
H3C
OH
H3C
HO
HO
61
H3C
63
H
N
OH
OH HO
O
H3C
OH
OH
OH
OH
OH
R1
H
N
62 R1 =
CH3
R2 =
NH
OH
CH3
N
CH3
OH
H3C
R2
CH3
CH3
OH
64 R1 = OH, R2 = H
H
H3C
O O
O
O
HO
HO
CH3
NH
O
NH2
O
HO
CH3
CH3
NH2
O
H2N
HO
NH2
O
OH
O OH
CH3
O
H2N
NH2
OH
65
66
67
4.4. Antiviral
Virus is a small infectious agent that can replicate only inside the living
cells of organisms bringing most common (i.e. cold, influenza, chickenpox
and cold sores) to greatest human health risk (i.e. ebola, AIDS, avian
influenza and SARS). Researches over last 25 years have resulted in the
identification of many natural product templates significant to antiviral drug
discovery, however fewer are in clinical investigation.
22
Betulinic acid 68, a topoisomerase I inhibitor isolated from bark of

Betula pubescens [71], is currently in Phase I clinical development.
Bevirimat (PA-457) 69, obtained from Syzygium claviflorum, was evaluated
by Panacos in Phase IIb trial for development as combination therapy with
other standard antiviral drugs. Bevirimat 69 inhibits the final step of the HIV
Gag protein processing and thus blocks HIV maturation [72]. In January
2009, Myriad Genetics announced for the acquisition of all rights from
Panacos for 69. Ribavarin 70, a NP-derived compound structurally similar to
pyrazomycin and showdomycin, was marketed as Rebetol until 2005 by
Schering Plough with Valeant Pharmaceuticals in the US. Valeant
Pharmaceuticals are developing taribavirin (Viramidine, ribamidine) 71, a
liver-targeting prodrug of ribavirin 70 [73], is in various Phase II/III trials for
the treatment of chronic hepatitis C virus (HCV). In 2006, 71 failed to meet
the non-inferiority efficacy endpoints in Phase III trials by Valeant. In 2007,
Valeant initiated another Phase IIb trial for 71 with higher doses and reported
the final results in June 2009 against HCV.
MBI-3253 (celgosivir, 6-O-butanoylcastanospermine) 72, a glucosidase
inhibitor and semi-synthetic derivative of indolizine alkaloid castanospermine
73 isolated from Castanospermum australe seeds [74], is an investigational
antiviral drug under clinical development by MIGENIX. As of January 2009,
MIGENIX has completed Phase II clinical studies of 72 as a triple
combination (with peginterferon -2b and ribavirin 70) and a double
combination (with peginterferon -2b) in HCV patients. After discontinuation
of exclusive option agreement with United Therapeutics Corporation (UTC)
in April 2009, MIGENIX are seeking other strategic options for further
development of 72.
Cyclosporin 74, a cyclophilin inhibitor obtained from Beauveria nivea,
exerts significant antiviral activity. However, due to calcineurin-related and
immunosuppressive side effects development of 74 as antiviral drug is not
possible [75]. NIM 811 (SDZ NIM 811, cyclosporin 29, MeIle4-cyclosporin)
75, discovered by Sandoz (now Novartis) with 1700 times less
immunosuppressive activity than cyclosporin 74 [76], was evaluated in
Phase I trial for anti-HIV and HCV activity. Likewise, debio-025 (UNIL025,
MeAla3EtVal4-cyclosporin) 76, a cyclophilin inhibitor with 7000 times less
immunosuppressive activity than 74, is being evaluated by Debiopharm in
various phase IIb trials for the treatment of HCV [77,78]. In February 2010,
Novartis in-licensed the exclusive rights to develop and market 76, as
potential first-in-class antiviral agent except in Japan.
4-Methylumbelliferone (Heparvit) 77 is a naturally occurring coumarin
that is in Phase II development by MTmedical Institute of Health and
BioMonde for the treatment of HBV and HCV. 1,5-DCQA (1,5-di-O-
23
caffeoylquinic acid) 78, a HIV-1 integrase inhibitor extracted from Inula

Britannic, is under human clinical trials by Chinese Academy of Military
Medical Sciences for treatment of HIV/AIDS and hepatitis B [79].
WAP-8294A2 (JA-002) 79, produced by the Gram-negative Lysobacter
species exerts antibacterial activity by interacting selectivly to membrane
phospholipids and causes sever damage to bacterial membrane [80]. The
aRigen Pharmaceuticals are evaluating injectible, gel and cream of 79 in
various Phase I/II trials to treat MRSA and acne. In August 2009, New
Energy and Industrial Technology Development Organization (NEDO), Japan
has decided for funding two-thirds R&D costs to aRigen Pharmaceuticals until
February 2011 for development of 79 as first-line anti-MRSA product
candidate.
CH2
H3C
R
H
N
CO2H
CH3 CH3 H
H
RO
H3C
CO2H
CH3 HO
CH3 O
H3C
H3C
70 R = O
72 R =
71 R = NH
73 R = H
CH3
O
H3C
O
N
CH3
CH3
CH3
CH3 O
H H
N
CH3 O
CH3
CH3 O
H
N
N
H
CH3 O
76
CH3
74 R =
N
R
N
CH3 O
O
CH3CH3 O
CH3
H3C
CH3
H3C
O
CH3 O
CH3
H
N
N
N
N
O
N
H
H
O
H3C
CH3 O
CH3
H3C
N
HO
CH3
CH3 O
O
CH3
CH3 O
H H
N
CH3
H3C
OH
CH3
CH3
O
CH3
OH
OHOH
69 R =
N
HO
H
CH3
OR
CH3
68 R = H OH C CH
3
3
H3C
NH2
HO
CH3
CH3
CH3
75 R =
CH3
HO
CH3
CH3
CH3
N
O
CH3
CH3
HO
CH3
CH3CH3 O
H3C
CH3
CH3
N
77
N
H
HO
O
HO
HO2C
HO
O
78
OH
OH
24

NH2
HO
H
N
O
H
N
HN
O
O
NH2
H3C
O
H
N
NH2
H3C
N
H
H
N
H
O
O
N
H
H3C
OH H3C
O
CH3 HO
CH3
H
N
CH3
N
O
O
N
H
O
CH3
O
NH
OH
NH2
79
5. Neurological diseases
Historically, the alkaloids like morphine 80 isolated from Papaver
somniferum and physostigmine 81 extracted from Physostigma venenosum,
were used to treat sever pain and diseases of central nervous system (CNS).
(+)-huperzine A 82, a sesquiterpene alkaloid and acetylcholinesterase
(AChE) inhibitor extracted from Huperzia serrata [81], is being evaluated by
Chinese scientists against Alzheimers disease. The National Institute on
Aging (NIA) is evaluating orally administered formulation of 82 in Phase II
trials against Alzheimer's disease [82]. Morphine-6-glucuronide (M6G) 83,
produced by metabolism of artemisone (BAY 44-9585) 84 (obtained
through semi-synthesis from artemisinin 63) in human body, was evaluated
successfully by CeNeS Pharmaceuticals as significant analgesic in Phase III
trials in Europe. PAION in June 2008 acquired CeNeS Pharmaceuticals
and later in November 2008 disclosed for completion of two Phase III trials.
A spicamycin derivative KRN-5500 85, obtained from Streptomyces
alanosinicus [83], was evaluated by DARA BioSciences in Phase I trials
against neuropathic pain. DARA BioSciences are currently running Phase IIa
trials of 85 given intravenously (IV) to cancer patients suffering from
neuropathic pain [84].
Debio 9902 (ZT-1) 86, synthesized by Shanghai Institute of Material
Medica, is a prodrug of 82 licensed to Debiopharm. Debiopharm in June
2007 announced the positive results of a Phase IIa trial of 86 against mild
Alzheimers disease. As of October 2008, Debiopharm have started tablet
25
formulation bridging study of 86 as Investigational New Drug (IND) to treat

Alzheimers patients. Lobeline 87, a VMAT2 ligand [85] reducing the
methamphetamine induced dopamine release, is a significant tobacco smoking
cessation agent occurring in Hippobroma longiflora [86]. Lobeline 87 is being
evaluated by Yaupon Therapeutics and NIH as a dopamine modulating agent
under Phase II trials against ADHD and methamphetamine addiction.
Anabaseine 88, isolated from marine worms of the phylum
Rhynchocoela [87], stimulates the neuronal nicotinic receptors thus has been
considered significant in the treatment of Alzheimers disease as Alzheimers
brain loses many of its nicotinic receptors by the time of death [88]. The 3(2,4-dimethoxybenzylidene)-anabaseine (DMXBA; also called GTS-21) 89, a
synthetic derivative of 88, was evaluated against Alzheimers disease in a
sponsored research by Taiho Pharmaceutical to Kems University of Florida.
HO
O
H
CH3
H3C
H3C
H
N
O
N
O
CH3
N H
RO
CH3
80 R = H
81
HO
83 R =
OH
OH
CO2H
CH3
H3C
H
N
H3C
H
H
N
O
O
O
O
H3C
H2N
CH3
82
84
H3C
N
H
85
OH
HO
H
N
O
HO
N
H
OH
NH
N
26

CH3
O
OCH3
OH
H
N
H3C
CH3
O
N
OCH3
87
HO
N
H3CO
Cl
86
88
89
The University of Florida licensed 89 to Osprey Pharmaceuticals whose

assets were purchased by CoMentis (previously Athenagen) in April 2006.
CoMentis are currently assessing 89 in various Phase I/II trials for safety
assessment and cognitive improvement in ADHD patients.
Tetrodotoxin (TectinTM, Wex Pharmaceuticals) 90, extracted from the
puffer fish [89], blocks the action potentials in nerves through binding to
sodium channels in cell membrane [90]. Wex are evaluating 90 in colaboratin
with Chinese medical institute against cancer pain and management of opiate
withdrawal symptoms in Phase III and I trials, respectively. Also a Phase IIa
trial of 90 against neuropathic pain caused by cancer chemotherapy is
underway by Wex Pharmaceuticals.
Capsaicin 28 and related compounds (called as capsaicinoids) are
produced by chili peppers as irritants against certain herbivores and fungi.
Among capsaicinoids, Xen-2174 91, obtained from venom of Conus
marmoreus targeting norepinephrine transporter (NET) was discovered by
researchers at the University of Queensland. Xenome are associated with
Phase II development of 91 against acute post-operative and chronic pain in
cancer patients resistant to morphine and hydromorphone. Anesiva are
evaluating capsaicin 28 (coded 4975, ALGRX 4975, AdleaTM) in various
clinical trials against pain indications such as severe post-surgical pain, posttraumatic neuropathic pain and musculoskeletal diseases [91]. Anesiva in
December 2008, disclosed to meet primary end point in a phase III trial of 28
against acute pain following orthopedic surgery. Winston Laboratories
are associated with Phase III trials of civamide (cis-capsaicin, zucapsaicin,
WL-1001) to treat episodic cluster headache and knee osteoarthritis. Winston
in October 2008 filed a NDS to Canada for Civanex (civamide 0.075%) to
use against osteoarthritis pain. In February 2009, an orphan drug designation
to Civanex was given by FDA with NON release to Winston Pharmaceuticals in
October 2009.
27
Phlorizin 92, a flavonoid that belongs to the group of dihydrochalcones

obtained from bark of pear (Pyrus communis), apple, cherry and other fruit
trees (family-Rosaceae), is a sodium glucose co-transporters (SGLTs)
inhibitor that lowers glucose plasma level and improves insulin resistance
[92] but has poor intestinal absorption and become inactive by lactasephlorizin hydrolase. Dapagliflozin (BMS-512148) 93, a 92 derivative that
selectivly inhibits SGLT2, is under clinical development by Bristol-Myers
Squibb (BMS) in collaboration with AstraZeneca for the treatment of 2
diabetes. In October 2009, the BMS announced the positive results of
Phase III placebo controlled trial of 93.
O
HO
H2N
O
N
HN H
HO
OH
NGVCCGYKLCHOC
OH
90
OH
91
Resveratrol 94, a triphenolic stilbene occurring in many plants is

significant against clinical indications such as cancer, ischemic injuries and
cardiovascular disease [93]. Resveratrol 94 is an agonist of Saccharomyces
cerevisiae silent information regulator (Sir2) protein, a class III histone
deactylase whose presence causes extention of lifespan in S. cerevisiae,
Caenorhabditis elegans and Drosophila melanogaster [94]. Italian scientists
in 2006 observed 56% increase in median life span of Nothobranchius furzeri
[95], a fish when supplemeted with 94. SRT-501, a formulation of 94 by
Sirtris Pharmaceuticals, acts by increasing mitochondrial activity and is under
clinical investigations against diabetes and obesity. Sirtris has announced the
positive results of Phase IIa trial in which oral doses of 1.25 or 2.5 grams of
SRT501 was found safe at twice daily dosing for 28 days in type 2 diabetes.
A similar Phase IIa cancer trial with SRT501 is under way.
Cannabinoids are a group of secondary metabolites responsible for
pharmacological properties of Cannabis sativa (cannabis plant) [96].
CP 7075 (IP 751, ajulemic acid, CT-3) 95, a synthetic cannabinoid,
suppressing IL-1 and matrix metalloproteinases (MMPs) through a
peroxisome proliferator-activated receptor (PPAR) -mediated mechanism
[97], was investigated by Indevus Pharmaceuticals in pre-clinical studies. In
October 2007, the drug was licensed by Cervelo Pharmaceuticals for Phase I
trials in neuropathic pain.
28

HO
HO
HO
OH
HO
HO
HO
O
OH
OH
OH
Cl
O
OH
CH3
93
92
CO2H
OH
OH
HO
H3C
OH
94
H3C
CH3
H3C CH3
95
6. Cardiovascular and metabolic diseases

Natural products have played an important role in development of drugs
against cardiovascular and metabolic diseases. Simvastatin (Zocor, Merck),
a lipid-lowering statin obtained from fermentation product of Aspergillus
terreus, inhibits 5-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA)
reductase. Orlistat (Xenical), a lipstatin derivative isolated from
Streptomyces toxytricini [98], inhibits pancreatic lipases and used for the
treatment of obesity. Captopril, ramipril and quinapril are the examples of
some antihypertensive angiotensin-converting enzyme (ACE) inhibitors
derived from the snake venom. An endopeptidase (NEP) inhibitor, ilepatril
(AVE-7688) 96, is being developed by Sanofi-Aventis in various Phase IIb/III
trials to treat hypertension while phase II trial for diabetic nephropathy.
(+)-1-Deoxygalactonojirimycin 97 and (+)-galactonojirimycin 98
obtained from Streptomyces species [99] display strong inhibitory activity
toward several -galactosidases. Miglustat (Zavesca) was used earlier to
treat Type 1 Gaucher disease (GD1) by Actelion. Migalastat (AmigalTM,
AT1001, 1-deoxygalactonojirimycin, 1-deoxygalactostatin) 97, a semisynthetic derivative of 98, stabilizes protein structures and restores correct
folding through binding with them. (+)-1-Deoxygalactonojirimycin 97 is an
orphan designated drug by European Commission in May 2006 to use in
treatment of Fabry disease. As of January 2010, the 97 is being evaluated
under Phase III trials by Amicus Therapeutics in collaboration with Shire
29
Pharmaceuticals against Fabry disease. Isofagomine (PliceraTM, AT2101) 99,

an aza-sugar that mimics the carbocation transition state used by glycosidases
[100], is under clinical development by Amicus Pharmaceuticals to treat
Gauchers disease [101]. Amicus in October 2009 announced the positive
results of a Phase II trial for two dose regimens consisting of 225 mg of 99
given three days on/four days off and seven days on/seven days off.
Ruboxistaurin (LY333531) 100, inhibiting protein kinase C (PKC), is
being developed by Eli Lilly to use in the treatment of microvascular
complications in diabetes mellitus [102]. In February 2006, Lilly submitted a
NDA for use of 100 in diabetic peripheral retinopathy. In August 2006, the
FDA essued an approvable letter to Lilly while suggesting another Phase
III trial for additional efficacy. SCH 530348 (TRA) 101, a PAR-1 antagonist
[103] similar to himbacine 102 obtained from Galbulimima baccata,
is in Phase III clinical trials by Schering-Plough for the treatment of
cardiovascular diseases such as atherosclerosis, ischemia, myocardial
infarction and stroke.
H
N
CO2H
O
NH
H3C
H
N
HO
H
N
HO
OH
HO
OH
OH
OH
SAc
H3C
97 R = H
98 R = OH
96
H
N
H 3C
H
N
99
O
O
CH3
O
H3C
H3C
N
CH3
O
N
H3C
CH3
F
100
101
102
30
Genaera Corporation are associated with clinical development of

trodusquemine (MSI-1436) 103 and squalamine 104 extracted from Squalus
acanthias [104]. MSI-1436 103 is a protein tyrosine phosphatase 1B inhibitor
[105] that is being evaluated by Genaera in a second Phase I trial using an
ascending single dose in overweight type 2 diabetics under the obesity IND.
Ouabain (g-strophanthin) 105, a cardiac glycoside occurring in ripe seeds
of Strophanthus gratus and bark of Acokanthera ouabaio, involves binding to
and inhibition of the plasma membrane Na+/K+-ATPase attainable in vitro or
with intravenous dosage [106]. Likewise, digoxin 106, isolated from Digitalis
lanata (foxglove plant) [107], also exists in the human adrenal gland and is
significant in atrial fibrillation and atrial flutter. Rostafuroxin (PST 2238)
107, an ouabain antagonist, is under Phase II development by Sigma-Tau to
use in the treatment of chronic arterial hypertension.
H3C
OSO3H
CH3
H
CH3
H
N
R
N
H
N
H
CH3
H3C
H
OH
103 R =
N
H
104 R = H
NH2
O
HO
CH3
HO
OH
H
CH3
HO
H3C
OH
OH
CH3
105
OH
O
HO
O
O
106
CH3
OH
CH3
H3C
H3C
H3C
OH
O
CH3
HO
O
HO
CH3
O
N
O
OCH3
O
CH3
CH3
H3C
H3C
O
H
OH
OH
OH
OH
CH3
CH3
OH
CH3
NH2
CH3
HO
OH
H3C
CH3
107
H3C
H3C
H3C
OH
OH
108
109
31
Mitemcinal (GM-611) 108, an agonist of motilin that lacks the antibiotic

properties of 51 and increases the amplitude & frequency of antral
contractions and initiates gastric contractions, was discovered by Chugai
Pharma. Phase I trials of 108 in Japan has been completed by Chugai while
Phase II trials in US are still running against diabetic reflux oesophagitis and
idiopathic gastroparesis [108]. Chugai are also conducting Phase II trials of
108 against irritable bowel syndrome (IBS).
Pyridoxamine (PyridorinTM) 109, consisting of a pyridine ring bearing
hydroxyl, methyl, aminomethyl, and hydroxymethyl substituents, is a vitamin
B6 analogue [109] that was evaluated by BioStratum in two phase II
trials demonstrating retardation of diabetic nephropathy. In October 2006,
BioStratum licensed 109 to NephroGenex, which has initiated a new Phase
IIb trial in patients with type 2 diabetes. Taisho Pharmaceutical is evaluating
109 (coded as K-163) in Phase II trials against diabetic nephropathy. In
January 2009, the FDA ruled for regulation of 109 as a pharmaceutical drug
and awarded a fast track drug designation.
7. Immunological, inflammatory and related diseases

Autoimmune and inflammatory disease condition arises through aberrant
reactions of the human adaptive or innate immune systems. Aspirin,
discovered in the late 1890s, is still a significant analgesic and antiinflammatory drug. Salbutamol, a 2-adrenergic receptor agonist, is marketed
by GlaxoSmithKlinen to treat asthma and chronic obstructive pulmonary
disease. Cyclosporin 74 (1983), tacrolimus (1993), sirolimus 10 (1999),
mycophenolate sodium (2003) and mycophenolate mofetil (1995) are among
some important immunosuppressive drugs sourced from natural products.
Everolimus (LuveniqTM or LX211) 110, a derivative of 10 inhibiting
mTOR, is marketed by Novartis as immunosuppressant Certican in organ
transplantation.
Voclosporin (ISA-247, R1524, LX211) 111, a derivative of 74 inhibiting
calcineurin [110], is under Phase IIb trial to prevent kidney graft rejection
and Phase III trial against psoriasis. Voclosporin 111 was licensed by Lux
Biosciences from Isotechnika for ophthalmic indications. As of March 2009,
Lux Biosciences have completed Phase III trials of 111 oral capsules against
uveitis. In February 2010, Lux Biosciences filed a NDA to the FDA and
MAA to the EMA for 111 under LuveniqTM against non-infectious uveitis,
which were accepted by respective agencies in March 2010. Eupatilin 112, a
flavone isolated from Korean traditional medicine Artemisia argyi possess
efficacy against chronic diarrhea [111]. DA-6034 113, a synthetic 112
derivative, is being developed by Dong-A Pharmaceuticals in Phase I and II
trials against dry eye and gastritis, respectively.
32
H3C
O
CH3
O
OH
O
CH3
O
HO
CH3
O
N
CH3
O
CH3
CH3
O
CH3
O
O
HO
O
H3C
CH3
110
H3C
CH2
CH3
HO
CH3
CH3
H H
N
H3C
N
O
CH3
CH3
CH3
CH3
O
CH3
O
H
N
CH3
CH3
H3C
CH3
N
H
CH3
N
CH3
CH3
CH3
H3C
CH3
N
H3C
N
H
O
CH3
CH3
111
OCH3
HO
OCH3
O
O
O
OCH3
HO
OCH3
H3CO
OH
112
OCH3 O
113
8. Oncological diseases
8.1. Small-molecule anticancer agents
8.1.1. Plant-derived compounds
Camptothecin 114, a topoisomerase I inhibitor isolated from
Camptotheca acuminata [112], exhibits significant anticancer activity.
Among other camptothecin class of drugs, belotecan (Camptobell, CDK-602)
was developed and launched in 2004 by Chung Kun Dong in Korea [113].
BNP-1350 (Karenitecin) 115 is an investigational drug under clinical
33
development by BioNumerik Pharmaceuticals for cancer chemotherapy. As of

February 2008, BioNumerik are running the Phase III trial of 115 against ovarian
cancer [114]. Diflomotecan (BN80915) 116, a 115 analogue [115], is being
developed by Ipsen under Phase II trials to treat advance metastatic cancers.
Gimatecan (ST-1481) 117, an oral topoisomerase I inhibitor, is currently in Phase
II development by Novartis against solid tumors [116]. Elomotecan (BN-80927,
LBQ707, R-1559) 118, inhibiting topoisomerase I and II, is a promising Phase I
pipeline by Ipsen in oncology (e.g. colon, breast and prostate cancer) [117]. DRF
1042 105 is a 114 derivative evaluated by Dr. Reddys Laboratories in Phase I
trials to use in the treatment of various cancers [118]. Dr. Reddys Laboratories in
September 2006 collaborated with ClinTec International for joint Phase II/III
development of 119. In September 2006, Sonus Pharmaceuticals initiated a
Phase I study of SN2310 120, a prodrug of SN-38 121 to address cancer is
presently ongoing. In May 2008, Sonus murged with OncoGenex Technologies
and the new company, OncoGenex Pharmaceuticals has included 120 as a strong
oncology pipeline.
R
F
O
N
N
F
114 R = H
N
O
H3C
HO
H3C
Si(CH3)3
117 R =
HO
115 R =
116
C(CH3)3
H3C
OH
N
O
O
H3C
O
N
Cl
N
O
H3C
CH3
CH3
CH3
HO
HO
118
H3C
119
CH3
H3C
CH3
O
H3C
O
CH3
CH3
O
N
120
N
O
H3C HO
34
Combretastatin A-4 phosphate (ZybrestatTM, CA4P) 121, a prodrug of

combretastatin A-4 122 obtained from South African Bush Willow
Combretum caffrum [119], is a reversible tubulin depolymerizing agent that
causes tumour-associated endothelial cells to change from a flat to a round
shape, thus by plugging the blood vessels deprives the tumour from oxygen
and nutrients. Oxigene are evaluating 121 as a vascular disrupting agent
(VDA) [120] and as on September 2008, Phase III trial against anaplastic
thyroid cancer (ATC) is underway. Oxigene in November 2009 disclosed the
positive results of a Phase II trial of 121 in non-small cell lung cancer
(NSCLC). Ombrabulin (AVE8062) 123, another 122 derivative licensed to
Sanofi-Aventis from Ajinomoto, is under Phase III trials in advanced STS
patients. Combretastatin A-1 diphosphate (OXi4503) 124, a pro-drug of
combretastatin A-1 125 that is capable of binding to proteins and nucleic
acids [121], is under various Phase I trials by OXGENE to use in the
treatment of advanced-stage solid tumors. Noscapine (CB3304, noscapine)
126, a benzylisoquinoline alkaloid occurring in the plants of family
Papaveraceae, is a microtubule targeting antitussive currently in Phase I/II
trials by Cougar Biotechnology for the treatment of multiple myeloma [122].
Vinblastine (Alkaban-AQ, Velban) 127, a microtubule inhibitor
isolated from Catharanthus roseus [123], has been found significant when
given intravenously to patients suffering from Hodgkin's disease, nonHodgkin's lymphoma, Kaposi's sarcoma, choriocarcinoma, TCL, breast,
testicular, lung, neck and head cancers. Vinflunine (Javlor) 128, a
fluorinated vinca alkaloid [124] discovered by Laboratoires Pierre Fabre, was
submitted for registration with the EMEA in June 2008, after positive Phase
III trial for metastatic treatment of bladder cancer. In June 2009, Pierre Fabre
received a positive opinion with recommendation for marketing authorization
of 128 in the metastatic treatment of bladder cancer.
Paclitaxel (TaxolTM, AbraxaneTM) 129, isolated from Taxus brevifolia
[125], is a mitotic inhibitor that stabilizes microtubules and interferes with
the normal breakdown of microtubules during cell division. Bristol-Myers
Squibb (BMS) are associated with commercial development of 129.
Cabazitaxel (XRP6258) 130 and larotaxel (XRP9881) 131 have been
designed by Sanofi-Aventis as poor substrates for membrane-associated
P-glycoprotein (P-gp), overexpressed in taxane resisting cells [126] and are
in Phase III trials against pancreatic and hormone-refractory prostate
cancers [127]. Luitpold Pharmaceuticals are developing DHA-paclitaxel
(Taxoprexin) 132, a fatty acid conjugate of 129, in Phase III trials against
metastatic melanoma [128]. Spectrum are associated with Phase I/II
development of intravenous/oral ortataxel (IDN-5109, BAY-59-8862) 133, a
third generation taxane with toxicity/tolerance profile similar to 129. As of
35
June 2009, the 133 is under Phase II trials in taxane-refractory solid tumors [129].
Milataxel (MAC-321, TL-00139) 134, a poor substrate for P-gp, is under Phase II
clinical development by Wyeth Pharmaceuticals to use in the treatment of
colorectal neoplasms [130]. Tesetaxel (DJ-927) 135, an orally administered semisynthetic taxane, was evlaulated by Genta in various Phase I/II trials against
advanced gastric and breast cancer [131]. The Phase II clinical trials of 135 are
running for the treatment of patients with advanced melanoma having a normal
serum lactate dehydrogenase (LDH) and have progressed after one chemotherapy
regimen. Other taxanes that are in Phase II clinical development, i.e. TPI-287 136
by Tapestry Pharmaceuticals and BMS-188797 137 by Bristol-Myers Squibb, to
treat patients suffering from pancreatic and advanced malignancies, respectively
are currently in Phase II clinical development [132].
R
OCH3
H3CO
NH2
H
N
O
OCH3
OH
OCH3
H3CO
OCH3
OCH3
OCH3
121 R = OPO3Na2
122 R = OH
123
OR
OR
O
OCH3
H3CO
CH3
O
H3C
OCH3
OCH3
H3C
124 R = PO3Na2
125 R = H
126
OH
CH3
N
OAc
H
CH3 CO2CH3
N
OAc
H
CH3 CO2CH3
128
CH3
CH3
O
H3C
H3C
O
CH3OH
CH3
NH
H3CO
H3C
CH3
O
HO
O
CH3
CH3 O
CH3
CH3
OH
CH3
OH
O
O
129
CH3
OH
H3CO
127
N
H
H3CO2C
CH3
OH
H3CO
F
CH3
N
H
H3CO2C
NH
CH3
HO
O O
O
O
H3C
O H3C
130
36
Acronycine 138, an alkaloid isolated from Acronychia baueri, exhibits

activity against various solid tumors such as S-180 and AKR sarcomas,
X-5563 myeloma, S-115 carcinoma and S-91 melanoma. S23906-1 139, a
benzoacronycine derivative inhibiting DNA synthesis and S-phase cell cycle
arrest, is currently in Phase I trials for the treatment of solid tumors [133].
CH3
CH3
O
O
CH3
NH
H3C
H3C
NH
CH3
CH3
HO
H3C
CH3
O
O
H3C
132
CH3
CH3
O
H3C
O
O
CH3 OH
H3C
CH3
CH3
NH
H3C
CH3
OH
HO
O
O
O
OH H3C
O
CH3 O
CH3
O
O
CH3
133
HO
H3C
O
OH
NH
CH3
131
O
CH3 OH
CH3
O
O
H3C
CH3
CH3
CH3
H3C
H3C
O
OH
O
H3C
H3C
134
Homoharringtonine (omacetaxine mepesuccinate, Ceflatonin) 140, a

myelosuppressive alkaloid isolated from Cephalotuxus fortuneii, inhibits
Mcl-1 protein synthesis and induces apoptosis [134]. The European
Commission in October 2004 granted orphan designation to Stragen France
SAS for 140 against acute myeloid leukemia (AML) that was later transferred
to ChemGenex Europe SAS in January 2009. The FDA in January 2009
designated 140 as orphan drug against myelodysplastic syndromes (MDS).
In September 2009, a NDA for 140 under Omapro (omacetaxine
mepesuccinate) was submitted by ChemGenex to the FDA for use in the
treatment of chronic myelogeneous leukemia (CML) patients having T315I
37
mutation or failed in imatinib therapy. ChemGenex are also running Phase II

trial of 140 for treatment of refractory or relapseed AML patients failed to
intensive chemotherapy.
CH3
CH3
O
CH3
CH3
O
NH
CH3
CH3
CH3
N
CH3
CH3
CH3
N
O
CH3
H3C
H3C
O
NH
O
O
CH3 O
H3C
CH3
CH3
OH
CH2
OH
HO
H3C
CH3
HO
O
O
NH
O
H3C
136
H3C
H3C
H3C
135
O
CH3 OH
OCH3
CH3
O
CH3
OH
HO
N
O
O
H3CO
137
CH3
CH3
R
CH3
R
138 R = H
139 R = OAc
3-O-methyl-nordihydroguaiaretic acid (NDGA) 141, a lignan isolated

from Larrea divaricatta exhibits significant anticancer activity by retardation
of tumor cell proliferation through inhibition of insulin-like growth factor
receptor (IGF-1R) and the c-erbB2/HER2/neu receptor. Terameprocol 142, a
synthetic 141 derivative that induces apoptosis in cancer cells through
inactivation of maturation promoting factor, was licensed by Erimos from
The Johns Hopkins University. Terameprocol 142 is currently in various
Phase I/II trials by Erimos against solid tumors, glioma and leukemia [135].
Epipodophyllotoxin (F11782) 143, a non-intercalating dual inhibitor of both
topoisomerases I and II, was originally isolated from root of Podophyllum
peltatum [136]. Tafluposide 144, a 143 derivative, is being developed by
Pierre Fabre under Phase I/II trials for various tumor types [137]. Ingenol
145, isolated from the sap of Euphorbia peplus, is under clinical development
by Peplin Biotech for topical treatment of basal cell carcinomas and
squamous cell carcinomas [138]. Afer merger of Peplin with LEO Pharma in
November 2009, ingenol mebutate (PEP005) 146, a 145 derivative that
38
activates PKC, is currently in Phase III trials against actinic keratosis (AK).
In December 2009, LEO Pharma disclosed the positive results of 146 in two
Phase III trials against AK lesions on head (including the face and scalp)
while announced to meet the primary endpoint in February 2010 with
disappearance of AK lesions in non-head locations.
Daidzein 147, an isoflavone occurring in Pueraria Mirifica, soybeans
and soy products, exhibits clinical indication against tumors [139].
Phenoxodiol 148 is a synthetic 147 derivative that was licensed by Marshall
Edwards from Novogen for development as combination therapy against
ovarian cancer and as mono-therapeutic agent for the treatment of prostate
and cervical cancers, resistant to standard chemotherapy [140]. Phenoxodiol
148 is supposed to inhibit sphingosine-1-phosphate and is under Phase III
development by Marshall Edwards to restore chemosensitivity in patients
with ovarin cancer resisting platinum drugs. A phase II trial of 148 against
castrate and noncastrate prostate cancer is also uderway. Triphendiol
(NV-196), an orally-delivered chemosensitizing derivative of 148 that was
licensed to Marshall Edwards by Novogen, is under Phase I trials for use in
combination therapy against cholangiocarcinoma, advanced prostate cancer
and melanoma. An orphan drug status was granted to 148 by the FDA for
cholangiocarcinoma, prostate cancer and stage IIb-IV malignant melanoma.
In January 2009, FDA granted IND approval to 148. Genistein 149, a
soy-derived antineoplastic phytoestrogen, inhibits protein-tyrosine kinase and
induces cell differentiation, is under Phase I/II trials by Astellas, Bausch
& Lomb for treatment of tumors. Genistein 149 is also supposed to
inhibit topoisomerase-II, resulting in DNA fragmentation and apoptosis.
(-)-Gossypol (AT-101) 150, a pan-Bcl-2 inhibitor isolated from the
cottonseed plant of genus Gossypium [141], is under Phase I/II clinical
development by Ascenta Therapeutics to address prostate, brain and lung cancers.
In October 2009 Ascenta announced the results of Phase I trial for two
combination regeims containing 150 for the treatment of malignant brain tumor.
OH
H3C
CH3
H3C
OH
CH3
O
OH
O
OCH3
H3C
O
O
H3CO
OR
OR
O
H
OR
H3CO
OCH3
OCH3
141 R = H
140
142 R = CH3
143
39
ASA 404 (vadimezan, AS1404 and DMXAA) 151, a tumor-VDA and

derivative of flavone-8-acetic acid 152, was discovered at Auckland Cancer
Society Research and later in-licensed by Antisoma. Novartis AG in April
2007 signed an agreement with Antisoma for worldwide rights and co-selling
of 151 in the US. As of April 2008, the 151 is currently in Phase III clinical
trials by Novartis as a second line treatment for NSCLC. The -Lapachone
(ARQ-501) 153, isolated from Tabebuia avellanedae, induces expression
of cyclin dependent kinase inhibitor 1A (CDKN1A or p21) and exerts
anti-tumor effect by sustained increase of the pro-apoptotic protein E2F-1
[142]. The -Lapachone 153 is currently in Phase II trials by ArQule as a
combination therapy against pancreatic and ovarian cancer.
Alvocidib (Flavopiridol, HMR 1275) 154, a CDK inhibitor and synthetic
derivative of rohitukine 155 isolated from Dysoxylum binectariferum [143],
is being developed by Sanofi-Aventis in collaboration with NCI. As on May
2009, the 154 is under late Phase III clinical development by Sanofi-Aventis
against NSCLC while Phase IIb trial for the treatment of chronic lymphocytic
leukemia (CLL).
F
CH3
CH3
F
H3C
F
F
H3C
H3C
RO HO
HO
HO
O
O
145 R = H
CH3
146 R =
CH3
O
H
OH
R
H3CO
OCH3
OH
O
P
144
OH
HO
147
Curcumin 156, isolated from Curcuma longa roots, can interfere with the
p53 tumor suppressor pathway and is under various Phase I/II trials world
wide while a Phase III trial for the treatment of metastatic colon cancer
(MCC) is underway [144]. RTA 402 (CDDO-Me, Bardoxolone methyl) 157,
40
an IkB alpha kinase activation inhibitor and synthetic derivative of oleanolic

acid 158 [145], is being evaluated by Reata Pharmaceuticals under Phase I/II
trials against prostate cancer and Phase II trials for the treatment of type 2
diabetes with chronic kidney disease (CKD). RTA 402 157 is an orphan drug
by the FDA against prostate cancer. In January 2010, Kyowa Hakko Kirin
gained exclusive rights from Reata Pharmaceuticals to develop and
commercialize 157 in Japan and other selected Asian regions to treat type 2
diabetes with CKD.
O
OH
OH
OH HO
OH
OH
HO
HO
HO
HO
H3C
CH3
H3C
CH3
148
OH
CH3
149
150
OH
H3C
Cl
O
HO
O
OH
CH3
CH3
HO2C
O
N
HO2C
151
H3C
152
CH3
CH3
154
153
CH3
O
O HO
O
N
HO
OH
155
CH3
H3CO
O
OCH3
HO
OH
156
Betulinic acid (ALS-357) 68, a topoisomerase I inhibitor isolated from

Betula pubescens [146], is an orphan drug (by the FDA) in Phase I trial by
Advanced Life Sciences for the treatment of malignant melanoma. Silybin
159, a flavonolignan isolated from Silybum marianum, is the active
constituent of IdB 1060 (silybin-phosphatidylcholine complex, Siliphos).
Silybin 159 is currently in Phase II trials by American college of
gastroenterology for chemoprevention of cancer [147].

H3C
41
CH3
H3C
CH3
OH
OCH3
OH
CH3
CH3
CO2CH3 CH
3
CH3
O
H3C
H
HO
H
CH3
CH3
H3C
CO2H
O
HO
CH3
OH
H
CH3
OH
157
159
158
8.1.2. Microorganism-derived compounds

8.1.2.1. Actinomycetes
Pladienolide D 160, obtained from fermentation broth of Streptomyces
platensis Mer-11107, exerts significant antiproliferative activities against
variety of cancer cell lines. E7107 161, a synthetic 160 derivative that binds
with spliceosome-associated protein 130 (SAP130) and inhibits the splicing
of pre-mRNA, is in various Phase I trials by Eisai against solid tumors [148].
Chartreusin (U-7257) 162 isolated from Streptomyces chartreuses and
elsamicin A (BMY-28090, elsamitrucin) 163 isolated from actinomycete
strain J907-21, are the antibiotics that inhibit RNA synthesis and result
in single-strand scission of DNA [149]. Elsamicin A 163 is also a
topoisomerase I/II inhibitor being developed by Spectrum Pharmaceuticals in
Phase II trials to use in the treatment of advanced solid tumors.
OR
OH
CH3
OH
H3C
H3C
O
O
CH3
H3C
OH
160 R = Ac O
161 R =
OH
CH3
Doxorubicin 164, an anthracycline antibiotic capable of intercalating

with DNA, was isolated from bacteria occurring in soil samples taken from
Castel del Monte, an Italian castle. Doxorubicin 164 is an orphan drug by the
FDA against acute lymphocytic leukemia (ALL) and AML. Valrubicin
(Valstar), a semi-synthetic 164 derivative was approved in 1999 for
the treatment of bladder cancer but was withdrawn in 2002 due to
some manufacturing issues and has been relaunched in September 2009.
L-annamycin 165, a topoisomerase II inhibitor that was developed at the MD
Anderson Cancer Center, is currently in Phase I/IIa trials by Callisto
42
Pharmaceuticals for the treatment of younger and adults with refractory or

relapsed ALL or AML. Berubicin (RTA744, WP744) 166, a DNA
intercalator capable of crossing the BBB, hence is significant for the
treatment of primary brain tumor. Reata Pharmaceuticals are associated with
Phase II development of 166 against malignant gliomas. Likewise,
sabarubicin (MEN-10755) 167 [150], a topoisomerase II inhibitor and
disaccharide analogue of 164, is currently in Phase II clinical trials by
Menarini Pharmaceuticals against solid tumors [151]. Nemorubicin
(MMDX, PNU-152243A) 168, a 3-deamino-3[2-(S)-methoxy-4morpholinyl] derivative of 164, is a topoisomerase I inhibitor exhibiting
activity against selected tumors resistant to current treatment. Nerviano
Medical Sciences are evaluating 168 in Phase I/II trials.
Distamycin A 169, a DNA minor grove binder (MGB) and lead
compound of brostallicin (PNU-166196) 170, was originally developed by
Nerviano [152]. Nerviano had transferred the exclusive world right of 170 to
Systems Medicine Inc (SMI) which was taken over by the Cell Therapeutics.
Currently, the 170 is in phase II trials by Cell Therapeutics as monotherapy
against metastatic or advanced stage STS.
H3C
H3C
O
OH
HO
OH
OH
HO
CH3
O
CH3
HO
H3C
OH
H3C
H3C
OH
OH
CH3
O
O NH2
OH
164 R = H
166 R =
163
O
H3C
162
H2N
O OH
OH
OH
CH3
O
O
O
CH3
O
OH
OH
O
O
OH
OH
O
OH
OH
OH
OH
OH
O
OH
O
O
OH
H3C
HO
HO
O
O
HO
CH3
H3C
OH
H3C
O
H3C
CH3
O
OH N
O
OH
165
H3C
NH2
167
168
43
Geldanamycin 171 is an antineoplastic benzoquinone ansamycin

antibiotic and was discovered from broth and mycelium of Streptomyces
species [153]. Tanespimycin (17-AAG, KOS-953, NSC-330507) 172, a
comparatively less toxic antibiotic derived from 171, can bind to HSP90 and
interrupts the MAPK pathway. As on November 2009, Kosan have
completed a Phase II/III trial of 172 in combination with Velcade against
relapsed-refractory multiple myeloma. Alvespimycin (17-DMAG, KOS1022, NSC-707545) 173, a second generation HSP90 inhibitor [154] is in
clinical development by Kosan to use in the treatment of solid tumors. As on
January 2008, 173 is in Phase I trials in combination with trastuzumab &
paclitaxel (Taxol) against solid tumors, Phase II monotherapic trials against
HER2-positive metastatic breast cancer and Phase I trials for the treatment of
solid tumors. Retaspimycin (IPI-504, 17-AAG hdroquinone salt) 174, a
HSP90 inhibitor, is being developed by Infinity Pharmaceuticals in Phase I/II
clinical trials to address certain cancers. Currently, the Infinity are evaluating
174 in a Phase II trial against NSCLC while enrolling patients for another
Phase II trial in combination with Herceptin against breast cancer.
H
NH
O
H
N
Br
H2C
CH3
H
N
O
N
NH
CH3
H
N
NH2
169
N
CH3
H
N
O
O
CH3
NH
H
N
O
N
CH3
H
N
NH
N
CH3
170
H
N
N
H
N
CH3
NH2
Deforolimus (AP23573, MK-8669) 175, is an mTOR inhibitor codeveloped by Merck and ARIAD Pharmaceuticals to address several tumor
types including sarcoma. The name of 175 was changed to ridaforolimus by
ARIAD in May 2009 and as on December 2009, the enrollment for a Phase
III study in patients with metastatic STS and bone sarcomas has been
completed by ARIAD. Besides, ARIAD are also running several Phase I/II
trials of 175 as a single agent and in combination therapies. Salinosporamide
A (NPI-0052) 176, a proteasome inhibitor produced by a marine bacterium
44
Salinispora tropica [155], exerts activity by modifying the threonine residues

of the 20S proteasome. Nereus are associated with Phase I clinical
development of 176 to use in the treatment of solid tumors and lymphomas.
As on April 2008, Nereus Pharmaceuticals are enrolling patients for a Phase
Ib trial of 176 in combination with vorinostat (Zolinza, Merck & Co.)
against selected solid tumor malignancies.
O
H
OH
O
CH3
N
H
O
H3C
H3C
171 R = OCH3
172 R =
CH3
OH
Cl
O
CH3
173 R =
NH2
H3C
N
H
N
H
CH3
H2C
CH2
OH
CH3
H3C
H3C
CH3
CH3
OH
O
H3C
O
CH3
O
O
174
NH2
Staurosporine 177, isolated from bacterium Streptomyces staurosporeus

[156], a precursor of protein kinase inhibitors like enzastaurin (LY317615) 178
and midostaurin (PKC-412, CGP 41251, 4-N-Benzoyl-staurosporine) 179, has
significant anticancer potenticals. Enzastaurin (LY317615) 178 is a
serine/threonine kinase inhibitor [157] that was evaluated in Phase II trials by Eli
Lilly to use in the treatment of NSCLC patients. As of April 2010, the 178 is
under Phase III trials for the treatment of diffuse large B-cell lymphoma.
Midostaurin (PKC-412) 179 inhibits protein kinases including FLT3 [158] and is
in Phase II trials by Novartis to treat AML patients carrying FLT3 mutations.
K252a 180, an alkaloid isolated from Nocardiopisis species, is the lead
compound of lestaurtinib (CEP-701, KT-5555) 181 that inhibits FLT3 and
tyrosine phosphorylation of Trk A. As of 2008, the 181 is in Phase II trials
against myeloproliferative disorders and Phase III trials for the treatment of
AML. Likewise, KRX-0601 (UCN-01, KW-2401) 182, inhibiting a broad
spectrum of kinases including CDKs, is being developed by Keryx (Kyowa
Hakko) in Phase II clinical trials under sponsorship of NCI against melanoma,
TCL and SCLC.
Diazepinomicin (ECO-4601, TLN-4601) 183, a dibenzodiazepine
alkaloid isolated from the culture of a marine actinomycete of the genus
Micromonospora [159], can bind to peripheral benzodiazepine receptor
(PBR) and inhibits the Ras/MAP kinase signaling pathway involved in
cellular proliferation and migration [160]. ECO-4601 183 was found safe and
well-tolerated in Phase I/II trials conducted by the NCI and Thallion. ECO-4601
183 can cross the BBB and as on September 2008, Thallion are enrolling
patients for Phase II trial of 183 as a second line treatment for GBM.
45
CH3
OH
CH3
O
OH
O
OH
HO
P
O
O
CH3
CH3
O
CH3
O
N
CH3
HN
Cl
CH3
CH3
O
CH3
O
H
N
176
O
HO
O
H3C
CH3
H
N
175
N
CH3
N
N
N
CH3
N
OCH3
NHCH3
178
177
H
N
H
N
CH3
OCH3
CH3
OH
CH3
180 R = CO2CH3
179
181 R = CH2OH
H
N
OH
O
CH3
CH3
N
N
HO
CH3
HN
CH3
OCH3
HO
OH
HN
CH3
182
CH3
183
46
8.1.2.2. Eubacteria
Prodigiosin (Streptorubin B) 184, a Bcl-2 inhibitor and lead compound of
obatoclax (GX15-070) 185, is produced by strains of the bacterium Serratia
marcescens [161]. Gemin X are developing intravenous infusion of 185 in
multiple Phase I/II trials as a monotherapy in hematological and solid tumors
while in combination with carboplatin & etoposide to treat SCLC and with
bortezomib (Velcade) against mantle cell lymphoma (MCL). In March
2009, Gemin X launched a Phase II study of 185 as first-line treatment for
SCLC while disclosed the results of a Phase Ib trial in May 2009 against
extensive-stage SCLC.
8.1.2.3. Myxobacteria
Patupilone (epothilone B, EPO-906) 21, produced by the myxobacterium
Sorangium cellulosum, is a microtubule-stabilizing agent currently in Phase
III trials by Novartis against ovarian cancer [162]. Sagopilone (ZK-EPO,
ZK-219477) 186, a synthetic 21 derivative, can retain activity in MDR cancer
cells overexpressing the P-gp [163]. As of February 2010, Schering AG is
evaluating 186 in Phase II trials for the treatment of lung, ovarian and
prostate cancers. Epothilone D (desoxyepothilone B) 187, a natural
polyketide inhibits the disassembly of microtubules by binding to tubulin.
9,10-Didehydroepothilone D (KOS-1584) 188 [164], a 187 derivative, being
evaluated by Kosan Pharmaceuticals to use in the treatment of multiple solid
tumors. In Phase I dose escalation trials by Kosan, 188 has demonstrated
efficacy and tolerability against patients with ovarian cancer and NSCLC. As of
February 2007, Kosan were planning to initiate Phase II clinical development
of 188 against multiple solid tumors in collaboration with Roche.
OCH3 H C
3
N
H
OCH3
N
N
H
HN
HN
H3C
184
185
CH3
47
8.1.2.4. Fungi
NPI-2350 (halimide, phenylahistin) 189 is a tubulin-depolymerizing
agent isolated from a marine fungi Aspergillus ustus [165]. Nereus are
developing plinabulin (NPI-2358) 190, a synthetic 189 analog in various
clinical trials for the treatment of NSCLC [166]. In November 2009, Nereus
announced the positive results of a Phase II trial in NSCLC patients.
Irofulven (MGI-114, HMAF) 191 is a DNA synthesis inhibitor and analog of
illudin S 192, a sesquiterpene toxin found in mushrooms of the genus
Omphalotus [167]. Eisai (MGI Pharma) are currently evaluatig 191 in various
Phase II/III trials in patients with advanced-stage prostate cancer and GI solid
tumors.
8.1.3. Marine-derived compounds
Plitidepsin (Aplidin) 193, extracted from Aplidium albicans [168], is
being evaluated by PharmaMar in Phase II trials to use in the treatment of
hematological and solid tumors. Plitidepsin 193 inhibits the vascular
endothelial growth factor (VEGF) and is currently in Phase II trials by
PharmaMar as a first-line monotherapy treatment and in combination with
dacarbazine for advanced unresectable melanoma [169]. Halichondrin B 194,
isolated from Halichondria okadai sponge [170], was identified as a
significant anticancer agent by NCI. Eribulin mesylate (E7389, ER-086526,
NSC-707389) 195, a 194 analog, is being developed by Eisai against advanced
breast cancer patients. Eribulin 195 is a microtubule dynamics inhibitor and in
March 2010, Eisai has submitted regulatory applications to agencies in Japan,
US and EU for approval of 195 to use in the treatment of locally advanced or
metastatic breast cancer. Hemiasterlin 196, derived from marine sponges [171],
is capable of inhibiting tubulin assembly and disrupts normal microtubule
dynamics by depolymerizing the microtubules. E7974 197, a synthetic
analogue of 196, can bind to - and -tubulin and is under Phase I clinical
development by Eisai against a variety of human tumor xenografts.
Psammaplin A 198, an inhibitor of key several enzymes that control gene
expression, DNA replication and angiogenesis, was originally isolated from
the marine sponge Psammaplinaplysilla. Panobinostat (LBH-589) 199, a
synthetic 198 analog and pan-deacetylase inhibitor that induces death of
tumor cell lines but not the normal cells, is in Phase Ib/II clinical trials by
Novartis to use as monotherapy and in combination with chemotherapy
and/or targeted therapy against Hodgkins lymphoma, malignant melanoma,
AML/MDS and other hematological malignancies [172]. Currently, Novartis
are enrolling patients for Phase III trial in relapsed malignant melanoma.
48
CH3
CH3
H3C
H3C
O
OH
OH
CH3
H3C
CH3
H3C
H3C
H3C
CH3
CH2
O
OH
OH
186
S
H3C
CH3
187
CH3
O
OH
NH
H3C
H3C
N
NH
CH3
HN
CH2
CH3
O
H3C
OH
CH3
CH3
189
188
O
OH
CH3
NH
CH3
OH
N
NH
HN
O
CH3
HO
CH3
H3C
CH3
H3C
CH3
OH
HO
H3C
192
191
190
OCH3
H3C
CH3
H3C
NH
CH3
O
O
O
H3C
H3C
OH
NH
H3C
N
H
O
CH3
CH3
H3C
H3C
CH3
193
O
O
CH3
49
CH3
H
HO
H
H
O
O
H
CH3
H
O
H
CH2
O
CH3
CH3
OH
O
HO
194
H3C
H2C
H
O
H
HO
O
O
H
NH2
H3C
CH3
H3C
O
CH3
CH3
CH3
CH2
O
O
CH3
CO2H
N
H
O
O
H3C
HN
CH3
CH3
CH3
H3C
H2C
195
196
OH
H3C
CH3 H C
3
O
N
N
H
CH3
CH3
CH3
N
O
H3C
Br
CO2H
CH3
CH3
HO
N
H
H
N
Br
O
198
197
H3C
H3C
HO
OAc
O
O
N
H
H
N
OH
O
HO
O
OCH3
HN
HO O
OCH3
CH3
O
O
H3C
200
OH
H3C
H3C
CH3
199
OH
OH
50
Bryostatin 1 200, a macrolide lactone isolated from Bugula neritina

collected from the Gulf of California and Mexico, inhibits PKC [173] and
was granted with orphan drug status by the FDA (2001) and a similar
designation by the EU (2002) for use as combination therapy with TaxolTM
against esophageal cancer. In 2001, Arizona State University licensed
200 to GPC Biotech, which are associated with current Phase I/II trials
under the guidance of the NCI. Jorumycin 201, isolated from Jorunna
funebris produces cytotoxic effects through binding to DNA [174] and is
the lead compound of Zalypsis (PM00104/50) 202 being developed
by PharmaMar in Phase I trials for the treatment of solid tumors or
lymphoma. As on November 2009, the 202 is in Phase II trials for treating
cervical and endometrial cancer patients previously treated with standard
chemotherapy.
Dolastatin 15 203, is an antimitotic agent structurally related to dolastatin
10 205, a five-subunit peptide obtained from Dolabella auricularia [175].
Tasidotin (synthadotin, ILX-651) 204, an analog 203, induces G2/M phase
cell cycle arrest by inhibiting tubulin polymerization was evaluated by
Genzyme in Phase I/II trials against solid tumors. In May 2009, Genzyme
signed an agreement with Ergomed for the co-development of 204 as an
antineoplastic agent. Soblidotin (YHI-501, TZT-1027, auristatin PE) 206, a
derivative of 205, inhibits tubulin polymerization and is under Phase II trials
by Yakult Honsha for treatment of solid tumors. Kahalalide F 207, obtained
from the Hawaiian sea slug Elysia rufescens, can alter lysosomal membrane
function [176] and is in Phase II trials since October 2008 for the treatment
of severe psoriasis. In June 2009, the 207 was licensed by Medimetriks
Pharmaceuticals from PharmaMar for uses outside of oncology and
neurology. PM02734 (Irvalec) 208, another 207 derivative, is in Phase II
trials against solid tumors by PharmaMar. As on February 2010, PharmaMar
are recruiting patients for Phase I trial of 207 in combination with erlotinib
against advanced malignant solid tumors.
8.2. NP-antibody anticancer conjugates

Anticancer agents conjugated with various supports (antibodies,
polymers, liposomes and nanoparticles etc.) have been extensively explored
during the last few decades [177]. Zinostatin stimalamer (ZSS), conjugated
with a molecule of neocarzinostatin (NCS) chromoprotein and two molecules
of polystyrene-co-maleic acid [178], was launched by Yamanouchi (now
Astellas) in Japan against hepatocellular carcinoma.
Gemtuzumab ozogamicin (Mylotarg) 209 linked to calicheamicin 210
(obtained from Micromonospora echinospora), was co-developed by Wyeth and
51
OCH3
O
H
H3C
H3CO
O
CH3
OH
OAc
NH
N
H
N
O
CH3
CF3
CH3
OH
202
CH3H3C
OCH3
O
H3C
CH3O
203
H3C
H3C
CH3H3C
CH3H3C
N
H
N
CH3
CH3
N
CH3
H
N
CH3
CH3
CH3
204
H3C
CH3 H3C
CH3 H3C
CH3
CH3
H3C
N
N
H
N
CH3
N
CH3 OCH3 O
OCH3 O
H
N
205
H3C
CH3 H3C
CH3 H3C
CH3
CH3
H3C
N
N
H
N
CH3
N
O
CH3
C
CHH
33
CH3
H3C
CH3
201
H3C
HO
CH3
H3C
H3C
OCH3
CH3 OCH3 O
206
OCH3 O
H
N
52

CH3
H2N
CH3
CH3
O
N
H
CH3
N
H
O
H3C
O
H3C
H3C
HN
H3C
HO
NH
CH3
H3C
H3C
NH
CH3
CH3
207 R =
NH
O
CH3
CH3
O
O
CH3 HN
NH
H3C
NH
O H3C
H3C
NH
CH3
H
N
O
208 R =
O
CH3
O
NH
R
UCB Pharma. Likewise, inotuzumab ozogamicin (CMC-544), a calicheamicinantibody conjugated with CalichDMH and hydrazone linker attached to
humanized IgG4 anti-CD22 [179], is being developed by Wyeth and UCB
Pharma in Phase II/III trials against non-Hodgkins lymphoma in combination
with rituximab, a chimeric human IgG1 antibody that targets another B-lymphoid
lineage-specific molecule, CD20 [180].
Maytansine 211, isolated from plants of the genus Maytenus, is a
microtubule inhibitor that failed to show significant activity at non-toxic
concentrations in Phase I/II trials. ImmunoGen are associated with clinical
development of IMGN-242 (HuC242-DM4) 212, a maytansinoid DM4 and
huC242 conjugate, currently in Phase II trials for CanAg-expressing cancers. In
June 2009, ImmunoGen discontinued the development of 212 and are seeking for
out-licensing. ImmunoGen are also evaluating IMGN-901 (HuN901-DM1) 213,
a maytansinoid DM1 and huN901 congugate targeting CD56 expressing tumors,
is under Phase I trials against multiple myeloma while Phase II trial for the
treatment of SCLC. The FDA in March 2010 awarded orphan drug designation to
213 for use against merkel cell carcinoma (MCC).
9. Conclusion
Natural products have been the major sources of chemical diversity for
starting materials while driving pharmaceutical discovery over the past century.
53
H
N
O
hP67.6
O
H3C
CH3
H3C
N
H
O
H3C
S
O
H3CO
H3C
OH
OCH3
H3C
O
N
H3CO
209
CH3
HO
O
H3C
H3C
O
H3C
HO
H3CO
O
O
H3C
OCH3
N
H
N
H HO
OCH3
O
O
HN
H3CO
OH
210
CH3
H3C
Cl H3C
H3CO
O
O
CH3
N
CH3
HO
N
H
CH3
OCH3
211
S
S
H3C
S
OH
H3C
OH
OCH3
N
H HO
OCH3
N
H
H3C
HO
HO
CH3
O
OCH3
54
CH3
H3C
H3C
CH3
S
S
N
H
huC 242
O
O
Cl H3C
H3CO
O
CH3
N
CH3
HO
CH3
OCH3
N
H
O
3-4
212
CH3
H3C
CH3
S
O
O
Cl H3C
H3CO
H
N
HuN901
O
CH3
N
CH3
HO
N
H
CH3
OCH3
3-4
213
Today, NPs are finding increasing use as probes to interrogate biological systems
as part of chemical genomics and related researches. The modification of natural
products in an effort to alter their biochemical capacity is a common technique
utilized by synthetic and medicinal chemists. There have been remarkable
achievements in the field of natural products drug discovery during last three
decades and several compounds having profound biological activities
have been searched out with the help of modern and sophisticated techniques.
The quality of leads arising from NP discovery is better and often more
bio-friendly, due to their co-evolution with the target sites in biological
systems.
55
The large number of NP-derived compounds in various stages of clinical

development indicates that the use of NP templates is still a viable source of
new drug candidates. In future, the natural products drug discovery will be
more holistic, personalized and involve wise use of ancient and modern
therapeutic skills in a complementary manner so that maximum benefits can
be accrued to the patients and the community.
Acknowledgement
Authors are grateful to Prof. Dr. Richard R. Schmidt, Department of
Chemistry, Universitat Konstanz, Germany for his useful discussions during
the preparation of manuscript. Financial assistance from DST, New Delhi has
been greatly acknowledged.
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Research Signpost
37/661 (2), Fort P.O.
Trivandrum-695 023
Kerala, India
ISBN: 978-81-308-0448-4
2. Natural products in discovery of potential

and safer antibacterial agents
1
Girija S. Singh1 and Surendra N. Pandeya2
Chemistry Department, University of Botswana, Gaborone, Botswana; 2Pharmaceutics Department

Saroj Institute of Management & Technology, Lucknow-226001, India
Abstract. This article describes the significance of natural products in

discovery of potential and safer antibacterial agents. The introductory
paragraph is followed by various classes of compounds depending
on the structural class. These include -lactams, macrolides
and ketolides, lincosamides, furanomycin, pyrrolidinediones,
tetrahydropyrimidinones, biphenomycins, tuberactinomycin and
capreomycin, glycopeptides, lysobactins, enopeptin depsipeptides,
tetracyclines and aminoglycosides. A brief historical development
of each class is described followed by its mechanism of
action. Several semi-synthetic compounds are described. Synthetic
methods are described in selected cases.
1. Introduction
Today, infectious diseases are the second major cause of death
worldwide and third leading cause of death in economically advanced
countries [1]. Bacterial pathogens are responsible for several serious
diseases (Table 1). Strains are getting resistant to antibiotics in clinical use
and hence posing threat to mankind (Table 2). The ability of bacteria to
deceive any kind of conventional therapy has become apparent and
pathogens resistant to one or more antibiotics are emerging and spreading
worldwide [2]. Unnecessary use of antibiotics has further fuelled this problem.
Correspondence/Reprint request: Prof. Girija S. Singh, Chemistry Department, University of Botswana, Gaborone
Botswana. E-mail: singh_gs_57@yahoo.co.in
64
Girija S. Singh & Surendra N. Pandeya
Table 1. Key bacterial pathogens and related diseases.
Pathogens
S. aureus
S. pneumonia
Infectious Diseases
Skin and wound infections, endocarditis
Upper respiratory tract infection, pneumonia,
sinusuitis, meningitis
Pharyngitis, tonsillitis, skin and soft tissue infection
S. pyogenes
E. faecalis
Endocarditis, urinary tract infection
E. faecium
Peritonitis, endocarditis, bacteremia
E. coli
Urinary tract infection, bacteremia, gastrointestinal
infection
Bacteremia, pneumonia
K. neumoniae
H. influenza
Respiratory tract infection, sinusuitis, meningitis
P. aeruginosa Bacteremia, burn infection
M. tuberculosis Tuberculosis
Table 2. Prevalence of resistance in hospital-acquired infections in USA (2004).
Antibiotics
Methicillin
Vancomycin
Cephalosporins (3rd generation)
Imipenem
Quinolones (synthetic)
Pathogen
S. aureus
Enterococci
Enterobacter sp.
P. aeruginosa
E. coli
K. pneumoniae
P. aeruginosa
P. aeruginosa
Resistance (%)
59.5
28.5
31.1
31.9
5.8
20.6
21.4
29.5
The discovery of vancomycin resistant S. aureus (VRSA) and multiresistant

S. aureus has generated worldwide concern. It has thus become evident that
there is urgent need for novel antibacterial drugs with broader spectrum,
lesser side effects, and without cross-resistance to antibiotics in use. More
initiative is required to foster the responsible and appropriate use of antibiotic
that is another issue.
The traditional medicine system based on natural products continues to
play an important role in treatment of many diseases especially the infectious
diseases. According to the WHO estimation, approximately 80% of the
worlds population relies mainly on traditional medicine for their primary
health. Indian traditional medicine system relied on plants and their parts to
treat various infectious diseases (Table 3). Hundreds of herbs are known to be
used for various diseases including many infectious diseases. Acacia, garlic,
turmeric, neem, ginger, clove, plum and pomegranate are only few to name.
Antibacterial natural products
65
Table 3. Some traditionally used herbs and their bioactivity.

Plant
Triphala i.e. three fruits viz.
Haritaki (Terminalia chebula),
Bibhitaka (Terminalia belerica)
& Amalaki (Emblica
officinalis)
Turmeric (Curcuma longa)
Plant Parts
90 % ethanolic &
aqueous extracts of
Triphala
Bioactivity
Significant activity on
S. aureus, E. coli and
P. areoginosa
Leaf extract
Fenugreek (Methika)
(Trigonella faenum- graecum)
Wildrue (Peganum harmal)
Seed extract
Antibacterial and
antifungal activity
Antibacterial and
antifungal activity
Antibacterial and
antifungal activity
Antibacterial and
antifungal activity
Gokarna (Clitoria Ternatea)

Sharapunkha (Tephrosia
puropurea)
Brahmi (Bacopa monnieri)
Tulsi (Ocimum sanctum)
Aqueous seed
extract
Hexane &
methanolic root
extract
Root extract
Ethanolic extract
of aerial parts
Leaf extract
Antibacterial and
antifungal activity
Antihelminth activity
Antibacterial and
antifungal activity
Later on extracts from many of such plants and herbs have been screened by
investigators in quest for potential and safer antibacterial agents [3-8]. Many
comprehensive review articles have been published on the role of natural
products in the discovery of antibacterial agents [9-12]. The plethora of
literature in the area indicates an urgent need for a coordinated effort for
meaningful research and discovery of novel antimicrobial agents.
Most of the antibacterial agents in use today are either natural products or
their semi-synthetic variations or improved subclasses. The success of
natural products as guideposts to new drugs is most obvious in antibacterials
(Table 4). Over 75% of new chemical entities submitted between 1984 and
2004 were based on natural product lead structures [13].
This article focuses on significance of selected natural products in
discovery of new antibacterial agents with broader spectrum and least side
effects. The sections are classified according to established structural
characteristics. A brief historical development of each class is described
followed by its mode of action. Selected recent examples of synthetic
modification of natural antibiotics are discussed. More emphasis is given on
-lactam antibiotics as it consists of several subclasses such as penems,
cephems etc. As mentioned in the preceding paragraph there are plethora of
66
Table 4. Some potential antibiotics and their targets.

Class
Representative example
H
N
-Lactams
Target
Cell wall
N
O
COOH
Penicillin G (1)
OH
NMe2
OH
Polyketides
Protein
biosynthesis
NH2
OH
OH
OH O
Tetracycline (2)
O2 N
NHCOCHCl2
OH
Phenylpropanoids
Protein
biosynthesis
OH
Chloramphenicol (3)
NH2
Aminoglycosides
HO
NH2 O
HO
Protein
biosynthesis
H2N
NH2
OH
OH
HO
NH2
Tobramycin (4)
O
Macrolides
Protein
biosynthesis
OH
HO
OH
HO
NMe2
OMeO O
OH
Erythromycin A (5)
OH NH
Glycopeptides
H3C
O
CH3
O
HO
O
O
NH
HO
N
H
Cl
H
N
OH
Cl OH
OH
N
H
O
H
N
O
O
NH2
OH
OH
HO
Cell wall
OH
O
Vancomycin
N
H
H
N
CH3
CH3
CH3
67
Table 4. Continued
Streptogramins
Me
N
NMe2
O
O
HN
O
O
N
O
O
Protein
biosynthesis
N
O
NH
O
NH
OH
Pristinamycin IA (7)
OH O
N
O
NH
O
O N
O
Pristinamycin IIA (8)
literature available in the area and each class has been reviewed by several
others from different angles it was considered pertinent to bring a concise
account of the material for the convenience of readers.
2. -Lactam antibiotics
The group of antibiotics containing four-membered cyclic amides
(azetidin-2-ones) is commonly known as -lactam antibiotics. It is the first
class of antibiotics to be used as a therapeutic treatment for bacterial
infections (Figure 1). The first member penicillin was discovered by Fleming
from the cultures of Penicillium notatum in 1928. Since then this group has
maintained its charm among synthetic and medicinal chemists [14,15].
About half of the antibacterial drugs prescribed today belong to this class.
Their broad antibacterial spectrum, clinical efficacy and excellent safety
profile make them preeminent in pharmaceutical drug discovery. As a
result of extensive research cephalosporins have reached their fourth
generation (cefepime). The main approaches in design of the new cephem
derivatives involve structural modifications at positions C-3 and C-7,
and the development of cephem prodrugs. The compounds with a
methoxy [16], carbamoyloxy [17] or heteroaryl ring such as tetrazole [18]
or thiazole [19] in the C-3 side chain are known to have potent antibacterial
activity.
68

OH
H
H
N
R
O
N
O
CO2H
OH
H
O
N
oxapenem
H
N
R
O
N
CO2H
H
N
R
R
oxacephem
Cl
CO2H
N
O
H2N
N
CO2-
Cefepime
H
N
R
N
OMe
H
N
S
CO2H
CO2H
sulbactam
carbapenem
NH
CO2H
NH
CO2H
oxapenam
NH2 OH
H
O
S
O
CO2H
penem
penam
OH
carbacephem
S
N
OAc
O
CO2H
cephem
H
N
R
O
N SO H
3
monobactam
Figure 1
Natural penicillin G, the first therapeutic antibiotic and lead structure of

this class still had a few critical features such as narrow antibacterial
spectrum, instability in acidic and alkaline environments, limited solubility
and pronounced sensitivity to hydrolyze by bacterial penicillase enzymes that
are needed to be improved. For about thirty years, penicillins (penams) and
cephalosporins (cephems) remained the only examples of -lactam
antibiotics. Many related subgroups such as the monobactams (aztreonam),
oxacephems (moxalactam), carbacephems (loracarbef), oxapenams
(clavulanic acid), penems (faropenem), carbapenems (imipenem), and
oxapenems (AM-112) were discovered during 1970s and 1980s either from
microbes or by synthetic efforts. Numerous structural variations of these lactam scaffolds provided derivatives with increased potency, low host
toxicity, improved physicochemical and pharmacokinetic profiles.
The -lactam antibacterials act on bacteria by inhibiting the final step of
the bacterial cell wall biosynthesis. Although several mechanisms might be
operating in this inhibition, the most important is probably the inhibition of
the terminal peptidoglycan cross-linking. Bacteria have a cytoplasmic
membrane similar to that of eukaryotes. This membrane is surrounded by a
periplasmic space, which is in turn enclosed by a peptidoglycan layer, and
finally the outer membrane. The peptidoglycan layer is a cross-linked
polymer that forms a net-like structure, which provides structural rigidity to
69
the organism, and allows it to survive in mediums to which it may be strongly

hypertonic. As a bacterium grows, a series of covalent cross-links must be
formed between adjoining peptidoglycan strands in the cell wall. These
cross-links are stitched together by transpeptidase enzymes in the cell
membrane through the replacement of a terminal D-alanine unit on one
peptidoglycan strand with a glycine residue on a neighboring peptidoglycan
strand. The initial cleavage of the D-alanine residue by transpeptidase occurs
by a nucleophilic addition of an active site serine onto the amide
functionality, as shown in Scheme 1. In a subsequent amidation step, the
resulting enzyme-linked peptidoglycan is converted to the cross-linked
material, which releases serine for further catalysis.
Penicillins, cephalosporins and related -lactam drugs possess an unusual
ability to interrupt this crucial cross-linking process by an irreversible
acylation of the hydroxy group of the catalytic serine unit within the enzyme
active site resulting in the formation of a catalytically inactive stable
enzyme-drug adduct (Scheme 2). The net result is decrease in the number of
cross-linked residues within the cell wall making it weak and prone to
rupture. Inhibition of the transpeptidase thus inhibits the bacterial growth.
The sequences leading to cidal action are still not clearly understood. Nicks
may be produced at the growth site of the cell wall. If these nicks are
sufficiently severe, the protoplast may protrude into the medium and burst
resulting in bacterial death.
The major limitation to the potentials of -lactam antibacterials is the
ability of bacteria to produce a family of enzymes called -lactamases. These
enzymes hydrolyze the -lactam ring which is required for antibacterial
activity. There are different types of -lactamases and their efficacy in
ngtp
H
N
CO2-
O
N
H
Me
Me
ngtp
OH
H
N
O
O
Me enzyme-serine
ngtp
H
N
Me
N
H
H
N ptgn
O
OH
enzyme serine
ptgn = Peptidoglycan
enzyme serine
Scheme 1
ROCHN
ROCHN
S
N
CO2H
OH
enzyme-serine
HN
OO
CO2H
enzyme-serine
Scheme 2
Bacterial-death
70
hydrolyzing the ring varies widely. There are four distinct classes of lactamases, of which class A enzymes are the most common. In order to
counter the hydrolysis by -lactamases, some antibiotics are administered in
combination with a -lactamase inhibitor drug. For example, amoxicillin is
administered in combination with clavulanic acid, itself also a -lactam
(oxapenam). However, the discovery of new variants of -lactamases, which
are resistant to known -lactamase inhibitors, has caused great concern
worldwide.
The major thrust areas in research on -lactams have been the
development of new stereoselective methodologies to construct the -lactam
ring, and structural modifications in compounds, especially carbapenems and
cephems with known activity, to design and develop new molecules with i) a
broad spectrum of activity, specially against resistant strains and, ii) least side
effects. The succeeding paragraphs describe the synthesis of some new
cephems. The biological activity is discussed in selected examples.
A series of -[(Z)-2-(2-aminothiazol-4-yl)-2-hydoxyiminoacetamido]-3[(E) and (Z)-2-substituted vinyl] cephalosporin derivatives 9,10 have been
synthesized using palladium-catalyzed coupling reaction of a 3methanesulfonoxy-3-cephem and an E substituted vinyl stannane (Scheme 3)
or Wittig reaction of a 3-triphenylphosphoniummethyl cephem and an
aldehyde (Scheme 4) as key steps [20].
OH
N
H
N
N
O
H2N
S
N
O
CO2H
9,10
R = H (Cefdinir), -CH2CH2OMe, -CH2CH2OTMS, -CH2CH2OH, CH2CH2OCONH2, CH2CH2OAc

a
O
e
O
N
N
N

OHCHN
71
OHCHN
n-Bu3Sn
a, b, c
N
OMs
CO2CHPh2
H2N
H2N
R
CO2CHPh2
i)
R
CO2CHPh2
c
H2N
COCl
iii)
S
OAc
N
H
N
vi), vii)
O
H2N
BocHN
R
CO2CHPh2
OAc
N
N
S
N
ii)
CO2CHPh2
iv), v)
CO2CHPh2
iv)
TFA
H2N
N
1. iii)
H2N
H
N
OH
N
COCl
OAc
H2N
2. v)
CO2CHPh2
CO2H
9a,c-e
Reagents and conditions: i) Pd(CH3CN)2Cl2, LiBr, DMF; ii) cHCl, MeOH; iii) BSA, CH2Cl2;
iv) TFA, anisole, CH2Cl2; v) NaHCO3, NH4Cl, MeOH-H2O; vi) Boc2O, MSA, THF; vii)
MeCOCl, Et3N, CH2Cl2
Scheme 3
BocHN
O
BocHN
R-CHO
a,b,f,m
S
PPh3I
BocHN
S
N
i)
CO2CHPh2
R
CO2CHPh2
CO2CHPh2
OH
N
a,e,m
a, e-m
b
1. iv) or v)
2. vi)
3. vii)
c
H
N
N
O
H2N
j,m
j,m
S
N
ii)
iii)
9j,m = E isomerCO2H
10a,c,d,f-l = Z isomer
Reagents and conditions: i) a. 1N NaOH, aq. NaCl, CH2Cl2, b. separation; ii) cHCl, MeOH;
iii) a. Cl3CONCO, CH2Cl2, b. SiO2, CHCl3, MeOH; iv) TFA, anisole, CH2Cl2; v) HCO2H, cHCl;
vi) BSA or MSA, (Z)-2-(2-aminothiazol-4-yl)-2-acetoxyiminoacetyl chloride hydrochloride, CH2Cl2;
vii) NaHCO3, NH4Cl, MeOH, H2O
Scheme 4
The research findings at Lilly in the nineteen seventies that

2,5-dichlorophenylthioacetamido at C-7 as a lipophilic side chain conferred
excellent Gram-(+) activity to the cephem class have been exploited further
[21]. Through a series of optimization at C-3 and C-7, four cephalosporins
11, 12, 13 and 14 possessing a 2,5-dichlorophenylthioacetamido group at
C-7 and a polar thiopyridinium group at C-3 with potent in vitro and in vivo
72
anti-MRSA activity have been reported [22]. The C-3 thiopyridinium ring
was substituted with amino acid and pyruvic acid groups that were designed
to provide aqueous solubility as required for IV formulation. These
compounds have excellent in vitro activity against a variety of Gram-(+)
bacteria including resistant strains such as penicillin-resistant S. pneumoniae,
methicillin-resistant S. epidermitis and S. haemolyticus (Table 5).
Furthermore, all of them were efficacious in a systemic murine model of
infection with PD5os ranging from 4.8-9.6 mg/kg. The aqueous solubility of
13 and 14 was much more (23 and 40 mg/mL, respectively at pH 7) in
comparison to 11 (2-3 mg/mL at pH 7 and at room temperature).
H
N
R
O
S
N
H
N
R
S
O
CO2
N
O
CO2H
NH3
11, R = diClPh (double zwitterion)

12, R = diClpyr
O2C
N
O
CO2
13, R = diClPh
14, R = diClpyr
Table 5. Antibacterial Activity of Cephalosporin Derivatives.

Organism
S. aureus/Hetero
MR
S. aureus/ + 50%
calf serum
S. aureus/Hetero
MR
S. aureus/Hetero
MR
S. aureus /Homo
MR
S. aureus /Hetero
MR
S. aureus /Homo
MR
S. aureus /Homo
MR
S. aureus /Homo
MR
S. aureus / MR, PPBP2a
IC50 (g/mL)
A No.
A27218
11
0.5
12
1
13
0.5
14
1
M
32
IM
1
A27218
0.5
NT
A27217
0.5
0.5
0.5
64
A25795
128
A27223
128
32
A27223
16
64
NT
A27621
64
16
A27295
128
64
A27226
64
A27225
128
NT
28
NT
10
4.5
100
NT
MIC in g/mL, MR = methicillin-resistant, P- = penicillin negative, M = methicillin,

IM = imipenem, NT = not tested.
73
The in vitro and in vivo activity of 7--methoxy-cephems and 7-methoxy-oxacephems 15-18 and their demethoxy congeners 15a-18a on
H. felis and H. pylori, human pathogens associated with type B gastritis,
peptic ulcer disease and gastric cancer have been studied that showed the
significance of 7--methoxy substituents in dealing with these bacteria [23].
The in vivo antibacterial activity was studied on a mouse helibacter infection
model after oral administration, in which mice were infected with H. felis.
All of the compounds except 18 and 18a exhibited very similar MICs for
both H. felis (0.25-0.5 mg/L) and H. pylori (0.5-1.0 mg/L). Compounds 18
and 18a had lower MIC for H. felis (0.13 mg/L) than for H. pylori (1.0-2.0
mg/L). Even though the MICs of all four pairs of compounds were within 1to
2-fold dilution for H. felis and H. pylori, the 7--methoxy compounds were at
least 4-fold more active at bacterial eradication than their demethoxy
counterparts (Table 6). Intravenous administration of flomoxef resulted in
extremely low eradication activity compared with oral administration. These
results, together with the fact that flomoxef is not absorbed orally, indicated
that the compound had direct access to the bacteria in the stomach after oral
administration.
H Y
N
R1
O
X
N
O
15-18
Compounds
15 Flomoxef
X
O
15a Dimethoxyflomoxef O
R2
CO2H
Y
OMe
R1
F
F
16 1-thiaflomoxef
OMe
16a
Dimethoxy-1thiaflomoxef
17
Cefmetazole
17a
dimethoxycefmetazole
18 M-1
OMe
OMe
18a H-1
N N
HO
NC
R2
N N
74
The activity of RWJ-54428, a new parenteral cephalosporin originally

developed by the R. W. Johnson Pharmaceutical Research Institute, NJ, USA,
against recent isolates of Gram-(+) bacteria, including staphylococci with
decreased susceptibility to vancomycin 6 has been reported [24]. This compound
has been shown to be active against a wide range of multiply resistant Gram-(+)
pathogens, including oxacillin-resistant S. aureus (MRSA), E. faecalis (MIC90 =
0.5 mg/L), vancomycin-resistant E. faecalis (MIC90 = 0.25 mg/L), and penicillinresistant pneumococci and streptococci (MIC90 = 1 mg/L). The only group of
organisms for which the MIC90 was >2 mg/L was ampicillin-resistant E. faecium.
Reinert and coworkers have evaluated cefditoren against penicillin-susceptible
strains of S. pneumoniae and penicillin-intermediate strains of S. pneumoniae
isolated from patients with respiratory tract infections and suggested it as a
promising agent for the treatment of infections caused by pneumococci with
reduced penicillin susceptibility [25].
Gerber and coworkers have reported cefepime, considered as fourth
generation cephalosporin, having an excellent CSF penetration with level
ranging between 10 and 16 mg/L after two intravenous injections (100
mg/kg). The bactericidal activity of cefepime was superior to ceftriaxone and
vancomycin in the treatment of rabbits with meningitis caused by an isolate
highly resistant to penicillin [26]. Schito and coworkers have studied the
activity of many cephalosporins against some common respiratory tract
pathogens such as S. pneumoniae, H. influenzae and M. catarrhalis etc.,
isolated from the patients in Italy, Spain, and Austria [27]. Cefpodoxime has
been reported as a suitable choice for use.
Table 6. Clearance and Eradication Doses of compound.
Compounds
15 Flomoxef
15a Demethoxyflomoxef
16 1-thia-flomoxef
16a Demethoxy-1-thiaflomoxef
17 Cefmetazole
17a Demethoxycefmetazole
18 M-1
18a H-1
Amoxicillin
15 Flomoxef (iv)
a
50% Clearance
dose (mg/kg/dose)a
1.00
4.00
0.97
3.84
1.00
5.79
0.47
3.59
1.92
>15.0
50% Eradication
dose (mg/kg/dose)b
3.67
17.4
3.38
>60
3.67
58.8
Not tested
Not tested
9.12
58.8
Compounds were administered orally twice a day for 1 day and mice were killed on the
following day.
b
Compounds were administered orally twice a day for 5 days and mice were killed after
14 days.
75
Some amides 19 and imines 20 containing 5-nitrofuryl and 3-methoxy-2nitrophenyl groups from 7--aminocephalosporanic acid and 7-aminodesacetoxycephalosporanic acid have been synthesized and evaluated
for antibacterial activity [28]. Many compounds, especially with 5-nitrofuryl
moiety, exhibited an activity equal to or better than those of ampicillin or
cephalexin against the majority of Gram-(+) organisms tested. None of the
compounds showed appreciable activity against E. coli.
ArOCHN
O
S
N
CH2OCOMe
CO2Na
19
ArHC N
O
S
N
CH2OCOMe
CO2Na
20
Ishikawa and coworkers are involved in the development of new

cefozopran (CZOP) 21 derivatives for use against MRSA [29,30]. They
observed that the CZOP with lipophilic alkoxyimino groups in the C-7 acyl
moiety showed potent anti-MRSA activity. Cyclopentyloxyimino derivatives
with amino-based substituent(s) in the C-3 azole moiety had anti-MRSA
activity comparable to vancomycin. In order to further increase the activity
they have modified the C-3 linked spacers of cephem derivatives bearing a
1-methylimidazo-[1,2-b]-pyridazinium-6-yl group at the C-3 position and a
2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(Z)-cyclopentyloxyiminoacetyl group at
the C-7 position [31]. They have found that the optimal spacers are (E)-2vinyl and (E)-2-thiovinyl groups. The anti-MRSA activity of the compounds
22a,b bearing these spacers were 16-32 times higher than CZOP. Taking
these two spacers they have modified the alkoxyimino group in the C-7 acyl
moiety and the 1-alkylimidazo-[1,2-b]-pyridazinium moiety at C-3 and
discovered compound 22c with anti-MRSA activity comparable to
vancomycin both in vitro and in vivo, high affinity (IC50 = 2.7 mg/mL) for
PBP2 of MRSA and potent activity against Gram-(-) bacteria as well.
Hakimelahi and coworkers are working on the concept of using
antibacterial prodrugs. They have reported the synthesis, antibacterial and
-lactamase inhibitor activity of clavunate derivatives of amoxicillin.
The compounds screened by them showed better antibacterial activity than
amoxicillin and clavulanic acid combination augmentin [32].
Since the discovery of thienamycin (23a) from the fermentation broth of
soil bacteria Streptomyces cattleya in 1976 several carbapenem derivatives
have been synthesized and evaluated for their antibacterial activity.
Thienamycin itself has excellent antibacterial activity against both Gram-(+)
and Gram-(-) bacteria, and is resistant to -lactamases. Numerous methods
76
N
N
OMe
H
N
H2N
S N
O
O
OMe
H
N
H2N
S N
CO2Na
21
CO222a
N
N
OR
H
N
O
O
N Me
H2N
S N
R2
N N
CO2-
22b, R1 = cyclopentyl; R2 = Me 65b

22c, R1 = CH2F; R2 = Me 65c
are described in literature for the total synthesis of thienamycin [33]. Currently,
two 1-H carbapenems, imipenem (23b) and panipenem (23c), and one methyl carbapenem, meropenem (24) are available in the market for clinical
use [34-36]. Although arbapenems have a broad antimicrobial spectrum and
potent bactericidal activity [37] most of them have some limitations as well
from the view point of clinical application. For example, imipenem is unstable
to the renal dehydropeptidase-I (DHP-I) and has epileptic side effect.
Meropenem has good stability to DHP-I due to steric hindrance of -methyl
group at C-1 and an excellent spectrum against Gram-(-)bacteria, but it is
relatively less active against Gram-(+) bacteria than imipenem.
The studies on carbapenems from a pharmaceutical point of view in the
previous decade have been devoted mainly to the synthesis and evaluation of
1--methylcarbapenem derivatives as antibacterials. New methodologies for
construction of the carbapenem skeleton are under investigation by Mori and
Me
OH
H H
X
R
N
O
CO2H
X = 23a-c: H; 24: Me
R = 23a: -SCH2CH2NH2 (Thienamycin)
23b: -SCH2CH2NHCH=NH (Imipenem)
23c:
Me (Panipenem)
NH
Me2NOC
24
S
NH
(Meropenem)
77
Kozawa [38,39]. A new method involving palladium-catalyzed C-N bond

forming reaction (Scheme 5) in azetidinone 25 leading to the synthesis of
carbapenem 26 with a carboxylic group on C-3 of the fivemembered ring
has been reported by them [40].
10 mol% Pd(OAc)2
15 mol% DP Ephos
PhMe, 100 oC, base (2 equiv.)
OSi
H H
NH
O
OSi
H H
CO2Et
CO2Et
26
25
Scheme 5
3. Macrolide and ketolide antibiotics

Macrolide antibiotics belong to the subgroup of polyketide natural
products and constitute an important therapeutic class. They act against
community-acquired respiratory infections such as community-acquired
pneumonia, acute bacterial exacerbations of chronic bronchitis, acute
sinusitis and tonsillitis [41,42]. Macrolides account for 20% of all the
antibiotics prescribed.
The principal representative of macrolides erythromycin A 5 was first
isolated from Streptomyces erythreus at the Lilly in 1952 [43]. Its absolute
configuration was established by NMR spectroscopic studies and X-ray
crystallographic data [44,45]. It helps against the major respiratory
pathogens, is considered safe and is widely prescribed for children. However,
it has a limited antibacterial spectrum and limited solubility in acidic medium.
The second generation macrolide antibiotics, 27-29 have gradually replaced
erythromycin A because of their higher potency, broader spectrum of activity,
improved physicochemical and pharmacokinetic profiles, and attenuated side
effects [46]. However, similar to erythromycin A, the second generation
variants also have poor activity against macrolide resistant pathoges.
The mode of action of macrolide antibiotics involves blocking bacterial
protein biosynthesis by binding to the 23S ribosomal RNA of the 50S subunit
and interfering with the elongation of nascent peptide chains during
translation [47]. Located in domain V, near the peptyl transferase site,
macrolide antibiotics obstruct the peptide exit tunnel without affecting
peptidyl transferase activity.
Ketolides [48] are derived from 14-membered macrolides by removal of
L-cladinone under acidic conditions and selective oxidation of the resulting
3-hydroxy group to the corresponding carbonyl group. The first semisynthetic
78

O
N
OMe
OMe
HO
OH
HO
OH
HO
OH
NMe2
OMeO O
NMe2
OMeO O
O
OH
HO
OH
O
Roxithromycin (28)
Clarithromycin (27)
Me
N
N
OH
HO
OH
HO
O
O
O
O
HO
NMe2
O
OMeO O
O
OH
OH
Azithromycin (29)
O
O
NMe2
O
Telithromycin (30)
ketolide RU-64004 (HMR 3004) was synthesized at Roussel Uclaf [49]. This
ketolide was stable in acidic media, showed good intracellular penetration,
and demonstrated potent activity against erythromycin A resistant and
penicillin resistant streptococci and H. influenzae. Systematic SAR studies
led to the discovery of several ketolide lead structures such as telithromycin,
cethromycin, and EP-013420 with potent activity and improved pharmacokinetic
profile. Telithromycin 30 was the first ketolide to be approved in Europe
(2001), Japan (2003) and in the US (2004) for the once daily oral dose for
treatment of respiratory tract infections. It was synthesized from clarithromycin
in eight steps [50].
4. Lincosamides
Lincomycin 31 and its semi-synthetic congener clindamycin 32 were
introduced into clinical use as oral antibiotics in 1960 and 1969, respectively
[51]. They exhibit a similar spectrum as macrolides, including activity
against most gram-(+) organisms and the anaerobes, but not the Gram-(-) and
enterococci [52]. Now a day they are not in much use due to their limited
antibacterial spectrum, the emergence of resistance, and severe side effects of
this class.
79
Clindamycin is a semi-synthetic derivative of the natural product

lincomycin, which is produced by fermentation of Streptomyces lincolnensis
[53]. This transformation involves selective transformation of only one secalcoholic group of the three present (Scheme 6).
Me
N
HO
O
N
H
HO
SMe
NCS, PPh3
THF, , 18h
OH
Me
N
Cl
N
H
HO
SMe
OH
OH
OH
84%
32
31
Scheme 6
The mode of action of lincosamide involves binding to the ribosome and

inhibiting bacterial protein synthesis. Specifically, macrolides, lincosamides,
and streptogramin B type antibiotics bind to adjacent sites on the 50S
ribosomal subunit. The complexes of bacterial ribosomes with these
antibiotics have been studied by X-ray crystallography [54,55].
Several methods for semi-synthetic modification of lincomycin and
clindamycin have been published in addition to the methodologies developed
during the total synthesis of natural product [56,57]. Substitution of the
7-hydroxy group by a methyl group in conjunction with novel amides resulted
into discovery of VIC-105404 33 and VIC-105555 34 (Scheme 7) [58,59]. The
latter compound has rapidly been progressed into preclinical development.
Hopefully these achievements will translate into clinical benefit.
5. Furanomycin
L-(+)-Furanomycin 35 is a low molecular weight (157 g/M) antibacterial
natural product. It is a -amino acid isolated by Katagiri and coworkers in
1967 from the fermentation broth of Streptomyces threomyceticu L-803
(ATCC15795) [60]. It inhibits the growth of bacteria such as M. tuberculosis,
E. coli, B. subtilis, and some Shigella- and Salmonella species in the M
range. Its initially assigned absolute configuration was later on revised to (+)(S,2R,5S) by synthesis starting from D-glucose and by X-ray crystallographic
study of the N-acetyl derivative [61-63].
Furanomycin 35 is accepted as a substrate by isoleucyl aminoacyl-t-RNA
synthetase and its antibacterial activity results from a substitution for isoleucine
during the bacterial protein translation [64]. Therefore, the antibacterial activity of
furanomycin is antagonized by isoleucine. Furanomycin hampers the formation
80

Me
N
HO
O
TMSO
O
N
H
HO
SMe
i), ii), iii)
BocHN
OH
TMSO
OH
SMe
iv), v)
OTMS
OTMS
31
BocHN
TMSO
SMe
H2N
vi), vii), viii)
BocHN
OTMS
OTMS
HO
Boc
N
CO2H
SMe
OH
H3C(H2C)4
ix), x), xi)
xii), xiii)
NH
Boc
N
OH
HO
HN
BocHN
HO
SMe
HN
BocHN
H3C(H2C)4
OH
OH
HO
33 VIC-10555
34 VIC-105404
SMe
OH
OH
Reagents and conditions: i) N2H4, H2O; II) (Boc)2O, Et3N, MeOH; iii) BSTFA, Et3N, DMF;
iv) DMSO, (COCl)2, Et3N, CH2Cl2, -70 - 40oC; (v) PPh3Me+Br-, t-BuOK; vi) Dowex H+, MeOH;
vii) H2 (65psi), Pd/C; viii) TFA/H2O (9:1); ix) HBTU, Et3N; x) TFA/H2O (9:1); xi) oxirane, Et3N;
xii) HBTU, Et3N; xiii) TFA/H2O (9:1)
Scheme 7
of isoleucine-tRNA in E. coli, whereas other aminoacyl-tRNA are not

affected [65,66]. Aminoacyl-tRNA synthetases are essential in all living
organisms and have attracted considerable interest as novel targets in
bacterial protein synthesis [67-70].
O
H2N
CO2H
35
O
H2N
CO2H
36
H2N
CO2H
37
81
Several approaches have been developed towards the synthesis of

Furanomycin 35. Many syntheses start from carbohydrates such as D-glucose
[51], D-ribose [71], D-glucosamine [72], L-xylose [73], and D-mannitol [74].
Glycine [75], serine [76], furans [77], and dimethyl L-tartarate [78], have also
been used as substrate for the synthesis of Furanomycin 35. Approaches
involving amino acids are appealing as they involve lesser steps.
A SAR was studied using many synthetic isomers and derivatives of natural
products. Unfortunately, all of them showed either no activity or poor activity
against a panel of selected Gram-(+) and Gram-(-) pathogens including
S. aureus, and E. coli. Only L-(+)-dihydrofuranomycin 36 showed borderline
MIC (32-64 g/mL) against S. aureus. The chiral carbon analogue 37 exhibited
weak antibacterial activity (4 g/mL) against an efflux-pum-deficient E. coli.
Furanomycin, thus, proved an insufficient lead and could not be a valuable
compound as a starting point for an antibacterial drug discovery program.
6. Pyrrolidinedione antibacterials
Komura and coworkers in 1987 isolated natural peptide antibiotic
compound andrimid 38 from cultures of a symbiont of the brown planthopper
Nilaparvata lugens [79]. Later on moiramide 39 was discovered in a marine
isolate of Pseudomonas fluorescens obtained from a tunicate collected in
Moira Sound at Prince of Wales Island, Alaska [80]. The structures of these
metabolites contain four characteristic elements: a pyrrolidinedione head
group, a valine derived -ketoamide, a (S)--phenylalanine moiety, and an
N-terminal polyunsaturated fatty acid. Various diastereoselective and
asymmetric total syntheses of andrimid and moiramides are known that allow
ready access to these antibiotics [81,82].
Pyrrolidinedione antibiotics act by inhibiting the first committed step
in bacterial fatty acid biosynthesis, a reaction catalyzed by the
carboxyltransferase subunit of the multimeric bacterial enzyme acetyl-CoA
carboxylase [83]. For most living organisms, fatty acid biosynthesis is a vital
metabolic process, but the pathway in bacteria and mammals are different.
Acetyl-CoA carboxylase is essential for microbial growth and is broadly
conserved amongst bacteria [84].
O
O
O
NH
n N
H
N
H
n = 2: Andrimid 38; n = 1 moiramide B 39
82
Using (S)-(-)-methylsuccinic acid 40 as a substrate, the synthesis of

a pyrrolidinedione antibacterial 41 is shown in Scheme 8. Through
this route, and also by solid-phase synthesis starting with polymer bound
(S)--phenylalanine, a wide variety of pyrrolidinedione antibacterial are
known in literature [85,86]. Broad structural variations were tolerated at the
fatty acid side chain without adversely affecting the bioactivity. Inhibitory
values (IC50) remained in the nM range for the E. coli and S. aureus
acetyl-CoA carboxylase enzymes with polar and lipophilic side chains
as well.
CO2H
O
i), ii)
HO2C
CO2H
40
86%
iii)
iv), v), vi)
NHBoc
N OBn
N OBn
40%
BocHN
68%
O
O
NC
N
H
vii)
NH
H2N
CO2H
66%
O
N
H
NC
NH
N
H
41
Reagents and conditions: i) MeCOCl, 4h, 60 oC; ii) O-benzylhydroxylamine, CDI, CH2Cl2, 12h, rt; iii) 1. NBoc-(2S)-cyclopentyl glycine, CDI, THF; 2. LiHMDS, THF, 15 min, -65 oC; 3. conc. aq. NH4Cl,- 65 oC - rt;
iv) H2, Pd/C (10%), EtOH, 1h, rt; v) 2'-bromoacetophenone, Et3N, cat DMAP, MeCN, 20h, rt; vi) 4N HCl in
1,4-dioxane, 2h, rt; vii) HATU, iPr2EtN, CH2Cl2, DMF, 10h, 0 oC - rt.
Scheme 8
Apparently, the side chain is not involved in key interactions with the
enzyme and could be used for tuning the physicochemical profile. On the
other hand, the nature of the side chain has a significant effect on
antibacterial activity. A comparison of compounds 41a and 41b demonstrated
that despite excellent target activity of 41a and the benefit of a polar
substituent for other parameters, such as solubility, reasonable lipophilicity
was required for penetration into bacterial cells and for good MIC values.
Replacement of the (S)--phenylalanine by non-aromatic -amino acids led
to a loss in activity. On the other hand significant activity was observed by
varying the leads -ketoamide part, for example, by replacing (S)-valine
with (2S)-cyclopentyl glycine, whereas aromatic amino acids in this position
rendered the molecule inactive.
83
O
O
HO2C
O
N
H
O
O
NH
N
H
O
N
H
41a
NH
N
H
41b
7. Tetrahydropyrimidinone antibiotics
The titled class of antibiotics was first isolated by scientist from the Takeda
Foundation in Japan from Flexibacter species found in soil samples of the Nachi
mountain area of the Wakayama prefecture of Japan [87]. The structures of
TAN-1057A-D 42-45 were disclosed in 1993 [88]. The epimeric
tetrahydropyrimidinones TAN-1057A/B 42,43 were isolated from Flexibacter
species, PK-74, whereas the epimeric dioxo diazepans TAN 1057C/D 44,45
resulted from Flexibacter species PK-176. Total synthesis endeavors and
medicinal chemistry optimization focused on TAN-1057A/B 42,43 due to
instability problem of TAN 1057C/D 44,45. The antibacterial activity of TAN1057A 42 was studied in detail. Its in vitro antibacterial activity against Gram-(+)
organisms such as S. aureus and S. pneumoniae was mediocre under standard
conditions (6.25-12.5 g/mL). However, its in vivo activity was reported superior
to vancomycin and imipenem in a murine S. aureus sepsis model.
NH
O
NH
H2N
N
H
Me
N 5
NH2 O
42 TAN-1057A (5S)
43 TAN-1057B (5R)
H2N
NH O
N
H
NH2
N
H
HN
N Me
2
NH H
N
HN
44 TAN-1057C (2R)
O
45 TAN-1057D (2S)
O
NH2
TAN-1057A/B acts by blocking bacterial protein biosynthesis [89].

The detailed studies found that it inhibited bacterial growth through
binding to the 50S subunit of ribosomes [90]. The synthesis of TAN-1057A/B
42,43 was reported by Yuan and Williams which involved a rather linear
approach starting from triple-protected -homoarginine (Scheme 9) [91]. Meijere
and coworkers published a more convergent synthesis shortly after [92].
The attractive antibacterial properties and the structure of natural
antibiotics TAN-1057A/B attracted several synthetic research groups.
Systematic SAR exploration required novel routes to -lysine and
-homolysine derivatives. The synthetic pyrimidinones (n = 1) 46 and (n = 2)
47 readily accessible on large scale exhibited improved cytotoxicity and
tolerability while retaining eminent potency of the natural compound.
84
8. Biphenomycin
An antibiotic with unusual biological properties, LL-AF283a was
isolated by fermentation of S. filipinesis at the Lederle Laboratories in 1967
[93,94]. Later on the discovery of peptide antibiotic biphenomycin A 48
(WS-43708A) was reported by scientists from Fujisawa in 1984 [95-97]. In
1991, Border and coworkers found that the two antibiotics were identical
[98]. Biphenomycins have unique structure with a cyclic tripeptide
containing a biphenyl moiety in a 15-membered ring.
The in vitro activity of biphenomycin A was almost limited to
Cornbacterium xerosis. It could not affect the growth of other bacteria,
such as S. aureus, E. coli, or S. pyogenes up to 200 mg/mL. However, it was
NH
Z
N
H
OH
N
Z
NH
NH
i), ii), iii)
+
Me
H
N
51%
N
H
Me
N
N
Z
NH O
79%
NHMe
N
H
CO2H
Z
NPhth
CO2tBu
NH2 O
iv)
NH
Z
N
H
Me
N
N
Z
H
N
SMe
NHBoc
, v)
NHZ
52%
CO2H
NH O
Z
NH
Z
vi), vii)
TAN-1057A/
TAN-1057B
42,43
Me
N
N
Z
N
H
66%
NH
Z
O O
MeS
H
N
NHBoc
O
N
H
NHZ
Reagents and conditions: i) BOPCl, 16h; ii) MeNH2, MeOH, 5min; iii) TFA/anisole 25:1, 0 oC to rt, 1h; iv)
Boc2O, Et3N, H2O/dioxane (1:1), 16h; v) EDC, DMAP, CH2Cl2, 16h; vi) TFA/anisole 10:1, 15 min,
evaporation, then Et3N, THF, 10 min; vii) PdCl2, H2, MeOH/CH2Cl2, 2:1, 99%.
Scheme 9
Me
N 5
H2N
NH O
NH2 O
N
H
O
46 n = 1, 47 n = 2
HO
OH
H
N
H2N
O
R
CO2H
N
H
OH
NH2
48 R = OH, Biphenomycin A
49 R = H, Biphenomycin B
NH2
85
highly effective in vivo in a murine sepsis model. It protected mice from an

otherwise lethal infection against S. aureus Smith (ED50 1.0 mg/kg) and was
five times more effective than vancomycin on subcutaneous administration.
The reason for this discrepancy between in vitro and in vivo activity is yet not
clear.
Although both the biphenomycins and vancomycin have a biphenyl
group, there is no evidence of binding of to the cell-wall analogues of N-AcD-Ala-D-Ala. Instead, bacterial protein biosynthesis appeared to be the target
of these antibacterials [99].
The biphenomycins represented an attractive starting point for an
optimization program in medicinal chemistry and need for its synthesis was
felt. The first total synthesis of Biphenomycin B 49 was reported by Schmidt
and coworkers in 1991 [100]. Its sequence involved 1) synthesis of (S,S)isotyrosine, 2) formation of an ansa-tripeptide, 3) macrocyclization, and 4)
removal of protecting groups (Scheme 10). However, besides the total
synthesis of natural biphenomycins A and B, neither derivative nor close
analogues had been prepared. A first series of simplified amide and ester
derivatives, including derivatization at the peptide backbone have been
reported recently [101,85]. The novel congeners 50 and 51 of biphenomycin
B 49, showed improved in vitro activity (Table 7) [102].
Table 7. In vitro antibacterial activity of 50 and 51 against Gram-(+) pathogens, MIC
(g/mL).
Compd No.
50
51
S. aureus
1.5
0.1
E. faecalis
1.0
3.0
B. catarrhalis
1.0
1.0
Thus, the route for the total synthesis of natural compounds and their
congeners with improved in vitro efficacy has been established. A further
insight into the molecular target of these compounds will definitely pave the
way for further development.
HO
OH
H
N
H2N
O
HO
OH
N
H
O
OH
NH2
H
N
H2N
O
NH2
50
N
H
O
OH
NH2
51
OMe
86
BnO
OBn i), ii)
CHO
BnO
OBn
94%
BocHN
BocHN
CO2Me
vii), viii),
ix
88%
BocHN
CO2TMSE
ZHN
CO2Bn
BocHN
CO2Bn
CO2H
O
OBn
OBn
OHC
69%
BnO
iii), iv), v), vi) BnO
BnO
x), xi)
N
Z
ZHN
ETMSO
71%
OBn
O
BocHN
O
N
H
O
CO2Bn
N
Z
xii), xiii)
HO
OH
H
N
H2N
O
H
CO2H
N
H
OH
81%
BnO
xiv), xv)
ZHN
60%
C6F5O
NH2
Biphenomycin B (49)
OBn
O
BocHN
O
N
H
OH
CO2Bn
NHZ
Reagents and conditions: i) methyl N-tert-butoxycarbonyl(dimethoxyphosphoryl)glycinate, LiCl, DBU, MeCN, rt,

1h; ii) Et3N, C, EtOH/CHCl3, (1:1), rt, 2days; iii) LiOH, H2O, dioxane, rt, 12h; iv) [Rh(cod)dipamp)]BF4, H2,
MeOH, rt, 72h; v) BnOH, DCC, DMAP, EtOAc, - 15 to 20oC, 12h; vi) PPTS, acetone, H2O, , 6h; vii) Nbenzyloxycarbonyl(dimethoxuphosphoryl)glycine tromethyl-silyl ester, LiCl, DBU, MeCN, rt, 2h; viii) Et3N, C,
EtOH/CHCl3, (1:1), rt, 2 days; ix) [Rh(cod)dipamp)]BF4, H2, MeOH, rt, 72h; x) HCl, dioxane, 20oC, 2h; xi) EDC,
HOBt, CH2Cl2, 15-20oC, 14h; xii) AcOH/H2O (9:1), 50oC; xiii) Bu4NF, DMF, rt, 1h, C6F5OH, EDC, CH2Cl2, - 15
to 20oC, 14h; xiv) HCl, dioxane/CH2Cl2 (1:1) 0oC, evaporation, CHCl3, NaHCO3, 20oC, 5min; xv) trimethylsilyl
trifluromethanesulfonate, thioanisole, TFA, rt, 30 min.
Scheme 10
9. Tuberactinomycins and capreomycins

Tuberactinomycins 52-55 and capreomycins 56-59 are closely related
cyclic homopentapeptides. The first member viomycin (Tuberactiinomycins
B) 52 was discovered in 1951 [103] and marketed by Ciba and Pfizer as a
tuberculostatic agent in the 1960s. The capreomycins were isolated from the
fermentation of Streptomyces capreolus as a four-component mixture, with
56 and 57 as the major and 58 and 59 as the minor components [104].
Both subclasses showed good activity against Mycobacteria including
multi-drug resistant strains but had only limited activity against other
species [105].
87
R1
H2N
OH
H
N
H
N
N
H
NH2 O
NH
O
OH
O
H
N
HN
H
N
O
NH
R2
N
H
NH2
NH
52 R1 = H, R2 = O, Tuberactinomycin B; 53 R1 = R2 = OH, Tuberactinomycin A

(viomycin); 54 R1 = OH, R2 = H, Tuberactinomycin N; 55 R1 = H, R2 = H, Tuberactinomycin O
Tuberactinomycins exert their antibacterial activity as potent

inhibitors of the translation step of prokaryotic protein biosynthesis by
inhibiting both the initiation and elongation steps. A detailed report on the
interaction of tuberactinomycin at the target level illustrated how these
compounds interacted with RNA [106]. When tested against a panel of
M. tuberculosis strains in vitro, capreomycins compared favorably with
streptomycin, cycloserine, and kanamycin [107]. No cross resistance of
tuberactinomycins with kanamycin, lividomycin, or paronomycin was
observed [108]. The in vivo efficacy of these compounds was low after
oral dosing, but good after subcutaneous administration in experimental
murine tuberculosis models [109]. Although tuberactinomycins were not
devoid of toxicological problems their toxicity profile after i.v. and
preoral administration was quite favorable [110]. These biological
features warranted further evaluation of this class for their clinical use as
antitubercular agents.
R1
O
H2N
H
N
N
H
NH
O
O
H
N
O
NH
HN
N
H
O
H
N
R2
NH2
O
N
NH
H
56 R1 = OH, R2 = -lysyl, Capreomycin IA; 57 R1 = H, R2 = -lysyl, Capreomycin IB;
58 R1 = OH, R2 = H, Capreomycin IIA; 59 R1 = R2 = H, Capreomycin IIB
88
Most of the derivatives prepared for biological testing have been

obtained through fermentation and semisynthesis. The acetylation of the
terminal amino group 60 or both amino groups of -lysine (Scheme 11) led
to a complete loss in activity [111]. Acylation with uncharged or acidic
amino acids at the same position also produced inactive compounds whereas
introduction of a basic amino acid 61 maintained the original MIC [112].
Similarly, blocking of the serine hydroxyl groups 62 left the activity
unaltered [113]. Surprisingly, hydrolysis of the urea functionality produced
63 with comparable in vitro activity as observed for viomycin 52. Oxidation
of 52 yielded the inactive bisamide 64 [114]. Finally, reductive opening of
the capreomycidine ring of 52 forming 65 led to a complete loss in
activity [115].
O P(OiPr)2
O
H
N
O
H2N
NH2
N
H
NH2 O
61
i)
N
H
iv)
OH
H
N
H
N
H2N
O
62
ii), iii)
N
H
NH2 O
NH
NH2 O
O
60
O
HN
H
N
O
NH
vii)
HO
NH2
N
H
HO
65
vi)
O
P(OiPr)2
N
H
OH
O
H
N
NH2
O
NH
v)
52
HN
HN
OH
O
64
63
Reagents and conditions: i) N-acetoxysuccinimide, Et3N, carbonate buffer, dioxane, 1h; ii) Z-D-Orn(Z)OSuc, Et3N, carbonate buffer, THF, 0oC, 12h; iii) H2, Pd, DMF; v) HOH; vi) KMnO4 ; vii) NaBH4
Scheme 11
89
Similar modifications of tuberactinomycine N 54, tuberactinomycine O

55, and capreomycins did not result in an improved activity against
Micobacteria or an extension of the antibacterial spectrum.
The 3,4-dichlorophenylamino analogue 66 of viomycine exhibited good
MICs against the animal pathogens P. multocida (MIC = 0.39 g/mL) but
only mediocre activity against E. faecium and E. faecalis (MIC = 25 and
12.5 g/mL, respectively). Further variation of the substituted ureido
analogues of capreomycines IA/IB (mixture of 56 and 57) yielded novel
compounds with activities against several multidrug resistant gram-(+) pathogens
and gram-(-) E.coli [116]. Despite proven in vitro and in vivo efficacy of the
novel analogues no further clinical development in this class has taken place.
H2N
OH
H
N
H
N
N
H
NH2 O
NH
O
O
HN
H
N
OH
O
H
N
O
NH
Cl
Cl
HO
N
H
NH
66
10. Glycopeptide antibiotics

Vancomycine 6, the first gylcopeptide introduced into clinical practice in
1959 was isolated from Streptomyces orientalis from soil samples by the
Lilly in the mid 1950s [117]. Its structure was unequivocally established in
the early 80s [118, 119]. Teicoplanin 67 is the only additional member of this
class that is available for human use. Both drugs are unaltered natural
antibiotics of the large dalbaheptide group that is produced by various
actinomycetes. Their common structural element is a linear heptapeptide
backbone (configuration R,R,S,R,R,S,S) in which some aromatic amino acids
residues are cross linked (biphenyl and diphenyl ether motives) and build a
rigid concave shape.
Glycopeptides inhibit bacterial cell walls biosynthesis by recognizing and
strongly binding to the L-Lys-D-Ala-D-Ala termini of peptidoglycan
precursor strands at the external side of the membrane. In this way,
transpeptidases are prevented from executing their cross linking job [120].
90
Glycopeptides antibiotics are restricted to treating gram-(+) infections as

they cannot penetrate the outer membrane of gram-(-) bacteria. With the rise
of MRSA infections in hospitals, vancomycines became the antibiotic of last
resort but, due to its frequent use, resistant gram-(+) pathogens, in particular
vancomycine-resistant enterococci (VRE) has emerged and worryingly
spread. By 2003, more than half of the clinical VRE isolates in the US had
become resistant to glycopeptides.
Three semi-synthetic second generation drugs oritavancin (Ly-333328)
[121], dalbavancin (Bi397) [122], and telavancin (TD-6424) [123] have been
advanced to clinical developments [124].
O
OH
O
N
H
OH
OH
O
Cl
OH
HO
HO
O
Cl
O
O
NH
O
O
HN
H
N
N
H
H
N
N
H
NH2
N
H
CO2H
HO
HO
O
OH
OH
OH
O
OH
OH
OH
67
11. Lysobactins
The lysobactins are good examples of structurally exciting natural
products that were isolated from urban soil organisms. Ketanosin B 68 was
isolated from the fermentation broth of Lysobacter sp. SC-14076 (ATCC
53042) by scientists from Squibb [125,126]. So far its total synthesis has not
91
been published. Recently, katanosin A has been found as a minor metabolite

of the Lysobacter sp. ATCC53042 starin [127]. Lysobactin and katanosin A
are highly active against Gram-(+) bacteria such as staphylococci and
enterococci. Their excellent in vitro antibacterial activity was maintained in
vancomycin-resistant enterococci. Promising therapeutic in vivo efficacy
has been demonstrated in a systemic murine S. aureus infection model
(ED50 = 1.8 mg/kg, i. v., CFU = 105) [128].
The primary target of lysobactin antibiotics appears to be the bacterial
biosynthesis. These compounds inhibit consumption of the cell-wall
precursor [14C]GlcNAc, a very good indicator for interference with the
peptidoglycan biosynthesis. The inhibition of peptidoglycan formation is
most likely induced by the binding of lysobactin to lipid intermediates (not
through binding to biosynthetic enzymes) that occurs as biosynthetic
precursors downstream of the muramyl pentapeptide.
The lysobactins are interesting antibacterial lead structures with
promising in vitro activity and in vivo efficacy. So far, the knowledge about
this class is based on these natural products and some semi-synthetic Edman
derivatives. A preliminary SAR has been established for the amino acid
position 1 within the liner segment. Further investigations need to be done for
a real assessment of the potential of this class.
H2N
O
HO
OH
H
N
H
N
NH
O
O
H2N
N
H
NH
HN
N
H
HO
NH
O
O
NH
HO
N
H
NH2
HN
NH
68
92
12. Enopeptin depsipeptide antibiotics

The name of the family was derived from two depsipeptides enopeptin A
69 and depsipeptides enopeptin B 70 isolated in 1991, from a culture broth of
Streptomyces sp. RK-1051, found in a soil sample collected in Tsuruoka city
of Japan [129]. They consist of a 16-membered lactone ring made up of five
(S)-amino acids and a lipophilic polyene side chain attached to the serine
N-terminus [130]. About a decade ago the Eli Lilly published the isolation
of a similar depsipeptide antibiotic A54556 71,72, a complex of eight
depsipeptide factors A-H, which was produced by aerobic fermentation of
Streptomyces hawaiiensis (NRRL 15010) [131].
Mode of action studies with B. subtilis demonstrated impaired bacterial
cell division and induction of filamentation. It has been shown by using RG
techniques that lead structures inhibited bacterial growth by binding to
caseine lytic protease [132,133].
R
O
N
O
O
NH
N
O
N
O
N
H
HO
O
HN
N
H
69 R = Me, Enopeptin A
70 R = H, Enopeptin B
71 R = Me, A54556A
72 R = H, A54556B
The natural enopeptin depsipeptides antibiotics had promising in vitro

activity against enterococci and streptococci but only moderate in vitro
potency against staphylococci and were inactive against Gram-(-) bacteria.
Both lead structures were not effective in vivo in standard lethal bacterial
infection models in mice and their ADME profile was critical. Their chemical
stability proved to be rather limited. Their solubility was insufficient for
parenteral application and they were readily cleared from the body. The
in vivo antibacterial activity of some natural and synthetic enopeptins is
described in Table 8.
93
O
R
O
O
O
NH
O
O
N
H
HN
NH
C6H11
N
H
HN
O
77
73-76
O
O
NH
O
N
O
N
H
O
O
HN
C4H9
RO
NH
O
N
O
N
H
HN
C4H9
O
O
79, 80
78
Table 8. Antibacterial activity of some natural and synthetic enopeptins.

No.
71
72
73
74
75
76
77
78
79
80
R
Me
H
H
3-F
3,5-F2
3,4,5-F3
H
COCH2NMe2
S. aureus
8
16
> 64
1
0.5
8
1
0.125
0.25
0.5
S. pneumoniae
0.5
1
> 64
0.25
0.125
2
0.125
0.125
0.125
0.125
E. faecium
1
2
> 64
0.125
0.125
2
0.125
0.125
0.125
0.125
E. faecalis
1
2
> 64
0.125
0.125
1
0.125
0.125
0.125
0.125
13. Tetracycline antibiotics

Tetracyclins belong to the group of polyketides. They are old-known
class of broad-spectrum antibiotics whose use has been reduced in recent
times with the onset of bacterial resistance. They consist of an
octahydrotetracene-2-carboxamide skeleton [134]. The first member of the
group, chlorotetracycline 81 was discovered in 1940s from a golden-colored
fungus-like, soil-dwelling bacterium called Streptomyces aureofaciens. Soon
after oxytetracycline 82 was discovered from a similar soil bacterium called
Streptomyce srimosus. The structure of oxytetracycline was determined by
94
Woodword and coworkers [135], which led to its synthesis by Conover and
coworkers. Doxycycline 83 is the most commonly known semi-synthetic
drug of this class.
In 2005, tigecycline [136] 84 belonging to the subclass of
glycylcyclines was introduced to treat infections that were resistant to other
antimicrobics including conventional tetracyclins [137]. Newer versions of
tetracyclins are currently in trials. Tetracyclins inhibit the protein
biosynthesis by inhibiting the binding of aminoacyl-tRNA to the mRNAribosome complex. Tetracyclins have also been found to inhibit matrix
metalloproteinase. This mechanism does not contribute to their antibiotic
activity, but has led to extensive research on chemically modified
tetracyclins. Tetracyclins inhibit cell growth by inhibiting translation.
It binds to the 16S part of the 30S ribosomal subunit and prevents the
aminoacyl tRNA from binding to the A site of the ribosome. The binding is
reversible in nature [138].
OH
O
OH
OH OH
O
OH
OH OH
H2N
H2N
O
N
HO
OH Cl
81
OH
82
OH O
OH O
OH
O
NH2
H
OH
OH
83
N
H
N
O
N
H
OH
O
OH O
84
OH
OH O
NH2
OH
95
14. Aminoglycoside antibiotics

Aminoglycoside antibiotics are among the oldest known class of
antibiotics [139, 140]. Although much has been written on this class it would
be worth mentioning the representatives of this class streptomycin
85, neomycin 86 and gentamycin 87 for completeness of the article.
The well-known streptomycin was isolated by Waksman and coworkers in
1944 from cultures of Streptomyces griseus. It was the first effective drug for
treatment of tuberculosis. Aminoglycosides are often administered into veins
or muscle to treat serious bacterial infections. Some aminoglycosides are also
used orally to treat intestinal infections or topically to treat eye infections.
Among several modes of actions some are protein biosynthesis inhibitors and
thus compromising the structure of the bacterial cell-wall. The mechanism of
resistance to aminoglycoside antibiotics has been investigated [141]. Recent
advances in structure, molecular mechanism, SAR, aminoglycoside mimetics
have been reviewed by Silva and Carvalk [142]. The current efforts to develop
new aminoglycoside derivatives with modification and reconstruction on each
sugar ring and advances in SAR have also been reviewed by Zhou and coworkers
[143].
The recent emergence of infections due to Gram-(-) bacterial strains with
advanced patterns of antimicrobial resistance has prompted reevaluation of
the use of aminoglycoside antibacterial agents [144]. This revived interest has
brought back to light the debate on the two major issues related to these
compounds, namely the spectrum of antimicrobial susceptibility and toxicity.
Current evidences show that aminoglycosides retain activity against the
majority of Gram-(-) clinical bacterial isolates in many parts of the world.
OH
Me
OHC
O
HO
O
HO
HO
OH
OH
H2N
NH2
N
O
O
N
H
OH
HO
HO
Me
HO
NH2
NH2
NH2
HO
H2N
O
OH
OH
85
NH2
NH2 H2N
OH
Me
HN
Me
OH
H2N
O
OH
O
H2N
HO
OH
87
O
NH2
Me
NH2
86
NH2
96
However, the relatively frequent occurrence of nephrotoxicity and ototoxicity

during aminoglycoside treatment makes physicians reluctant to use these
drugs in everyday practice. Recent advances in the understanding of the
effect of various dosage schedules of aminoglycosides on toxicity have
provided a partial solution to this problem, although more research still needs
to be done in order to overcome this problem entirely [145].
15. Concluding remarks

Pathogenic bacteria are increasingly evading the standard treatment for
antibacterial infections as resistance to multiple antibiotics is spreading
worldwide. Resistant pathogens lead to higher expenditure on treatments due
to extended stay in hospitals and expensive medicines. There is an urgent
need for a sustainable supply of new, potential and safer antibacterial drugs
having no cross-resistance to currently used antibiotics.
Nature so far has been proved the treasure of potential remedies for
diseases. It is more relevant for treatment of infectious diseases. Most of the
antibacterials in use today could be discovered on the basis of information
obtained from the study of natural products from microbes, marines and
plants. Medicinal chemists do systematic studies and provide the tools for
optimization of natural products to obtain drug molecules with improved
pharmacokinetic, physicochemical and toxicological properties. The diligent
selection of natural antibiotics lead structures for medicinal chemistry
programs and guideposts for valid targets can reveal pathways for future
therapies. Advance studies have been conducted on many classes in order to
identify the drug target and mechanism of action.
There is still a plenty of scope even within the known antibiotics. Many old
classes have not been thoroughly investigated and only partial SAR
information is available on their backbone structures. Macrolides are an
excellent example that many old classes have not been completely explored.
Undoubtedly, more could be done to fully exploit the weapons against bacteria.
-Lactams continue to be one of the most important classes in antibacterial
research and development. Their efficacy and compatibility has allowed their
broad therapeutic application. Further investigations on second generation
lincosamides might be useful for successful treatment of infections by
enterococci. Biphenomycins appear promising for the future studies. Further
work is anticipated on lysobactins for a real assessment of this class.
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Research Signpost
37/661 (2), Fort P.O.
Trivandrum-695 023
Kerala, India
ISBN: 978-81-308-0448-4
3. Anti-tubercular activity of natural

products: Recent developments
L. N. Rogoza, N. F. Salakhutdinov and G. A. Tolstikov
Department of Chemistry, Russian Acad Sci, Siberian Div, Vorozhtsov Inst Organ Chem
Pr Akad Lavrenteva 9, Novosibirsk 630090, Russia
Abstract. An increasing incidence of deaths due to tuberculosis and

the known drawbacks of the current existing drugs including the
emergence of multi drug-resistant strains have led to a renewed interest
in the discovery of new anti-tubercular agents. The recent researches
focused on natural products have shown a useful way to obtain a
potentially rich source of drug candidates. This review covers the most
active naturally occurring compounds with antitubercular properties at
minimal inhibitory concentrations (MICs) of 5 mg/mL or less. The
literature from January 2001 to 2009 is reviewed. The compounds are
presented in order of chemical type, namely alkynes, heterocyclic
compounds, phenols and quinones, peptides, alkaloids, terpenoids and
steroids.
1. Introduction
Tuberculosis is a chronic infectious disease, one of the major enemies of
the humanity from times immemorial. Today it still remains one of the most
serious medical and social problems. It is responsible for 3 million deaths per
year and around 8 million cases of first-recorded disease. The advances in the
chemotherapy of tuberculosis in the mid-20th century have recently given
way to anxiety over the evolution of drug resistance based on the genetically
Correspondence/Reprint request: Prof. L. N. Rogoza, Department of Chemistry, Russian Acad Sci, Siberian
Div, Vorozhtsov Inst Organ Chem, Pr Akad Lavrenteva 9, Novosibirsk 630090, Russia
E-mail: rogoza@nioch.nsc.ru
104
L. N. Rogoza et al.
fixed mutations of M. tuberculosis. Moreover, nearly all drugs used for the
treatment of tuberculosis and possessing different mechanisms of activity are
able to cause adverse side effects on the human organism. Therefore, it is
extremely important to search for new, low-toxic substances superior to the
available drugs in their activity and efficiency. This primarily concerns the
agents possessing activity against M. tuberculosis strains with multidrug
resistance.
Modern tuberculosis is generally associated with M. tuberculosis and
M. bovis, mycobacteria that are pathogenic to the human organism. Because
of slow growth and pathogenicity of M. tuberculosis H37Rv, many research
groups used fast-growing and/or nonpathogenic mycobacteria including
M. tuberculosis H37Ra, M. smegmatis, M. aurum, and others as organisms to
be tested. The antimycobacterial activity was also investigated on M. avium
and M. intracellulare, which cause bird tuberculosis and are associated with
human diseases in advanced countries (AIDS patients and immunocompromised
individuals), to find compounds with a wide range of activity.
A special group of research works includes investigations on
M. tuberculosis clinical isolates and strains possessing multidrug resistance.
Multidrug-resistant tuberculosis (MDRTB) is strictly defined as
M. tuberculosis strains showing resistance simultaneously against isoniazid
and rifampicin [1, 2]. Tuberculosis with a different drug resistance (DDRTB)
involves M. tuberculosis strains displaying mono- or polyresistance not
including associated resistance against isoniazid and rifampicin [3]. The
M. tuberculosis strains may be sensitive (inhibited by first series drugs such
as isoniazid) or resistant (not inhibited by isoniazid). Since researchers use
different analytical procedures and/or organisms under test, care should be taken
in comparing the biological activities obtained by different authors.
The review covers publications from 2001 to first half 2009; the selected
structures have minimum inhibiting concentrations (MIC) of 5 g/mL or less.
Due to this limitation, the most effective compounds were analyzed within one
review. For better insight into the "structure-property" relationship, we
occasionally gave structures with higher MIC values. The review includes the
introduction section, two chapters on synthetic and natural compounds with an
antimycobacterial activity, and the conclusions section. To reveal possible
"structure-activity" relationships, we grouped the data according to chemical
structures.
2. Alkynes and heterocyclic compounds

The metabolite of several strains of the endophytic fungus of the
genus Phomopsis, 3-nitropropionic acid (1), actively inhibited growth of
Anti-tubercular natural products
105
M. tuberculosis H37Ra (MIC 0.4 g/mL). Although the high neurotoxicity of

this compound was a hindrance to its use as a pharmaceutical, it could be
used as a model for the synthesis of new inhibitors of isocitratelyase, an
enzyme essential to the catabolism of fatty acids and virulence of
M. tuberculosis [4]. Linoleic acid (2) that inhibits growth of M. phlei (MIC 2
g/mL) is extracted from the plant Humulus lupulus. An example of
polyacetylene compounds is 3S,8R stereoisomer (3) isolated from Anethum
graveolens and having MIC 2-4 g/mL when tested on a group of fast
growing mycobacteria (M. fortuitum ATCC 6841, M smegmatis ATCC
14468, M. phlei ATCC 11758, M. aurum Pasteur Institute 104482, and
M. abscessus ATCC 19977; for ethambutol, MIC 0.5-4 g/mL) [5].
However, cytotoxicity of this class of polyacetylene compounds can lower
the interest in their biological activity [6]. Compounds (4a) and (4b), the
synthetic analogs of the natural antibiotic thiolactomycin, inhibit growth of
M. tuberculosis with MIC 1-16 g/mL, including drug-resistant strains [7].
OH
HOOC
NO2
CO2H
3
(2)
(1)
C7H15
(3)
OH
O
O
C8H17
OH
R
(4a), R = O(CH2)10Br
(4b), R = O(CH2)8SCH2COOMe
(5)
(6)
Investigation of the components of the plant Cinnamomum kotoense led to

the isolation of a number of compounds, of which lincomolide (5) with MIC
2.8 g/mL had the highest antituberculosis activity [8]. Micromolide (6), which is
a -lactone derivative of oleic acid, was isolated from the stem bark of
Micromelum hirsutum and has MIC 1.5 g/mL against M. tuberculosis (H37Rv).
Further evaluation of activity on J774 mice cells infected with a more
virulent strain of M. tuberculosis Erdman gave MIC 5.6 g/mL [9].
2-Substituted furans (7a,b) and (8a,b) isolated from the roots of Polyalthia
evecta possess activity against M. tuberculosis (MIC 3.1 and 6.25 g/mL,
respectively) [10]. The synthesized natural compound pamamycin-607 (9)
inhibits growth of M. bovis BCG, M. smegmatis and M. tuberculosis (MIC 0.54.7 g/mL). It does not show cross resistance to isoniazid and rifampicin [11].
106
L. N. Rogoza et al.
R
O
(CH2)13
O
n
OR
(7a) R = H
(7b) R = Me
(8a) n = 5 R = C15H31
(8b) n = 3 R = (CH2)13OH
NMe2
O
O
O
O
O
O
(9)
3. Phenols and quinones

Phenylpropanoids (10) and (11), metabolites of Pimpinella sp.,
inhibit growth of a number of mycobacteria, including M. intracellulare,
M. smegmatis, M. aurum, and M. phlei (MIC 1.25-10 g/mL) [12]. The
tricyclic diphenol ether engelhardion (12) is very active against M. tuberculosis
H37Rv (MIC 0.2 g/mL) [13]. (-)-4-Hydroxy-1-tetralone (13, MIC 4.0 g/mL)
& 3-methoxycarboxy-1,5-dihydroxyantraquinone (15, MIC 3.125 g/mL) were
isolated as an antituberculosis component of Engelhardia roxburghiana [13].
As is known, the level of the intra- and extracellular inhibition of
M. tuberculosis by 7-methyljuglon (14) (MIC 0.5 g/mL) extracted from the
plant . natalenis, is comparable to that of streptomycin and ethambutol
(MIC 1 and 2 g/mL, respectively). Its derivatives, namely, 5-hydroxy-,
5-alkoxy-, and 5-acetoxy-8-substituted naphthoquinones, are less active (MIC
2.5- >20 g/mL) and possess low antituberculosis selectivity, probably
because of their nonspecific activity with various disulfide reductases found
in mammal cells. Optimization of the specificity of these compounds for
mycothiol disulfide reductase, which is one of the several biological targets
for the antituberculosis activity of naphthoquinones of this structure, is
required [14].
107
HO
OH
O
O
O
H
O
(11)
(10)
O
(12)
OH
OCH3
H
OH
OH
(13)
OH
O
(15)
(14)
Marine metabolites pseudopyronines A and B (16a,b, MIC 0.78-3.125

g/mL) inhibit the growth M. tuberculosis H37Rv [15]. Pyrone (17, MIC 4
g/mL) is a component of Piper sanctum that is active against M. tuberculosis
H37Rv [16]. Ferulenol (18a) isolated from the Sardinian giant fennel Ferula
communis is effective against M. smegmatis (MIC 0.5 g/mL), as well as
M. fortuitum, M. phlei and M. aurum (MIC 2 g/mL). The analogs of this
compound, (18b-d), were isolated from the same plant; compound (18b) with
a benzyloxy group retained its activity against M. smegmatis and M. phlei,
and, to a lesser extent, against M. fortuitum and M. aurum, while the activity
of (18c) and (18d) with the hydroxy and acetoxy groups is considerably
lower [17]. Ostruthin (19), the metabolite of Peucedanum ostruthin Koch,
inhibit the growth M. aurum (IC 3.4 g/mL) [18].
OH
OMe
O
(16a), R = C5H11
(16b), R = C7H15
(17)
OH
R
O
O
(18a), R = H
(18b), R = OBz
(18c), R = OH
(18d), R = OAc
HO
O
(19)
108
L. N. Rogoza et al.
OH
R
OH
OH
O
O
O
OH
HO
R2
O
O
(21)
R1
(20a)
(20b)
(20c)
(20d)
(20e)
(20f)
(20g) R = O
(20h) R = OH
R1 = OH, R2 = H
R1 = OH, R2 = H, saturated
R1 = O, R2 = H
R1 = O, R2 = H, saturated
R1 = O, R2 = OMe, saturated
R1 = O, R2 = OH, saturated
HO
OH
OH
OH
OR
OAc
(22c)
RO
OR
O
AcO
OAc
OH
O
HO
OR
OAc
OH
OH
(22a) R = Ac
(22b) R = H
Compounds (20a-h), isolated from the lichen fungus Microsphaeropsis

sp., show different activities against M. tuberculosis H37Ra (MIC 25, 3.12,
3.126.25, 6.25, 12.5, 25, 1.56-3.12, 50 g/mL, respectively), but are also
characterized by cytotoxicity [19]. The dibenzofuran derivative, usnic acid
(21), which is a secondary metabolite of lichen, inhibits growth of
M. tuberculosis (MIC 2.55 g/mL) [20]. One of the xanthone dimers
isolated from the endophytic fungus of the genus Phomopsis, phomoxanthone
A (22a), is very active against M. tuberculosis H37Ra (0.5 g/mL), while its
deacetylated derivative (22b) is inactive. Phomoxanthone B (22c) is less
active (MIC 6.25 g/mL). Both active compounds are cytotoxic [21].
The anthraquinone celastramycin B (23), isolated from the unknown
species Streptomyces, is active against M. Vaccae (MIC 3.1 g/mL) [22]. The
anti-HIV agent (+)-calanolide A (24) was tested for the antituberculosis
activity; a combination of the anti-HIV and antituberculosis activities in one
agent is very attractive in view of the concurrence of these diseases. This
compound, isolated from the tropical tree Calophyllum lanigerum, also has
an antituberculosis activity against M. tuberculosis (MIC 3.13 g/mL) and a
number of drug resistant strains (MIC 816 g/mL) [23].
OH
109
OH
Cl
O
O
(23)
OH
OH
(24)
4. Peptides
Four cyclic peptides, namely, enniatins H (25a), I (25b), B (25c), and B4
(25d), which are the components of the pathogenic fungus Verticillium
hemipterigenum, inhibit growth of M. tuberculosis H37Ra (MIC 3.126.25
g/mL) [24]. Syringomycin E (26), isolated from Pseudomonas syringae pv.
Syringae, is active against M. smegmatis (MIC 1.5 g/mL) [25].
The metabolite of Nocardia sp. (ATCC 202099), namely, the thiazole
peptide nocathiacin (27) shows activity against M. tuberculosis ATCC 35828,
M. avium A26778, and M. avium A26640 (MIC 0.008, 0.06, and
0.25 g/mL, respectively). Unfortunately, compounds from this class
typically show poor pharmacokinetics and solubility (the latter problem can
be solved by synthesizing analogs with higher solubility in water) [26].
5. Alkaloids
Two compounds, namely, the known antibiotic pyrrolnitrin (28) and
banegasine (29), isolated from the zoobacterium Aristabacter necator, act
synergetically against M. smegmatis (MIC (29) >0.5 g/mL, (28) 0.3 g/mL,
(28) + (29) 0.075 g/mL) [27]. Their analog celastramycin A (30), which is a
dichloropyrrole metabolite of the Streptomyces strain, has a broad spectrum
of antimycobacterial activity (MIC 0.05-3.1 g/mL against M. smegmatis,
M. aurum, M. vaccae, and M. fortuitum) [22]. The bis-1-oxaquinolizidine
alkaloid ()-araguspongine (32), isolated from the sea sponge
Xestospongia exigua, inhibits growth of M. tuberculosis H37Rv (MIC 1.9
g/mL) [28].
In the series of quinolone alkaloids (33a-d), isolated from the fruits of
Evodia rutaecarpa, compounds with usaturated aliphatic chains at 2-position
exhibited better antimycobacterial activity as compared with saturated chain
compounds [18]. Agelasine E (33a) and agelasine D (33b) were previously
isolated from the sea sponge Agelas nakamurai. While agelasine is
inactive, its methoxy analogs (33c-g), having different terpenoid side chains,
110
L. N. Rogoza et al.
R1
O
O
N
R6
R4
O
O
HN
NH2
H
N
NH2
N
OH
O
O
NH
O
O
HO
(CH2)2NH2
(26):R = NHCOCH2CH(OH)(CH2)8CH3
OH
NH
N
H
S
H
N
NH
H
N
O
H
N
CH2OH
N
H
HO
Me2N
NH
R1 = R2 = R3 = R5 = R6 = i-Pr; R4 = s-Bu;
R1 = R2 = R3 = R6 = i-Pr; R4 = R5 = s-Bu;
R1 = R2 = R3 = R4 = R5 = R6 = i-Pr;
R1 = i-Bu; R2 = R3 = R4 = R5 = R6 = i-Pr
MeO
NH
H2N(H2C)2
R5
O
(25a)
(25b)
(25c)
(25d)
R2
CH(OH)CH2Cl R
O
CH(OH)CO2H
HN
H
N
O
N
OH
O
(27)
Cl
Cl
OH
NH2
NO2
NH
Cl
Cl
N
H
N
H
(28)
OH
COOH
(30)
(29)
(31a)
N
(31b)
(31c)
(31d)
HO
H
O
N
N
O
H
OH
(32)
Cl
111
demonstrate high activity against M. tuberculosis H37Rv (MIC 3.13, 1.56, and
3.13 g/mL respectively). Possibly, the presence of an alkoxy group at the
terminal nitrogen atom is a very important factor for the antimycobacterial
activity of these compounds. However, there is only slight difference
between the activities of agelasine D (33b) and its alkoxy derivatives (33f)
and (33g) [29]. It is interesting that the simpler analog of the compounds,
9-methyladenine (33h), has MIC of 6.25 g/mL [30].
The tetracyclic alkaloid cryptolepine (34a), isolated from Cryptolepis
sanguinolenta, is active against a number of fast-growing mycobacteria,
including M. aurum (MIC 2 g/mL), M. phlei (MIC 4 g/mL), and
M. fortuitum (MIC 16 g/mL) [31]. Metabolite of Allium neapolitanium (34b)
R1O
NH2
R
N
N
N
HN
NH2
N
N
Me
Me
Me
(33a) R = c
(33b) R = d
(33c) R = a, R1 = Me
(33d) R = b, R1 = Me
(33e) R = c, R1 = Me
(33f) R = d, R1 = Me
(33g) R = d, R1 = t-Bu
(33h)
a
n
Me
N
R
N
H
(34a)
(34b), R = H
(34c), R = OH
112
L. N. Rogoza et al.
displayed enhanced activity against the M. smegmatis (mc22700), when

compared with that for (34c) (MIC 2-8 g/mL). Furthermore, the activity of
(34b) was greater against M. smegmatis (mc2 2700) than M. smegmatis
(ATCC 14468) (MIC 16 g/mL for (34c) and 8 g/mL for (34b) [32].
The metabolites of the Thailand pathogenic fungus Hirsutella nivea BCC
2594 hirsutellones A-D (35a-d) inhibit growth of M. tuberculosis H37Ra
(MIC 0.78, 3.125, 0.78, 0.78 g/mL, respectively). Compound (35d) exhibits
moderate in vitro cytotoxicity, while other compounds are less cytotoxic [33].
Hirsutellone F (35e), which is a new dimer alkaloid isolated, together with
known hirsutellones A, B, and C, from the seeds of the fungus Trichoderma
sp. BCC 7579 shows a weaker antituberculosis activity against M. tuberculosis
H37Ra (MIC 3.12 g/mL) than hirsutellones , , and [34].
The known alkaloid ecteinascidin 770 (36a) and the new one,
ecteinascidin 786 (36b), both isolated from Ecteinascidia thurstoni, inhibit
growth of M. tuberculosis H37Ra (MIC 0.1 and 1.6 g/mL, respectively) [35].
Manzamine alkaloids isolated from sea sponges are promising from the
viewpoint of their antituberculosis activity. Manzamines (37a), (37c),
and F (37d) and their hydroxyl derivatives 6-hydroxymanzamine (37e) and
O
NH
NH
(35a) R = H
(35b) R = Me
OH
OH
OH
NH
N
H
(35d)
H
(35c)
O
O
(35e)
NH
O
113
(+)-8-hydroxymanzamine (37b) show activity against M. tuberculosis

H37Rv (MIC 1.5, 3.8, 2.6, 0.4, and 0.9 g/mL, respectively) [36].
Manadomanzamines A (38a) and B (38b) inhibit growth of M. tuberculosis
H37Rv (MIC 1.9 and 1.5 g/mL, respectively) [37].
OMe
R
HO
OAc
N
N
R2
N
H
N
O
O
MeO
OH
R1
R1
N
O
NH
HO
(37a), R = R1 = H
(37b), R = H, R1 = OH
(36a) R1 = CN, R2 = none

(36b) R1 = CN, R2 = O
R
N
N
H
OH
H
R1
22
O
N
H
N
N
H H
H
OH
N
HN
O
(37c), R = R1 = H
(37d), R = H, R1 = OH
(37e), R = OH, R1 = H
(38a), 22H
(38b), 22H
114
L. N. Rogoza et al.
6. Terpenes
Compound (39), isolated from Indigofera longeracemosa, is active
against M. tuberculosis (MIC 0.38 g/mL) [39]. Diterpenes (40) and (41)
from Calceolaria pinnifolia [40], the structurally related lecheronol A
(42), isolated from Sapium haematospermum (MIC 4 g/mL) [40], and
metabolite of Melica volkensii 6-hydroxyculactone (43) [18] have the
same value of MIC against M. tuberculosis H37Rv. Ugandensidial (44,
from Warbugia ugandensis) inhibit growth of M. aurum and M. phlei at
this value of MIC [18].
OMe
O
O
OAc
CH2OCOCH2COOH
MeOOCH2COCOH2C
(40)
(39)
(41)
OH
OHC
OH
CHO
OH
O
H
OCOCH3
O
H
OH
(42)
(43)
(44)
The diterpenes diaportheines A (45a) and B (45b) were isolated from the
fungus Diaporthe sp. Compound (45b) has antituberculosis activity against
M. tuberculosis H37Ra (MIC 3.1 g/mL) and cytotoxicity, while compound
(45a) is much less active and cytotoxic (MIC 200 g/mL) [41]. These data
indicate that the presence of a carbonyl group is important for the
antituberculosis activity. A metabolite of the African tree Combretum
imberbe, traditionally used in folk medicine is imberbic acid (46), which
shows activity against M. fortuitum (MIC 1.56 g/mL) [42].
115
HOOC
HO
OH
O
OH
OH
R2
R1
OH
HO
Me
(46)
(45a) R1 = OH, R2 = H
(45b) R1 + R2 = O
The chemical modifications of the parent structure of ursolic acid (at the
C-3 position to cinnamate-based esters) resulted in an 4-fold increase in
antimycobacterial activity ((47), MIC 3.13 g/mL for M. tuberculosis
H37Ra, for ursolic acid MIC 12.5 g/mL) [43].
COOH
O
CO
R
MeO
(47a) R = OAc
(47b) R = OH
O
R1
COOH
R2
MeO2C
MeO2C
HO
(48)
(49a) R1 + R2 = O
(49b) R1 = H, R2 = OH
116
L. N. Rogoza et al.
24
OH
H
H
HO
(51) saturated
(52) unsaturated
(50a) 24R
(50b) 24S
R2
O
O
O
O
R1O
HO
(54a) R1 = CO(CH2)16Me, R2 = Me
(54b) R1 = H, R2 = Me
(54c) R1 = H, R2 = Et
(53)
H
H
O
HO
H
CHO
OH
(54d)
(55a) R = Me
(55b) R = Et
Triterpene (48), isolated from Elateriospermum tapos, is active against

M. tuberculosis H37Ra (MIC 3.13 g/mL, for isoniazide and kanamicin
sulfate MIC 0.05 & 1.25 g/mL, respectively) [44]. Aegicerin (49a) and
protoprimulagenin A (49b) were isolated from Aegiceras sp., Embelia
Schimperi, and the Peruvian plant Clavija procera. Aegicerin (49a) was
tested on 37 different strains of tuberculosis (MIC 1.6-3.1 g/mL against one
strain of H37Rv, 21 sensitive clinical strains, two clinical isolates resistant to
isoniazid, and 13 MDR clinical strains). The inactivity of protoprimulagenin
117
A (49b) (MIC 200 g/mL) demonstrates that as in the case of (45a) and
(45b), the presence of a carbonyl group is critical for the antituberculosis
activity. For the first time, an oleane type triterpene shows uniformly high
activity against a wide range of both sensitive and resistant strains.
Regretfully, for many MDR strains, its excellent antituberculosis activity (for
comparison, MIC is 4-32 g/mL for isoniazid and 2-16 g/mL for rifampicin)
has not yet been effected [45].
7. Steroids
Saringosterol, isolated from brown seaweeds Sargassum ringgoldianum
and Lessonia nigrescens in the form of a 1:1 mixture of the 24R isomer (50a)
and 24S isomer (50b), inhibits growth of M. tuberculosis H37Rv (MIC
0.25 g/mL) and has low cytotoxicity. In pure form these isomers possess
different levels of activity (MIC is 0.125 g/mL for the 24R isomer and
1 g/mL for the 24S isomer) [46].
Lipids that inhibit growth of M. tuberculosis H37Rv were isolated from
an extract from Morinda citrifolia (Rubiaceae), traditionally used in folk
medicine in the Philippines for the treatment of tuberculosis and respiratory
diseases. The highest activity was found for a mixture of (51) and (52) (MIC
<2.0 g/mL for the 2:1 mixture) and endoperoxide (53) (MIC 2.5 g/mL)
[47]. Sterines (54a-d), isolated from an extract from the Argentinian plant
Ruprechtia triflora, are active against M. tuberculosis (MIC 2-4 g/mL) [39].
Synthetic analogues (55a,b) of 5(67) abeo-sterol from the Caribbean
Sea sponge Svenzea zeai inhibit growth of M. tuberculosis H37Rv
ATCC 27294 (MIC 3.8 & 3.9 g/mL, respectively) but possess moderate
cytotoxicity [48].
8. Conclusions
More than 50 % of the medicines introduced in world medical practice
are connected with natural compounds to some extent. It can be as native
metabolites and synthetically the modified derivatives. Enthusiastic examples
of discovery of natural compounds with remarkable pharmacological
properties such as antitumoral metabolite taxol or antimalarial metabolite
artemisinin and also tens other compounds of a plant and animal origin with
various high biological activity allow to hope for prompt discovery of highly
effective low-toxic natural compound which will be leader in struggle against
a tuberculosis.
118
L. N. Rogoza et al.
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Research Signpost
37/661 (2), Fort P.O.
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ISBN: 978-81-308-0448-4
4. Scope of natural products in

fighting against leishmaniasis
B. B. Mishra, R. R. Kale, V. Prasad, V. K. Tiwari and R. K. Singh
Department of Chemistry & Biochemistry, Faculty of Science, Banaras Hindu University
Abstract. Leishmaniasis, a group of tropical diseases resulting from

infection of macrophages by obligate intracellular parasites of genus
Leishmania, is a major health problem worldwide. Growing incidence
of resistance for the generic pentavalent antimony complex for
treatment in endemic and non-endemic regions has seriously hampered
their use. The second line drugs such as amphotericin B, paromomycin
and miltefosine are the other alternatives, but they merely fulfill the
requirements of a safe drug. The recent researches focused on natural
products have shown a wise way to get a true and potentially rich
source of drug candidates against leishmaniasis. The present review
initially highlights the current status of leishmaniasis, synergy of the
disease with HIV, therapeutic options available and in later
sections summarizes natural products that have shown significant
antileishmanial activities. In order to highlight any possible mechanism
based action, the review has been organized according to chemical
structural classes.
1. Introduction
The Leishmania are Kinetoplastid protozoans that cause four main clinical
syndromes: Cutaneous Leishmaniasis; Muco-cutaneous Leishmaniasis (also
known as espundia); Visceral Leishmaniasis (VL; also known as kala-azar); and
Difuse Leishmaniasis. Leishmaniasis continues to be one of the six entities
Correspondence/Reprint request: Dr. Rakesh K Singh, Department of Chemistry & Biochemistry, Faculty of
Science, Banaras Hindu University, Varanasi-221005, India. E-mail: rakeshbhu@yahoo.com
122
B. B. Mishra et al.
on the World Health Organization tropical disease list [1]. Leishmania

species are transmitted by 30 species of sand fly and essentially requires two
different hosts: an invertebrate insect vector, Phlebotomus (in the OldWorld)
or Luztomiya (in the NewWorld) sandfly-mosquito and a vertebrate host
(human, dog or even a wild vertebrate) [2].
Leishmaniasis is prevalent in tropical and temperate regions of world,
ranging from rainforests in Central and South America to deserts in West
Asia and the Middle East. Current epidemiological reports estimate about 350
million populations at risk with 12 million people affected worldwide, while
1.5-2 million new cases being recorded each year. The visceral leishmaniasis
has an estimated incidence of 500,000 new cases and 60,000 deaths each year
with more than 90 % of cases are centralized to India, Bangladesh, Nepal,
Sudan, and Brazil [3].
There are a growing number of reports of Leishmania/human
immunodeficiency virus (HIV) co-infections across the world. LeishmaniaHIV co-infection has been globally controlled in Southern Europe since 1997
by highly active anti retroviral therapy (HAART), but it appears to be an
increasing problem in other countries such as Ethopia, Sudan, Brazil or India
where both infections are becoming more and more prevalent [4]. The
situation is particularly alarming in southern Europe, where 50-75% of adult
VL cases are HIV positive and among the 45 million people infected by HIV
worldwide, an estimated one-third lives in the zones of endemic Leishmania
infections [5]. To date, the greatest prevalence of Leishmania/HIV
co-infection has been in the Mediterranean basin. Among more than 2,000
cases notified to the WHO, 90 % of them belong to Spain, Italy, France and
Portugal [6].
The symptoms of leishmaniasis include fever, weight loss, enlarged
spleen, swollen glands, skin sores (changing in size and appearance over
time), splenomegaly, lymphadenopathy, hepatomegaly, pancytopenia,
progressive anemia, and hypergammaglobulinemia with hypoalbunemia.
Leishmaniasis is always fatal when left untreated and some times patients
(50% in Sudan and 1-3% in India) develop post kala-azar dermal
leishmaniasis (PKDL) [7].
The present review briefly illustrates the current status of Leishmaniasis,
occurrence and treatment around the world, and also critically discusses the
key points in natural products based drug discovery protocols. Finally, a
comprehensive coverage of natural products with significant activity against
Leishmania species has been given in detail. In order to highlight any
possible structure-activity relationships, the review has been organized
according to chemical structural class.
Scope of natural products against leishmaniasis
123
2. Taxonomy of Leishmania spp.

The Leishmania are protozoa belonging to the order Kinetoplastida and
family Trypanosomatidae. Earlier various classifications have been successively
applied to the genus Leishmania; however the simplest one can be
summarized from Figure 1.
ORDER Kinetoplastida
FAMILY Trypanosomatidae
GENERA Leishmania
Viannia
SUB-GENERA Leishmania
L. tropica L. mexicana L. aethiopica L. lainsoni

L. donovani L. major
L. tropica L. mexicana
L.
major
L.
donovani
SPECIES
L. killicki
L. amazonensis
L. chagasi
L. garhani
L. infantum
L. archibaldi
L. braziliensis L. guyanensis
L. braziliensis L. guyanensis
L. peruviana
L. panamiensis
L. pifanoi
L. venezuelensis
Figure 1. Classification of Leishmania parasite.
3. Morphology and life cycle

Leishmania are the obligate intracellular parasites existing in two
morphologic forms: promastigotes and amastigotes. Promastigotes are found
in digestive tract of sandfly and are long spindle-shaped with a single delicate
flagellum (15-28 M long) attached to cytoplasmic organelle called,
kinetoplast containing intertwined circular DNA (kDNA) molecules known
as maxicircles and minicircles, which make up 5-10% of total DNA [8].
A fully developed promastigote measures about 114.3 to 20 M in length and
1.5 to 1.8 M at their widest part [9]. The small, round to oval bodies called
amastigotes (2 to 3 M in length) are the non-infective Leishmania parasites
occurring in monocytes, polymorphonuclear lecucocytes or endothelial cells
of vertebrates (hosts) while promastigotes represent the infective stage in
sandfly (vector).
124
B. B. Mishra et al.
Figure 2. Life cycle of Leishmania parasite.
The Leishmania promastigotes are transmitted by sandfly to vertebrate

hosts e.g. canines, marsupials, edentates and rodents. Once inside the
bloodstream of reservoirs for the disease, promastigotes are phagocytosed by
the mononuclear phagocytic cells and are transformed to amastigotes that
multiply by means of binary fission. On lyse of host cell, the free parasites
spread to new cells and tissues of different organs including the spleen, liver
and bone marrow. Amastigotes in the blood as well as in the monocytes are
ingested during a blood meal by female sandfly. Once ingested, the
amastigotes migrate to the midgut of the sand fly and transform into the
promastigotes. After a period of four to five days, promastigotes move
forward to the oesophagus reach to salivary glands of the sandfly. Infected
sandfly during the second blood meal regurgitates the infectious promastigotes
from its pharynx into the bloodstream of the host vertebrates and the life
cycle is repeated [10].
4. Chemotherapy of leishmaniasis
The leishmanicidal agents with the most favorable therapeutic index are
the antimony compounds known as antimonials. Pentostam (sodium
stibogluconate) and Glucantime (meglumine antimoniate), able to interfere
with the bioenergetics of the Leishmania amastigotes [11], are the mainstay
therapy for VL. They bind to and inhibit enzymes involved in the glycolysis
and oxidation of fatty acids. Since ADP phosphorylates to ATP using NADH
generated by glycolysis and citric acid cycle, the intracellular ATP levels
125
essential for the survival of Leishmania are depleted. However, due to high
cost (approx 200 USD per patients) of branded sodium stibogluconate, a
generic sodium antimony gluconate (SAG, Albert David Ltd, India, 13USD
per patients) was used to treat patients satisfactorily without any significant
difference in final cure. However, due to serious side effects (pain at the site
of injection, stiff joints, gastrointestinal problems, cardiotoxicity, hepatic and
renal insufficiency) and declining efficacy, the SAG is no longer used in VL
hyper endemic regions of India.
COOH
COOH
HO
OH
.3 Na
.9 H2O
O
Sb
HO
H
OH
Sb
O
OH
H OH
Sodium stibugluconate
Pentamidine (1) that hampers replication and transcription at the
mitochondrial level in pathogen was the first drug used for the treatment of
patient refractory to Sbv [12]. Biophysical analysis, foot-printing studies and
the crystal structure has proved that the charged amidinium groups of
pentamidine establish hydrogen bonding with O2 of thymine or N3 of adenine
and form complexes with the minor groove of DNA. However, the efficacy
of 1 has gradually declined over the years and now it cures only 70% of
patients producing serious adverse events like shock, hypoglycemia and
death in significant proportion.
HN
O
H2N
H2N
O
HN
1
Amphotericin B (2) is a pollen antibiotic that was recommended as first

line drug in India by National Expert Committee for Sbv refractory regions of
VL. At doses of 0.75-1.0 mg/kg for 15 infusions on alternate days its cures
more than 97% of patients. The drug can perturb both parasitic and
mammalian cells, but the selective lethality of 2 for parasitic cells is the result
of its great affinity towards 24-substituted sterols, called ergosterol, the major
cell membrane sterols [13].
126
B. B. Mishra et al.
Miltefosine (3) originally developed as anti tumor agent, was approved in

India at 50100 mg (~2.5 mg/kg) doses for four weeks against VL patients
including children. The drug 3 blocks Leishmania proliferation, alters
phospholipid and sterol composition and activates cellular immunity.
However, due to high cost and serious side effects, medical advisors
generally avoid 3 in their prescriptions [14].
OH
OH
H3C
HO
CH3
OH
O
HO
HO
HO
HO
COOH
H3C
O
CH3
HO
OH
NH2
Paromomycin (4), an amino glycoside antibiotic originally identified as

an antileishmanial drug in the 1960s, acts synergistically with antimonials
in vitro, and was demonstrated significant (93% cure rate) at a dose of
16 mg/kg when given intramuscularly for 21 days to VL patients in India.
Like other amino glycosides, the drug 4 acts by impairing the macromolecular
synthesis and alters the membrane properties of Leishmania [15].
O
CH3
P
H3C
HO
H3C
O
O
H3N
OH O
O
NH3
OH
CH3
CH3
NH3
HO
CH3
NH3
N
O
H3N
OH
O
OH
N
HN
OH
OH
H3C
CH3
127
Sitamaquine (5), an orally active analog of 8-aminoquinoline, is in

clinical development by the Walter Reed Army Institute in collaboration with
GlaxoSmithKline (formerly SmithKline Beecham) to use for the treatment of
VL. In a randomized, open label and multicenter Phase II trial in India and
Kenya, the drug 5 was found efficacious and well tolerated at various dose
levels [16]. As on March 2002, the drug 5 is currently in Phase III trials for
the treatment of VL.
5. Natural products as folk medicines for treatment of

leishmaniasis
Utility of natural products in drug discovery and development is not
surprising as many of medicinal plants i.e. Cinchona calisaya (bark),
Strychnos pseudoquina (bark), Deianira erubescens (roots and leaves) and
Remijia ferruginea (bark) were historically used against different parasitic
diseases. Ancient records as well as recent literature reports have established
the effectiveness of natural products as potentially rich sources of new and
selective agents for the treatment of important tropical diseases caused by
protozoans and other parasites. In 1970s, artemisinin, an important
antimalarial drug was identified from traditional Chinese medicine Artemisia
annua and since then many artemisinin derivatives were prepared and
evaluated in various pre-clinical and clinical trials to use for the treatment of
malaria. Likewise, paromomycin 4 (HumatinTM, King Pharmaceuticals),
obtained from Streptomyces krestomuceticus, is an orphan drug that was
approved by Drug-Controller General of India in September 2006 against
VL. Paromomycin 4 was originally developed by the Institute for OneWorld
Health and is an off-patent antibiotic marketed in the US to treat intestinal
parasites also.
Natural products literature provides a growing research on plant derived
antileishmanial agents and several natural products so far have been
discovered with excellent activity against leishmania parasites, however,
none of them have been clinically evaluated in studies or projected to reach
the clinical applications in near future. This review is focused to cover the
entire formal and constant research on leishmanicidal natural products from
the mid-1980 to June 2010 with special attention on structure-activity
relationship (SAR) based activity and mechanism of action.
6. Alkaloids
The alkaloids constitute an important class of natural products
exhibiting significant anti-leishmanial activities. The quinoline alkaloids,
128
B. B. Mishra et al.
2-n-propylquinoline (6), chimanine-D (7) and chimanine-B (8), isolated from

Galipea longiflora (Rutaceae), exhibit antileishmanial activity against
L. braziliensis promastigotes with an IC90 values of 50, 25 and 25 g/mL,
respectively. Oral in vivo studies using 6 in BALB/c mice demonstrates
99.9% suppression of liver parasites while subcutaneous treatment with
7 causes 86.6% parasite suppression when given for 10 days at 0.54 mmol/kg
[17]. However, oral treatment with 7 for 5 days results in 72.9% parasite
suppression only. Likewise, dictylomide-A (9) and B (10), isolated from the
bark of Dictyoloma peruviana (Rutaceae), causes total lyses of L. amazonensis
promastigotes at 100 g/mL concentration [18].
6.1. Indole alkaloids

Dihydrocorynantheine (11), corynantheine (12) and corynantheidine
(13), isolated from the bark of Corynanthe pachyceras (Rubiaceae), are the
respiratory chain inhibitors exhibiting IC50 of 3 M against L. major.
Pleiocarpine (14) isolated from stem bark of Kopsia griffithii (Apocynaceae),
shows in vitro antileishmanial activity with an IC50 < 25 g/mL against
L. donovani promastigotes. Gabunine (15), a bis-indole alkaloid obtained
from stem bark of Peschiera van heurkii (Apocynaceae), exhibits in vitro
activity with an IC50 25 g/mL against L. amazonensis amastigotes [19].
6.2. Isoquinoline alkaloids

O-methylmoschatoline (16) and liriodenine (17), isolated from Annona
foetida (Annonaceae), display in vitro activity against promastigote forms of
O
N
CH3
CH3
CH3
CH3
HO
CH3
9
10
129
L. braziliensis with an IC50 < 60 M [20]. The SAR study among these
oxoaporphine alkaloids reveals that 17 bearing methylenedioxy moiety is
eight times more active against L. braziliensis and L. guyanensis than the
16. Berberine (18), occurring in many plant species of Annonaceae,
Menispermaceae and Berberifaceae, exhibits in vivo leishmanicidal activity
with an IC50 value of 10 g/mL against L. major. Isoguattouregidine (19)
isolated from Guatteria foliosa (Annonaceae), shows activity at 100 g/mL
N
N
H
N
H
H
C2H3
C2H5
H
OCH3
OCH3
H3COOC
H3COOC
H
12
11
N
N
N
H
C2H5
H
N
H
H
OCH3
H3COOC
H
H
14
13
N
H3CO
COOCH3
COOCH3
N
H
H3COOC
CH3
H3COOC
H
N
N
H
15
CH3
130
B. B. Mishra et al.
OCH3
HO
O
H3CO
CH3
NH
OH
H
OCH3
OH
OCH3
17
16
18
OCH3
H3CO
O
N
H3CO
CH3
O
O
21
20
19
O
O
N
H3CO
NH
N
H
HN
O
22
23
24
concentrations against L. donovani and L. amazonensis. Anonaine (20)

isolated from Annona spinescens (Annonaceae), exhibits activity against
promastigotes of L. braziliensis and L. donovani [21].
The alkaloids, (+)-neolitsine (21) and cryptodorine (22), isolated from
Guatteria dumetorum (Annonaceae), display significant activity against
promastigotes of L. maxicana at 15 and 3 M concentrations, respectively.
Xylopine (23), an aporphine alkaloid isolated from Guatteria amplifolia
(Annonaceae) shows activity against promastigotes of L. mexicana (IC50
value 3 M) and L. panamensis (IC50 value 6 M) [22]. Unonopsine (24), a
dimeric aporphine alkaloid isolated from the Unonopsis buchtienii
(Annonaceae), displays antileishmanial activity (IC100 value 25 g/mL)
against L. donovani promastigotes [23].
131
6.3. Naphthylisoquinoline alkaloids

Among the naphthylisoquinoline alkaloids, ancistroealaine-A (25)
isolated from Ancistrocladus ealaensis (Ancistrocladaceae), exhibits activity
against L. donovani promastigotes with an IC50 value 4.10 g/mL.
Ancistrocladinium A (26) and B (27) isolated from yet un-described
Congolese Ancistrocladaceae species, require 2.61 and 1.52 g/mL
concentrations, respectively to reach the IC50 towards L. major promastigotes.
An apoptosis-like death pathway is the possible mode of action for
compounds 26 & 27. Ancistrocladidine (28), isolated from Ancistrocladus
tanzaniensis (Ancistrocladaceae) shows relatively weak activity by a factor of
2 against L. donovani when compared to ancistrotanzanine-B (29) (IC50 = 1.6
g/mL), while by a factor of 10 in comparison to miltefosin (positive
control). Likewise, ancistrotanazanine-A (30), exhibits activity against
promastigotes of L. donovani. SAR based studies among the alkaloids
suggest that the compound bearing C,C-biaryl axis connecting the naphthyl
and isoquinoline moiety shows weak or no leishmanicidal activity [24].
6.4. Bisbenzylisoquinolinic alkaloids

Daphanandrine (31) isolated from Albertisia papuana (Menispermaceae),
obaberine (32) obtained from Pseudoxandra sclerocarpa (Annonaceae),
gyrocarpine (33) produced by Gyrocarpus americanus (Hernandiaceae) and
limacine (34) isolated from Caryomene olivasans (Menispermaceae), display
OCH3 OCH3
CH3
H3CO
CH3
H3CO
CH3
CH3
H3CO
TFA OH
TFA
OCH3
OCH3 CH3
OCH3 CH3
CH3
OCH3
OCH3
H3C
OCH3 CH3
26
25
27
OCH3 OCH3
H3CO
H3CO
CH3
OCH3 OH
N
CH3
H3C
CH3 H3CO
H3CO
OCH3 CH3
CH3
N
CH3
OCH3 CH3
28
CH3
HO
29
OCH3 CH3
30
132
B. B. Mishra et al.
activity against L. donovani, L. braziliensis and L. amazonensis with an IC100

of ~50 g/mL. SAR studies among these alkaloids demonstrate that alkaloids
with methylated nitrogen are more active than those with non-substituted or
aromatic nitrogens while quaternization of one or more nitrogen atoms results
in the loss of antileishmanial activity [25].
OCH3 H3CO
HN
OCH3 H3CO
N
HO
CH3
H3C
N
H
CH3
O
OCH3
OCH3
32
31
OCH3 H3CO
OCH3 HO
H 3C
H3CO
O
H3CO
O
CH3 H3C
OH
CH3
O
OCH3
OCH3
33
34
6.5. Steroidal alkaloids

Among the alkaloids, holamine (35), 15--hydroxyholamine (36),
holacurtine (37) and N-desmethylholacurtine (38), obtained from Holarrhena
curtisii (Apocynaceae), the metabolite 35 exhibits strongest activity against
L. donovani (1.56>IC50>0.39 g/mL) in compared to 36, 37 and 38
(6.25>IC50>1.56 g/mL) [26].
O
O
CH3
CH3
CH3
CH3
CH3
CH3
H
OH
H2N
H2N
35
O
CH3
CH3
36
CH3
H
CH3
H
CH3
O O
H
NH
H3C
H
OCH3
37
CH3
CH3
OH CH3
O O
H
H
NH2
H
OCH3
38
H
OH
133
6.6. Benzoquinolizidine alkaloids

Klugine (39), cephaeline (40), isocephaeline (41) and emetine (42),
demonstrating significant leishmanicidal activity against L. donovani have
been isolated from Psychotria klugii (Rubiaceae). Among these metabolites,
the compound 39 (IC50 of 0.40 g/mL) and 41 (IC50 0.45 g/mL) exhibit
<13- and <15-fold less potent activity in compared to 40, while compound 40
with IC50 of 0.03 g/mL demonstrates >20- and >5-fold more in vitro activity
against L. donovani when compared to pentamidine and amphotericin-B,
respectively. The alkaloid 42, exhibits activity against L. donovani with an
IC50 value 0.03 g/mL, however produces toxicity in treatment of cutaneous
leishmaniasis caused by L. major [27].
H3CO
R1
N
H3CO
H3CO
CH3
CH3
R2
OCH3
H
OCH3
HN
HN
OH
41 R = OCH3
42 R= OH
39 R1 = OH; R2 = OH
40 R1 = OCH3; R2 = H
6.7. Diterpene alkaloids

The alkaloids, 15,22-O-Diacetyl-19-oxo-dihydroatisine (43), azitine (44)
and isoazitine (45), isolated from Aconitum, Delphinium and Consolida
species, show significant leishmanicidal activities. The metabolite 45 exhibits
strongest activity against promastigotes of L. infantum with IC50 values 44.6,
32.3 and 24.6 M at 24, 48 and 72 h of culture, respectively. The compound
44 and 43 with IC50 values of 33.7 and 27.9 M at 72 h of culture,
respectively, exhibit activity against promastigotes of L. infantum [28].
CH2
CH2
CH2
OAc
OAc
OH
HN
OH
CH3
CH3
O
43
CH3
CH3
44
45
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B. B. Mishra et al.
6.8. Pyrrolidinium alkaloids

The pyrrolidinium alkaloid (2S,4R)-2-carboxy-4-(E)-p-coumaroyloxy1,1-dimethylpyrrolidinium inner salt (46), isolated from Phlomis
brunneogaleata (Lamiaceae), display activity with an IC50 of 9.1 g/mL
against axenic amastigotes of L. donovani [29].
6.9. Acridone alkaloids

The rhodesiacridone (47) and gravacridonediol (48) isolated
from Thamnosma rhodesica (Rutaceae), exhibit 69% and 46% inhibition
at 10 M concentration, respectively against promastigotes of L. major.
The compounds also display activity against L. major amastigotes and cause
over 90% and 50% inhibition at 10 and 1 M concentration, respectively
[26].
O
OH
OH
O
OOC
N
H3C
N
CH3
O
CH3
46
R
47 R = C(OH)(CH2OH)COCH3
48 R = C(OH)(CH3)CH2OH
6.10. -Carboline alkaloids

The harmaline (49), isolated from Peganum harmala (Nitrariaceae),
exhibits amastigotespecific activity (IC50 of 1.16 M). Harmine (50) isolated
from same plant species reduces spleen parasite load by approximately
40, 60, 70 and 80% in free, liposomal, niosomal and nanoparticular forms,
respectively in mice model. Canthin-6-one (51) and 5-methoxycanthin-6-one
(52) occurring in plant species of Rutaceae and Simaroubaceae, demonstrate
in vivo activity against L. amazonensis in BALB/c mice model. Nhydroxyannomontine (53) and annomontine (54) isolated from Annona
foetida (Annonaceae), show efficient leishmanicidal potentials. The SAR
studies suggest that the metabolite 54 (IC50 = 34.8 M) displays 6 times more
activity compared to 53 against L. braziliensis promastigotes. The compound
53 also exhibits activity against promastigotes of L. guyanensis while 54
remain inactive [25].
135
N
N
H3CO
N
H
R N
O
R
R
H2N
49 R = H
50 R = CH3
51 R = H
52 R = OCH3
53 R = OH
54 R = H
6.11. Alkaloids from marine sources

Many marine sponges e.g. Amphimedon viridis, Acanthostrongylophora
species, Neopetrosia species, Plakortis angulospiculatus and Pachymatisma
johnstonii serve as rich sources of alkaloids with significant antileishmanial
potentials. Renieramycin A (55) isolated from Neopetrosia species, is a
La/egfp (expressing enhanced green fluorescent protein) inhibitor that shows
efficient antileishmanial activity against L. amazonensis with IC50 0.2 g/mL.
Araguspongin C (56), isolated from a marine sponge Haliclona exigua,
displays leishmanicidal activity against promastigotes as well as amastigotes
at 100 g/mL concentrations [30].
N
OCH3
H
CH3
O
H
H3C
CH3
H3CO
O
OH
CH3
O
O
H
N
CH3
55
56
Among the ciliatamides A-C (57-59) isolated from Aaptos ciliate, the
peptide 57 and 58 at 10.0 g/mL concentrations inhibit 50% growth L. major
promastigotes [31]. The lipopeptides, almiramides A-C (60-62) isolated
from cyanobacterium Lyngbya majuscule, exhibit significant in vitro
antileishmanial activity against L. donovani. The SAR studies among these
peptides suggest that 61 and 62 exhibit strong activity against L. donovani
136
B. B. Mishra et al.
with EC50 values of 2.4 and 1.9 M, respectively. The metabolites 61 and 62
also display weak cytotoxicity to mammalian Vero cells at 52.3 and 33.1 M
concentrations, respectively [32]. Dragonamide A (63), E (64) and herbamide
B (65), isolated from same cyanobacterium strain, exhibit in vitro activity against
L. donovani with EC50 values of 6.5, 5.1 and 5.9 M, respectively [33].
Viridamide A (66) isolated from Oscillatoria nigro-viridis, shows
activity against L. mexicana with EC50 of 1.5 M [34]. Venturamides A (67)
and B (68) obtained from cyanobacterium Oscillatoria species, exhibit
activity against L. donovani with EC50 >19.0 M. Valinomycin (69), a
dodecadepsipeptide isolated from Streptomyces strains, exhibits activity against
promastigotes of L. major with EC50 < 0.11 M, but at the same time shows
cytotoxicity to 293T kidney epithelial cells and J774.1 macrophages [35].
7. Quinones
Primin (2-methoxy-6-pentylcyclohexa-2,5-diene-1,4-dione), occurring in
Primula obconica and other species (Primulaceae), shows significant
leishmanicidal activity against L. donovani with an IC50 of 0.711 M.
Diospyrin (70), a bis-naphthoquinone inhibiting topoisomerase I, isolated
from the bark of Diospyros Montana (Ebenaceae), demonstrates
antileishmanial activity against L. donovani promastigotes with an MIC of
1.0 g/mL [36]. The hydroxylated derivative of 70 at 3 M concentration
eliminates 73.8% of amastigotes in infected macrophages [37]. Plumbagin
(72), originally isolated from Plumbago zylenica, shows leishmanicidal
activity against amastigotes of L. donovani (IC50 = 0.42 g/mL) and
L. amazonensis (IC50 = 1.1 g/mL). At a concentration of 10 g/mL, the
O
H
N
H
N
HN
N
O
HN
N
O
CH3
60 R =
61 R =
62 R =
H3C
CH3
CH3
59
57 R = (CH2)7CHCH2
58 R = (CH2)6CH3
O
CH2
CH3
N
O
H3C
O
N
CH3
CH3
CH3
H
N
O
H3C
CH3
N
CH3
CH3
CH3
O
NH2

CH3
H3C
O
CH3
H3C
O
CH3
N
O
CH3
CH3
H3C
CH3
CH3
CH3
H
N
Cl3C
NH2
N
CH3
CH3
CH3
137
CH3
O
N
63 R =
65
64 R =
CH3
CH3
OCH3
HC
H
N
H3C
N
H3C
NH
N
O
HN
H3C
N
CH3
S
NH
HN
S
O
H3C
CH3
H3C
68
O
NH
N
H
CH3
CH3
HN
O
CH3
H3C
67
H3C
N
H
CH3
OH
N
H
CH3
H3C
H3C
COOCH3
66
CH3
O
H3C
CH3
CH3
N
H
O
H3C
CH3
CH3
CH3
H3C
O
CH3
H3C
CH3
CH3
CH3
CH3
CH3
O
HN
H3C
H3C
NH
CH3
CH3
CH3
CH3
N
H
CH3
H3C
CH3
69
compound 72 presents an amastigote survival index (SI) of 16.5% against

L. amazonensis with the absence of toxic effects against the macrophages.
The metabolite 72 also shows in vivo activity against L. amazonensis and
L. venezuelensis at concentrations 2.5 and 5 mg/kg/day, respectively.
The mechanism of the action of compounds 72 and 71 involves generation of
oxygen free radicals from which the parasites remain unable to defend.
The dimeric products 3,3-biplumbagin (73) and 8,8-biplumbagin (74),
isolated from the bark of Pera benensis (Euphorbiaceae), display significant
138
B. B. Mishra et al.
antileishmanial activity. Among these, the metabolite 73 shows lower

activity (IC90 = 50 g/mL) compared to 72 and 75 (IC90 = 50 g/mL) against
L. braziliensis, L. amazonensis, and L. donovani promastigotes [38,39].
Lapachol (75), a prenylated hydroxynaphthoquinone isolated from Tecoma
species (Bignoniaceae), displays activity with mechanism of action similar to
71 and 72 against L. donovani amastigotes in peritoneal mice macrophages.
The metabolite 3,4-dihydronaphthalen-1(2H)-one (76), isolated from the bark
of Ampelocera edentula (Ulmaceae), exhibits leishmanicidal activity (IC90
of 10 g/mL) against L. braziliensis, L. amazonensis and L. donovani
promastigotes. The metabolite 76 demonstrates strong in vivo activity on
subcutaneous treatment in BALB/c mice infected with L. amazonensis or
L. venezuelensis when compared to Glucantime (25 mg/kg/day vs 56 mg
SbV/kg/day). However, the use of tetralones is limited due to cytotoxic,
carcinogenic and mutagenic properties in animals [40].
Jacaranone (77), a quinone isolated from the leaves of Jacaranda copaia
(Bignoniaceae), exhibits a strong activity with an ED50 of 0.02 mM against
L. amazonensis promastigotes but at the same concentration shows toxicity to
peritoneal mice macrophages. The prenylated dihydroquinone hydropiperone
(78), isolated from Peperomia galioides (Piperaceae), shows activity at a
concentration of 25 g/mL against promastigote forms of L. braziliensis,
L. donovani and L. amazonensis. At 100 g/mL concentration the metabolite
78 causes total lysis of the parasites [41].
OH
OCH3 OH
OH
H3C
OCH3 OH
CH3
H3C
O
H3C
OH
H3C
OH
OH
72
71
70
O
OH
O
CH3
O
OH
OH
O
H3C
CH3
CH3
O
OH
O
H3C
CH3
O
OH
73
O
74
75
139
The anthraquinone-2-carbaldehydes, 79 and 80, isolated from the roots of

Morinda lucida (Rubiaceae), shows leishmanicidal potential selective to
L. major promastigotes. SAR studies suggest that presence of an aldehyde
group at C-2 and a phenolic hydroxy group at C-3 in both structures, are
essential for their antiprotozoal activity [42].
O
O
H3C
HO
CH2COCH3
OH
76
77
O
R1
CH3
CH3
CH3
CH3
OH
CH3
O
OH
78
OH
CHO
OH
O
79 R1 = OCH3
80 R1 = H
CH2OH
O
81
The aloe-emodin
(81) isolated from Stephania dinklagei
(Menispermaceae), shows leishmanicidal activity at IC50 values of 185.1 and
90 M against L. donovani promastigotes and amastigotes, respectively [43].
Vismione D isolated from Vismia orientalis (Clusiaceae) exhibits activity
against axenic amastigotes of L. donovani with an IC50 value of 0.37 g/mL but
shows cytotoxicity when tested on human L6 cells (IC50 of 4.1 g/mL) [29].
8. Terpenes
8.1. Iridoids
Iridoids, a class of monoterpenoid glycosides often serve as intermediates
in the biosynthesis of indole alkaloids are well known for significant
leishmanicidal activity. The arbortristosides-A (82), B (83), C (84) and 6-hydroxyloganin (85), isolated from Nyctanthes arbortristis (Oleaceae) exhibit
in vitro activity against L. donovani amastigotes. The in vivo studies using
intraperitoneal and oral treatment (10 and 100 mg/kg concentrations for
5 days) of hamsters infected with L. donovani, the metabolite 82 displays
significant leishmanicidal activities [44]. Picroside I (86) and kutkoside (87),
140
B. B. Mishra et al.
obtained from Picrorhiza kurroa, exhibits a high degree of protection against

the infection of promastigotes of L. donovani in hamsters [45]. Picroliv, a
standardized fraction of iridoid glycosides 86 and 87, increases the
nonspecific immune response and induces a high degree of protection against
the infection of promastigotes of L. donovani in hamsters. Picroliv is an
adjuvant proposed to increase the efficacy of leishmanicidal drugs and has
demonstrated excellent therapeutic index in Phase I and II clinical trials [46].
HO
OH H CO2CH3
O
R1O
O
O
R1O
H
OR2 O
O
OH
OH
OH
86 R1 = Vanilloyl, R2 = H
87 R1 = H, R2 = Cinnamoyl
OH
CH3H O
O
HO
OH
OH
82 R1 = p-Methoxycinnamoyl
83 R1= Caffeoyl
84 R1 = Coumaroyl
85 R1 = H
Amarogentin (88), a secoiridoid glycoside isolated from Swertia chirata

(Gentiaceae), produces leishmaincidal effect at a concentration > 60 M against
L. donovani through inhibition of catalytic activity of topoisomerase I [47].
The metabolite 88 exerts inhibitory effect with a mechanism of action similar to
Pentostam i.e. by binding to the enzyme and preventing the formation of a
binary complex with DNA. The evaluation of 88 in the form of liposomes and
niosomes shows an enhanced leishmanicidal activity (without toxic effects) than
those observed for free 88 when tested in hamsters [48].
O
OH
H
CH2
O
O
HOH2C
O
O
C
HO
OH
HO
OH
88
141
8.2. Monoterpenes
Espintanol (89), isolated from the bark of Oxandra espintana
(Annonaceae), shows antileishmanial activity against promastigotes of twelve
Leishmania species. However, the metabolite 89 exhibits only a weak activity
in vivo in mice infected with L. amazonensis. Grifolin (90) and piperogalin
(91) obtained from Peperomia galoides, causes total lysis of L. braziliensis,
L. donovani and L. amazonensis promastigotes at 100 g/mL concentrations.
At 10 g/mL concentration, metabolite 91 causes more than 90% lysis of the
promastigotes [49].
CH3
OH
CH3
CH3
CH3
OH
H3C
90
OH
CH3
CH3
CH3
OH
OH
H3C
H3CO
H3C
CH3
OCH3
CH3
89
H3C
CH3
91
8.3. Sesquiterpenes
A sesquiterpene lactone, dehydrozaluzanin C (92), isolated from the
leaves of Munnozia maronii (Asteraceae), shows activity at concentrations
between 2.5-10 g/mL against promastigotes of eleven Leishmania species.
The in vivo test using the metabolite 92 in BALB/c mice results in reduction
of the lesions caused by L. amazonensis [50].
Sesquiterpene dilactone, 16,17-dihydrobrachycalyoxide (93), isolated
from Vernonia brachycalyx (Asteraceae), exhibits activity (IC50 = 17 g/mL)
against L. major promastigote but also inhibits the proliferation of human
lymphocytes [51]. Kudtriol (94), a sesquiterpene alcohol isolated from the
aerial parts of Jasonia glutinosa (Asteraceae), shows toxic activity against
promastigotes of L. donovani at 250 g/mL concentration. SAR study with
metabolite 94 indicates that the presence of a C-5 hydroxy group in the
-orientation is essential for the expression of the leishmanicidal activity
[52]. The (+)-curcuphenol (95), isolated from sponge Myrmekioderma styx,
exhibits in vitro anti-leishmanial activities against L. donovani with an EC50
of 11.0 M [53].
142
B. B. Mishra et al.
H2C
H
C2H5
HO
H
CH2
H
O
O
CH3
O
H3C O
93
OH
CH3
CH3
OH H3C
OH
H3C
94
O
O
92
OH
CH2 H3C
OH
O
O CH
3
O
H2C
H2C
CH3
95
8.4. Diterpenes
A phorbol diester, 12-O-tetradecanoyl phorbol-13-acetate (TPA) 96, also
known as phorbol 12-myristate 13-acetate (PMA), was originally identified
from the croton plant, which at a concentration of 20 ng/mL displays ability
to cause a variety of structural changes in the parasites of L. amazonensis by
activation of protein kinase C, an important enzyme in the development of
several cellular functions [54]. Among the other diterpenoids isolated from
Euphorbiaceae species with leishmanicidal potentials are jatrogrossidione
(97) and jatrophone (98). These metabolites possess toxic activity against
the promastigote forms of L. braziliensis, L. amazonensis and L. chagasi.
SAR studies with these metabolites revealed that 97 with IC100 value of
0.75 g/mL displays activity higher than 98 (IC100 = 5 g/mL), but remains
inactive in vivo [55].
The 15-monomethyl ester of dehydropinifolic acid (99), obtained from
the stem bark of Polyalthia macropoda (Annonaceae), and ribenol (100), an
ent-manoyl oxide derivative isolated from Sideritis varoi (Lamiaceae), show
in vitro activity against promastigotes of L. donovani [56]. Also the different
derivatives of this metabolite, obtained through chemical or biological
transformations, exhibit strong leishmanicidal activity. Additionally, 6-hydroxyrosenonolactone (101), a diterpene isolated from the bark of
Holarrhena floribunda (Apocynaceae), has a moderate and weak activity
against promastigotes and amastigotes of L. donovani, respectively [57].
143
H3C
H3C(H2C)12OCO
H3C
OCOCH3
CH3
H3C
HO
O HO
CH3
H
HO
H
CH3
CH3
CH2
H3C
H
O
H3C
CH2OH
CH3
H2C
CO2CH3
CH3
CH3
97
96
98
H3C
CH3
CH3
CH3
CH3
O
CH2
CH2
CH3
CH2
O
CH3
HO
H
H3C CO2H
99
H
O
H3C CH3
H
CH3
100
OH
101
8.5. Triterpenes
The ursolic acid (102) and betulinaldehyde (103), obtained from the bark
of Jacaranda copaia and the stem of Doliocarpus dentatus (Dilleniaceae),
respectively show activity against the amastigotes of L. amazonensis.
However, the metabolite 103 exhibits toxicity to peritoneal macrophages in
mice while 102 displays limited activity in vivo.
The triterpenes, (24Z)-3-oxotirucalla-7,24-dien-26-oic acid (104) and
epi-oleanolic acid (105), isolated from the leaves of Celaenododendron
mexicanum (Euphorbiaceae), display leishmanicidal activity against
L. donovani with IC50 values of 13.7 and 18.8 M, respectively. The
quassinoids, simalikalactone D (106) and 15--heptylchaparrinone (107),
obtained from species of Simaroubaceae family show activity against
promastigotes of L. donovani but at the same time exhibit toxicity to
macrophages [58]. Triterpene glycosides obtained from marine sources e.g.
holothurins A (108), isolated from the sea cucumber Actinopyga lecanora,
causes 73.2 6.8% and 65.8 6% inhibition of L. donovani promastigotes
and amastigotes, respectively at 100 g/mL concentration. The other isomer
B (109) obtained from same source shows 82.5 11.6% and 47.3 6.5%
inhibition against promastigotes of L. donovani at 100 and 50 g/mL
concentrations, respectively [59].
144
B. B. Mishra et al.
CH2
CH3
H3C
H3C
H
CH3
H
CH3
CO2H
CH3
CH3
CHO
CH3
CH3
HO
HO
H
CH3
H3C
H
CH3
H3C
102
103
H
CH3
CO2H
CH3
CH3
CH3
H
CH3
H3C
CH3
H3C
CO2H
CH3
CH3
CH3
O
CH3
H3C
H
CH3
CH3
105
104
OH
OH
HO
OH
CH3
HO
OH
CH3
CH3
O
(CH2)6CH3
OCOCH(CH3)C2H5
O
H
O
H
CH3
O
CH3
CH3
107
106
CH3
HO
CH3
O
OH
CH3
CH3
O
O
H3C
CH3
O
NaO3SO HO
H3C
O
OH
OH
OH
HO
O
HO
OR
108 R1 = HO
MeO
109 R2 = H
HO
OH
CH3
145
9. Saponins
The -hederin (110), -hederin (111) and hederagenin (112), obtained
from the leaves of Hedera helix (Araliaceae), show lishmanicidal activity
against L. infantum and L. tropica. Among these, the metabolite 112 also
shows significant activity against the amastigote forms while both 110
and 111 exhibit strong anti-proliferative activity on human monocytes [60].
The saponins 110-112 appear to inhibit the growth of Leishmania
promastigotes by acting on the membrane of the parasite with induction of a
drop in membrane potential [61]. The hederecolchiside-A1 (113), isolated
from Hedera colchica, shows strong activity against the promastigotes and
amastigotes of L. infantum, but also displays a notable activity on human
monocytes.
The saponin, mimengoside-A (114), isolated from the leaves of Buddleja
madagascariensis (Loganiaceae) [62], exhibits activity against promastigotes
of L. infantum. Muzanzagenin (115), obtained from the roots of Asparagus
africanus (Liliaceae), displays activity with an IC50 value 31 g/mL against
the L. major promastigotes. However, the metabolite 115 also inhibits the
proliferation of human lymphocytes [63].
10. Phenolic derivatives

10.1. Chalcones
The chalcone, (E)-1-[2,4-hydroxy-3-(3-methylbut-2-enyl)phenyl]-3-[4hydroxy-3-(3-methylbut-2-enyl)phenyl]-prop-2-en-1-one
(116)
shows
toxicity to promastigotes of L. donovani, while 2,6-dihydroxy-4methoxychalcone (117), isolated from inflorescences of Piper aduncum
(Piperaceae), exhibits significant in vitro activity against promastigotes and
amastigotes of L. amazonensis by affecting the ultrastructure of the parasite
mitochondria without causing damage or inducing NO production in the
macrophages [64,65].
The metabolite 117 with an IC50 value of 0.5 g/mL shows strong
antileishmanial activity against the promastigotes of L. amazonensis, while
exhibit lower activity (IC50 = 24 g/mL) against amastigote forms.
Encapsulated formulation of 117 when administered at 1.0 g/mL causes the
reduction in the level of L. amazonensis infected macrophages by 53% [66].
Ultrastructural studies suggest that 117 produces selective toxicity to the
intracellular amastigotes without affecting macrophage organelles even when
exposed to 80 g/mL concentration. The licochalcone-A (118), isolated from
roots of the Chinese licorice plant Glycyrrhiza species (Fabaceae), shows in vitro
146
B. B. Mishra et al.
H3C
CH3
CH3
CH3
CO2H
CH3
R1O
CH3
R2H2C
110 R1 = Ara 2-1 Rha, R2 = OH

111 R1 = Ara 2-1 Rha, R2 = H
112 R1 = H, R2 = OH
113 R1 = Ara [Glc 4-1] 2 Rha, R2 = H
Ara: -L-arabinopyranose
Glc: -D-glucopyranose
Rha: -L-rhamnopyranose
Fuc: -D-fucopyranose
H3C
CH3
O
CH3
CH3
CH3
RO
H3C
OH
114 3-0- -L-rhamnopyranosyl-(1-4)- -D-glucopyranosyl(1-3)-[ -D-glucopyranosyl-(1-2)]- -D-fucopyranoside
of 16-dehydroxysaikogenin G
H
H
OH
CH3
CH3
CH3
H
H
HO
O
115
O
CH3
147
OH
CH3 H3CO
HO
OH
CH3
H3C
CH3
OH
OH
117
116
CH2
HO
OH
H3C
H3C
HO
OH
HO
OCH3
118
119
OH
HO
R1O
OH
HO
OH
OH
OH
120
OR2
121 R1 = H, R2 = H
122 R1 = H, R2 = OCH3
123 R1 = OCH3, R2 = OCH3
activity against L. major and L. donovani promastigotes. The intraperitoneal

administration of 118 prevents the development of lesions in BALB/c mice
infected with L. major [67,68]. The intraperitoneal and oral administration of
118 significantly reduces the parasite load in the spleen and liver of hamsters
infected with L. donovani. The compound 118 appears to affect the parasite
respiratory chain without damaging the organelles of macrophages or
phagocytic function by altering the ultrastructure and function of
mitochondria only. However, at lower concentrations 118 inhibits the
proliferation of human lymphocytes. Subsituents that hinder free rotation in
chalcones have been demonstrated to be inactive. The introduction of polar
chemical moieties (like hydroxyl and glycosyl groups) led to a reduction of
the antileishmanial activity. The modification at the ,-double bond in
chalcones results in marginal reduction of the leishmanicidal activity
compared to parent compounds, thus this part is just a chemical spacer
148
B. B. Mishra et al.
necessary only. The sulfuretin (2-[(3,4-dihydroxyphenyl)methylene]-6hydroxybenzofuran-3(2H)-one) (119), is an aurone, a group of metabolites

related biosynthetically to the chalcones, exhibit activity with EC50 values of
0.09-0.11 g/mL against promastigotes of Leishmania species. The metabolite
119 with an EC50 value of 1.24 g/mL displays activity against L. donovani
amastigotes, but remains non-toxic to bone marrow-derived macrophages [69].
10.2. Flavonoids
The compound 5,7,4-trihydroxyflavan (120) shows activity against the
amastigotes of L. amazonensis [70], while the biflavonoids amentoflavone
(121), podocarpusflavone A (122) and B (123), isolated from the leaves of
Celanodendron mexicanum, exhibit weak activity against L. donovani
promastigotes. The flavones fisetin (124) (isolated from Acacia greggii and
A. berlandieri), 3-hydroxyflavone (125), luteolin (126) (isolated from Salvia
tomentosa), and quercetin (127) (isolated from plants of family Alliaceae)
exhibit potent antileishmanial activity against the intracellular forms of the
L. donovani with IC50 values 0.6, 0.7, 0.8 and 1.0 g/mL, respectively.
Biochanin A (128), an O-methylated isoflavone occurring in legumes, shows
activity against L. donovani with an IC50 value of 2.5 g/mL [3].
O
HO
OH
OH
OH
HO
OH
OH
OH
O
OH
124
125
O
126
OH
HO
HO
O
OH
OH
O
127
OH
OH
OCH3
128
10.3. Lignans
The lignans (+)-medioresinol (129), (-)-lirioresinol B (130) and (+)nyasol (131), show activity against the amastigotes of L. amazonensis,
whereas 131 also exhibits high selectivity in its activity against the
promastigotes of L. major. Dyphillin, isolated from Haplophyllum
bucharicum (Rutaceae), modulates phagocytosis of macrophages and
selectively inhibits the amastigotes of L. infantum with an IC50 value
0.2 g/mL [71].
149
R2
HO
HO
H3CO
H
H
OCH3
H2C
OH
OH
R1
129 R1 = R2 = OCH3
130 R1 = R2 = CH3
131
10.4. Coumarins
The coumarin isomers 2-epicycloisobrachycoumarinone (132) and
cycloisobrachycoumarinone (133), isolated from Vernonia brachycalyx
(Asteraceae), display selective activity against promastigotes of L. major.
R1
CH3
R2
CH3
HO
OH
R1
R2
CH3
O
132 R1 =CH3, R2 = H
133 R1 =H, R2 = CH3
CH3
OH
134 R1 = R2 = OCH3
135 R1 = H, R2 = OCH3
136 R1 = H, R2 = H
10.5. Curcumins
The curcumins, curcumin (134), desmethoxycurcumin (135) and
bis-desmethoxycurcumin (136), isolated from the rhizomes of Curcuma
longa, show significant anti-leishmanial activity against promastigotes of
L. major. However, these metabolites also inhibit the proliferation of human
lymphocytes [72].
11. Other metabolites

Acetogenins like senegalene (137), squamocine (138), asimicine (139)
and molvizarine (140), isolated from the seeds of Annona senegalensis
(Annonaceae), show activity against promastigotes of L. major and
L. donovani at concentrations that vary between 25 and 100 g/mL.
However, these metabolites also show cytotoxicity greater than that of
150
B. B. Mishra et al.
vinblastine against KB and VERO cell lines [73]. Other acetogenins such as
rolliniastatin-1 (141), isolated from Rollinia emarginata (Annonaceae),
annonacin A (142) and goniothalamicin (143), obtained from Annona glauca
(Annonaceae), display promicing activity against the promastigote of
L. braziliensis, L. donovani, L. amazonensis, however a clear SAR has not
been established [74].
O
O
OH
OH
OH
O
H3C
(CH2)3CH3
7
OH
O
O
137
R1
OH
O
H3C
R2
OH
O
(CH2)5CH3
threo-trans-threo-trans-*
138 R1 = H, R2 = OH, n = 10, *= erythro
139 R1 = OH, R2 = H, n = 10, *= threo
140 R1 = OH, R2 = H, n = 8, *= erythro
OH
CH3
OH
O
H3C
CH3
6
141
R2
H3C
OH
R1
O
OH
n
O
CH3
OH
142 R1 = OH, R2 = H, n = 5, m = 8, *= erythro

143 R1 = H, R2 = OH, n = 3, m = 10, *= threo
Future prospectives
Despite the advances in the parasitological and biochemical researches
using various species of Leishmania, the treatment options available against
leishmaniasis are far from satisfactory. In current situation, development of
new drugs to combat leishmaniasis require increased input from the
disciplines of chemistry, pharmacology, toxicology and pharmaceutics to
complement the advances in molecular biology that have been made in past
21 years.
Natural products are potential sources of new and selective agents for the
treatment of important tropical diseases caused by protozoans and other
parasites. The tremendous chemical diversity present in natural products and
the promising leads that have already been demonstrated significant against
parasitic diseases are needed to be addressed also against leishmania
151
parasites. The development of antileishmanial natural products or their

analogs in accordance to the considerations outlined above would have a
dramatic positive impact on the treatment of leishmaniasis. A safe, non-toxic
and cost-effective drug is urgently required to eliminate this problem from
every corner of world. A safer, shorter & cheaper treatment, identification of
the most cost effective surveillance system and control strategies, suitable
vector control approach are among some important aspect for the control and
complete eradication of this deadly disease.
Acknowledgement
Financial assistance from DST, New Delhi is greatly acknowledged.
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Planta Med., 1994, 60, 8.
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41. Mahiou, V., Roblot, F., Hocquemiller, R., Cave, A. J. Nat. Prod., 1996, 59, 694.
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T.G., Cornett, C., Watkins, W.M., Kharazmi, A., Lemmich, E. Planta Med.,
1998, 64, 559.
52. Villaescusa-Castillo, L., Diaz-Lanza, A.M., Gasquet, M., Delmas, F., Olliver, E.,
Bernabe, M., Faure, R., Elias, R., Balansard, G. Pharm. Biol., 2000, 38, 176.
53. Gul, W., Hammond, N.L., Yousaf, M., Peng, J., Holley, A., Hamann, M.T.
Biochim. Biophys. Acta, 2007, 1770, 1513.
54. Vannier-Santos, M.A., Pimenta, P.F.O., Souza, W. J. Submicrosc. Cytol. Pathol.,
1988, 20, 583.
55. Schmeda-Hirschmann, G., Razmilic, I., Sauvain, M., Morretti, C., Munoz, V.,
Ruiz, E., Balanza, E., Fournet, A. Phytother. Res., 1996, 10, 375.
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Castilla-Calvente, J.J., Osuna, A. J. Nat. Prod., 1997, 60, 13.
57. Loukaci, A., Kayser, O., Bindseil, K.U., Siems, K., Frevert, J., Abreu, P.M. J.
Nat. Prod., 2000, 63, 52.
58. Camacho, M., Mata, R., Castaneda, P., Kirby, G.C., Warhurst, S.C., Croft, S.L.,
Phillipson, J. D. Planta Med., 2000, 66, 463.
59. Singh, N., Kumar, R., Gupta, S., Dube, A., Lakshmi, V. Parasitol. Res., 2008,
103, 351.
60. Majester-Savornin, B., Elias, R., Diaz-Lanza, A.M., Balansard, G., Gasquet, M.,
Delmas, F. Planta Med., 1991, 57, 260.
61. Delmas, F., Giorgio, C.D., Elias, R., Gasquet, M., Azas, N., Mshvildadze, V.,
Dekanosidze, G., Kemertelidze, E., Timon-David, P. Planta Med., 2000, 66, 343.
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Balansard, G. Planta Med., 1996, 62, 92.
64. Christensen, S.B., Ming, C., Andersen, L., Hjorne, U., Olsen, C.E., Cornett, C.,
Theander, T.G., Kharazmi, A. Planta Med., 1994, 60, 121.
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Research Signpost
37/661 (2), Fort P.O.
Trivandrum-695 023
Kerala, India
ISBN: 978-81-308-0448-4
5. Naturally occurring antihyperglycemic

and antidyslipidemic agents
T. Narender, T. Khaliq and G. Madhur
Medicinal and Process Chemistry Division, Central Drug Research Institute
Lucknow-226 001, U.P., India
Abstract. Diabetes mellitus is an independent risk factor for the

development of coronary artery diseases, myocardial infarction,
hypertension, and dyslipidemia. Clinically diabetic patients are
characterized by marked increase in blood glucose level followed by
mild hyperlipidemia. Non-insulin dependent diabetes mellitus
(NIDDM) accounts for approximately 8090% of all cases and it is the
fastest growing global threat to public health. If the current trend
continues, it is likely to result in an estimated 215 million sufferers
from NIDDM worldwide by the year 2010. When carbohydrates are in
low supply or their breakdown is incomplete, fats become the preferred
source of energy. Fatty acids are mobilized into the general circulation
leading to secondary triglyceridemia in which total serum lipids in
particular triglycerides as well as the levels of cholesterol and
phospholipids increases.This rise is proportional to the severity of the
diabetes. Uncontrolled diabetes is manifested by a very high rise in
triglycerides and fatty acid levels. These conditions are responsible for
one third of deaths in industrialized nations Plants have always been a
rich source of drugs and many of the currently available drugs have
been derived either from natural products or its templates. We here
in present a precise description of naturally occurring compounds
possessing potential antihyperglycemic action or antidyslipidemic
action against specific drug targets.
Correspondence/Reprint request: Dr. T. Narender, Medicinal and Process Chemistry Division, Central Drug
Research Institute, Lucknow-226 001, U.P., India. E-mail: t_narendra@cdri.res.in
156
T. Narender et al.
1. Diabetes mellitus
Diabetes is a disease in which the body does not produce or properly use
insulin. Insulin is a hormone that converts sugar, starch and other food into
energy needed for daily life. The causes of diabetes are not known clearly,
although both genetics and environmental factors such as obesity and lack of
exercise appear to play roles. Diabetes mellitus and glucose intolerance are
common in adolescent and adult patients with cystic fibrosis. Diabetes is
invariably associated with pancreatic exocrine dysfunction (malabsorption).
The prevalence in patients over 20 years of age may be as high as 53% [1].
The major types of diabetes include type-I and type-II diabetes. The former
results from the body's failure to produce insulin, the hormone that "unlocks"
the cells of the body, allowing glucose to enter and fuel them while the latter
results from insulin resistance, a condition in which the body fails to properly
use insulin combined with relative insulin deficiency. Type-II insulin-resistant
diabetes mellitus accounts for 90-95% of all diabetes. This heterogeneous
disorder afflicts an estimated 6% of the adult population in western society; its
worldwide frequency is expected to continue to grow by 6% per annum,
potentially reaching a total of 200-300 million cases in 2010 [2].
2. Drug targets
At present, therapy for type-II diabetes relies mainly on several
approaches intended to reduce the hyperglycemia itself.
Table 1. Current therapeutic agents for type-II diabetes.
Drug class
Molecular target
Site(s) of action
Insulins
Insulin receptor
Liver, muscle, fat Hypoglycemia,

weight gain
Pancreatic -cell Hypoglycemia,
weight gain
Sulphonylureas
SU receptor/ K+
(e.g.
ATP channel
glibenclamide)
plus nateglinide &
repaglinide
Biguanides
Unknown
Metformin
Acarbose
-glucosidase
Thiazolidinediones PPAR
Rosiglitazone,
Pioglitazone
Liver (muscle)
Intestine
Fat, muscle, liver
Adverse events
Gastrointestinal
disturbances,
lactic acidosis
Gastrointestinal
disturbances
Weight gain,
anemia,
oedema,
157
3. Antihyperglycemic isolates from nature

Plants have always been an exemplary source of drugs and many of the
currently available drugs have been derived directly or indirectly from them.
The ethnobotanical information reports that about 800 plants may possess
anti-diabetic potential [3]. Several such herbs have depicted antidiabetic
activity while assessed using currently available experimental techniques [4].
A wide array of plant derived active principles representing numerous
chemical compounds has demonstrated activity consistent with their possible
use in the treatment of non-insulin dependent diabetes mellitus (NIDDM) [5].
Amongst these are flavonoids, alkaloids, glycosides, polysaccharides,
peptidoglycans, hypoglycans, guanidine, steroids, carbohydrates,
glycopeptides, terpenoids and amino acids. Even the discovery of widely
used hypoglycemic drug, metformin was developed on the basis of the
natural products lead isolated from Galega officinalis [6]. Thus, plants are a
potential source of anti-diabetic drugs. Herein, is presented a precise
description of naturally occurring compounds possessing potential
antihyperglycemic action.
3.1. Flavonoids
The flavonoids are polyphenolic compounds possessing 15 carbon atoms;
two benzene rings joined by a linear three carbon chain. Flavonoids
constitute one of the most characteristic classes of compounds in higher
plants. Many flavonoids are easily recognized as flower pigments in most
angiosperm families (flowering plants). However, their occurrence is not
restricted to flowers but include all parts of the plant. They show wide variety
of activities including antihyperglycemic activity. Bio-flavonoids with
promising anti-diabetic potential: A critical survey by Goutam Brahmachari
will give comprehensive information on the flavonoids and their
antihyperglycemic activity.
3.2. Triterpenoids and steroids

There are at least 4000 known triterpenes, which are derived from
mevalonic acid pathway. Triterpenes are precursors to steroids in both plants
and animals. Steroids are hormonal substances in animals, but they are
components of membranes in most organisms. Many triterpenes and sterols
occur free, but others occur as glycosides or in special combined forms.
Momordica charantia belongs to the family of Cucurbitaceae the fruits of
the plant is also known as bitter melon or bitter guard. Cucurbitane class of
158
T. Narender et al.
triterpenoids isolated from M. charanta such as 5-,19-epoxy-3-,25dihydroxycucurbita-6,23-(E)-diene (1) and 3-,7-,25-trihydroxycucurbita5,23-(E)-dien-19-al (2) have blood hypoglycemic effects in the diabetesinduced male ddY mice strain at 400 mg/kg [7].
Hypoglycemic activity guided fractionation together with chemical
analysis on the stem of Agarista mexicana led to the isolation of 12-ursene
(3) and 23,24-dimethyl-24-ethyl-stigmast-25-ene (4) from the chloroform
fraction. The isolated triterpenes showed hypoglycemic activity in normal
and alloxan-diabetic CD1 mice at a dose of 50 mg/kg body weight.
Comparison was made between the action of the triterpenes and a known
hypoglycemic drug, tolbutamide (50 mg/kg). The 12-ursene (3) was found to
be less potent than tolbutamide where as 23,24-dimethyl-24-ethyl-stigmast25-ene (4) was shown to be more effective than tolbutamide [8].
20
17
20
OH
17
OHC
OH
H
O
HO
OH
HO
From the roots of Salacia oblonga a friedelane-type triterpene,

kotalagenin 16-acetate (5), maytenfolic acid (6), 3,22-Dihydroxyolean-12en-29-oic acid (7) and a unique thiosugar sulfonium sulfate named Salacinol
(60) was isolated. They were screened for inhibitory activity on aldose
reductase and were found to be responsible components for the inhibitory
activity [9].
Bioassay-guided isolation work on Cabernet Sauvignons grape skin
yielded antihyperglycemic active compounds which were identified as,
oleanolic acid (8) and oleanolic aldehyde (9). These compounds were assayed
for insulin production using an INS-1 cell assay. In a dose-response study,
159
COOH
COOH
OH
OH
H
OAc
OH
HO
HO
oleanolic acid stimulated insulin production of INS-1 cells by 20.23, 87.97, 1.13
and 6.38 ng of insulin/ mg of protein at a dose of 6.25, 12.5, 25 and 50 g/mL
respectively. The activity was similar to the dose-dependent insulin production of
INS-1 cells by glucose. Oleanolic aldehyde also showed a dose-dependent insulin
production in the same assay [10]. Our activity guided fractional and isolation
work on the plant Ficus racemosa yielded moderately active antihyperglycemic
principle, -amyrin acetate (10). Several ester derivatives of -amyrin were
prepared to study their structure activity relationship [11].
CHO
COOH
O
HO
HO
10
Triterpenoid and steroidal glycosides referred to collectively as saponins

are bioactive compounds present naturally in many plants and known to
possess potent hypoglycemic activity [12]. Glucuronide saponin named
betavulgaroside (11) was isolated from the roots and leaves of Beta vulgaris
L. (sugar beet) exhibited hypoglycemic effect in rats [13].
H
CO2H
HO2C
O
HO
HO2C
HO2CH2CO
O
O
OH
11
160
T. Narender et al.
The root cortex of Aralia elata provided another triterpnoid glycoside,

Elatoside E (12), which was shown to affect the elevation of plasma glucose
level by oral sugar tolerance test in rats [14]. Hypoglycemic activity-guided
fractionation on the rhizomes of Anemarrhena asphodeloides yielded
steroidal glycosides, pseudoprotoimosaponin AIII, (13) and prototimosaponin
AIII, (14). These compounds exhibited hypoglycemic effects in a
dose-dependent manner in streptozotocin-diabetic mice but showed no effects
on glucose uptake and insulin release, suggesting that the hypoglycemic
mechanism may be due to inhibition of hepatic gluconeogenesis and/or
glycogenolysis [15].
HO
HO
O
R=
O
HO
RO
OH
OH
COO
O
O
OH
O
OH
OH
OH
O
OH
OH
OH
12
HO
OGlu
OGlu
Glu GalO
2
Glu
13
GalO
2
14
Charantin (15) a steroidal saponin, obtained from Momordica charantia

is known to have an insulin-like activity [16]. Charantin stimulates the release
of insulin and blocks the formation of glucose in the bloodstream. Similar
steroidal saponin (16) was isolated from the fruiting bodies of Ganoderma
applanatum, which exhibits Rat lens aldose reductase (RLAR) inhibiting
activity. The same plant also produced few other class of compounds (65-67)
with RLAR inhibiting property [17].
A steroidal saponin, chloragin (17) was isolated from the aerial part of
Chlorophytum nimonii (Grah) Dalz. The saponin characterized as tigogenin3-O--L-rhamnopyranosyl-(1 4)--D-glucopyranosyl-(1 3)--Dxylopyranosyl-(1 4)--D-glucopyranosyl-(1 4)--D-xylopyranoside
showed potent antihyperglycemic activity in streptozotocin induced diabetic
rats [18].
OH
OH
OH
161
OH
O
OH
OH
O
OH
O
O
OH
O
16
15
O
H
OH
OH
OH
O
OH
O
OH
O
OH
O
OH
OH
O
OH
OH
O
OH
O
OH
OH
17
Yoshikawa and co-workers isolated elatoside G (18), H (19) and I (20)

from a garnish foodstuff "Taranome," the young shoot of Japanese Aralia
elata were found to exhibit potent hypoglycemic activity in rats [19].
From Gynostemma pentaphyllum Makino (Cucurbitaceae) a gypenoside
saponin, named phanoside (21,23-epoxy-3--20,21-trihydroxydammar-24ene-3-O-([-D-rhamnopyranosyl-(12)]-[-D-glucopyranosyl-(13)]--Dlyxopyranoside) (21), has been isolated. Phanoside is a dammarane-type
saponin and found to stimulate insulin release from isolated rat pancreatic
islets. Phanoside (40 and 80 mg/mL) improved glucose tolerance and enhanced
plasma insulin levels at hyperglycemia, when given orally to rats [20].
Coagulin C (22), 17-hydroxywithanolide K (23), withanolide F (24),
coagulanolide (25) and coagulin L (26), isolated from the fruits of Withania
somnifera, showed significant inhibition on postprandial rise in
hyperglycemia post sucrose load in normoglycemic rats and in
streptozotocin-induced diabetic rats. Coagulin L (26) showed significant fall
in peripheral blood glucose profile and also improved the glucose tolerance
of db/db mice [21].
Methanolic extract of the leaves of Boussingaultia baselloides yielded
four nor-saponins and a saponin with hypoglycemic activity (27-31).
Amongst these, boussingoside A1 (31) exhibited very strong hypoglycemic
activity in rats [22].
3.3. Diterpenoids
Diterpenoids are composed of four isoprene units and have the molecular
formula C20H32, which are derived from geranylgeranylpyrophosphate
pathway. Andrographolide (32), a diterpenoid lactone, obtained from
162
T. Narender et al.
COOH
COOH
OH
COOH
O
O
OH
OH
COOH
CH2OH
CH2OH
O
CH2OH
OH
OH
OH
OH
OH
OH
OH
19
18
O
HO
OH
COOH
OH
COOH
O
O
O
CH2OH
O
O
CH2OH
OH OH
OH
OH
OH
CH2OH
OH
OH
OH
OH
OH
OH
OH
HO
OH
OH
20
21
OH
HO
O
H O
O
OH
H O
O
OH
H O
OH
OH
23
22
24
OH
OH
H O
O
OH
H
H
OH
25
OH
OH
H O
O
OH
HO
HO
OH
O
OH
OH
26
163
H
CO2R1
R3
HO
HO
O
HO
HO
O
R2
OH
CO2
H
HOOC
O HO
O
O
O
OH
OH
27- R1 = H, R2 = Me, R3 = CO2H

28- R1 =-D-glucosyl, R2 = Me, R3 = CO2H
29- R1 =-D-glucosyl, R2 = R3 = CH2OH
30- R1 = H, R2 = CH2OH, R3 =CO2H
31
Andrographis paniculata was found to possess significant hypoglycemic

activity [23]. A modified diterpene, saudin (33) was isolated from the leaves
of Cluytia richardiana (Euphorbiaceae) growing in Saudi Arabia. It is related
to the labdane-type of diterpenes with a novel rearrangement of lactone
groups, was found to possess hypoglycemic activity when tested in alloxan
induced diabetic rats [24]. Bioassay-guided fractionation of the EtOH extract
of Maprounea africana, on noninsulin-dependent diabetes mellitus db/db
mouse model, resulted in the isolation of a daphnane-type diterpenoid,
maprouneacin (34) which showed potent glucose-lowering properties by the
oral route [25].
O
O
HO
O
O
CH2
O
O
HO
CH2OH
32
O
H
Me
O
O H
O
O
O
33
OH
OH
OH
34
164
T. Narender et al.
3.4. Sesquiterpenoids
Sesquiterpeniods consist of three isoprene units and have the molecular
formula C15H24. A sesquiterpene lactone, lactucain C (35) and furofuran
lignan, lactucaside (77), were isolated from Lactuca indica which showed
in vivo antihyperglycemic activity profile -22.74 12.53% and -17.95
5.63% using STZ-diabetic rats at a dose of 1 M/kg [26].
O
14
13'
1
3
H
11'
12'
O
6'
5'
1'
H
R
15
12
15'
13
14'
35: R =
16
O C
H2C
18
21
OH
3.5. Alkaloids
An alkaloid is a naturally occurring nitrogenous organic molecule that
has a pharmacological effect on humans and other animals. Berberine (36) is
known to have potent hypoglycemic activity. It was obtained from the
traditional medicinal plant Tinospora cordifolia [27]. The mode of its
antihyperglycemic activity was investigated in the Caco-2 cell line. Berberine
effectively inhibited the activity of disaccharidases in Caco-2 cells, decreased
sucrase activity after pre-incubation with Caco-2 cells for 72 h but failed to
produce any significant effect on gluconeogenesis and glucose consumption
of Caco-2 cells, suggesting that the antihyperglycemic activity of berberine is
at least partly due to its ability to inhibit -glucosidase and decrease glucose
transport through the intestinal epithelium [28].
Other alkaloids such as catharanthine (37), vindoline (38) and
vindolinine (39) obtained from Catharanthus roseus also lower blood sugar
level [29]. Arecoline (40), an alkaloid isolated from Areca catechu was
investigated and reported to have hypoglycemic activity in an animal model
of diabetes upon subcutaneous administration [30].
165
Cryptolepine (41) is a rare example of a natural product whose synthesis

was reported prior to its isolation from Cryptolepis sanguinolenta.
Cryptolepine and its salts form lower blood glucose in rodent models of type
II diabetes. To optimize this natural product lead, a series of substituted and
hetero substituted cryptolepine analogs was synthesized [31].
Aegeline (42), an alkaloidal-amide from the leaves of Aegle marmelos,
was isolated by our group and was found to have antihyperglycemic activity
as depicted from the lowering of the blood glucose levels by 12.9% and
16.9% at 5 and 24 h, respectively, in sucrose challenged streptozotocin
induced diabetic rats (STZ-S) model at the dose of 100 mg/kg body weight.
The reasonable mapping of compound to a validated pharmacophoric
hypothesis and 3D QSAR model with an estimated activity (283 nM)
suggested that aegeline might be a 3-adregenic receptor (AR) agonist [32].
Hypoglycemic activity of trigonelline (43) and 4-hydroxyisoleucine (57)
isolated from seeds of Trigonella foenum graecum viz was evaluated in
alloxan induced diabetic mice. The combination of 4-hydroxyisoleucine and
trigonelline [4-HIT, 40: 30, 120 mg/kg] was administered orally in alloxan
induced diabetic mice. After 28 days treatment with 4-HIT, there was
significant decrease in blood glucose level. 4-HIT increased the glucose
threshold as compared to only alloxan treated group. Histology of pancreas
showed formation of new islets near the vicinity of the pancreatic duct.
Glyburide was used as a standard antidiabetic drug and its effect on
pancreatic cell was also studied. The pancreatic cells of glyburide treated
mice did not show any islets in the vicinity of pancreatic duct. LD50 was
found to be more than 5000 mg/kg. These results suggested that 4-HIT
showed hypoglycemic effect in alloxan induced diabetic mice. The presence
of the pancreatic islets in the vicinity of duct suggested that 4-HIT might act
by regeneration of new islets [33].
The therapeutic potential of Galega officinalis for the management of
diabetes was defined in the first half of the twentieth century. G. officinalis is
a rich source of guanidine and related molecules, which account for its
biological effects. The toxicity of guanidine precludes its use clinically, and
experiments by Georges Tanret in the years immediately before the Great
War identified a less toxic guanidine-like alkaloid, galegine (44) [34]. The
synthetic biguanides such as metformin (45) and its analogues were
synthesized on the basis galegine chemical structure.
3.6. -Carbolines
The -carboline alkaloids Harmane (46), norharmane (47) and pinoline
(48), were found to increase insulin secretion two to three-fold from isolated
166
T. Narender et al.
O
N
+
N
OCOCH3
OCH3
N
H
OCH3
COOC
H3
36
COOCH3
OH
N
H
H3CO
38
37
O
N
O
N
N
COOCH3
N
H
41
40
39
O
OH
H
N
+
N
MeO
NH
O_
42
H2N
NH
N
H
44
43
NH
N
H
NH2
45
human islets of langerhans. Harmane and norharmane obtained from Tribulus

terrestris may account for the hypoglycemic property of the plant [35].
Harmane stimulates insulin secretion in a glucose-dependent manner. The
results strongly substantiated the claim of -carbolines as potent insulin
secretagogues [36]. Harmine (49) is found in Syrian rue (Peganum harmala)
and other plants. Recently Waki and co-workers through a small-molecule
library screen has identified it as a proadipogenic that acts by inducing
PPAR expression. Obese (db/db) mice treated with harmine show a delay in
the onset of diabetes, coincident with increased oxygen consumption and
thermogenesis. A 2-fold increase in PPAR levels was selectively seen in
white adipose tissue, while there was a 50% decrease in PPAR levels in the
liver and no change in muscle, brown adipose tissue, or kidney. The effect of
harmine on PPAR expression in the brain and pancreas is currently
unknown [37].
NH
H3CO
N
H
N
H
N
H
46
47
48
N
H
MeO
49
167
3.7. Carbohydrates
Two hypoglycemic principles, ganoderan B (50) and C (51), isolated
from the fruit bodies of Ganoderma lucidum were shown to be
peptidoglycans with mol wts of 7400 and 5800, respectively.
Physicochemical and chemical studies demonstrated that the backbone
and side chains of ganoderan B contain D-glucopyranosyl -13 and
-16-linkages while those of ganoderan C contain D-glucopyranosyl
-13 and -16-linkages and a D-galactopyranosyl -16-linkage
[38].
-D-Glcp1
3)- -D-Glcp-(1
6 -D-Glcp1
6 -D-Glcp
3)- -D-Glcp-(1
[
(1
50
3)- -D-Glcp-(1
]5 [
3)- -D-Glcp
6)- -D-Galp-(1
]1
51
3.8. Amino acids

FR225659 (52) and four related compounds (53-56) are gluconeogenesis
inhibitors that consisted of an acyl-group and three unusual amino acids.
They were isolated from the culture broth of Helicomyces sp. and purified by
absorptive resin and reverse-phase column chromatography. They were found
to be potent inhibitors of gluconeogenesis in primary cultured rat hepatocytes
and thus may be useful as anti-diabetic agents [39]. T. foenum-graecum
(Leguminosae family) is an annual herbaceous plant commonly known as
fenugreek and is widely distributed across Asia, Africa, and Europe. Fowden
[40] was the first to isolate and identify the unusual amino acid,
4-hydroxyisoleucine (57). Christophe et al. [41] discovered that the major
isomer 2S,3R,4S of 4-hydroxyisoleucine induces insulin secretion through a
direct effect on pancreatic cells in rats and humans. Recent studies by our
group also confirm the antihyperglycemic activity [42]. The plant Blighia
sapida belongs to sapindacae family, which is known for its poisonous
properties. Two unusual amino acids such as hypoglycin A (58) and
hypoglycin B (59) isolated from this plant possess antihyperglycemic
activity [43].
168
T. Narender et al.
NH2
HO
N
OH
HN
NH2
COOH
NH
Cl
57
NH
O
R1
R2
NH2
R2
R3
52
-OH
-CH3
53
54
-OH
-OH
-OH
-H
55
-OCH3
-OH
-CH3
-CH2CH3
-OCH3
-OH
-CH3
56
58
COOH
R1
-OH
COOH
R3
HN
NH2
HN
-CH2CH3
COOH
COOH
59
3.9. Miscellaneous
Salacinol (60) has been isolated from an antidiabetic ayurvedic
traditional medicine, Salacia reticulata, through bioassay-guided separation
and was found to be most potent natural -glucosidase inhibitor [44].
Allicin (thio-2-propene-1-sulfinic acid S-allyl ester) (61), a sulphur
compound isolated from garlic (Allium sativum) has resulted in pronounced
hypoglycemia in mildly diabetic rabbits upon oral administration (0.25
mg/kg) [45]. S-allyl cysteine sulphoxide (62), a sulphur containing amino
acid which is the precursor of allicin and garlic oil, has been found to show
significant antidiabetic effects in alloxan diabetic rats at a dose of 200 mg/kg
body weight [46]. Leporin B (63), a demethylated analog of leporin A (64)
was isolated from a taxonomically unidentified fungal strain to discover
compounds with the ability to increase expression levels of the enzyme
hexokinase II [47].
O
O
HO
HO
-O3SO
S+
CH2OH
H
H
CH2OH OH
60
S
S
H2N
OH
N
O
O
61
RO
62
63 R=Me
64 R=H
169
Rat lens aldose reductase (RLAR) inhibitors (65-67 and 16) from the
fruiting bodies of Ganoderma applanatum were isolated, protocatechualdehyde
(67) was the most potent RLAR inhibitor (IC50 = 0.7 g/mL) equivalent to that
of the positive control TMG (IC50 = 0.6 g/ml) [17].
O
CHO
OH
OMe
HOOC
HO
HO HO
n
65
NH
O
HO
OH
n = 12-15
66
n = 7-9
OH
OH
67
2-Arylbenzofuran, puerariafuran (68) was isolated from MeOH extract of

the roots of Pueraria lobata as active constituent, using an in vitro bioassay based
on the inhibition of advanced glycation end products (AGE). The compound (68)
and coumestrol (69) exhibited a superior inhibitory activity against AGEs
formation with IC50 values of 0.53 and 0.19 M, respectively, compared to a well
known positive control, aminoguanidine (IC50 value of 473 M) [48].
Two compounds viz, kodaistatin A (70) and kodaistatin C (71) were
isolated from cultures of Aspergillus terreus. The kodaistatins are effective
inhibitors of the glucose-6-phosphate translocase component of the glucose6-phosphatase system (EC 3.1-3.9), an enzyme system which is important for
the control of blood glucose levels. The IC50 was 80 nM for kodaistatin A and
130 nM for kodaistatin C [49].
H
HO
OH
HO
O
H3CO
68
69
HO
O
HO
HO
OH
HO
O
O
R
OH
70 R = H
71 R = OH
OH
170
T. Narender et al.
The glucose lowering effect of mangiferin (72), a xanthone glucoside,

isolated from the leaves of Mangifera indica was studied in streptozotocininduced diabetic rats. Hypoglycemic activity of mangiferin (10 and 20 mg/kg, i.p.
once daily for 28 days) at different time intervals in STZ induced diabetic rats and
improvement in oral glucose tolerance in glucose-loaded normal rats upon
chronic administration (10 and 20 mg/ kg, i. p.) for 28 days was observed [50].
O
HO
O
HO
HO
HO
OH
OH
OH
O
OH
OH
72
OH
73
A xanthone, which is close analogue of mangiferin was isolated from the

hexane fraction of the plant, Swertia chirayita, identified as 1,8-dihydroxy3,5-dimethoxyxanthone (swerchirin: 73). It has a very significant blood sugar
lowering effect in fasted, fed, glucose loaded, and tolbutamide pretreated
albino rat models. The ED50 for 40% blood sugar lowering in CF male albino
rats (body weight 140-165 g) is 23.1 mg/kg/oral [51].
Various active components like ()-epicatechin (74), the benzofuranone,
marsupsin (75) and the stilbene, pterostilbene (76) isolated from the bark and
heartwood of Pterocarpus marsupium were evaluated for their putative
antihyperglycemic activity against streptozotocin-induced hyperglycemic rats
and were found to possess blood sugar lowering activity. The phenolic
constituents viz, marsupsin (75) and pterostilbene (76) significantly decreased
the plasma glucose level of STZ-induced diabetic rats by -33% and
-42% respectively. The antidiabetic activity of pterostilbene (-42%) was
comparable to that of the reference compound, metformin (-48%) [52].
OH
HO
HO
O OH
OH
OH
OH
OH
OH
74
OCH3 O
75
MeO
OMe
76
171
A furofuran lignan, lactucaside (77) along lactucain (35) was isolated

from Lactuca indica which showed in vivo antihyperglycemic activity profile
-17.95 5.63% using STZ-diabetic rats at a dose of 1 M/kg [26].
OCH3
OH
O
9' 7
HO
8'
1'
O
4'
HO
R = glucose
3'
OCH3
77
HO
OH
MeO
78
H
79
Ferulic acid (78) is polyphenolic compound found in many medicinal

plants such as Curcuma longa. Ohnishi and co-workers from Japan
demonstrated its antihyperglycemic activity in insulin dependent (IDD) and
non-insulin dependent diabetes milletus models (NIDDM) [53]. Similar class
of compound that is cinnamaldehyde (79) was isolated from Cinnamonum
zeylanicum (cinnamon) exhibits potent antihyperglycemic activity in
streptozotocin (STZ)-induced male diabetic wistar rats [54]. Both the
compounds also possesses hypolipidemic properties [51,52].
4. Dyslipidemia
Dyslipidemia is elevation of plasma cholesterol, triglycerides (TGs), or
both, or a low high density lipoprotein level that contributes to the
development of atherosclerosis. Causes may be primary (genetic) or
secondary. Diagnosis is by measuring plasma levels of total cholesterol, TGs,
and individual lipoproteins. When carbohydrates are in low supply or their
breakdown is incomplete, fats become the preferred source of energy in
diabetic patients. As a result, the fatty acids are mobilized into the general
circulation leading to secondary triglyceridemia in which total serum lipids in
particular triglycerides as well as the levels of cholesterol and phospholipids
172
T. Narender et al.
increase. This rise is proportional to the severity of the diabetes. Uncontrolled

diabetes is manifested by a very high rise in triglycerides and fatty acid
levels. An increase in plasma lipids, particularly cholesterol, is a common
feature of atherosclerosis, a condition involving arterial damage, which may
lead to ischemic heart disease, myocardial infarction, and cerebrovascular
accidents. These conditions are responsible for one-third of deaths in
industrialized nations [55].
5. Current therapeutics
Current antidyslipidemia drugs include statins, fibrates, niacin,
ezetimibe, and bile acid binding resins (Table-2).
These drugs target one component of the lipid profile, with smaller
additional effects on other parameters. For instance, statins and fibrates
produce sizable reductions primarily in plasma LDL-C and TG, respectively.
Meanwhile, niacin has the greatest HDL-C raising capacity. However, many
high CHD risk patients fail to reach strict guideline target levels with currently
Table 2. Currently available pharmaceuticals for dyslipidemia.
Medication
Effects on lipid
parameters
LDL-C, TG
Minimal effects on
HDL-C
(rosuvastatin can
increase HDL-C
levels)
Adverse effects
Fibrates
(PPAR-
agonists)
LDL-C, TG,
HDL-C (mild)
Myalgias, Rhabdomyolysis
Cholelithiasis, Elevations in serum creatinine
Ezetimibe
(intestinal
cholesterol
absorption
inhibitor)
Niacin
LDL-C, TG
Myalgias (very rare)

Rhabdomyolysis (very rare)
Statins
(HMG-CoA
reductase
inhibitors)
Myalgias, Myositis/rhabdomyolysis
Transaminitis
TG, HDL-C, Flushing/vasodilation

Impair insulin sensitivity
LDL-C, LP (a)
Gout, gastric
Bile acid
LDL-C
resins
(inhibitors of
enterohepatic
circulation)
TG
Bloating, constipation
Interference with absorption of other,
medications such as levothyroxine, warfarin,
digoxin, statins
173
available drugs. A small but clinically relevant proportion of patients experience

adverse effects. Thus, additional pharmaceutical strategies are required to fill
these gaps in efficacy and tolerability. Plants have always been an exemplary
source of drugs and many of the currently available drugs have been derived
directly or indirectly from them.
5.1. Sterols and triterpenoids

A number of studies, both in animal models and human clinical trials,
have shown that guggulipid (80,81) isolated from the Resin of the gum of
the guggul tree, Commiphora mukul, has beneficial effects on serum
lipoprotein profiles [56]. A pregnane glycoside roylenine (82) was isolated
from Marsdenia roylei. The glycoside (82) and its acetylated derivative
showed singnificant antioxidant and antidyslipidemic activities [57].
H3C
OH
OH
O
80
H3C
81
OH
82
OH
H3C
AcO
AcO
O O
A
steroidal
saponin,
chloragin
(17)
[tigogenin-3-O--Lrhamnopyranosyl-(1 4)- -D-glucopyranosyl-(1 3)--D-xylopyranosyl(1 4 )--D-glucopyranosyl-(1 4)--D-xylopyranoside] was isolated
from the aerial part of Chlorophytum nimonii (Grah) which showed potent
antidyslipidemic activities in albino rats [18]. Coagulin L (26) isolated from
Withania somnifera showed significant fall in peripheral blood glucose
profile and also improved the glucose tolerance of db/db mice. It also showed
antidyslipidemic activity in db/db mice that is comparable to median
effective dose of fenofibrate i.e., 50 mg/kg body weight [21].
Sudhahar and co-workers reported hypercholesterolemia in lupeol (83)
and linoleate ester of lupeol (84) [58]. We have also prepared several ester
derivatives of lupeol and studied their structure activity relationship. Some of
the derivative showed potent activity than the lupeol. Lupeol nicotenate (85)
was found to be the most potent triglyceride lowering agent in addition to
antihyperglycemic activity [59].
174
T. Narender et al.
O O
HO
N
84
83
85
Wiedendiol-A (86) and B (87), sesquiterpene-hydroquinones which

inhibit cholesteryl ester transfer protein (CETP), have been isolated from the
marine sponge Xestoepongia wiedenmayeri [60]
HO
HO
HO
HO
OCH3
CH3
OCH3
CH3
H
87
86
Statins are currently marketed drugs used to lower the plasma cholesterol
levels in humans. Natural statins obtained from different genera and species
of filamentous fungi. Lovastatin (88) is mainly produced by Aspergillus
terreus strains and mevastatin (89) by Penicillium citrinum. Pravastatin (90)
was obtained by the biotransformation of mevastatin by Streptomyces
carbophilus and simvastatin (91) by a semi-synthetic process, involving the
chemical modification of the lovastatin side chain. The hypocholesterolemic
effect of statins lies in the reduction of the very low-density lipoproteins
(VLDL) and LDL involved in the translocation of cholesterol, and in the
increase in the high-density lipoproteins (HDL), with a subsequent reduction of
the LDL- to HDL-cholesterol ratio, the best predictor of atherogenic risk [61].
HO
O
O
O
HO
HO
O
O
O
O
COOH
OH
HO
O
O
O
HO
88
89
90
91
175
Several synthetic statins such as atorvastatin (92), cerivastatin (93),

pitavastatin (94) and rosuvastatin (95) were developed on the basis of
structures of natural statins.
HO
COOH
OH
HO
F
F
HO
COOH
OH
COOH
OH
O
92
COOH
OH
HN
HO
S
O
93
94
N
N
O
95
A diterpene, (96) which has close structural features of statins was

isolated from the leaves of Polyalthia longifolia [62]. This compound showed
significant antidyslipidemic activity in high diet (HFD) fed dyslipidemic
hamsters at different doses.
O
OH
96
5.2. Polyphenolic compounds

Few naturally occurring flavanones and their glycosides such as
hesperetin (97), hesperidin (98), naringenin (99), and naringin (100) have
been reported as potential agents for improving the cholesterol metabolism in
diet-induced hypercholesterolemic animals [63]. We also isolated three
modified furano-flavonoids (101-103) and a rare flavonol glycoside (104) as
an antidyslipidemic agents from the aerial parts of Indigofera tinctoria [64].
Flavonoid mixture (101 and 102) showed potent triglyceride lowering
activity in high fat fed hamster model.
176
T. Narender et al.
O
O
H
O
H
H
R2
RO
O
101
OH O
H
O
R1
O
H
H
O
102
OR
O
97: R= H; R1=OH; R2=OCH3
98: R= -D-Rutinoside; R1=OH; R2= OCH3
O
99: R = H; R1=H; R2=OH
100: R=Neohespiridoside; R1=H; R2=OH
RO
O
O
O
OH
O
OH O
O
103
104: R= Rhamnose
Eriocitrin (105) (eriodictyol 7-O--rutinoside) is the main flavonoid in

lemon fruit (Citrus). Eriocitrin was effective in lowering effect on serum and
hepatic lipids in high-fat and high-cholesterol fed rats [65].
OH
HO
OH
OH
H3C
OH
O
HO
OH
OH
OH O
105
Pterosupin (106) and liquiritigenin (107) were isolated from the

heartwood of Pterocarpus marsupium showed hypolipidemic activity in
Triton model. Both the compounds lowered the serum cholesterol and
LDL-cholesterol. Pterosupin also lowered the triglycerides [66].
OH
HO
OH
HO
Glc
OH O
106
OH
O
107
177
Rutin (108) is flavonoid glycoside found in many plants and is also an

important dietary constituent of food and plant-based beverages. Several
studies demonstrated lipid lowering effect of rutin. Recently Amir and
co-workers reported its anti-hyperchloesterolaemic effect (plasma cholesterol
and LDL-C) in rat model [67]. Odbayar and co-workers from Japan studied
the effect of quercetin (109) and its glycoside (rutin) and their studies
indicated that quercetin better than the rutin in reduction of hepatic
lipogenesis (hypolipidemic effect) [68].
OH
OH
HO
OH
HO
O
OH
OH
OH
OH
O
H3C
O
HO
HO
HO
O
OH
OH
OH
O
109
108
Tso-Hsiao Chen and co-workers studied about 40 flavonoids for their

HMG-Co-Enzyme reductase activity. Astilbin (110) was the only effective
HMG-Co-Enzyme reducates inhibitor in their studies, which demonstrates its
hypochelestereamic activity [69].
OH
O
HO
OH
O
HO
OH
OH
O
CH3
OH
110
Kurarinol (111) is a prenylated flavanone, which is known for its alpha

glucosidase, beta amylase and diacylglyceral transferase activity. Kuraridinol
(112) is a prenylated chalcone. Both were isolated from the Sophora
flavescens showed significant hyperlipidemic and hypercholesterolemic
effect. Kuraridinol was more potent than kurarinol in their studies [70].
178
T. Narender et al.
OH
OH
OH
OH
HO
HO
OH
OH
OH
O
O
112
111
Resveratrol (113), a naturally occurring stilbenoid commonly available in

red wine act as a free-radical trap to halt the progression of LDL oxidation.
It is very strong antioxidant and mild lipid lowering agent, which certain
extent prevents the development of atherosclerosis [71]. Resveratrol
derivatives such as, pterostelbene (76) and trimethylated resveratrol (114)
and its analogue Piceatannol (115) have been studied for their PPAR alpha
activity and in-vivo hyperlipidemic activity. Pterostelbene showed good
PPAR alpha agonist activity and hypolipidemic activity than other
compounds [72]. Polydatin (116) is glycoside of resveratrol isolated from
Polygonum cuspidtum also has been reported for its lipid lowering effect in
high fat diet fed hamster [73].
OH
HO
HO
MeO
OH
OH
OH
OMe
113
OH
OMe
114
115
Mangiferin (72) a xanthone glucoside, isolated from the leaves of

Mangifera indica showed significant antihyperlipidemic activity at a dose of
10 and 20 mg/kg, i.p. Further, in streptozotocin-induced diabetic rats it
showed antiatherogenic activities as evidenced by significant decrease in
plasma total cholesterol, triglycerides, low-density lipoprotein cholesterol
(LDL-C) levels coupled together with elevation of high density lipoprotein
cholesterol (HDL-C) level and diminution of atherogenic index in diabetic
rats [50].
179
HO
H
OH
H
OH
H O
HO H
HO H
O
H
H
OH
116
Bergenin (117) is commonly available in many plants of Euphorbiaceae,

Saxifragaceae and Myrsinaceae. It is a C-glucoside of 4-O-methylgallic acid.
Oral administration of bergenin isolated from the leaves of Flueggea
microcarpa reduced the serum cholesterol, triglycerides, low-density
lipoprotein (LDL) and very low-density lipoprotein (VLDL)-cholesterol
levels were significantly [74].
OH
OH
OH O
MeO
OH
O
HO
O
117
5.3. Alkaloids
Berberine (36), a natural plant alkaloid isolated from the root of
Berberis oblonga. In vitro and in vivo studies have showed its effects on
hyperglycemia and dyslipidemia [75].
Our activity guided fraction and isolation work o the leaves of
A. marmelos led to isolate an alkaloidal-amide, Aegeline (42) and found to
have antihyperglycemic activity as well as hypolipidemic activity [32].
Aegeline has strong triglyceride lowering activity in our studies and the
activity was comparable with the marketed drug i.e. fenofirbrate. Hsu and
coworkers showed that arecoline (40) inhibited adipogenesis as determined
180
T. Narender et al.
by oil droplet formation and adipogenic marker gene expression. There

further studies indicated that arecoline induced lipolysis in an adenylyl
cyclase-dependent manner [30].
5.4. Amino acid

We isolated an unusual amino acid 4-hydroxyisoleucine (57) from the
seeds of T. foenumgraecum, which significantly decreased the plasma
triglyceride levels by 33% (P < 0.002), total cholesterol (TC) by 22% (P <
0.02), and free fatty acids by 14%, accompanied by an increase in HDLC/
TC ratio by 39% in the dyslipidemic hamster model [11]. 4-Hydroxyisoleucine is
also very good insulin releasing agent.
5.5. Miscellaneous
C60-polyprenol (118) was isolated from the chloroform fraction of the
ethanol extract of Coccinia grandis. It significantly decreased serum TG by
42%, total cholesterol (TC) 25% and glycerol (Gly) 12% and increased
HDL-C/TC ratio by 26% in high fat diet (HFD)-fed dyslipidemic hamsters at
the dose of 50 mg/kg body weight as compared to the standard drug
fenofibrate at the dose of 108 mg/kg [76].
O
S
7
OH H2N
OH
O
118
119
S-methyl cysteine sulfoxide SMCS (119) isolated from Allium cepa was
investigated for its lipid lowering action in SD rats. SMCS at a dose of
200 mg/kg body weight for 45 days enhanced the hyperlipidemic condition.
Concentrations of cholesterol, triglyceride and phospholipids were
significantly reduced with respect to control [77]. Itokawa and co-workers
also reported the lipid lowering activity in S-methyl cysteine sulfoxide
(SMCS) and S-allylcysteine sulfoxide (62) [78].
Ferulic acid (78) and cinnamaldehydes (79) which are commonly
available in many medicinal plants have been reported for their lipid lowering
activity as well as anthyperglycemic activity [51,52].
181
6. Conclusion
Type-II diabetes poses a lethal threat to mankind in the present health
scenario. The more alarming situation has raised owing to the secondary
complications such as atherosclerosis, (ischemic heart disease, myocardial
infarction, and cerebrovascular accidents) associated with this silent killer.
So, there is an urgent need for broad based drugs which can ameliorate this
complex menace. Natural products have always been the inexhaustible source
of new drugs from the time immemorial. Notwithstanding the significant
headways in synthetic chemistry in the management of hyperglycemia and
hyperlipidemia, chemical entities emanating from the natural source still hold
promise in alleviating the blood glucose levels and lipids and its concurrent
ailments. More has been done but much has remained unexplored in the drug
discovery paradigm of natural products attributed with therapeutic virtues.
Some targets have been identified for the active principles but unless, their
mechanism of action is not determined and clinical studies not performed,
their potential as antihyperglycemics and antidyslipidemics will remain
unearthed. Moreover, the combination of plant based drugs and synthetic
pharmaceuticals for correcting this metabolic error could pave way for costeffective therapies. The scope of plant drugs lies in the rectifying the problem
of adverse side effects generated by synthetic drugs, cost-effectiveness and
minimal side-effects. The resurgence of natural products in the drug
discovery and development may hold the key in the proper utilization of
biodiversity for the management of hyperglycemia and hyperlipidemia.
Acknowledgements
The authors are grateful to the Director, CDRI, Lucknow for constant
encouragement for the program on Indian medicinal plants, CSIR, New Delhi
for financial support.
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6. Bio-flavonoids with promising antidiabetic potentials: A critical survey

Goutam Brahmachari
Department of Chemistry, Visva-Bharati University, Santiniketan-731 235
West Bengal, India
Abstract. Bio-flavonoids comprise a group of phenolic secondary

plant metabolites that are widespread in nature. Major flavonoids
that have well categorized structures and well defined structure
function-relationships are: flavans, flavanones, flavones, flavonols,
flavanols, flavanonols, cetechins, anthocyanidins and isoflavones.
Bio-flavonoids are well-known for their multi-directional
biological activities including anti-diabetic efficacy. Numerous
studies have been carried out to explore their potential role in the
treatment of diabetes. A good number of studies have already
demonstrated the hypoglycemic effects of flavonoids using
different experimental models and treatments - the drug candidates
have been shown to exert such beneficial effects against the disease
manifestation, either through their capacity to avoid glucose
absorption or to improve glucose tolerance. It has also been
demonstrated that flavonoids can act per se as insulin
secretagogues or insulin mimetics, probably by influencing the
pleiotropic mechanisms, to attenuate the diabetic complications;
besides, the drug candidates have been found to stimulate glucose
uptake in peripheral tissues, and regulate the activity and/or
expression of the rate-limiting enzymes involved in carbohydrate
metabolism pathway. As a result, bio-flavonoids are now-a-days
regarded as promising and significantly attractive natural
substances to enrich the current therapy options against diabetes.
Correspondence/Reprint request: Dr. Goutam Brahmachari, Department of Chemistry, Visva-Bharati
University, Santiniketan-731 235, West Bengal, India. E-mail: brahmg2001@yahoo.co.in
188
Goutam Brahmachari
The purpose of this resume is to represent promising anti-diabetic flavonoid

candidates highlighting their absorption and metabolism along with their mode of
action in regulating diabetic symptoms.
1. Introduction
Diabetes mellitus is the most prevalent metabolic syndrome world-wide
with an incidence varying between 1 to 8% [1,2]. The disease arises when
insufficient insulin is produced, or when the available insulin does not
function properly. Thus diabetes is characterized by hyperglycaemia
(elevation in blood sugar levels) resulting in various short-term metabolic
changes in lipid and protein metabolism and long-term irreversible vascular
changes. The long-term manifestation of diabetes can result in the
development of some complications, broadly classified as microvascular or
macrovascular disease. Microvascular complications include neuropathy
(nerve damage), nephropathy (renal disease) and vision disorders
(retinopathy, glaucoma, cataract and corneal diseases), while macrovascular
complications include heart disease, stroke and peripheral vascular disease,
which can lead to ulcers, gangrene and amputation [3]. These complications
are also found in non-diabetic population, but have a two to five-fold increase
in diabetic subjects [4]. The last century has seen a rapid increase in the
global prevalence of coronary artery disease (CAD) [5,6].
Current estimates from different countries in Europe and the United
States have shown that diabetes and its complications account for 8-16% of
the total health costs for society and this will increase dramatically unless
major efforts are made to prevent the ongoing epidemic. There are two major
categories of diabetes - insulin dependent diabetes mellitus (IDDM, Type 1
diabetes mellitus) and non-insulin dependent diabetes mellitus (NIDDM,
Type-2 diabetes mellitus). Type 1 diabetes occurs due to almost 95%
destructions of -cells of islets of Langerhans in the endocrine pancreas
caused by an autoimmune process, usually leading to absolute insulin
deficiency, this type has an early onset, most often between the ages of 10
and 16 yrs. Insulin resistance in peripheral tissue and an insulin secretive
defect of the -cells characterizes Type-2 diabetes mellitus (NIDDM). It is
the most common form of diabetes mellitus constituting above 90% of the
diabetic population and highly associated with a family history of diabetes,
older age, obesity and lack of exercise [3]. The global prevalence of diabetes
is estimated to increase, from 4% in 1995 to 5.4% by the year 2025 [7]. The
World Health Organization (WHO) has predicted that the major burden will
occur in the developing countries, there will be a 42% increase from 51 to
72 million in the developed countries while 170% increase from 84 to
Anti-diabetic bio-flavonoids
189
228 million, in the developing countries [8]. Prevalence of the complications

is greater among the lower socio-economic people due to lack of good
control of glycaemia and hypertension and also due to behavioral factors. The
direct and indirect costs involved in the treatment of the chronic disease
especially when associated with the vascular complications are enormous.
The overall global scenario urges to implement cost-effective and at the same
time efficacious preventive measures against diabetes to reduce the high
morbidity and mortality [4].
2. Currently available therapies

Currently available therapies for diabetes include insulin and various oral
anti-diabetic agents such as sulfonylureas, biguanides, -glucosidase
inhibitors, and glinides, which are used as monotherapy or in combination to
achieve better glycemic regulation. Many of these oral anti-diabetic agents
suffer from various adverse effects, thus, managing diabetes without any side
effects is still a challenge to the workers [9], and hence the search for more
effective and safer therapeutic agents in eradiating diabetic syndromes has
continued to be an important area of investigation. Both fasting and
postprandial impaired glucose tolerance are associated with an increased risk
of developing Type-2 diabetes mellitus and therefore form an important
target group for interventions aimed at preventing diabetes [10]. The
pharmacological agents with the greatest effect on postprandial
hyperglycemia include insulin lispro, amylin analogues, and -glucosidase
inhibitors. In hyperglycemia associated with diabetes, the use of aldose
reductase inhibitors has been reported for the treatment of diabetic
complications [11]. Aldose reductase as a key enzyme in the polyol pathway
has been reported to catalyze the reduction of glucose to sorbitol. Sorbitol
does not readily diffuse across cell membranes, and the intracellular
accumulation of sorbitol has been implicated in the chronic complications of
diabetes such as peripheral neuropathy, retinopathy, and cataracts [12]. A
recent study reported that aldose reductase may also be involved with another
signal transduction pathway in the pathogenesis of diabetic nephropathy [13].
3. Back to the plant kingdom

The use of ethnobotanicals has long folkloric history for the treatment of
blood sugar abnormalities. In the India, indigenous remedies have been used
in the treatment of diabetes since the time of Charaka and Sushruta (6th
century B.C.) [14]. Plants have always been exemplary source of drugs and
many of the currently available drugs have been derived directly or indirectly
190
Goutam Brahmachari
from them. The ethnobotanical information reports about 800 plants that may
possess anti-diabetic potential [15]. Many of such plants have exhibited
anti-diabetic activity when assessed using presently available experimental
techniques [17-20]. It may be mentioned in this connection that the discovery
of widely used hypoglycaemic drug, metformin came from the traditional
approach of using Galega officinalis. In spite of all these, the indigenous
system has not yet gained enough momentum in the scientific community.
The reasons may be many including lack of belief among the practitioners of
conventional medicine over alternative medicine, alternative form of
medicine are not very well-defined and natural drug may vary tremendously
in content, quality and safety. To cope with severe problems associated with
using of synthetic anti-diabetic drugs, there is a need to look for more
efficacious drugs with lesser side effects and also of low cost. It is the high
time to turn our attention to the plant kingdom in search of natural drugs for
diabetes following an integrated approach and using correct procedures. The
hypoglycemic effect of several plants used as anti-diabetic remedies has
already been confirmed, and the mechanisms of hypoglycemic activity of
these plants are being studied; if even a single plant material stands the acidtest of efficacy comparable to commonly used synthetic oral drugs already
marketed, it will herald the discovery of cheap and relatively nontoxic drug.
4. Purpose of the present review

A number of review articles on the uses of various plants (different parts
of plant materials, crude extracts, herbal formulations, etc.) as anti-diabetic
agents have been published time to time [22-26]. Naturally occurring
chemotypes of varying structural skeletons have also been reported to possess
anti-diabetic properties [27,28], and the purpose of this resume is to represent
promising anti-diabetic bio-flavonoids highlighting their absorption and
metabolism along with mode of action in regulating diabetic symptoms.
5. Anti-diabetic bio-flavonoids of promise

Bio-flavonoids comprise a group of phenolic secondary plant metabolites
that are widespread in nature. Major flavonoids that have well categorized
structures and well defined structure function-relationships are: flavans,
flavanones, flavones, flavonols, flavanols, flavanonols, cetechins, anthocyanidins
and isoflavones. Bio-flavonoids are well-known for their multi-directional
biological activities including anti-diabetic efficacy [29-32]. Numerous studies
have been carried out to explore their potential role in the treatment of diabetes
[27,28,33]. A good number of studies have already demonstrated the
191
hypoglycemic effects of flavonoids using different experimental models and

treatments - the drug candidates have been shown to exert such beneficial effects
against the disease manifestation, either through their capacity to avoid glucose
absorption or to improve glucose tolerance. It has also been demonstrated
that flavonoids can act per se as insulin secretagogues or insulin mimetics,
probably by influencing the pleiotropic mechanisms, to attenuate the diabetic
complications; besides, the drug candidates have been found to stimulate glucose
uptake in peripheral tissues, and regulate the activity and/or expression of the
rate-limiting enzymes involved in carbohydrate metabolism pathway. As a result,
bio-flavonoids are now-a-days regarded as promising and significantly attractive
natural substances to enrich the current therapy options against diabetes. This
present section embodies the information on promising anti-diabetic efficacies of
certain bio-flavonoids.
Choi et al. [34] demonstrated that intraperitoneal administration of
prunin (naringenin 7-O--D-glucoside) produces a significant hypoglycemic
effect in diabetic rats. Anti-hyperglycemic effects have also been
demonstrated for various flavonoids including chrysin and its derivatives,
silymarin, isoquercetrin and rutin [35-37]. Long-term studies carried out with
rutin orally administered to diabetic rats showed that it decreased the plasma
glucose levels by up to 60% when compared to the control group. However,
oral administration of rutin to normal rats did not show any significant effect
on fasting plasma glucose levels [38]. Chronic treatment with hesperidin and
naringin was found to lower the blood glucose level of db/db mice compared
with the control group [39].
Myrciacitrins I, II, III, IV and V (1-5) isolated from the dried leaves of
Myrcia multiflora DC. (family: Myrtaceae) were reported to possess significant
rat lens aldose reductase inhibitory activity [40], the IC50 values for the flavonoids
1-5 were determined as 3.2 x 106, 1.5 x 105, 4.6 x 105, 7.9 x 107, 1.6 x 105
and 1.3 x 105 M, respectively [40,41]. Hence, myrciacitrin IV (4) exhibited the
most potent activity, although it had less activity than epalrestat, a commercially
available synthetic aldose reductase inhibitor (IC50 = 7.2 x 108 M) [40].
Kawabata et al. [42] isolated five 6-hydroxy-flavonoids (6-10) from the
methanol extract of Origanum majorana L. (family: Lamiaceae) leaves and
studied their -glucosidase enzyme inhibitory activity, three of these
flavonoids: 6-hydroxyapigenin (scutellarein) (6), 6-hydroxyapigenin-7-O-D-glucopyranoside (7), 6-hydroxyluteolin-7-O--D-glucopyranoside (8)
are previously known [43-47], and the other two feruloylglucosides namely,
6-hydroxyapigenin-7-O-(6-O-feruloyl)--D-glucopyranoside (9) and 6hydroxyluteolin-7-O-(6-O-feruloyl)--D-glucopyranoside (10) are novel
compounds. All the isolates showed rat intestinal -glucosidase inhibitory
activity, at an equal concentration of 500 M, the flavonoid candidates 6-10
192
Goutam Brahmachari
inhibited the enzyme activity by 81%, 44%, 55%, 25% and 26%,
respectively.
The respective IC50 values for 6-10 were determined as 12, >500, 300,
>500 and >500 M. Another flavonoid, 6-hydroxyluteolin (11) [48], was also
found to exhibit potent -glucosidase inhibitory activity (92% inhibition at a
concentration of 500 M) with an IC50 value of 10 M [42]. The same group
[49] also evaluated 5,6,7-trihydroxyflavone (baicalein, 12), the flanonoid
constituent of Scutellaria baicalensis, as an important inhibitor against rat
intestinal -glucosidase (IC50 = 32 M).
The investigators also observed that apigenin (5,7,4-trihydroxyflavone,
13) and luteolin (5,7,3,4-tetrahydroxyflavone, 14), both lacking the
6-hydroxyl substituent, showed negligible activity (12% and 22% inhibition at
500 M, respectively) in the -glucosidase inhibitory assay. From their study, the
present investigators suggested that 5,6,7-trihydroxyflavone skeleton is crucial for
high -glucosidase inhibitory activity regardless of B-ring hydroxylation, in
addition, glycosation of 7-hydroxyl substituent as well as acylation of the sugar
reduces the enzyme inhibitory activity [49].
Haraguchi et al. [50] isolated C-glucosidic flavone derivative named
as isoaffineyin (5,7,4,3,5-pentahydroxyflavone-6-C-glucoside, 15) from
Manikara indica (family: Sapotaceae), the flavonoid candidate exerted promising
inhibition against porcine lens aldose reductase activity with an IC50 value of
4.6 M (epalrestat was used as positive control, IC50 = 0.87 M).
193
The genistein derivatives (16-19) isolated from an EtOAc-soluble

partition of the MeOH extract of a branch of Tetracera scandens (family:
Dilleniaceae) were evaluated to possess promising activities on Type-2
diabetes mellitus treatment since the test compounds significantly stimulated
the uptake of glucose, adenosine monophosphate-activated kinase (AMPK),
glucose transport protein-4 (GLUT4) and GLUT1 mRNA expressions and
protein tyrosine phosphatase 1B (PTP1B) inhibition in L6 myotubes [51].
The IC50 values for isofavonoids 16-19 in inhibiting PTP1B activities were
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Goutam Brahmachari
determined as 31.75 0.27, 28.13 0.19, 20.63 0.17 and 37.52 0.31 M,
respectively (ursolic acid was used as positive control with IC50 value of
5.13 0.45M). No muscle cell toxicity was reported with compounds
17-19, while compound 16 reduced muscle cell viability with IC50 value of
18.69 0.19 M. The investigators, thus, demonstrated that the isoflavonoids
constituents (16-19) of T. scandens stimulate glucose-uptake in basal and
insulin-stimulated L6 myotubes in a dose-dependent manner - AMPK
activation, GLUT4 and GLUT1 expressions and PTP1B inhibition by these
bioactive constituents appeared to be involved in the mechanism of the
stimulation of basal and insulin-responsive glucose-uptake. Hence,
compounds 16-19 may be possible candidates of a novel therapeutic strategy
for Type-2 diabetes mellitus treatment, although further studies will
be required to clarify the molecular mechanism of these bioactive
constituents [51].
Isoorientin (20), isolated from the water and butanolic extracts of

Cecropia obtusifolia (family: Ceropiaceae), exhibited potent hypoglycemic
activity comparable to that of glibenclamide at a dose of 3 mg/kg body
weight in diabetic rats [52].
Kim et al. [53] isolated a new flavonol glycoside, quercetin
3-O--L-arabinopyranosyl-(12)--D-glucopyranoside (21) along with the
known flavonoid glycosides such as kaempferol 3-O--D-glucopyranoside
195
(astragalin) (22a) and quercetin 3-O--D-glucopyranoside (isoquercetin)

(22b) from the leaves of Eucommia ulmoides (family: Eucommiaceae), these
flavonoid constituents were found to be glycation inhibitors having
comparable activity to that of aminoguanidine, a known glycation inhibitor.
The IC50 values for the test compounds 21, 22a and 22b were determined as:
2.95 x 107, 4.86 x 107, and 3.20 x 107 M, respectively (aminoguanidine was
used as positive control, IC50 = 4.45 x 107 M) [53].
Tabopda et al. [54] reported that six unusual C-4-prenylated flavonols,

dorsilurins F-K (23-28), isolated from the roots of Dorstenia psilurus (family:
Moraceae), were found to exhibit glycosidase enzyme inhibitory activity
against -glucosidase, -glucosidase, and -mannosidase. Compound 23,
with three unmodified prenyl groups, showed the best -glucosidase
inhibitory activity (IC50 4.13 M), while compound 28, with only one
unmodified prenyl group, showed the least -glucosidase inhibitory activity
(IC50 43.95 M). Thus, it was suggested that -glucosidase inhibitory activity
of the compounds increased with the number of unmodified prenylated
groups present. These compounds (23-28) showed very weak enzyme
inhibitory activities against -glucosidase and -mannosidase [54].
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Goutam Brahmachari
Two dihydroflavonol glycosides such as engeletin (29) and astilbin (30),

isolated from the leaves of Stelechocarpus cauliflorus (family: Annonaceae),
exhibited inhibitory activity against a recombinant human aldose reductase,
the inhibitory activity of 29 (IC50 = 1.16 M) was found to be twice that of
quercetin (positive control, IC50 = 2.48 M), and 23 times greater than that of
30 (IC50 = 26.7 M) [55].
Flavonoid glycosides (FG 1 and FG 2), isolated from Phyllanthus

fracternus (family: Euphorbiaceae), at a dose of 100 mg/kg p.o. were found
to be hypoglycaemic in alloxanised rats (20 and 25%) at 3 hrs, however, no
197
blood sugar lowering was observed in normal rats [56]. A neoflavonoid,

coutareagenin [5-hydroxy-7-methoxy-4-(3,4-dihdroxyphenyl)-2H-benzo-1pyran-2-one] isolated from the bark of Hintonia latiflora (family: Rubiaceae),
exhibited promising anti-diabetic efficacy in streptozotocin-induced Wistar
rats as well as in menopausal diabetic women [57,58].
Kaempferol-3,7-O-()-dirhamnopyranoside (kaempferitrin, 31), isolated
from the n-butanol fraction of the leaves of Bauhinia forficata (family:
Leguminosae), exhibited significant hypoglycemic effect in normal and
alloxan-induced diabetic rats on oral administration. In normal rats, reduction
in blood glucose level was noticed only with the higher dose of 31 (200 mg/kg)
at 1 h after treatment, whenever such efficacy of the test compound in diabetic
rats was evident at all doses administered (50, 100, and 200 mg/kg), and this
profile was found to be maintained throughout the period studied for both
higher doses. However, in glucose-fed hyperglycemic normal rats,
kaempferitrin could not down-regulate blood glucose levels [59]. Kaempferol3-neohesperidoside, a glycosylated flavonoid structurally very similar to
kaempferitrin, was also shown to demonstrate promising hypoglycemic effect
in both oral and intraperitoneal treatments in diabetic rats, in addition,
kaempferol-3-neohesperidoside-VO(IV) complex showed potent hypoglycemic
efficacy throughout the post-treatment period studied when compared with zero
time [60]. When complexed with vanadium, quercetin also demonstrated much
promising insulin-enhancing activity in STZ-diabetic mice with no effect on the
blood glucose level of normal mice, which is in agreement with the results for
kaempferitrin and kaempferol-3-neohesperidoside- VO(IV) complexes [60,61].
Quercetin itself was evaluated to possess anti-diabetic effect by reducing the
blood glucose level of diabetic rats in 8-10 days of treatment [62], in the same
study by Vessal and his group, the test compound exerted no effect on the
glucose tolerance curve either in normoglycemic or in STZ-diabetic rats [62].
These results support the views of Shetty et al. [63] for hypoglycemic effects of
quercetin in diabetic rats.
Three prenylated flavanones (33-35) isolated from stem barks of
Erythrina abyssinica (family: Liguminosae) exhibited inhibitory activity
against protein tyrosine phosphatase 1B (PTP1B) in dose-dependent manner
with IC50 values >60, 18.91.9 and 15.70.4 M, respectively [64], hence,
the flavanone (32) bearing a 2,2-dimethylpyran moiety on B ring is less
potent than the other two (33 & 34) in the series. The investigators, thus,
suggested that substitution of prenyl groups on flavonoids may be important
for in vitro PTP1B inhibitory activity and cyclization between a hydroxy
group and the prenyl group in B ring without prenyl or methoxy groups may
reduce the activity [64]. One more isoprenyl flavonoid (35) isolated from the
root barks of Erythrina mildbraedii were also found to exhibit inhibitory
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Goutam Brahmachari
activity against PTP1B enzyme in dose-dependent manner with IC50 values

21.21.6 M. The present investigators argued that substitution of isoprenyl
groups on ring-B might be important for PTP1B inhibitory activity in vitro, and
introduction of one more hydroxyl group to C-5 of ring-A or one of the isoprenyl
groups in ring-B might be responsible for a loss of such activity [65].
Isorhamnetin 3-O--D-glucoside (36) isolated from the ethylacetate
fraction of Salicornia herbacea (family: Chenopodiaceae) was evaluated to
possess significant inhibitory activity against rat lens aldose reductase
(RLAR) in vitro with an IC50 value of 1.4 mM, which is similar to that of
tetramethylene glutaric acid (IC50 = 1.7 mM) [66]. The flavonol glycoside
(36), when administered orally at 25 mg/kg in streptozotocin (STZ)-induced
diabetic rats, caused not only a significant inhibition of serum glucose
concentration but also sorbitol accumulation in the lenses, red blood cells
(RBC), and sciatic nerves, thereby, advocating the test compound from
S. herbacea as a leading compound for further study as a new drug for the
prevention and/or treatment of diabetes and its complications [66].
Luteolin 6-C-(6-O-trans-caffeoylglucoside) (37) isolated from
Phyllostachys nigra (family: Gramineae) showed inhibitory efficacy against
advanced glycation end products (AGEs), hence, this compound could be
offered as a leading compound for its further study towards development of
new natural products drug for diabetic complications [67]. Jang et al. [68]
reported two flavan-3-ol derivatives (38 and 39) from the roots of Actinidia
arguta (family: Actinidiaceae) that were found to exhibit inhibitory activity
in vitro on the formation of advanced glycation end products with IC50 values
of 13.5 and 17.9 g/mL, respectively.
Few more advanced glycation end products (AGEs) inhibitors such as the
dihydroflavonol glycosides (40 and 41) [55], isoflavone C-glucosides (42 and
43) [69] and the 2,3-dioxygenated flavanone erigeroflavanone (44) have also
been reported [70]. The isoflavone C-glucosides (42 and 43) isolated from
the roots of Pueraria iobata (family: Pueraria) showed more potent in vitro
inhibitory activity against AGEs formation with IC50 values 8.7 and
24.9 g/mL, respectively [69]. The present investigators [69] suggested that
the compound (42) is worthy of consideration as a therapeutic agent for
diabetic complications or related diseases. Yoo et al. isolated the 2,3dioxygenated flavanone, erigeroflavanone (44) from the flowers of Erigeron
annuus (family: Asteraceae/Compositae), and evaluated its inhibitory activity
against AGEs formation with an IC50 value 22.7 M [70].
A flavone xylopyranoside, 4',5-dihyroxy-6,7-dimethoxyflavone-3-O-D-xylopyranoside (45), isolated from the roots of Euphorbia leucophylla
(family: Euphorbiaceae) by Satyanarayana et al., was found to reduce the
199
blood glucose levels (BGLs) and increase the serum insulin levels in normal
and diabetic rats [71]. One flavone [1(R)-5,4,1-trihydroxy-6,7-(3,3dimethylchromano)flavone, 46] and one flavanone [(2S)-4-O-methyl-6methyl-8-prenylnaringenin, 47) both isolated Eysenhardtia platycarpa
(family: Leguminosae) were evaluated to possess promising anti-
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Goutam Brahmachari
hyperglycemic activity by decreasing glucose level of streptozotocin (STZ)induced diabetic rats (31 mg/kg of body weight, P < 0.05) [72].
Matsuda et al. [12] examined a variety of flavonoids for their rat lens aldose
reductase inhibitory activity to study structure-activity relationships. Among the
flavone constituents, 3,4-dihydroxyflavone (48), 3,4,7-trihydroxyflavone (49),
luteolin (50), and luteolin 7-O--D-glucopyranoside (51) were found to possess
potent inhibitory activity with IC50 values of 0.37, 0.30, 0.45 and 0.99 M, the
flavonoid glycosides, quercitrin (52), guaijaverin (53) and desmanthin-1 (54) also
showed the most potent activity against the enzyme with respective IC50 values of
0.18, 0.18 and 0.082 M [12]. The activity of desmanthin-1 (54) was equivalent
to that of a commercially available synthetic aldose reductase inhibitor, epalrestat
(IC50 = 0.072 M). From their detailed studies, Matsuda et al. suggested the
following structural requirements of flavonoids for aldose reductase inhibitory
activity - (i) the 5-hydroxyl moiety has no effect, (ii) the 3-hydroxyl and 7-Oglucosyl moieties reduce the activity, (iii) the 2-3 double bond enhances the
activity, and (iv) the flavones and flavonols having the catechol type moiety at the
B ring (the 3,4-dihydroxyl groups) exhibit stronger activities than those of
pyrogallol-type moiety (the 3,4,5-trihydroxyl groups) [12].
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6. Absorption and metabolism of flavonoids

6.1. Absorption of flavonoids
As far as reports are available, the absorption of dietary flavonoids may
be influenced by the matrix in which they are consumed, with enhanced
excretion in urine of easily recognized mammalian conjugates observed when
presented in foods with a higher fat content [73-78] - although certain reports
are there in contrast to [79-83]. However, an important factor in the
absorption efficiency of flavonoid glycosides in the intestine is the sugar
moiety, as demonstrated for quercetin glycosides, its aglycone and rutin
supplements in healthy ileostomy volunteers [84]. Flavonoid aglycones,
being hydrophobic in nature, can be transported across membranes by passive
diffusion, whereas in flavonoid glycosides the sugar moiety enhances the
hydrophilicity of the flavonoid molecules as a whole, thereby, reducing the
possibility of passive transport. Hence, it may be argued that flavonoids are
absorbed by active transport [85]. A good number of studies in human and
animals are in agreement with the fact that some dietary flavonoids such as
flavanols [86], quercetin-3-glucoside and quercetin-4-glucoside [87-89] can
be absorbed in the small intestine however, quercetin, quercetin-3galactoside, quercetin-3-rutinoside (rutin), naringenin-7-glucoside, genistein7-glucoside and cyanidine-3,5-diglucoside have been found not to be [89,90].
It has been suggested that before absorption flavonoids are cleaved by
specific enzymes either in the lumen or inside the cells of the gut. Lactasephlorizin hydrolase (LPH) is anchored in the brush-border membrane in the
small intestine and catalyzes extracellular hydrolysis of some glucosides
[91,92]. Another enzyme, located intracellularly and with broad specificity, is
the cytosolic -glucosidase (CBG). It is found in abundance in the small
intestine, liver and kidney of mammals and requires active transport of
hydrophilic glucosides into the cells [93]. Concerning LPH activity, it has
been shown that the enzyme cleaves some flavonol and isoflavone glycosides
such as quercetin-4-glucoside, quercetin-3-glucoside, quercetin-3,4glucoside, 3-methylquercetin-3-glucoside, genistein-7-glucoside, and
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daidzein-7-glucoside.
However,
quercetin-3-rhamnoglucoside
and
naringenin-7-rhamnoglucoside (naringin) are not substrates for this enzyme
[91,93]. In addition, -glucosidase activity is reported to act on flavonoid and
isoflavone glycosides according to the position and the structure of the sugar
moiety attached to the flavonoid aglycone [94]. Mechanism of absorption
have still not been completely elucidated but is believed to involve inter alia
interaction of certain glucosides with the active sugar transporter-1 (SGLT-1)
and luminal lactase-phlorizin hydrolysate (LHP), passive diffusion of the
more hydrophobic aglycones, or absorption of the glycoside and interaction
with cytosolic -glucosidase (CBG).
6.2. Metabolism of flavonoids

After being absorbed in body, flavonoids undergo three main types of
conjugations such as methylation, sulfation and glucuronidation [95-97]. The
most important enzymes involved in flavonoids metabolism are catechol-Omethyltransferase (COMT, EC 2.1.1.6), phenol sulfotransferase (P-PST,
SULT, EC 2.8.2.1) and UDP glucuronosyl transferase (UDPGT, UGT, EC
2.4.1.17). Catechol-O-methyltransferase methylates polyphenols and has the
highest activity in the liver and kidneys [98]. Phenol sulfotransferases are
cytosolic enzymes that transfer sulfate moieties to hydroxyl groups from
substrates such as iodothyronines, phenols and hydroxyarylamines mainly in
the liver [96,97,99]. UDP glucuronosyl transferase catalyzes the conjugation
of polyphenols to glucuronic acid in endoplasmic reticulum in the intestine,
liver and kidney. In humans, the liver has the greatest capacity for
glucuronidation while in rats, the highest level of glucuronyl transferase
activity was observed in the intestine [99-101]. Conjugation reactions with
glucuronic acid and/or sulfate appear to be the most common type of
metabolic pathways for the flavonoids first occurring in the gut barrier [85]
and these conjugates then reach the liver, where they are further metabolized
[81,99,102]. Otake et al. [103] showed that hepatic UDP-glucuronosyl
transferase isoforms were the main factors responsible for galangin
metabolism into two major glucuronides conjugated at the 7- and 3- positions
by using human liver microsomes. Also, Vaidyanathan and Walle [104]
demonstrated no glucuronidation of ()-epicatechin by human liver and small
intestinal microsomes. However, in rats, ()-epicatechin was efficiently
metabolized by liver microsomes with formation of two glucuronides. In the
same study, the authors concluded that sulfation also occurred in both the
liver and intestine in human and rats.
Three ()-epicatechin metabolites such as ()-epicatechin-3-Oglucuronide, 4-O-methyl-()-epicatechin-3-O-glucuronide, and 4-Omethyl()-epicatechin-5 or 7-O-glucuronide have been isolated from human
203
urine [105], whereas the exact fate of (+)-catechin is not known although
there is evidence for the formation of (+)-catechin sulfates, sulfoglucuronides, and 4-methylated conjugates in plasma and urine [76,106]. In
contrast, ()-epicatechin gallate and ()-epigallocatechin gallate appear to be
excreted in bile [79,86,107,108]. The ()-epicatechin gallate is extensively
methylated by human liver catechol O-methyl transferase at the 4-position
and to a lesser extent at the 3-position [109,110], while ()-epigallocatechin
gallate is metabolized first to the 4-methyl ether and then to the 4,4dimethyl ether [110].
Flavonoid glycosides that are not absorbed in the small intestine along
with the conjugated metabolites that are excreted in bile can be metabolized
by microflora when they reach the colon. Glycoside flavonoid-hydrolyzing
enzymes have been identified in fecal flora cultures. Bokkenheuser et al.
[111] recovered three enzyme-producing strains that, using -glucosidases,
-rhamnosidases, and/or -galactosidases, were capable of converting rutin
to quercetin. Also, it was shown that at least some of the bacterial
glycosidases are able to cleave glycosidic bonds and flavonoid-saccharide
bonds in the gut [91]. Genistein-7-glucoside and daidzein-7-glucoside have
not been found in human plasma [112] but the aglycones have been observed
[113]. Human metabolism of isoflavone glycosides produces genistein and
daidzein 7-glucuronides/7-sulfates and 4,7-diconjugates (including
diglucuronides and mixed conjugates), with monoglucuronides predominant
[114,115]. The profile of metabolites has been demonstrated in studies with
quercetin, rutin and naringin. The flavonoid metabolism produces aromatic
acids such as phenylvaleric, phenylpropionic, phenylacetic and benzoic acids
with easy absorption through the colonic barrier [116-118]. Flavonol
glycosides and quercetin aglycone have not been convincingly demonstrated
in plasma [119-121], although kaempferol aglycone has been detected [122].
The main kaempferol metabolite in human plasma is the 3-glucuronide [122].
The three major metabolites of quercetin are: quercetin-3-glucuronide,
quercetin-3-sulfate, and isorhamnetin-3-glucuronide. Apigenin glucuronides
have been detected in urine after volunteers consumed parsley [123], luteolin
aglycone administered to volunteers has been detected in plasma as a monoglucuronide accompanied by a trace of unconjugated luteonin [124,125].
Chrysin is transformed primarily to the 7-glucuronide with much smaller
yields of the 7-sulfate [126].
Metabolites of flavonoids in general (and also microflora metabolites),
aglycones, glycosides and conjugated metabolites which are not absorbed,
may follow two pathways of excretion: via the biliary or the urinary route.
Large conjugated metabolites are more likely to be eliminated in the bile
whereas small conjugates such as monosulfates are preferentially excreted in
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Goutam Brahmachari
urine [100]. When excreted in bile, the flavonoids are passed to the
duodenum and metabolized by intestinal bacteria, which results in the
production of fragmentation products and/or the hydrolysis of glucurono- or
sulfoconjugates [127]. The resulting metabolites which are released may be
reabsorbed and enter an enterohepatic cycle or being excreted in feces [128,129].
For each flavonoid, the beneficial effect will be dependent upon their absorption
and availability in the body. Thus, these factors should be considered in any
interpretation of the potential health effects of flavonoids.
7. Mode of action of flavonoids

Very recently, Cazarolli et al. [130] reviewed on the mode of action of
flavonoids including cellular and molecular mechanism. In their review, the
authors thoroughly discussed about the various effects of the drug candidates
in regulating diabetic syndromes. It has been demonstrated that flavonoid
compounds act against diabetes mellitus either through their capacity to avoid
glucose absorption or to improve glucose tolerance. In vitro studies have
shown that a soybean extract containing the isoflavones genistein and
daidzein inhibits glucose absorption into the intestinal brush border
membrane vesicles of rabbits [131]. Naringenin was also found to reduce
glucose uptake in the intestinal brush border membrane vesicles of diabetic
rats to a level similar to that of normal rats [132]. The ()-epicatechin gallate,
myricetin, quercetin, apigenin, ()-epigallocatechin gallate, and ()epigallocatechin demonstrated a marked reduction in glucose absorption,
when compared with the control, by competitive inhibition of sodiumdependent glucose transporter-1 [133]. The non-glycosylated flavonoids were
shown to reduce glucose absorption under sodium-dependent conditions
in vivo and in vitro in animal tissues [134,135]. Besides reducing glucose
absorption, another possible mechanism followed by flavonoid compounds to
control blood glucose levels is the inhibition of -glucosidase activity in the
intestine. Such inhibitory effects against -glucosidase activity were
observed when luteolin, kaempferol, chrysin and galangin were used both
in vitro and in vivo to study the potential role in the absorption and
metabolism of carbohydrates [136]. Kim et al. [137] also demonstrated the
-glucosidase inhibitory activity of flavonoids in a study, where it was shown
that luteolin, amentoflavone, luteolin 7-O-glucoside and daidzein are the
strongest inhibitors of the compounds tested.
It has also been demonstrated that flavonoids can act per se as insulin
secretagogues or insulin mimetics, probably by influencing the pleiotropic
mechanisms, to attenuate the diabetic complications, besides, the drug
candidates have been found to stimulate glucose uptake in peripheral tissues,
205
and regulate the activity and/or expression of the rate-limiting enzymes

involved in carbohydrate metabolism pathway. In an experimental study by
Liu et al. [138], genistein was found to act directly on pancreatic -cells,
leading to activation of the cAMP/PKA signaling cascade to exert an
insulinotropic effect.
Interestingly, it has found that epigallocatechin 3-gallate mimics the
effects of insulin on the gene expression reduction of phosphoenolpyruvate
carboxykinase and G-6-Pase in the mouse liver [139], like insulin, the drug
candidate enhances tyrosine phosphorylation of the insulin receptor and
insulin receptor substrate-1 (IRS-1), mitogen-activated protein kinase,
p70s6k, and PI3K activity, and reduces phosphoenolpyruvate carboxykinase
gene expression mediated by PI3K [140]. Furthermore, epigallocatechin 3gallate upregulates glucokinase mRNA expression in the liver of db/db mice
[141]. In another study, oral administration of rutin to diabetic rats resulted in
a decrease in plasma glucose and increase in insulin levels, and restored the
glycogen content and hexokinase activity. The activity of enzymes such as
G-6-Pase and fructose-1,6-bisphosphatase significantly decreased in the
liver and muscles of rutin-treated diabetic rats [142]. Kaempferol-3neohesperidoside has been shown to have the efficacy for prompt stimulating
of glycogen synthesis in rat soleus muscle by approximately 2.38-fold, it has
also been demonstrated that the phosphatidylinositol-3-kinase (PI3K) glycogen synthase kinase-3 (GSK-3) pathway and mitogen-activated protein
kinase (MEK) - protein phosphatase-1 (PP-1) pathway are involved in the
stimulatory kaempferol-3-neohesperidoside effect on the glycogen synthesis
[143]. Very recently, Cazarolli et al. [144,145] have reported on the mechanism
of action of the anti-diabetic effects of apigenin-6-C--L-fucopyranoside and
apigenin-6-C-(2-O--L-rhamnopyranosyl)--L-fucopyranoside the former
drug candidate was evaluated to stimulate insulin secretion and potentiated
glucose-induced insulin secretion in hyperglycemic rats, in addition, this
flavonoid stimulated glycogen synthesis in rat soleus muscle through
mechanisms well known to insulin signal transduction, thereby, establishing the
dual effects of apigenin-6-C--L-fucopyranoside as an anti-hyperglycemic
(insulin secretion) as well as an insulino-mimetic (glycogen synthesis) agent
[144]. In another study, the same group of investigators has characterized
apigenin-6-C-(2-O--L-rhamnopyranosyl)--L-fucopyranoside as both an
insulin secretagoge and an insulin-mimetic agent [145].
8. Conclusions
Diabetes mellitus has already emerged as an alarming disease worldwide affecting the public health much. Though presently available therapies
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Goutam Brahmachari
against the disease reduce the sufferings to some extent, still it remains
inadequate and at the same time is costly, and also associated with a lot of
side effects. Hence, there is an urgent need for search of more efficacious
drugs with no or minimum side effects. There has been a growing interest in
anti-diabetic agents from natural products, particularly those derived from
plants. Flavonoids are naturally occurring phenolic compounds with a broad
range of biological activities and the beneficial effects of flavonoids have
been studied in relation to diabetes mellitus, either through the inhibition of
intestinal -glucosidase enzyme or through their capacity to avoid glucose
absorption and/or to improve glucose tolerance. A good number of
bio-flavonoids reported over the past 15-20 years discussed in this review
clearly demonstrate that these exogenous substances represent an
unparalleled source of molecular diversity in relation to the drug discovery
process in the treatment of Type-2 diabetes. Although there has been
considerable scientific progress over the past few years in unraveling of the
effect and mechanism of action of flavonoids, we still need to define the
missing steps in the flavonoid-signaling network and elucidate the mechanism
of cross-talk based on the complex mechanism of insulin action, in order to
provide new insights into the potential role of flavonoids in diabetes treatment.
Further study is required concerning safety (assessment of toxic effect) and
human trial to develop potential anti-diabetic remedies of choice.
Acknowledgement
The author greatly appreciates financial support under Major Research
Grant from the University Grants Commission (UGC), New Delhi, India
[Project No. F.34-357/2008(SR) dt 02.01.2009].
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Research Signpost
37/661 (2), Fort P.O.
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ISBN: 978-81-308-0448-4
7. Marine natural alkaloids as

anticancer agents+
Deepak Kumar and Diwan S. Rawat
Department of Chemistry, University of Delhi, New Delhi-110007, India
Abstract. Alkaloids are naturally occurring nitrogen containing

biologically active heterocyclic compounds. Over the last few
years, a large number of biologically important alkaloids with
antiviral, antibacterial, anti-inflammatory, antimalarial, antioxidant
and anticancer activities have been isolated from marine source.
Present article summarizes the isolation and anticancer activity
evaluation of natural marine alkaloids and their synthetic analogues
that includes pyridoacridine, indole, pyrrole, pyridine,
Isoquinoline, guanidine and steroidal alkaloids.
Introduction
Since ancient times nature has been a source of medicines to cure many
deadly diseases. Majority of drugs in use today are either natural products (NP),
their derivatives (ND), natural products mimics (NPD) or semisynthetic
derivatives (SSD) [1-4]. In natural sources, plants, animals and microorganisms
have been the main source of biologically important molecules. Ocean has been
considered as the main source of medicines and during the past two decades
thousands of compounds and their metabolites with several different type of
biological activity such as antimicrobial, anti-inflammatory, antimalarial,
antioxidant, anti HIV and anticancer activity have been isolated from marine
microorganisms [5-12]. But till date only few anticancer drugs such as citarabine,
+
Dedicated to Dr. DS Bhakuni and Prof. Deepak Pental
Correspondence/Reprint request: Prof. Diwan S. Rawat, Department of Chemistry, University of Delhi

New Delhi-110007, India. E-mail: dsrawat@chemistry.du.ac.in
214
Deepak Kumar & Diwan S. Rawat
vidarabine etc have been commercially developed from marine compounds while
several others are currently in different stages of clinical trials [13]. Over 18000
compounds have been isolated from marine source and approximately 150
compounds are cytotoxic against the different tumor cells [14,15]. Some of the
prominent anticancer compounds which are in different stages of clinical trials
include aplidine, ecteinascidin-734 (Yondelis), bryostatin-1, squalamine, dolastatin10, ILX651, and KRN7000 (-galactosylceramide) [16].
The present article summarises the recent development in the area of marine
alkaloids that includes pyridoacridine, indole, pyrrole, pyridine, isoquinoline,
guanidine and steroidal alkaloids.
1. Pyridoacridine alkaloids
Pyridoacridines are highly coloured marine natural products having
polycyclic planar heteroaromatic 11H-pyrido[4,3,2,mn]acridine system (1) [17].
They are probably the largest class among marine alkaloids and are almost
universally isolated from sponges, ascidians as well as from a mollusc and a
coelenterate [18]. Pyridoacridine alkaloids show significant biological activity
primarily cytotoxicity and certain specific biological properties viz. fungicidal
and bactericidal properties, inhibition of topoisomerase II, anti HIV, intercalation
of DNA property, Ca+2 releasing activity, production of reactive oxygen species
[19-22]. These activities depends upon the substitution pattern of the basic
structure of pyridoacridine, therefore many synthetic analogues have also been
synthesized keeping the basic skeleton of pyridoacridine in mind. The synthesis
of these analogues and their biological activity evaluation revealed that in most of
the cases cytotoxicity of the analogues has improved compared to the parent
molecule [23, 24]. During the last few years, numerous additional compounds of
this family were isolated; most of them are polycyclic with different substituents
such as shermilamine, kuanoniamine, neoamphimedine, arnoamines and
styelsamines.
It has been observed that almost all the pyridoacridines shows promising
cytotoxicity against different type of tumors. Therefore a great interest was
developed to modify the pyridoacridine moiety for developing a new generation
of therapeutic agents. The first review article on marine pyridoacridines alkaloids
was published by Molinski in 1993 [25] followed by Ding et al. in 1999 [26]. The
cytotoxicity of the compounds of this family is a manifestation of their DNA
binding properties, topoisomerase II inhibition and the production of reactive
oxygen species.
Pyridoacridines vary structurally by attachment of different side chains or
fusion of different rings to ring C of the basic structure (1) and less often to the
acridine nitrogen. Halogen substitution in pyridoacridines is quite rare; even if it
is present, then it is always bromine at C2 in ring A. Oxidation states of the rings
are variable and in some cases ring D is partially saturated. Additional rings are
215
often attached to ring C. Pyridoacridines can be divided into tetracyclic,

pentacyclic, hexacyclic, heptacyclic and octacyclic alkaloids.
2
1
3
A
11a
11 HN
4
B
10a
4a
5
10
C
7a N
7
N
O
1.1. Tetracyclic alkaloids

In 1988, Kobayashi et al. reported the isolation of three tetracyclic alkaloids,
cystodytins A-C (3-5) from tunicate Cystodytes dellechiajei collected from
Okinawa [27]. Then in 1991, the same group reported six other novel tetracyclic
alkaloids of cystodytin family, cystodytins D-I (6-11) along with cystodytins A
(3) and B (4) [28]. Thus cystodytins A-C (3-5) are the first pyridoacridine
alkaloids isolated from a marine tunicate and therefore the first tetracyclic
member of this class. The common heterocyclic nucleus of cystodytins A-C (3-5)
is an iminoquinone substituted at C10 with a 2-amidoethyl side chain. The N-acyl
groups are derived from ,-dimethylacrylic, tiglic and 3-hydroxy-3methylbutanoic acids, respectively.
Cystodytins D-I (6-11) are chiral, levorotatory compounds. Cystodytins F-I
(7-10) are substituted with an O-methyl ether or O-9-octadecenoate ester. The
isomeric pairs of cystodytin ,-dimethylacrylate and tiglate amides could not be
separated and were characterized as 7:2 mixtures. Hydration of cystodytin A (3)
in presence of 6% aq. HC1 at 100 C gives cystodytin C (5). When treated
with diazomethane, it afforded monomethyl ether (12) with 23% yield. This
transformation is unusual as it constitutes a formal reductive methylation.
Cystodytin A (3) is readily reduced in the ionization stage of a mass spectrometer
as observed for quinones. Cystodytin A (3), when hydrogenated over Adams
catalyst in acetic acid yielded (13) by reduction of the side chain and disubstituted
benzene ring, but the iminoquinone part remains intact. Compounds 3, 4 and 5
showed potent cytotoxicity against L-1210 with IC50 values of 0.22, 0.22 and
0.24 g/mL, respectively. Cystodytins D-I (6-11) were also found to be cytotoxic
against murine lymphoma L-1210 cells with IC50 values of 1.1 (6 and 7), 0.068 (8
and 9) and 0.080 (10 and 11) g/mL and values of 1.4 (6 and 7), 0.078 (8 and 9)
and 0.092 (10 and 11) g/mL against human epidermoid carcinoma KB cells
in vitro.
216

O
R2
H
N
R1
H
N
X=
HN
O
O
N
N
Y=
O
3, R2 = X, R1 = H
O
4, R2 = Y, R1 = H
5, R2 = Z, R1 = H
6, R2 = X, R1 = OH
Z=
7, R2 = Y, R1 = OH
8, R2 = X, R1 = OMe
9, R2 = Y, R1 = OMe
10, R2 = X, R1 = OCO(CH2)7CH=CH(CH2)7CH3
11, R2 = Y, R1 = OCO(CH2)7CH=CH(CH2)7CH3
12
H
N
OH
OMe
O
N
13
Cystodytin J (14) was isolated from a ascidian Cystodytes sp. [29].

Cystodytin J (14) showed cytotoxic activity against HCT and xrs-6 with IC50
values of 1.6 and 135.6 M, respectively. It also inhibited the topoisomerase
(TOPO) II-mediated decatenation with IC90 value of 8.4 M. Recently, Appleton
et al. reported isolation of cystodytins K (15), a new member of cystodytins, from
the extract of an ascidian Lissoclinum notti collected near Leigh Harbour, Northland,
New Zealand [30]. Structure of compound was determined by spectroscopic
techniques, including 2D 1H-15N NMR experiments and was found to be 12-methoxy
derivative of cystodytin J (14). Cystodytins K (15) exhibited cytotoxic activity against
P-388 murine leukaemia cell line with IC50 value of 1.3 M.
Two bright crimson pigments, Varamine A (16) and B (17) were isolated
from the Fijian ascidian Lissoclinum vareau [31]. Varamines A (16) and B (17)
have parent tetracyclic aromatic ring system at the same oxidation level as the
methylation product of cystodytin A (12). Varamines also contain a methyl
thioether substituent at C9. Varamine A (16) was readily oxidised by aq. cerric
ammonium nitrate to imonoquinone (18) with 90% yield. Varamine A (16) and B
(17) exhibited cytotoxicity towards L-1210 murine leukemia cells with IC50
values of 0.03 and 0.05 g/mL, respectively.
In 1989, Ireland et al. isolated a new tetracyclic alkaloid, diplamine (19)
from the tunicate Diplosoma sp. collected from the Fiji Island [32]. The structure
was established by interpretation of spectral data and chemical analysis.
Diplamine (19) was found to be cytotoxic towards L-1210 murine leukemia cells
with IC50 value of 0.02 g/mL. Recently, two novel alkaloids, isodiplamine (20)
and lissoclinidine (21) along with known diplamine (19) were isolated from
an ascidian Lissoclinum notti collected near Leigh Harbour, Northland,
New Zealand.
All the compounds (19-21) were tested for their cytotoxicity against murine
leukaemia (P-388), human colon tumour (HCT-116) and non-malignant African
Green Monkey kidney (BSC-1) cell lines. Diplamine (19) was found to be the
217
most active compound among the three and it was observed that movement of the
thiomethyl group from C-9 (diplamine) to C-5 (isodiplamine) decreases
cytotoxicity against all the cell lines and the same pattern also observed, when the
thiomethyl group is cyclised into a benzoxathiole ring (lissoclinidine). These
results were also found to be consistent with the proposed mechanism of
cytotoxicity of diplamine, which includes DNA intercalation, inhibition of
topoisomerase II and other DNA processing enzymes and bioreductive activation.
Lissoclinidine (21) was also evaluated against the NCI 60 cell line panel and
demonstrated moderate activity and selectivity with panel average values of
GI50 = 1.0 mM, TGI = 6.9 mM and LC50 = 29 mM.
H
N
H
N
O
S
N
O
14
15
HN
16, R = CH2CH3
17, R = CH3
O
HN
R1
R2
R3
S
S
O
N
H CF3COO
21
20, R1 = H, R2 = SCH3, R3 = H
19, R1 = SCH3, R2 = H, R3 = H
18
N
OMe
O
H
N
HN
O
MeO
H
N
In 1998, Copp et al. reported the isolation of four new tetracyclic

pyridoacridine alkaloids, styelsamines A-D (22-25) from an extract of the
ascidian Eusynstyela latericius [33]. The structures of all the compounds
were determined on the basis of 1D and 2D NMR spectroscopy. Styelsamines
A-D (22-25) exhibited mild cytotoxicity toward the human colon tumor
cell line (HCT-116) with IC50 values of 33, 89, 2.6 and 1.6 M, respectively.
OH
H
HO
N
H
CF3COO
22, R =
NH3
23, R =
NHCOMe
24, R =
25, R =
CHO
NH3
CF3COO
218
1.2. Pentacyclic alkaloids

Amphimedine (26) was the first example of pyridoacridine alkaloids to be
fully characterized [34]. In 1983, Schmitz et al. isolated amphimedine as a
sparingly soluble yellow pigment from Amphimedon sp. The structure of
amphimedine was established on the basis of spectroscopic data analysis. High
resolution mass spectral analysis established the molecular formula C19H11N3O2
(m/e = 313.08547, + 0.35 mass error) for amphimedine. In mass spectrum very
few fragments were observed corresponding to loss of CH, CO, CHO and HCN.
The UV spectra of compound (26) in absolute ethanol showed absorption at max
210 nm (19690), 233 nm (39393), 281 nm (9099), 341 nm (6060). Significant
changes were observed upon addition of NaBH4 [max 235 nm (12879), 280 nm
(9090)], indicating the presence of ,-unsaturated ketone, which was further
supported by the strong absorbtion at 1690 cm-1. Further presence of amide
functionality was confirmed by IR and 13C NMR. No OH or NH absorptions were
observed in the IR and due to low solubility of compound (26) in common
organic solvents, NMR spectral data were obtained in trifluoroacetic acid-d4
and CDCl3 (2:1). The 2D NMR techniques (1H-1H correlation and 13C-13C
INADEQUATE NMR) were also used to confirm the structure of amphimedine (26).
In 1999, Ireland et al. reported the isolation of a new pyridoacridine,
neoamphimedine (27) along with amphimedine (26) from Xestospongia sp.
from the Philippines and Xestospongia cf. carbonaria from Micronesia [35].
He deduced the molecular formula for neoamphimidine as C19H11N3O2 by
high-resolution fast atom bombardment (FAB) mass spectral analysis. Both
amphimedine and neoamphimedine have the same molecular formula hence they
are isomers. Recently, deoxyamphimedine (28) along with two known compounds
(26 and 27) was isolated from two tropical Xestospongia sponges [36]. Amphimedine,
neoamphimedine and deoxyamphimedine have the same skeleton, but they differ in
biological activities and this is probably due to the the differences in their structures.
Literature servey reaveled that amphimidine relatively inactive compared to
neoamphimedine and deoxyamphimedine. Neoamphimedine inhibits topoisomerase
II while amphimedine is relatively nontoxic at the same dose level [37] and
deoxyamphimedine damages DNA independent of topoisomerase enzymes through
the generation of reactive oxygen species [38].
O
N
N
O
26
N
O
27
28
219
Schmitz et al. reported the isolation of three new alkaloids 29-31 from two
ascidians. The meridine (29) and a relatively stable tautomer of meridine i.e. 30
were isolated from Amphicarpa meridiana collected at Stenhouse bay, South
Australia [39]. The structure of meridine (29) was determined by X-ray analysis
while that of 31 was established by spectral analysis. The third alkaloid, 11hydroxyascididemin (31) was isolated from a Leptoclinides sp. from Truk
Lagoon. All three alkaloids (29-31) were found to be cytotoxic. Recently,
Menendez et al. synthesized a regioisomer of meridine named as 9
Hydroxybenzo[b]pyrido[4,3,2-de](1,10)-phenantrolin-8-one (32) from 5,8dimethoxy-6-nitro-4(1H)-quinolinone in eight steps with 23% overall yield [40].
Compound (32) was tested for cytotoxicity against different tumor cell lines and
exhibited mild to strong cytotoxic activity against P-388, A-549, HT-29 and
MEL-28 with IC50 values of 4.18, 0.03, 0.40 and 0.17, whereas IC50 values for
meridine were 0.08, 0.08, 0.84 and 0.08, respectively. Compound 32 and the
natural meridine (29) were also tested in vitro for Topoisomerase II inhibitory
activity. Meridine showed mild activity (IC50 = 3 mM), whereas compound 32
was found to be inactive even at the highest concentration (33 mM).
In 1988, a novel pentacyclic alkaloid, ascididemin (33) was isolated from
brown colored tunicate Didemnum sp. collected at Kerama Islands, Okinawa [41].
The structure of compound was elucidated on the basis of spectroscopic data.
Ascididemin (33) was found to be cytotoxic against L-1210 murine leukemia cells
in vitro with IC50 value of 0.39 g/mL. Delfourne et al. synthesized an isomer of
ascididemin, named as 9H-quino[4,3,2-de][1,7]phenanthroline-9-one (34) starting
from 1,4-dimethoxyacridine with an overall yield of 12% along with other derivatives
(35-39) of compound 34 [42]. These compounds were tested in vitro at six different
concentrations on 12 different human cancer cell lines such as glioblastomas, breast,
colon, lung, prostate and bladder cancers. Almost all the compounds showed
significant cytotoxic activity and compound 34 was found as much potent or slightly
less potent as the natural ascididemin (33). Ascididemin (33) and the isomer (34)
exhibited cytotoxicity against U-87MG (0.07, 0.8 M), U-373MG (0.5, 0.8 M), SW1088 (0.6, 3 M), T-47D (0.6, 0.7 M), MCF-7 (0.07, 0.9 M), Lovo (0.9, 0.7 M),
HCT-15 (0.06, 0.4 M), A-549 (0.2, 7 M), A-427 (0.06, 0.08 M), PC-3 (0.008,
0.09 M), T-24 (0.8, 0.1 M) and J-82 (0.3, 1 M), respectively.
A new pentacyclic alkaloid, cystodamine (40) was isolated from a mediterranean
ascidian Cystodytes dellechiajei collected near the bay of Gabes, at Skhira, Tunisia
[43]. The structure was determined by extensive 2D NMR data analysis and was
found to contain a phenanthroline unit fused with 7 aminopyridine moiety.
Cystodamine (40) showed cytotoxic activity against CEM human leukemic
lymphoblasts with IC50 value of 1.0 g/mL. Later, Delfourne et al. revised the
structure of cystodamine (40) to 11-hydroxyascididemin (31) by comparison of the
spectroscopic data with those of synthetic cystodamine, meridine and 11hydroxyascididemin [44]. 11-Hydroxyascididemin had been previously isolated by
Schmitz et al. from the other marine source Amphicarpa meridian.
220
In 1988, Scheuer et al. reported the isolation of a new pentacyclic alkaloid,

shermilamine A (41) from purple colonical tunicate Trididemnum sp. [45]. After
one year, shermilamine B (42) was reported by two groups simultaneously
Scheuer [46] and Kashman [47]. In 1994, McDonald et al. isolated shermilamine
C (43) from a Fijian ascidian Cystodytes sp. [48]. Shermilamine A (41) contains a
pentacyclic pyridoacridine thiazinone system while shermilamine B (42) is a
debromo analogues of shermilamine A (41).
Two novel shermilamine alkaloids, shermilamine D (44) and E (45) were
isolated from the Indian Ocean tunicate Cystodytes violatinctus collected at the
Mayotte Lagoon, Comoros Islands, northwest of Madagascar [49]. Shermilamine
D (44) exhibited cytotoxicity against P-388, A-549, HT-29 and MEL-28 cancer
cell lines with IC50 values of 0.53, 0.27, 2.66 and 0.53 M, respectively [50].
A new member of shermilamines, cycloshermilamine D (46) was isolated
from the same marine tunicate Cystodytes violatinctus [51]. The structure of
cycloshermilamine D (46) was established mainly on the basis of NMR
spectroscopic data and was found to be closely related to shermilamine D (44)
having hexacyclic structure.
Kuanoniamines A-D (47-50) were isolated along with the known
shermilamine B (42) from a tunicate and its prosobranch mollusc predator
Chelynotus simperi [52]. The structures were established by extensive NMR
analysis and correlations spectroscopy. Kuanoniamines C (49) and D (50) were
also isolated from another tunicate of the genus cystodytes collected in Pohnpei
[53]. Kuanoniamines B (48) and D (50) are homologues of kuanoniamine C (49)
having isovaleramide and acetamide side chains, respectively. Kuanoniamine A
(47) is structurally different from the other three alkaloids and lacks the
2-amidoethyl side chain and contains an iminoquinone moiety. In 1994,
221
McDonald et al. isolated dehydrokuanoniamines B (51) from a Fijian ascidian

Cystodytes sp. [54]. More recently, the N-deacyl derivative (52) was isolated
from the sponge Oceanapia sp. collected at Truk Lagoon, Micronesia along with
its two parent molecule (49) and (50) [55]. Kuanoniamines A-D (47-50) showed
weak cytotoxicity. Kuanoniamine A (47) was found to be the most active
compound of the group and inhibits the proliferation of KB (human pharyngeal
cancer) cell lines in vitro with IC50 value of 1 g/mL. Dehydrokuanoniamine B
(51) and kuanoniamines D (50) were found to have comparative potentials
in vitro against HCT (IC50 = 8.3 and 7.8 M) and xrs-6 cells (IC50 = 80 and 88.9
M). The N-deacyl derivative (52), kuanoniamine C (49) and D (50) were tested
in vitro against two human cancer cell lines, HeLa cells and MONO MAC-6
cells. Kuanoniamine C (49), D (50) and N-deacyl derivative (52) exhibited IC50
values of 5.1, 1.4 and 1.2 g/mL (HeLa) and values of 1.2, 0.8 and 2.0 g/mL
(MONO MAC-6).
H
N
N
O
N
H
R1
H
N
N
H
O
R2
41, R1 = Br, R2 = NHCOMe

42, R1 = H, R2 = NHCOMe
43, R1 = H, R2 = NHCOCH=C(Me)2
44, R1 = H, R2 = NMe2
45, R1 = H, R2 = N(O)Me2
NMe2
R
46
47
48, R = NHCOCH2CH(CH3)2
49, R = NHCOCH2CH3
50, R = NHCOCH3
51, R = NHCOCH=C(CH3)2
52, R = NH3
In 1988, Gunawardanda et al. isolated dercitin (53) from the deep water marine
sponge Dercitus sp. collected from Bahamas [56]. The structure of dercitin was
assigned on the basis of spectroscopic data. This structure (53) was subsequently
revised to structure (54) by the interpretation of the magnitude of long range protoncarbon coupling constants. Dercitin (54) exhibited in vitro antitumor activity against
P-388 (IC50 = 0.05 g/mL) and human tumor cells (HCT 8, A-549, T47D) with IC50
value of 1.0 g/mL. Dercitin (54) also showed in vivo activity against P-388 (T/C
170%, 5 mg/kg). One year later, the same group isolated three new pentacyclic
pyridoacridine alkaloids, nordercitin (55), dercitamine (56) and dercitamide (57) from
the extract of a red coloured sponge Stelletta sp. collected in Bahamas [57]. Later
dercitamide (57) was found to be identical to kuanoniamine C (49). Compounds (5557) inhibited the proliferation of P-388 murine leukemia cells in vitro with IC50 values
of 4.79, 26.7 and 12.0 M, respectively.
Two new pyridoacridine alkaloids, arnoamines A (58) and B (59) were
isolated from the ascidian Cystodytes sp. collected in the vicinity of Arno Atoll,
Republic of Marshall Islands [58]. They were supposed to be the first members of
222
pentacyclic pyridoacridine alkaloids having a pyrrole ring fused with the

pyridoacridine ring system. The structures of 58 and 59 were established on the
basis of spectroscopic data, particularly those obtained from HMBC and NOE
NMR experiments. The arnoamines A (58) and B (59) displayed a much
unexpected chemical reactivity. The pyrrole ring hydrogens labelled as Ha and
Hb showed duterium exchange, when NMR were recorded in CDCl3/TFA-d4.
Arnoamine A (58) exhibited cytotoxicity against the MCF-7, A-549 and HT-29
cell lines with GI50 values of 0.3, 2.0 and 4.0 g/mL, respectively, whereas
Arnoamine B (59) showed GI50 values of 5.0, 2.0 and 3.0 g/mL against the
MCF-7, A-549 and HT-29 cell lines, respectively.
The methanol extract of the ascidian Cystodytes dellechiaijei, collected in
Brazil yielded two novel alkaloids, sebastianine A (60) and B (61) [59]. The
structures of both the compounds were established by analysis of spectroscopic
data. Sebastianine A (60) was found comprising of a pyridoacridine system fused
with a pyrrole unit and sebastianine B (61) is having a pyridoacridine system
fused with a pyrrolidine system condensed with R-hydroxyisovaleric acid.
Sebastianine A (60) and B (61) showed cytotoxic activity against a panel of
HCT-116 colon carcinoma cells.
N
Ha
Hb
Y
N
H
N
R
NMe2
OR
55, R = N(CH3)2
56, R = NHCH3
57, R = NHCOCH2CH3
53, X = S, Y = N
54, X = N, Y = S
58, R = H
59, R = Me
O
N
H
N
OH
N
H
NH
H
N
CF3
60
61
62, R = H
63, R = OH
Recently, Davis et al. isolated two new pyridoacridine alkaloids, ecionines

A (62) and B (63) from Australian sponge Ecionemia geodides [60]. Both the
compounds were found to contain an imine moiety, which is very rarely found
in pyridoacridine class of compounds. Both the compounds were tested
against a panel of human bladder cancer cell lines (TSU-Pr1, TSU-Pr1-B1 and
TSU-Pr1-B2) and the superficial bladder cancer cell line 5637. Compound
(63) showed moderate cytotoxicity against all the cell lines, with IC50 values
223
of 6.48 mM (TSU-Pr1), 6.49 mM (TSU-Pr1-B1), 3.55 mM (TSU-Pr1-B2)

and 3.66 mM (5637), whereas Compound (64) showed cytotoxic effect on 5637
and TSU-Pr1-B2 cells at 10 mM, with cell growth inhibitions of 54% and
51% cells, respectively, but did not have any effect on TSU-Pr1-B1 cells
at 10 mM.
1.3. Hexacyclic alkaloids

The extracts of a deep violet sponge Dercitus sp. collected in the Bahamas
yielded a hexacyclic alkaloid cyclodercitin (64). The sixth ring in cyclodercitin
(64) is formally derived by cyclization of the 2-aminoethyl side chain to the
acridine nitrogen, while the pyridine ring is substituted with an N-methyl group.
Cyclodercitin (64) inhibited the proliferation of P-388 murine leukemia cells
in vitro with IC50 value of 1.9 M.
Recently, stellettamine (65) was isolated from a deep water marine sponge
Stelleta sp. [61]. The molecular formula, C20H14N4S was determined by high
resolution FAB mass spectroscopy. The structure of the compound was
established on the basis of 1H-13C correlation spectroscopy except the orientation
of thiazole ring. Therefore complete structure of stellettamine (65) was
determined by a single-crystal X-ray diffraction experiment.
1.4. Heptacyclic alkaloids

Eilatin (66) is the only known heptacyclic pyridoacridine alkaloid of the
marine origin [62]. Molecular formula of eilatin (66) was determined as C24H12N4
by high-resolution EIMS. 1H NMR spectrum showed only six aromatic protons
that could agree with the common six protons of the benzodiazaphenanthroline
system. The 13C NMR spectrum exhibited only 12 carbon lines (6 for
monoprotonated carbons and 6 nonprotonated carbons). This suggests a
symmetrical dimeric structure for eilatin (66). Various 2D NMR experiments
such as 1H-13C correlations and a HETCOSY experiment were failed to deduce
the structure and finally it was determined by a single-crystal X-ray analysis.
Eilatin (66) was found to exhibit cytotoxic activity against HCT cell line with
IC50 value of 5.3 M.
N
N
NMe2
64
65
66
224
1.5. Octacyclic alkaloids

In 1991, Faulkner et al. isolated two novel optically active octacyclic
alkaloids, eudistones A (67) and B (68) from the Seychelles tunicate Eudistoma
sp. [63]. Eudistone A (67) was obtained as an amorphous yellow powder. The
molecular formula C27H19N5O for eudistone A (67) was determined by high
resolution mass spectroscopy, which implies 21 degrees of unsaturation. The
13
C NMR signal at 191.8 ppm and an IR band at 1660 cm-1 indicated the presence
of an unsaturated ketone and the broad bands at 3360 and 3220 cm-1 attributed for
primary or secondary amines. The complete structure of the compounds was
determined on the basis of other correlations NMR techniques such as COSY,
NOE, HMBC and HMQC. Eudistone B (68) was obtained as a white amorphous
powder. The molecular formula C27H17N5O for eudistone B (68) was determined
and has one more degree of unsaturation than that present in eudistone A (67).
Therefore eudistone B (68) is a dehydrogenation product which was also
supported by air oxidation of eudistone A (67) to eudistone B (68). When air is
bubbled through a solution of eudistone A (67) in DMSO at 60oC for 48 hrs, the
dihydropyridine ring of eudistone A (67) is aromatized to yield eudistone B (68).
N
OCH2CH3
O
HN
HN
N
N
HN
N
N
NH
67
68
N
H
N
N
69
Recently, Demeunynck et al. synthesized an octacyclic analogue (69) of

eilatin [64]. The compound (69) was tested against two cancer cell lines, HT-29
(human colon adenocarcinoma) and A-431 (human epithelial carcinoma).
Unfortunately due to its low solubility in water, the compound could only be
tested at low concentration (5 M) and did not show any activity against HT-29
and 85% survival on A-431 cell lines.
2. Indole alkaloids
Indole-containing alkaloids have frequently been isolated from diverse
marine invertebrates including bryozoans, coelenterates, sponges, tunicates,
algae, symbiotic bacteria and fungi [65-72]. Indole alkaloids show different type
of biological activities such as cytotoxic, antitumor, antiviral, antimicrobial,
225
antiparasitics, antiserotonin and anti-inflammatory activities [73]. Due to the

interesting biological activities and unique structural features, the indole series
have become an attractive research field for the development of new
pharmacological lead compounds. In the past few years, some of the isolated
natural organic compounds and their derivatives have been synthesized by
chemists and evaluated for their biological activity to find new lead compounds
against different infectious diseases [74-79].
2.1. Bisindole alkaloids

In 1988, Kohmoto et al. isolated a bisindole alkaloid, dragmacidin (70) from
a deep water marine sponge Dragmacidin sp. [80]. Dragmacidin was found to
contain two indole groups joined by a piperazine ring system which had not been
found before in marine natural products. The molecular formula of dragmacidin
was deduced as C21H19Br3N4O from FAB HRMS data analysis. Several 2D NMR
experiments such as COSY, HETCOR, COLOC and HETCOSY were performed
in order to determine the structure of the compound. Dragmacidin (70), when
treated with excess acetic anhydride and pyridine overnight at room temperature
yielded the triacetate derivative (71). An ethanolic solution of dragmacidin (70)
on treatment with 10% Pd/C at room temperature under 20 psi of hydrogen gives
tridebromodragmacidin (72). Dragmacidin (70) exhibited in vitro cytotoxicity
with IC50 values of 15 g/mL against P-388 cell lines and 1-10 g/mL against
A-549 (human lung), HCT-8 (human colon) and MDAMB (human mammary)
cancer cell lines.
The pacific sponge Hexadella sp. collected from the coast of British
Columbia yielded two other members of dragmacidin family, dragmacidon A
(73) and dragmacidon B (74) along with a new alkaloid, topsentin C (75) [81].
The structures of the compounds 73-75 were proposed on the basis of
spectroscopic analysis. Dragmacidon A (73) showed in vitro cytotoxicity in the
L-1210 assay with ED50 value of 10 mg/mL, whereas topsentin C (75) and
dragmacidon B (74) were found to be inactive.
In 1995, Capon et al. reported the isolation of dragmacidin D (76) from a
deep water marine sponge Spongosorites sp. collected from the southern
Australian coast [82]. Dragmacidin D (76) was found to be active against human
lung tumor cell lines and inhibited in vitro growth of the P-388 murine and A-549
with IC50 values of 1.4 and 4.5 g/mL, respectively.
Four new bisindole alkaloids, nortopsentins A-D (77-80) were isolated from
the Caribbean deep sea sponge Spongosorites ruetzleri [83]. The structures of
nortopsentins A-D (77-80) were established mainly on the basis of NMR
spectroscopic data and were found to contain an imidazole ring between two
indole units. Compounds (77-80) exhibited cytotoxic activity against P-388 cells
with IC50 values of 7.6, 7.8, 1.7 and 0.9 g/mL, respectively.
226
The sponge Topsentia genitrix, collected from Banyuls (France) yielded two
bisindole alkaloids, topsentin (81) and bromotopsentin (82). They were found to
contain 2-acyl imidazole moiety inserted between two indole units with different
substitution on benzene rings [84]. In 1995, Capon et al. reported the isolation of
isobromotopsentin (83) from the deep water sponge Spongosorites sp. collected
from the coast of southern Australia [85].
R1
R4
N
R3
R2
N
H
Br
N
R5
HN
Br
N
H
N
H
70, R1 = OH, R2 = R3 = Br, R4 = Me, R5 = H

71, R1 = OAc, R2 = R3 = Br, R4 = Me, R5 = OAc
72, R1 = OH, R2 = R3 = H, R4 = Me, R5 = H
73, R1 = H, R2 = R3 = Br, R4 = Me, R5 = H
74, R1 = H, R2 = R3 = Br, R4 = Me, R5 = Me
H
N
O
R3
75
OH
H
N
R2
HN
R1
N
HN
NH
Br
N
HN
HN
76
H2N
NH
77, R1 = R2 = Br
78, R1 = Br, R2 = H
79, R1 = H, R2 = Br
80, R1 = R2 = H
Topsentin (81) inhibited proliferation of cultured human and murine tumor

cells. It exhibited in vitro activity against P-388 with IC50 value of 3 g/mL,
human tumor cell (HCT-8, A-549, T47D) with IC50 value of 20 g/mL and
in vivo activity against P-388 (T/C 137%, 150 mg/kg) and B16 melanoma (T/C
144%, 37.5mg/kg) [86]. Bromotopsentin (82) showed antiproliferative activity
against human bronocopuemonary cancer cells (NSCLC-N6) with an
IC50 = 12 g/mL [87]. Deoxytopsentin (84) was isolated from the sponge
Hexadella sp collected in Jervis Inlet, British Columbia [88]. In 1999,
bromodeoxytopsentin (85) and isobromodeoxytopsentin (86) were isolated from
sponge Spongosorites genitrix collected from Jaeju Island Korea by Shin et al.
[89]. Structurally topsentin (81) and deoxytopsentin (84) are the same except the
indole ring which is unsubstituted in case of deoxytopsentin (84). Deoxytopsentin
(84) showed the antiproliferative activity against human bronocopulmanary
cancer cells (NSCLC-N6) with an IC50 value of 6.3 g/mL. It also displayed
moderate activity against breast cancer and hepatoma (HepG2) with an IC50 of
10.7 and 3.3 g/mL, respectively.

O
NH
R1
N
H
NH
R3 R1
N
H
R2
227
R3
N
H
N
H
R2
84, R1 = R2 = R3 = H
85, R1 = Br, R2 = R3 = H
86, R1 = R2 = H, R3 = Br
81, R1 = R2 = H, R3 = OH
82, R1 = Br , R2 = H, R3 = OH
83, R1 = H , R2 = OH, R3 = Br
Recently, Kobayashi et al. isolated a new cytotoxic bis-indole alkaloid,

hyrtinadine A (87) from Okinawan marine sponge Hyrtios sp. [90]. The structure
elucidation was achieved on the basis of spectroscopic data. Hyrtinadine A (87)
was supposed to be the first example of a bisindole alkaloid with a 2,5-disubstituted
pyrimidine ring between two indole units. Hyrtinadine A (87) exhibited in vitro
cytotoxicity against murine leukemia L-1210 and human epidermoid carcinoma KB
cells with IC50 values of 1.0 and 3 g/mL, respectively.
Hyrtiosins A (88) and B (89) were also isolated together with known
5-hydroxyindole-3-aldehyde (90) from the Okinawan marine sponge Hyrtios erecta
[91]. Compound (90) exhibited cytotoxic activity against human epidermoid
carcinoma KB cells in vitro with IC50 value of 4.3 g/mL, while hyrtiosins A (88)
and B (89) were less cytotoxic than 5-hydroxyindole-3-aldehyde (90) and showed
21% and 16% inhibition, respectively, at 10 g/mL against KB cells.
HO
OH
HN
N
H
NH
87
OH
OH HO
HO
88
HO
N
H
89
N
H
N
H
90
2.2. Indolocarbazoles
Staurosporine (91) was first isolated from Streptomyces staurosporeus
Awaya (AM-2282) [92,93] and subsequently from other actinomycetes e.g.
Streptomyces actuosus [94] and Streptomyces species strain M-193 [95]. The
structure and stereochemistry of the compound in its MeOH-H2O solvate form
was deduced by X-ray crystallography. Staurosporine (91) exhibited in vitro
activity against several different type of tumors such as human neuroblastoma
cell line (NB-1), HeLa S3 cells, B16 melanoma cells and P-388 leukemia cells
[96,97]. Cordell et al. evaluated the cytotoxicity of staurosporine (91) towards the
murine P-388 lymphocytic leukemia and human carcinoma KB cell lines.
Staurosporine (91) showed potent cytotoxic activity with ED50 value of 0.0024
g/mL for the KB system and <0.08 g/mL for the P-388 system.
228
Schupp et al. isolated two new indolocarbazole alkaloids, 3-hydroxy-3demethoxy-3-hydroxystaurosporine (92) and 11-hydroxy-4-N-demethylstaurosporine
(93) from the marine ascidian Eudistoma toealensis and its predator, Pseudoceros sp.
along with four known congeners (94-97) and staurosporine (91) in their protonated
states [98]. Recently, a natural staurosporine analogue, ZHD-0501 (98) was isolated
from the fermentation broth of a marine-derived Actinomadura sp. 007 through a
bioassay-guided separation procedure [99]. ZHD-0501 (98) was supposed to be the
first example of staurosporine analogue carrying a heterocycle fused to the pyran ring.
Schupp et al. evaluated the potential of these staurosporine derivatives as
inhibitors of cell proliferation and macromolecule synthesis [100]. Compound (94)
was found to be the most active staurosporine derivative both as MONO-MAC-6 cells
inhibitor and inhibitor of RNA and DNA synthesis. The IC50 values of staurosporine
(91) and the derivatives, 94, 95 and 96 for inhibiting MONO-MAC-6 cells were 24.4,
13.3, 33.3 and 29.7 ng/mL, respectively, while those of 92 and 93 was >100 ng/mL
each. The percentage inhibition of RNA and DNA synthesis of compounds 91 and 94
were 93 and >98, 98 and >98, respectively. Compound (98) inhibited the proliferation
of human cancer A-549, BEL-7402, HL-60 cells and mouse leukemia P-388 cells
with the percentage inhibition of 82.6%, 57.3%, 76.1%, 62.2% in the SRB assay
[101]. It also inhibited the proliferation of mouse cancer tsFT210 cells with the
inhibition rates of 28.3% at 21 M and 20.5% at 2.1 M in the SRB assay. Analysis of
structure activity relationship demonstrated that hydroxylation of staurosporine at
position 3 of the indolocarbazole moiety causes an increase in antiproliferative
activity, while hydroxylation at 11th position resulted in a decrease in activity. All
these data suggested that not only the presence or absence of hydroxyl group, but also
the position of OH group is crucial to determine the antiproliferative properties of the
various staurosporine analogues.
H
N
H
N
H
N
R1
N O N
H
H
H
H
Me R4
R3
HH
R2
91, R1 = H, R2 = CH3, R3 = OCH3, R4 = H

92, R1 = OH, R2 = CH3, R3 = OH, R4 = H
93, R1 = H, R2 = H, R3 = OCH3, R4 = OH
94, R1 = OH, R2 = CH3, R3 = OCH3, R4 = H
95, R1 = H, R2 = CH3, R3 = OH, R4 = H
96, R1 = H, R2 = H, R3 = OCH3, R4 = H
N
H
N
H
N
O
97
N
Me
98
CHO
N
OH
OH
O
O
99
229
A novel carbazole alkaloid, coproverdine (99) was isolated from an unidentified

ascidian Anchorina sp. collected from the north Island of New Zealand [102].
The structure of 99 was established on the basis of extensive spectroscopic data
analysis. Coproverdine (99) was evaluated against a variety of murine and human
tumor cell lines such as P-388, A-549, HT-29, MEL-28 and DU-145 exhibiting IC50
values of 1.6, 0.3, 0.3, 0.3 and 0.3 M, respectively.
2.3. Ergoline alkaloids

Makarieva et al. isolated pibocin A (100) from the far-eastern ascidian
Eudistoma sp. [103]. Its structure and absolute stereochemistry were established
on the basis of spectroscopic and X-ray data analysis and was supposed to
represent the first example of marine ergoline alkaloids. Pibocin A (100)
exhibited moderate cytotoxicity against mouse Ehrlich carcinoma cells with ED50
value of 12.5 g/mL. Recently, pibocin B (101) was isolated from the colonial
ascidian Eudistoma sp. [104]. Its structure was established as (8)-2-bromo-N-Omethyl-6,8-dimethylergoline on the basis of NMR, FAB and MALDI TOF MS
data and chemical means. Pibocin B (101) exhibited moderate cytotoxic activity
against mouse Ehrlich carcinoma cell with an ED50 value of 25 g/mL.
H3C
CH3
H
H3C
CH3
H
Br
Br
N
OCH3
N
H
100
101
2.4. Peptidoindoles
Styelin D, a 32-residue, C-terminally amidated peptide was isolated from the
blood cells of the solitary ascidian Styela clava [105]. It was found to contain two
novel amino acids, dihydroxyarginine and dihydroxylysine, and two distinctly
unusual amino acids including, 6-bromotryptophan and 3,4-dihydroxyphenylalanine.
Styelin D exhibited cytotoxicity against HCT-116 cells with IC50 value of 10.1
g/mL, and human ME-180 cervical epithelial cells with ED50 value of 50 g/ mL.
Nakao et al. isolated kapakahine B (102) from the marine sponge
Cribrochalina olemda collected at Pohnpei, Micronesia [106]. Kapakahine B
(102) was found having a cyclic hexapeptide with an -carboline ring system and
showed moderate cytotoxicity against P-388 murine leukemia cells with an IC50
value of 5.0 g/mL.
230
O
N
N
H
O
NH
O
HN
HN
NH
O
O
NH2
NH
N
H
NH
N
HO
N
H
O
OH
O
H
N O
H
NH
NH
NH
N
O
HN
O
NH
O
HO
O
OH
O
H
NH
N O
H
NH
102
103
104
Two isomeric cycloheptapeptides, phakellistatin 3 (103) and isophakellistatin

3 (104), were isolated from the Western Indian marine sponge Phakellia carteri
[107]. They were supposed to represent the first examples of photo-Trp serving as
a natural peptide unit. A significant difference in the activity was also observed
with the photo-Trp indole ring juncture. Phakellistatin (trans-ring juncture)
exhibited inhibition of P-388 (ED50 = 0.33 g/mL) while isophakellistatin
(cis-ring juncture) showed no significant effects.
2.5. -Carbolines
Eudistomin K (105) was isolated from the Caribbean ascidian Eudistoma
olivaceum and found to exhibit antitumor activity against L-1210, A-549, HCT-8
and P-388 cell lines with IC50 of 0.01 g/mL against P-388 cell line [108].
Recently Kobayashi et al. reported the isolation and structure elucidation of a
new -carboline alkaloid, eudistomidin G (106) from the Okinawan marine
tunicate Eudistoma glaucus [109]. Eudistomidins G (106) exhibited significant
cytotoxic activity against L-1210 murine leukemia cells with IC50 value of
4.8 g/mL in vitro.
Adesanya et al. reported the isolation of two novel brominated
-carbolines, eudistalbin A (107) and B (108) from the marine tunicate
Eudistoma album along with the known compound eudistomin E (109) [110].
The cytotoxicity of these compounds was tested using the human
nasopharyngeal carcinoma KB cell lines. Eudistomin E (109) exhibited 100%
cytotoxicity at seven concentrations ranging from 10 to 0.005 g/mL (ED50
<5.0 ng/ml). Eudistalbin A (107) showed 100% cytotoxicity at 10, 92% at 5,
and 0% at 1 g/mL (ED50 = 3.2 g/mL), whereas eudistalbin B (108) exhibited
0% cytotoxic activity at 10 and 1 g/mL.

R1
N O
R2
N Me
H
N H
H HN
R4
R3
231
N
H
Br
105, R1 = H, R2 = H, R3 = Br, R4 = H
109, R1 = Br, R2 = OH, R3 = H, R4 = H
HN
106
N
H H N
2
Br
N
H
Br
108
107
Three new alkaloids, hyrtioerectines A-C (110-112) were isolated from a red
coloured marine sponge Hyrtios erectus [111]. The structure of the compounds
110-112 were established on the basis of their spectral data including 1D (1H and
13
C) and 2D (1H-1H COSY, NOESY, ROESY, HMQC and HMBC) NMR
experiments and compound 110 was found to contain 6-hydroxy -carboline and
6-hydroxyindole units linked through C3-C3 carbon bond. Hyrtioerectines A-C
(110-112) were evaluated for their cytotoxicity against HeLa cells and showed
moderate cytotoxic activity with IC50 values of 10, 5.0 and 4.5 g/mL,
respectively.
HO
H
N
OH
COOH
COOH
O
N
HO
N
H
110
NH
HO
N
H
111
CH3
OH
HO
N
H
112
Foderaro et al. reported the isolation of a new tetrahydro--carboline

alkaloid, bengacarboline (113) from the Fijian ascidian Didemnum sp. [112]. The
structure of the compound was determined by 1H, 13C NMR and HRMS-FAB
data analysis and was found to contain one indole and one tryptamine units
attached to C-1 of a tetrahydro--carboline system through C-3 and C-2 of the
indole and tryptamine moieties. Bengacarboline (113) was found to be cytotoxic
towards a 26 cell line human tumor panel in vitro with a mean IC50 value of
0.9 g/mL and also inhibited the catalytic activity of topoisomerase II at 32 M.
232
More recently, a new 1-imidazoyl-3-carboxy-6-hydroxy--carboline

alkaloid, named as hyrtiocarboline (114) was isolated from a marine sponge
Hyrtios reticulates [113]. The structure was elucidated on the basis of
spectroscopic data such as 1H-13C and 1H-15N HMBC NMR experiments.
Hytriocarboline (114) was tested for antiproliferative activity against 13 cancer
cell lines and showed selective activity against three cancer cells lines, non-small
cell lung (H522-T1), melanoma (MDA-MB-435) and lymphoma (U937) with
IC50 values of 1.2, 3.0 and 1.5 g/mL, respectively. Hyrtiocarboline (114) also
exhibited 57% inhibition of HeLa cells at 230 M.
Two new -carboline alkaloids, 6-hydroxymanzamine A (115) and 3,4dihydromanzamine A (116) were isolated from the marine sponge Amphimedon
sp collected from the Kerama Islands, Okinawa, Japan [114]. The structures of
the compounds were elucidated on the basis of NMR spectral data. Compounds
115 and 116 were found to be cytotoxic in vitro against L-1210 with IC50 values
of 1.5 and 0.48 g/mL, respectively and KJ3 cells with IC50 values of 2.5 and
0.61 g/mL, respectively.
O
H2N
OH
N
HO
NH
N
H
NH
N
H
N
H
N
H
NH
113
OH
OH
N
H
N
H
H
OH
N
H
114
N
H
115
116
Edrada et al. reported the isolation of four new manzamine congeners

117-120 and four known compounds 121-124 from the marine sponge
Xestospongia ashmorica collected from the shores of Mindoro Island,
Philippines [115].
The structures of the compounds were established on the basis
of NMR spectroscopic and mass spectrometric data analysis. The N-oxide
structures for compounds 118-120 were confirmed by conversion to the
corresponding tertiary bases by reduction with Zn/HCl. All compounds (117124) were tested for their in vitro cytotoxicity against L-5178 mouse lymphoma
cells using the microculture tetrazolium (MTT) assay at different concentrations
ranging from 0.3 to 20 g/mL. All the compounds, except 121 were found
to be active against L-5178 cell lines. From the activity profile, structure
activity relationship between the different manzamine derivatives was also
established. The N-oxide compounds 119 and 120 were the most active
compounds with ED50 value of 1.6 g/mL followed by compounds 117 and 122
N
H
N
H
OH
N
H
N
H
OH
233
N
H
N
H
OH
HN
N
H
N
H
OH
118
117
N
H
N
H
OH
HN
OH
N
H
N
H
120
119
OH
N
H
N
H
OH
OH
122
121
N
H
124
123
(ED50 = 1.8 g/mL each). The other compounds 118, 123 and 124 also exhibited
significant cytotoxic activity with ED50 values of 3.2, 6.6 and 2.3 g/mL,
respectively.
Two years later, three new manzamine congeners, manzamine M (125), 3,4dihydromanzamine J (126) and 3,4-dihydro-6-hydroxymanzamine A (127) were
isolated from the Okinawan marine sponge Amphimedon sp. [116]. The structures
and relative stereochemisty were determined on the basis of spectroscopic data.
Manzamine M (125), 3,4-dihydromanzamine J (126) and 3,4-dihydro-6hydroxymanzamine A (127) showed cytotoxicity against murine leukemia
L-1210 cells with IC50 values of 1.4, 0.5 and 0.3 g/mL, respectively.
OH
N
H
N
N
H
N
H
OR
OH
OH
HN
125
126
N
H
N
H
N
H
OH
127
234
2.6. Trisindole alkaloids

In 1994, Bifulco et al. reported the isolation of two tris-indole alkaloids,
Gelliusines A (128) and B (129) from a deep water new Caledonian sponge
Gellius or Orina sp. [117]. Gelliusin A (128) and B (129) were found to be
diastereomeric compounds made up by the coupling of three indole units.
In compounds 128 and 129, two 6-bromo tryptamine units are linked through
their aliphatic chains to the C-2 and C-6 position of a central serotonin moiety.
The coupling of the indole unit appears to be non stereoselective giving two
enantiomeric pairs, having different relative configuration at C-8 and C-8" named
() Gelliusines A (128) and B (129). Gelliusines A (128) and B (129) showed
cytotoxicity with an IC50 value of between 10 and 20 g/mL against KB, P-388,
P-388/dox, HT-29 and NSCLCN-6 cell lines.
NH2
HN
HO
NH2
N
H
Br
N
H
H2N
128 and 129,
Br
Gelliusine A and B
2.7. Miscellaneous indole alkaloids

Kondo et al. reported the isolation of two new indole alkaloids, isoplysin A
(130) and D6-bromohypaphorine (131) from the Okinawan marine sponge
Aplysina sp. [118]. The structures of both the compounds were established by
spectral and chemical means. Isoplysin A (130) was found to be weakly cytotoxic
against murine lymphoma L-1210 (IC50 = 11.5 g/mL) and human epidermoid
carcinoma KB cells (31% inhibition at 20 g/mL), while D-6-bromohypaphorine
(131) showed no significant effects.
In 2007, four new prenylated indole alkaloids, notoamides A-D (132-135)
were isolated from marine-derived fungus Aspergillus sp. which was separated
from the mussel Mytilus edulis collected off Noto Peninsula in the Sea of Japan
[119]. The structures and absolute stereochemistry of the compounds were
determined mainly on the basis of spectroscopic data analysis and were found
comprising of pyranoindole ring system. Compounds (132) and (133) contain the
bicyclo[2.2.2]diazaoctane ring system also. Notoamides A-C (132-134) exhibited
weak cytotoxicity against HeLa and L-1210 cells with IC50 values in the range
of 2252 g/mL but the IC50 value of notoamide D (135) was greater than
100 g/mL and it was believed that the dihydroxypyrano-2-oxindole ring system,
that is common to compounds 132-134 is responsible for the remarkable
differences in cytotoxic activity. Notoamide D (135) contains a pyrroloindole
235
instead of dihydroxypyrano-2-oxindole ring system. Recently, six new prenylated

indole alkaloids, notoamides F-K (136-141) were isolated from a marine-derived
Aspergillus sp [120]. Notoamide I (139) showed weak cytotoxicity against HeLa
cells with an IC50 value of 21 g/mL, whereas for notoamides F (136), J (140)
and K (141), the IC50 values were more than 50 g/mL.
Three new indole alkaloids, shearinines D-F (142-144) along with the known
shearinine A (145) were isolated from marine-derived fungus Penicillium
janthinellum [121]. The structures of all the compounds were established by 1D
and 2D NMR such as HSQC, HMBC, COSY, NOESY and HREIMS data
analysis. Shearinines A, D, E and F were tested for cytotoxicity against mouse
epidermal JB6 P+ Cl 41 cells using the MTS method. The compounds displayed
no cytotoxicity up to 200 M, whereas some of the compounds showed cancer
preventive and antileukemic properties. Shearinine E (143) inhibited
EGF-induced malignant transformation of JB6 P+ Cl 41 cells in a soft agar with
INCC50 (inhibition of number of the colonies) value of 13 M. The Shearinines A
(145), D (142) and E (143) induced apoptosis in human leukemia HL-60 cells at
100 M concentration by 10%, 39% and 34% of the apoptotic cells when
compared to control cells, respectively.
O
O
H3C
N
O
R2
N(CH3)
COO
N
NH
N(CH3)3
N
H
Br
130
O
H
HO
N
H
N
R1
136, R1 = R2 = H, R3 = OMe
137, R1 = OH, R2 = H, R3 = OMe
139, R1 = H, R2 = R3 = O
N
O
H
N
N
132, R1 = OH, R 2 = H
133, R1 = R2 = H
138, R1 = OH, R 2 = OH
131
N
R1
N
H
R2
N
H
O
N
H
N
H
N
H
O
H
O
H
OH
N
H
N
H
HO
135, R =
141, R =
N
O
R3
134
N
H
O
140
H1 OH
OH
N
H
OH
O
N
H
O
O
142, H and H1 trans

143, H and H1 cis
144
OH
O
O
O
N
H
O
O
145
236
Reyes et al. reported the isolation and structure elucidation of six new
bromoindole alkaloids, aplicyanins A-F (146151) from CH2Cl2/MeOH extract of
the tunicate Aplidium cyaneum collected in Antarctica [122]. Aplicyanins A-F
(146-151) were tested for cytotoxicity against three human tumor cell lines,
including colon (A-549), lung (HT-29) and breast (MDA-MB-231). Compounds
147, 149, 150 and 151 showed cytotoxicity against these cell lines, whereas
compounds 146 and 148 were found to be inactive. Compounds 147, 149, 150
and 151 demonstrated IC50 values of 0.66, 0.63, 8.70 and 1.31 (A-549), 0.39,
0.33, 7.96 and 0.47 (HT-29) and 0.42, 0.41, 7.96 and 0.81 (MDA-MB-231). From
the activity profile it is clear that compound 150 shows the least activity and this
was explained on the basis of presence of the acetyl group at N-16 in compounds
147, 149 and 151 which is crucial to exhibit the activity.
Amade et al. reported the isolation and structure elucidation of new bromine
containing oxindole alkaloid, matemone (152) along with a known compound,
6-bromoindole-3-carbaldehyde from the Indian Ocean sponge Iotrochota
purpurea [123]. Compound 152 showed weak cytotoxicity against NSCLC-N6
L16 strain18 (lung cancer), Mia PaCa-2 cell line (pancreas cancer) and DU145
cell line (prostatecancer) with IC50 values of 30, 24 and 27 g/mL, respectively.
Dendridine A (153), a unique C2-symmetrical 4,4-bis(7-hydroxy)indole
alkaloid was isolated from an Okinawan marine sponge Dictyodendrilla sp.
[124]. The structure of compound was elucidated by spectroscopic data including
2D NMR data such as the 1H-1H COSY, ROESY and HMBC spectra. Dendridine
A (153) exhibited moderate cytotoxicity against murine leukemia L-1210 cells
with IC50 value of 32.5 g/mL.
R1
H
N
N
HN
O
Br
R3
H
N
OH
N
R2
Br
146, R1 = R2 = R3 = H
147, R1 = Ac, R2 = R3 = H
148, R1 = R3 = H, R2 = OMe
149, R1 = Ac, R2 = OMe, R3 = H
150, R1 = H, R2 = OMe, R3 = Br
151, R1 = Ac, R2 = OMe, R3 = Br
N OMe
H
152
OH
Br
H2N
NH2
Br
OH
N
H
153
Four novel brominated indole alkaloids, arborexidines A-D (154-157) were

isolated from the extract of a marine tunicate Pseudodistoma arborescens [125].
Out of four, only arborescidine D (157) showed in vitro cytotoxic activity against
the growth of KB human buccal carbinoma cells with IC50 value of 3 g/mL.
237
N Me
H
N
N
H
Br
Br
N Me
H
N
Br
R1
154
R2
156, R1 = H, R2 = OH
157, R1 = OH, R2 = H
155
3. Pyrrole alkaloids
3.1. Bromopyrrole alkaloids
Kuramoto et al. isolated two novel alkaloids, cylindradines A (158) and B
(159) from the marine sponge Axinella cylindratus collected at the Seto inland
sea near Sada Cape in Ehime prefecture [126]. The chemical structures and
absolute stereochemistry of these compounds were assigned by spectroscopic and
X-ray data analysis. Cylindradines A (158) and B (159) displayed moderate
cytotoxicity against the murine leukemia cell line P-388 with IC50 value of 7.9
and 33 g/mL, respectively.
In 1993, a novel alkaloid, agelastatin A (160) was isolated from the deep
water marine sponge Agelas dendromorpha collected in the Coral Sea near New
Caledonia [127]. Agelastatin A (160) showed significant in vitro activity against
L-1210 and KB tumor cells [128]. He also studied the structure activity
relationship of agelastatins and found that the C-8a hydroxyl group and both NH
Br
H H
N
HO N
Br
HN
H H
N
Br
H2N
HN
Br
Br
H2N
HO
H
H
N
H
O
H
HO
158
R2
Br
R1
Br
R3
O N
H
H
N
O
NH2
NH
HN
Br
161, R1 = H, R2 = Me, R3 = Me
162, R1 = Br, R2 = H, R3 = H
NH
Br
NH2
N
Br
N
H
160
159
N
H
HN
N
H
H
N
O
NH2
O
163
164
HN
N
238
groups are necessary for optimal activity. Alkylation or acylation of these functional
groups, as well as removal of the C-1 pyrrole bromine, leads to a significant loss of
potency. Recently, Tilvi et al. isolated three related pyrrole-imidazole alkaloids,
named agelastatins E (161), F (162) and benzosceptrin C (163) along with agelastatin
A (160) from marine sponge Agelas dendromorpha [129]. The structures of the
compounds were established on the basis of spectroscopic data interpretation. The
compounds 160-163 were evaluated for cytotoxic activity against the KB cell lines.
All the compounds lacked significant bioactivity at 30 M except for agelastatin A
(160) which showed 100% activity at 30 and 3 M.
A new pyrrole alkaloid, clathrodin (164) was isolated from the MeOH extract
of the Caribbean sea sponge Agelas clathrodes [130] and showed significant
cytotoxicity against CHO-K1 cells with ED50 value of 1.33 g/mL.
The tetracyclic pyrrole-imidazole alkaloid, dibromophakellstatin (165) was
isolated from the marine sponge Phakellia mauritiana [131]. The structure and
absolute stereochemistry of the compound was determined by interpretation of
NMR and X-ray crystal data analysis. Dibromophakellstatin (165) showed inhibitory
activity against a panel of human cancer cell lines, ovary (OVCAR-3), brain (SF-295),
kidney (A-498), lung (H-460), colon (KM20L2) and melanoma (SK-MEL-5) with
ED50 values of 0.46, 1.5, 0.21, 0.62, 0.11 and 0.11 g/mL, respectively.
Br
Br
H H
N
N
H2
N
Br
O
N
H
Br
Br
N
H
NH
Br
N
H2N
X
N
H
HO
N
H
NH
O
O
O
Me
Cl
165
166, R = H
167, R = Br
168
169
Four new alkaloids 3-bromomaleimide (166), 3,4-dibromomaleimide (167),

12-chloro-11-hydroxydibromoisophakellin (168) and N-methylmanzacidin C
(169) were isolated from the marine sponge Axinella brevistyla collected in
western Japan [132]. Their structures were determined on the basis of
spectroscopic data analysis. Compounds 166-168 exhibited cytotoxicity against
L-1210 cells with IC50 values of 1.1, 0.66 and 2.5 g/mL, respectively, whereas
N-methylmanzacidin C (169) was found to be inactive.
Umeyama et al. reported the isolation and structure elucidation of a novel
bromopyrrole alkaloid (170) along with ()-171 and ()-longamide (172) from
the marine sponge Homaxinella sp. collected in japan [133]. Compounds 170 and
()-171 showed mild cytotoxic activity in vitro against P-388 lymphocytic
leukemia cells with ED50 values of 21.5 and 30 g/mL, respectively, while
compound (172) was inactive (ED50 = >100 g/mL).
239
Br
O
Br
Br
Br
Br
H
N
N
H
O
Br
Br
NH
NH
OCH3
Br
O
H3CO
171
170
NH
H3CO
N
HO
171
172
More recently, Hertiani et al. reported the isolation of 11 new brominated

pyrrole alkaloids from the Indonesian marine sponge Agelas linnaei [134]. These
alkaloids includes a new dibromophakellin derivative (173), 4-(4,5-dibromo-1methylpyrrole-2-carboxamido)-butanoic acid (174), agelanin A and B (175 and
176), agelanesins AD (177-180) and mauritamide B-D (181-183).
NH 2
Br
HN
HO
NH 2
N
Br
Br
N
O
173
OH
N
H
Br
O
N
Br
OH
Br
174
175
N
Br
O
N
H
Br
OH
OH
R1
H
N
R2
O
N
H
Br
O
177, R1 = H, R2 = Br
178, R1 = H, R2 = I
179, R1 = Br, R2 = Br
180, R1 = Br, R2 = I
176
Br
Br
O
N
H
O
S OH
O
HN
Br
O
N
H
N R
O
HN
181, R = H
182, R = CH 2CH3
Br
O
OH
S
O
NH
183
All the compounds (173-183) were tested for cytotoxicity against the murine
L-1578Y mouse lymphoma cell line. Agelanesins AD (177-180) showed
prominent activity while others were found to be inactive. The IC50 values for
agelanesins AD (177-180) were 9.55, 9.25, 16.76 and 13.06 M, respectively.
Compounds 177 and 178 were the most potent concluding that cytotoxicity of the
240
agelanesins is related to the degree of bromination of the pyrrole ring. Increase in

bromination decreases the activity as observed for 179 and 180 compared to 177
and 178. While the presence of an iodide substituent on the tyramine moiety
causes a small differences in activity as 177 and 179 have similar activity
compared to 178 and 180.
3.2. Pyrroloquinones
A new dipyrroloquinone, zyzzyanone A (184) was isolated from the
Australian marine sponge Zyzzya fuliginosa [135]. Zyzzyanone A (184)
showed mild cytotoxic activity against mouse Ehrlich carcinoma cells with
IC50 value of 25 g/mL. One year later Zyzzyanones B-D (185-187), three related
dipyrroloquinones were isolated from the same sponge Zyzzya fuliginosa along
with the known zyzzyanone A (184) [136]. The structures of the compounds
185-187 were established by extensive NMR spectroscopic data. Zyzzyanones
B-D (185-187) also pronounced weak cytotoxicity against mouse Ehrlich
carcinoma cells with IC50 value of 25 g/mL.
R
N
R
N
H
N
H
N
O
N
H2N
OH
184, R = Me
185, R = H
CHO
OH
186, R = Me
187, R = H
3.3. Pyrroloquinoline alkaloids

In 1986, Landini et al. reported the isolation of discorhabdins C (190) from
the extract of red-brown sponge Latrunculia du [137]. The structure of the
compound was determined by a single crystal X-ray diffraction study and it was
found to contain a new tetracyclic iminoquinone chromophore with a spiro 2,6dibromocyclohexadienone. Two years later, discorhabdins A (188) and (189) B
along with known discorhabdins C (190) were isolated from the three species of
Latrunculia sponge collected in New Zeeland [138]. A related compound,
discorhabdins D (191) was isolated from Latrunculia brevis collected in
New-Zeeland [139]. Discorhabdins A (188), B (189) and C (190) showed in vitro
cytotoxicity against P-388 assays with ED50 values of 0.05, 0.1 and 0.03 g/mL.
Discorhabdin A (188) and discorhabdin C (190) showed no cytotoxicity against
P-388 system in vivo but were found to be toxic to mice at about 2 mg per kg of
241
body weight. Discorhabdin B (189) showed some antitumour effect with a T/C of
117% at a dose of 0.25 mg/kg, but this did not reach the significance level of
120%. Discorhabdins C (190) was also found to be active toward L-1210 tumor
cells at very low levels (ED50 < 100 ng/mL). Discorhabdin D (191) exhibited
mild cytotoxicity against P-388 in vitro with IC50 value of 6 g/mL, however
in vivo it showed significant activity against P-388 (T/C 132% at 20 mg/kg).
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
NH
NH
NH
Br
Br
188
Br
Br
190
189
191
Two other members of discorhabdin family, discorhabdins L (192) and I

(193) were isolated from Latrunculia brevis and their structures were assigned on
the basis of spectroscopic data analysis and comparison with the known
discorhabdins A (188) and B (189) [140]. Discorhabdins L (192) and I (193) were
tested against a panel of 14 tumor cell lines including prostate (DU-145 and
LN-caP), ovary (SK-OV-3, IGROV and IGROV-ET), breast (SK-BR3),
melanoma (SK-MEL-28), endothelio (HMEC1), NSCL (A549), leukemia (K562), pancreas (PANC1) and colon (HT29, LOVO and LOVO-DOX). Both the
compounds exhibited potent cytotoxic activity in most of the cases. The HT-29
colon cell line was found to be the most sensitive with GI50 values of 0.12 and
0.35 M for compounds 192 and 193, respectively.
Recently, Lang et al. isolated a novel alkaloid, discorhabdin W (194) from a
marine sponge Latrunculia sp [141]. It is a symmetrical dimer of discorhabdin in
which two discorhabdin units are linked by a disulfide linkage. The structure and
stereochemistry were assigned by 1D and 2D NMR experiments and mass
spectrometry. Discorhabdin W (194) exhibited potent cytotoxicity against P-388
cells with IC50 value of 0.09 g/mL.
H
N
H
N
H
N
H
N
H
N
H
N
H
N
S S
N HO
H
192
NH
O
193
N
Br
N
Br
O
194
H
N
242
Sun et al. reported the isolation of three highly functionalized pyrroloquinoline

alkaloids, batzelline A-C (195-197) from the deep water sponge Batzella sp collected
in Bahamas [142]. The structure of 195 was determined by X-ray and those of 196
and 197 by comparison of their spectral data with that of 195 and by chemical
transformations. One year later same group isolated four related alkaloids,
isobatzellines A-D (198-201) from the sponge Batzella sp. [143]. Isobatzellines A-D
(198-201) were found to exhibit in vitro cytotoxicity against P-388 leukemia cell
lines, whereas batzellines (195-197) were inactive and it was explained on the basis
of difference in their structure. Both, isobatzellines and batzellines have the
same pyrrolo[4,3,2 de] quinoline ring system but isobatzellines contain an
aminoiminoquinone moiety that is different from the aminoquinone moiety in the
batzellines which could be responsible for the activity of isobatzellines.
In 1999, two novel batzelline analogues, named as secobatzellines A (202)
and B (203) were isolated from a deep water marine sponge Batzella sp. The
structure of the compounds were determined by NMR, HR FABMS data analysis
and chemical analysis and was found to contain pyrroloaminoiminoquinone
moiety previously reported in isobatzellines. Secobatzellines A (202) and B (203)
exhibited in vitro cytotoxicity against the cultured murine P-388 tumor cell line
with IC50 values of 0.06, 1.22 g/mL and against human lung carcinoma A-549
cell line with IC50 values of 0.04, 2.86 g/mL, respectively.
R
R3
R2
N
HN
R3
O R
2
O
Cl
N
H
195, R = Me, R2 = SMe

196, R = H, R2 = SMe
197, R = Me, R2 = H
H2N
N
R4
198, R3 = SMe, R4 = Cl
199, R3 = SMe, R4 = H
200, R3 = H, R4 = Cl
201, R3 = H, R4 = H
H2N
R1
Cl
202, R1 = NH, R2 = R3 = H
203, R1 = O, R2 = R3 = H
Kobayashi et al. reported the isolation and structure elucidation of a sulphur

containing alkaloid, prianosin A (204) from the Okinawan marine sponge
Prianos melanos [144]. Prianosin A (204) was found to be cytotoxic against
L-1210 and L-5178Y murine leukemia cells with IC50 values of 37 and 14 ng/mL
in vitro. In 1988, same group reported the isolation of three related alkaloids,
prianosins B-D (205-207) from the same sponge Prianos melanos [145]. All the
alkaloids were found having the same tetrahydrothiophene ring as prianosin A
(204). Prianosins B-D (205-207) were evaluated for cytototoxic activity in vitro
against murine lymphomas L-1210 and L-5178Y cells and human epidermoid
carcinoma KB cells. The IC50 values were found to be 2.0, 1.8 and >5.0 g/mL
(24% inhibition at 5.0 g/mL) for prianosins B (205), 0.15, 0.024 and 0.57 g/mL
for prianosins C (206) and 0.18, 0.048 and 0.46 g/mL for prianosins D (207).
H
N
H
N
H
N
H
N
S
N
Br
H
N
OH
H
N
N
O
205
OH
H
N
S
HO
H
N
S
S
N
Br
204
243
206
H
O
207
Radisky et al. reported the isolation and structure elucidation of seven novel
pyrroloiminoquinones, the makaluvamines A-F (208-213) from the Fijian sponge
Zyzzya cf. marsailis [146]. The makaluvamines A-F (208-213) exhibited potent
in vitro cytotoxicity against the human colon tumor cell line HCT-116,
topoisomerase II sensitive CHO cell line xrs-6, and also inhibited the catalytic
activity of topoisomerase II. Makaluvamine A (208) and C (210) also exhibited
in vivo antitumor activity against the human ovarian carcinoma ovcar-3 implanted
in athymic mice. Makaluvamine F (213) was found to be the most active
compound followed by makaluvamine E and A. Makaluvamine D and C were
less potent than A, E and F, whereas Makaluvamine B was found not active
against HCT-116. The same activity pattern was also observed against xrs-6, a
Chinese hamster ovary (CHO) cell line being makaluvamine F (213) the most
potent compound, while makaluvamine B least active. However, the metabolite
cytotoxicity trends are substantially different than the hypersensitivity factors
(HF) obtained by comparison of the cytotoxicity against xrs-6 versus BR1 (a
DNA-repair proficient CHO line). Makaluvamine A (208) exhibited the largest
hypersensitivity factor of 9, followed by makaluvamines F, E, C, and D. These
results give a clue about the mechanism of action of Makaluvamines that involves
DNA double-stranded breakage, an activity characteristic of topoisomerase II
inhibitors.
Makaluvamine G (214) was isolated from a sponge of the genus
Histodcnnella collected in Indonesia [147]. The structure of the compound was
determined on the basis of 1D and 2D NMR experiments. Makaluvamine G (214)
pronounced significant cytotoxicity to several tumor cell lines exhibiting an IC50
value of 0.50 g/mL against P-388 (murine leukemia), A-549 (human nonsmall
cell lung cancer), HT-29 (human colon cancer) and MCF-7 (human breast cancer)
and value of 0.35 g/mL against KB (human oral epidermoid carcinoma). It was
also found to be a moderate inhibitor of topoisomerase-I (IC50 = 3.0 M) and did
not significantly inhibit topoisomerase-II. It also inhibited RNA (IC50 = 15 M),
DNA (15 M) and protein (21 M) synthesis.
In 1997, a new related alkaloid, makaluvamine N (215) was isolated from the
Philippine sponge Zyzzya fuliginosa [148]. Compound 215 showed in vitro
cytotoxicity against the human colon tumor cell line HCT-116 with LC50 value
of 0.6 g/mL. Makaluvamine N (215) also demonstrated an ability to inhibit the
244

O
O
NH2
NH2
NH
NH
208
H
N
H
N
H
N
NH
OH
211
H
N
H
N
S
NH
OH
212
H
N
NH2
210
H
N
209
O
NH
H
N
213 Br
O
O
NH2
OH
H
N
Br
N
214
NH
OH
215
216
OH
catalytic activity of topoisomerase II and exhibited 90% inhibition of

topoisomerase II unwinding of pBR-322 at 5 g/mL. Casapullo et al. reported the
isolation of a new member of makaluvamine family, makulavamine P (216) from
the sponge Zyzzya cf. fuliginosa collected in the Vanuatu Islands [149]. The
compound was characterized on the basis of its spectral data and comparison with
the other related compounds. Makulavamine P (216) exhibited moderate
cytotoxicity to KB tumor cells (64% inhibition of cell growth at 3.2 g/mL).
Recently Shinkre et al. synthesized two series of makaluvamine analogs
217 (a-g) and 218 (c-g) by introducing different substituents at the 7-position
of the pyrroloiminoquinone ring present in makaluvamines. These
compounds were obtained in two steps by treatment of the
methoxypyrroloiminoquinone with different primary amine derivatives and
subsequent removal of tosyl protecting group [150]. Compounds 217 (a-g)
and 218 (c-g) were evaluated for their cytotoxicity against human breast
cancer cell lines MCF-7 and MDA-MB-468 and human colon cancer cell line
HCT-116 using etoposide and m-AMSA as standard drugs. HCT-116 cells
were shown to be the most sensitive to etoposide and m-AMSA with IC50
values of 1.7 and 0.7 M, respectively and MDA-MB-468 cells showed IC50
values of 13.6 and 8.5 M for etoposide and m-AMSA, respectively, whereas
MCF-7 cells were found to be the least sensitive with IC50 values of 35.6 and
21.7 M for etoposide and m-AMSA, respectively. Most of the
makaluvamine analogs have shown significantly better inhibition than the
control drugs in these assays. Compounds (217c, 218d, 218f, and 218g)
245
exhibited better activity (IC50 = 1.3, 0.5, 1.0 and 0.8 M, respectively)
against HCT-116 as compared to control drug etoposide (IC50 = 1.7 M).
Compound 218d exhibited better IC50 value against HCT-116 as compared to
m-AMSA (IC50 = 0.7 M). All the compounds exhibited better IC50 values
against MCF-7 and MDA-MB- 468 as compared to etoposide as well as
m-AMSA. Compounds 217 (a-g) and 218 (c-g) were also evaluated for their
ability to inhibit topoisomerase II enzymatic activity and found that five
makaluvamine analogs (217c, 217d, 217f, 218c and 218e) exhibited
inhibition of topoisomerase II comparable to etoposide and m-AMSA. Three
of these compounds (217f, 218c and 218e) showed the strongest inhibition of
catalytic activity of topoisomerase II.
In 1997, the methanol extract of the Fijian sponge Zyzzya fuliginosa yielded
a new pyrroloiminoquinone derivative, veiutamine (219) [151]. The structure of
the compound was determined by 1D and 2D NMR experiments and was found
bearing a p-oxy benzyl substituent at carbon 6 of the basic pyrroloiminoquinone
system. Veiutamine (219) exhibited cytotoxicity against the human colon tumor
cell line HCT-116 with IC50 value of 0.3 g/mL. Wakayin (220) was isolated
from the ascidian Clauelinu sp [152]. It was supposed to represent the first
example of pyrroloiminoquinone alkaloid to be isolated from an ascidian.
Wakayin (220) exhibited in vitro cytotoxicity against the human colon tumor
cell line (HCT-116) with IC50 value of 0.5 g/mL. Preliminary studies
such as Inhibition of topoisomerase II enzyme (250 M) and the observation
of a 3-fold differential toxicity toward the CHO cell line EM9 (sensitive to
DNA-damaging genotoxic agents) versus BR16 (resistant to BCNU) provided
evidences that the activity of wakayin could be related to interfering with or
damaging DNA.
H
N
Ts
N
H
N
R
R
NH
NH
217 (a-g)
R=
CH3
CH2CH3
H
N
218 (c-g)
H2CH2C
H2C
d
Br
H2CH2C
OH
OH
H2CH2C
Br
H2CH2C
NH
246
Two new bispyrroloiminoquinone alkaloids, tsitsikammamme A (221) and

tsitsikammamine B (222) were isolated from the South African Latrunculid
sponge Tsitsikamma favus [153]. Reinvestigation of the extracts of the sponge
Tsitsikamma favus yielded two N-18 oxime analogues of tsitsikammamine A and
B, 223 and 224 [154]. Compounds 223 and 224 exhibited significant cytotoxic
activity against human colon tumor (HCT-116) cell line with IC50 values of 128.2
and 16.5 M, respectively, when compared with their parent alkaloids 221 and
222 (IC50 = 1.4 and 2.4 mM, respectively).
Recently, two aza-analogs, 225 and 226 of tsitsikammamine and wakayin
were synthesized based on a 1,3-dipolar cycloaddition reaction between indole
4,7-dione and a diazo-aminopropane derivative in which the pyrrole ring of the
pyrroloquinoline moiety was replaced by a pyrazole ring [155]. The ability of the
compounds 225 and 226 to inhibit the DNA cleavage activities of human
topoisomerases I and II was assayed in a cell-free assay. Both the compounds
exhibited 0% inhibition of topoisomerase II. Compound 225 inhibited partially
topoisomerase I at 100 M, whereas no inhibitory activity was observed for
compound 226.
O
H
N
NH2
H
N
NH
NH
OH
220
H
N
219
O
H
N
NH
N
H
R2
N
R
N
H
N
221, R = H
222, R = CH3
H
N
N
N
N
N
N
H
R1
223, R1 = OH, R2 = H
OH
224, R1 = OH, R2 = Me
225
N
H
OH
N
H
226
3.4. Pyrroloacridine
Two novel alkaloids, plakinidine A (227) and B (228) were isolated from
Vanuatuan red sponge Plakortis sp. [156]. Their structures were determined by
1D and 2D NMR experiments and were found to contain a pyrrolo (2,3,4-kl)
acridine system. In the same year, IreIend et al. reported the isolation and
structure elucidation of a new compound plakinidine C (229) together with
plakinidine A (227) and B (228) from the MeOH extract of Plakortis sp. collected
247
in Fiji [157]. Plakinidine A-C (227-229) exhibited cytotoxic activity towards

L-1210 murine leukemia cell lines with IC50 values of 0.1, 0.3 and 0.7 g/mL,
respectively.
R
N
N
NH
227, R = H
228, R = CH3
229, R = H, 9,10-didehydro
3.5. Miscellaneous pyrrole alkaloids

Ircinamine B (230) was isolated from the marine sponge Dactylia sp.
collected at Cape Sada in Japan and showed moderate cytotoxic activity against
the murine leukemia cell line P-388 with IC50 value of 0.28 g/mL [158].
A novel tetracyclic alkaloid, perinadine A (231) was isolated from the
cultured broth of the fungus Penicillium citrinum separated from the gastrointestine
of a parrot fish Scalus ovifrons collected at Hedo Cape, Okinawa Island [159].
Perinadine A (231) exhibited mild cytotoxicity against murine leukemia L-1210
cell line with IC50 value of 20 g/mL.
In 1994, Perry et al. isolated a new alkaloid, Variolin B (233) from the
Antarctic sponge Kirkpatrickia varialosa. The structure was determined by X-ray
crystallography and interpretation of spectral data [160]. Variolin B (233) was
supposed to be the first examples of natural products with a pyridopyrrolopyrimidine
moiety. In the same year two other pyridopyrrolopyrimidine alkaloids, variolin A
(232) and N(3)-methyl tetrahydrovariolin B (234) were isolated from the same
sponge Kirkpatrickia varialosa [161]. Variolins (232-234) were tested in vitro
against P-388 cell lines. Variolin A (232) and variolin B (233) showed in vitro
activity against P-388 cell lines with IC50 value of 3.8 ng/mL and 210 g/mL,
respectively. Compound 234 was found to be inactive against P-388 but showed
in vivo activity against P-388 leukemia (T/C 125% at 10 mg/Kg). Compound 234
also showed significant in vitro activity against the HCT-116 cell line with IC50
value of 0.48 g/mL.
Kashman et al. isolated a novel bisquinolinylpyrrole alkaloids, halitulin (235)
from a marine sponge Haliclona tulearensis collected in Sodwana Bay, Durban,
South Africa [162]. Its structure was established mainly on the basis of spectroscopic
data and chemical means. Halitulin (235) was considered as the first natural
248
compound to be discovered that has a 7,8- dihydroxyquinoline system and found to be

cytotoxic against several tumor cell lines such as P-388 murine leukemia, A-549
human lung carcinoma, HT-29 human colon carcinoma and MEL-28 human
melanoma with IC50 value of 0.025, 0.012, 0.012 and 0.025 g/mL, respectively.
O
NH2
N
H
OH
O
N
H
232
231
OH
HO
N
OH
OH
NH2
OH
OH
N
N
NH2
NH2
N
OH
HO
230
16
N
N
N
N
NH2
NH2
233
234
Me
235
4. Pyridine alkaloids
In 1999, Kobayashi et al. isolated a novel pyridine alkaloid, pyrinodemin
A (236) from the Okinawan marine sponge Amphimedon sp. [163]. The
structure of compound 236 was assigned from 2D NMR data and EIMS
fragmentation and was found to contain two 3-alkyl-substituted pyridine
rings with a cis-cyclopent[c]isoxazolidine moiety. Pyrinodemin A (236)
demonstrated potent cytotoxicity in vitro against murine leukemia L-1210
and KB epidermoid carcinoma cells with IC50 values of 0.058 and 0.5 g/mL,
respectively. One year later, three new bis-pyridine alkaloids, pyrinodemins
B-D (237-239) were isolated together with pyrinodemin A (236) from the
same sponge Amphimedon sp. [164]. Pyrinodemins B-D (237-239) exhibited
potent cytotoxicity in vitro against murine leukemia L-1210 with IC50 values
of 0.07, 0.06 and 0.08 g/mL, respectively and KB epidermoid carcinoma
cells (IC50 = 0.5 g/mL each).
249
A novel pyridine alkaloid, pyrinadine A (240) was isolated from the marine
sponge Cribrochalina sp. collected from the Unten Port, Okinawa [165]. The
structure was established by spectroscopic data and chemical conversions. When
treated with zinc/acetic acid, pyrinadine A yielded compound (241), generated by
cleavage at the azoxy moiety of pyrinadine A. Pyrinadine A (240) exhibited
in vitro cytotoxicity against L-1210 murine leukemia (IC50 = 2 g/mL) and KB
human epidermoid carcinoma cells (IC50 = 1 g/mL).
H
H
236
H
H
O
237
H
H
O
238
N
H
H
O
239
N
N
240
NH2
N
241
In 2006, Takekawa et al. reported the isolation of amphimedosides A-E

(242-246) from a marine sponge Amphimedon sp. [166]. The structures of
compounds 242-246 were determined by NMR, FABMS data interpretation. The
site of glycosylation in compound 242 was confirmed by the 1H-15N HMBC
experiment and the location of the double bond in 246 was assigned on the basis
of tandem FABMS data. Amphimedosides (242-246) were the first examples of
-D-glucosylated 3-alkylpyridine alkaloids till the date and exhibited mild to
strong cytotoxicity against P-388 murine leukemia cells with IC50 values of 11,
11, 5.0, 0.45 and 2.2 g/mL, respectively.
250

HO
HO
HO
O
OH
OCH3
N
n
242, m = 3, n = 9
243, m = 3, n = 7
244, m = 1, n = 9
m
N
HO
HO
HO
OCH3
N
OH
245
HO
HO
HO
OCH3
N
OH
246
Echinoclathrines A-C (247-249), a new class of pyridine alkaloids having

4-aryl-2-methylpyridine unit, were isolated from an Okinawan sponge,
Echinoclathria sp. [167]. The structures of compounds were established by
interpretation of spectral data. Only echinoclathrine A (247) displayed weak
cytotoxicity (IC50 = 10 g/mL) against P-388, A-549 and HT-29 cell lines,
while others were found to be inactive.
OH
O
OR
O
N
H
N
H
247
N
SR1
248, R = H, R1 = Ac
249, R = R1 = H
5. Isoquinoline alkaloids
Two new isoquinolinequinones alkaloids, cribrostatins 1 (250) and 2 (251) were
isolated from a deep blue colored sponge Cribrochalina sp. [168]. The structures of
the compounds were determined by extensive NMR data analysis and single-crystal
X-ray diffraction experiment. Cribrostatins 1 and 2 were found to be active against
lymphocytic leukemia cell line (P-388) with ED50 values of 1.58 and 2.73 g/mL,
respectively. Pettit et al. reported the isolation of cribrostatins 3 (252), 4 (253) and 5
251
(254) from the same sponge Cribrochalina sp. [169]. Compounds 251-254 were
evaluated for cytotoxicity against several cancer cell lines. Mouse leukemia P-388 cell
line was found to be the most sensitive to Cribrostatins 3 (252), 4 (253) and 5 (254)
exhibiting with ED50 values of 2.5, 2.2 and 0.045 g/mL, respectively.
Cribrostatin 6 (255) was also isolated from the same marine sponge
Cribrochalina sp. [170]. The structure of compound was assigned on the basis of
1
H, 13C, 15N NMR and HRMS data interpretation and finally structure was confirmed
by X-ray crystal data analysis. Cribrostatin 6 (255) was found to inhibit the growth of
murine P-388 lymphocytic leukemia (GI50 = 0.29 g/mL) and a panel of human
cancer cell lines. Among human cancer cell lines, the best activity in terms of potency
was obtained against MCF-7 (GI50 = 0.21) followed by SF-268 (GI50 = 0.24) and
DU-145 (GI50 = 0.38), whereas GI50 value of >1g/mL was observed against
BXPC-3, NCI-H460 and KM20L2 cell lines.
A new isoquinoline alkaloid, jorumycin (256) was isolated from the mantle and
the mucus of the pacific nudibranch Jorunna funebris [171]. The structure of
compound was established on the basis of ESIMS data and of an extensive 2D NMR
analysis. Jorumycin (256) showed very interesting activity against NIH 3T3 tumor
cells (100% of inhibition at 50 ng/mL) and also exhibited promising cytotoxic activity
against P-388, A-549, HT-29 and MEL-28 with IC50 value of 12.5 g/mL each.
O
Me
Me
N
H2N
O
Me
250
251
HO
O
OCH3
CH3
N
H
N
O
H
O
252, R = H O
254, R = CH3
253
255
O
H
OH
O
N
O
Me
H
NCH3
H3CO
OCH3
CH3
H
NCH3
H3CO
O
H
O
OH
256
6. Guanidine alkaloids
In 1989, Kashman et al. reported the isolation of a novel guanidine alkaloid
ptilomycalin A (257) from the Caribbean sponge Ptilocaulis spiculifer and the red sea
sponge Hemimycale sp. [172]. Ptilomycalin A (257) consists of a pentacyclic
guanidine unit and a spermidine unit linked by a linear long-chain fatty acid.
252
Ptilomycalin A (257) exhibited significant cytotoxic activity against P-388, L-1210

and KB cell lines with IC50 values of 0.1, 0.4 and 1.3 mM, respectively.
H
N
N
O H
O
N
H O
CH3
CH3
H2N
H2N
N
O
257
Recently, Black et al. synthesized three novel analogues, 258, 259 and 260 of
ptilomycalin A (257) [173]. Compounds 258-260 were tested against four cancer
cell lines including human chronic myelogenous leukaemia (K-562), human
ovarian carcinoma (A-2780), human large cell carcinoma (H-460) and mouse
lymphoid neoplasm (P-388). Compound 258 showed the best activity against all
the cell lines comparable to the parent compound (257). The IC50 values of 0.52,
0.92, 0.52 and 0.69 g/mL were obtained against K-562, A-2780, H-460 and
P-388, respectively for compound 258, whereas compound 259 was found to be
less potent than compound 258. Compound 260 was the least active compound of
the three, which indicated that the presences of a spacer chain and spermidine
residue are essential for the compounds to demonstrate the biological activity.
H2N
H2N
O
NH
Cl 2CF3COOH
NH
O
O
NH
Cl 2HCl
NH
O
N
O
258
H2N
H2N
O
O
H
259
O
NH
H
N
H
NH
O
260
BF4
253
Seven new tricyclic guanidine alkaloids, netamines A-G (261-267) were

isolated from the extract of the poeciloscleridae sponge Biemna laboutei collected
near the Sainte-Marie Island on the east coast of Madagascar [174]. The
structures of compounds were determined on the basis of 1D, 2D NMR and
HRFABMS data interpretation. All the compounds 261-267 were evaluated for
cytotoxicity against three human tumor cell lines: NSCL (A-549), colon (HT-29)
and breast (MDA-MB-231). Only netamines C (263) and D (264) showed
promising activity against A549 (GI50 = 4.3 and 6.6 M) HT29 (GI50 = 2.4 and
5.3 M) and MDA-MB-231 (GI50 = 2.6 and 6.3 M), whereas other compounds
were found to be inactive or very less toxic.
NH
HN
NH
NH
HN
NH
NH
CH3
HN
CH3
CH3
261
CH3
263
262
NH
NH
HN
NH
NH
NH
HN
NH
CH3
CH3
265
HN
CH3
CH3
CH3
264
CH3
CH3
CH3
HN
NH
CH3
CH3
CH3
266
267
7. Aminoimidazole alkaloids
Ralifo et al. reported the isolation and structure elucidation of two novel
alkaloids, leucosolenamines A (268) and B (269) from the marine sponge
Leucosolenia sp. [175]. Compound 268 was found to contain a 2-aminoimidazole
unit substituted at C-4 and C-5 by an N,N-dimethyl-5,6-diaminopyrimidine-2,4dione and a benzyl group, respectively. Although, compound 269 has the same core
structure but C-4 is substituted by a 5,6-diamino-1,3- dimethyl-4-(methylimino)3,4-dihydropyrimidin-2(1H)-one moiety. This substitution pattern is unique and had
never been observed in imidazole alkaloid chemistry. Leucosolenamine A (268)
exhibited mild cytotoxicity against the murine colon adenocarcinoma C-38 cell line,
whereas compound 269 was inactive. In the same year the other group isolated two
new imidazole alkaloids, naamidines H (270) and I (271) from the marine sponge
Leucetta chagosensis collected in North Sulawesi, Indonesia [176]. The compounds
270 and 271 demonstrated weak cytotoxicity against HeLa cells with IC50 values of
5.6 and 15 g/mL, respectively.
254
O
H2N
H
N
CH3 CH3
N
N
O
CH3
N
O
H2N
CH3
H
N
CH3
HO
N
H
OMe
N
O
HN
HN
MeO
N
R
O
O
268
269
OMe
270, R = O
271, R = NMe
8. Steroidal alkaloids
Four novel steroidal alkaloids, plakinamine G (272), plakinamine H (273), 4Rhydroxydemethylplakinamine B (274) and tetrahydroplakinamine A (275) were
isolated from the marine sponge Corticium sp. [177]. The structures of these
compounds were established spectroscopically mainly by 1D, 2D NMR and mass
spectrometry (HR-EIMS). Compounds 272-275 were tested for cytotoxicity against
rat glioma (C6) and murine macrophages (RAW-264) cell lines. Compounds 272 and
275 found to be the most active against C6 cells with IC50 values of 6.8 and 1.4
g/mL, respectively, whereas they showed no activity against RAW-264 cell line.
Compounds 273 and 274 were cytotoxic against both the cell lines with compound
273 being more active against C6 cells (IC50 = 9.0 g/mL) than to RAW-264 (IC50 =
61 g/mL), while compound 274 showed greater value of IC50 (16.2 g/mL) against
RAW-264 cell line than to C6 cells (IC50 = 26.1 g/mL). One year later four new
related steroidal alkaloids, plakinamine I-K (276-278) and dihydroplakinamine K
(279) were isolated from the same sponge Corticium niger [178]. Compounds (276279) as their hydrochloride salts were evaluated for cytotoxicity against the human
colon tumor cell line (HCT-116). Compounds 278 and 279 were found to be the most
active in terms of potency with an IC50 value of 1.4 M each. Compounds 276 and
277 were moderately active with IC50 values of 10.6 and 6.1 M, respectively.
Ritterazines B (280) and C (281), two dimeric steroidal alkaloids were isolated
from the tunicate Ritterella tokioka collected off the Izu Peninsula [179]. Their
structures including absolute stereochemistry were assigned by spectral and chemical
methods. Ritterazines B (280) and C (281) displayed potent cytotoxicity against the
P-388 murine leukemia cells with IC50 values of 0.018 and 9.4 ng/mL, respectively.
Three novel steroidal alkaloids, cortistatins J-L (282-284) were isolated from the
Indonesian marine sponge Corticium simplex [180]. The structures of compounds
282-284 were established by 1D and 2D NMR (COSY, HMQC and HMBC) data
analysis. Cortistatin J (282) demonstrated potent cytostatic anti-proliferative activity
255
against human umbilical vein endothelial cells (HUVEC) with IC50 value of 8 nM and
also inhibited migration and tubular formation of HUVEC induced by VEGF or
bFGF, whereas cortistatins K (283) and L (284) were less potent than cortistatin J
(282) with IC50 values of 40 and 23 nM, respectively.
NH
HN
O
H2N
N
O
272
273
N
HN
H2N
H2N
OH
274
275
H
N
H
NH2
276
277
HN
HN
H
H
N
H
N
H
H
OAc
H
OAc
278
279
OH
H
H
HO
HO
OH
HO
280
282
O
OH
H
H
HO
H
O
OH
H
N
N
R
O
HO
HO
H
N
281
283, R = H
284, R = OH
256
9. Miscellaneous alkaloids
Four novel alkaloids 285-288, related to aaptamines were isolated from the
MeOH extract of the Indonesian marine sponge Xestospongia sp. collected
from Jakarta along with the known aaptamine (289), isoaaptamine (290),
demethyl(oxy)aaptamine (291) and its dimethylketal (292) [181]. Their structures
were determined on the basis of 1D and 2D NMR spectroscopic data. All the
compounds 285-292 were evaluated for cytotoxic activity against KB cell lines.
Compounds (289-292) exhibited moderate cytotoxicity against KB cells with ID50
values of 3.7, 0.5, 1.8 and 3.5, respectively, while compounds (285-288) were
less potent with ID50 value of >10 g/mL.
Four tetracyclic alkyl-piperidine alkaloids, Haliclonacyclamie E (293)
arenosclerins A (294), B (295) and C (296) were isolated from the marine sponge
Arenosclera brasiliensis [182]. All the compounds were tested for their
cytotoxicity against HL-60, B-16, U-138 and L-929 cancer cell lines. Compound
293-296 exhibited almost the same range of cytotoxicity with IC50 values of 4.23,
4.31, 4.07, 3.65 (HL-60), 1.82, 1.77, 1.76, 1.71 (B-16), 6.06, 3.83, 3.62, 3.60 (U138) and values of 3.89, 2.34, 2.24, 2.17 (L-929), respectively.
R2
O
R1
OCH3
OCH3
H3CO
H3CO
N
N
N
285, R1 = H, R2 = CH3
286, R1 = CH3, R2 = H
287
288
OCH3
OCH3
HO
OCH3
OCH3
N
R
N
N
289
N
N
290
291, R = CH(CH3)2
292, R = H
Four bis-piperidine alkaloids, madangamine F (297), haliclonacyclamine F

(298), arenosclerins D (299) and E (300) were isolated from the marine sponge
Pachychalina alcaloidifera [183]. Compounds 297-300 were evaluated for
cytotoxicity against SF-295 (human CNS), MDA-MB-435 (human breast), HCT8 (colon) and HL-60 (leukemia) cancer cell lines. Haliclonacyclamine F (298)
and arenosclerin D (299) were found to be the most active compounds with IC50
values of 4.5 and 5.9 g/mL (SF-295), 1.0 and 1.2 g/mL (MDA-MB-435), 8.6
and 6.2 g/mL (HCT-8), 2.2 and 6.2 g/mL (HL-60), whereas compounds 297
257
and 300 showed IC50 values of 19.8 and 8.7 g/mL (SF-295), 16.2 and 3.1 g/mL
(MDA-MB-435), 16.7 and 6.9 g/mL (HL-60), >25 and >25 g/mL (HCT-8) cell
lines.
H
N
N
H
H
H
H
H
H
H
H
H
297
HO
295
N
N
OH
298
HO
294
H
H
HO
293
296
N
H
H
N
HO
299
H
N
H HO
300
Matsunaga et al. reported the isolation and structure determination of two

new 3-alkylpiperidine alkaloids, tetradehydrohalicyclamine A (302) and 22
hydroxyhalicyclamine A (303) along with a known halicyclamine A (301) from a
marine sponge Amphimedon sp. collected in southern Japan [184]. Compounds
301, 302 and 303 were found to be cytotoxic against P-388 cells with IC50 values
of 0.45, 2.2 and 0.45 g/mL, respectively.
Three new diketopiperazine alkaloids, 6-methoxyspirotryprostatin B (304),
18-oxotryprostatin A (305) and 14-hydroxyterezine D (306) along with other
metabolites were isolated from the ethyl acetate extract of a marine-derived
fungal strain Aspergillus sydowi [185]. All the compounds were evaluated for
Cytotoxicity against A-549 and HL-60 cell lines. Compounds 304-306 exhibited
weak cytotoxicity against A-549 cells with IC50 values of 8.29, 1.28 and 7.31 M,
respectively. In addition, compound 304 also demonstrated significant
cytotoxicity against HL-60 cells with an IC50 value of 9.71 M.
Two novel pyrazine alkaloids botryllazine A (307) and botryllazine B (308)
along with the new imidazole alkaloid 2(p-hydroxybenzoyl)-4-(phydroxyphenyl)-imidazole (309) were isolated from the red ascidian Botryllus
leachi [186]. The structures of compounds 307-309 were elucidated by
interpretation of spectral data and botryllazine A (307) was supposed to
represents the first example of a marine alkaloid containing a pyrazine nucleus
derived from three tyrosine precursors. All the compounds (307-309) were
tested in vitro for cytotoxicity against P-388 mouse lymphoma, A-549 human
lung carcinoma, HT-29 human colon carcinoma and MEL-28 human
melanoma. Botryllazine A (307) was inactive with ED50 value of 10 g/mL each.
258
NH
NH
H
NH
N 2CHF3CO2
NH 2CHF3CO2
H
NH 2CHF3CO2
H
OH
301
302
O
MeO
303
H
HN
O
NH
HN
O
HN
O
N HO
H
N
O
MeO
304
305
306
Botryllazine B (308) exhibited weak cytotoxicity (ED50 = 5 g/mL) against

A-549 and MEL-28 cell lines, whereas compound 309 was mildly active against
all the four tumor cell lines with ED50 of 5 g/mL.
OH
OH
N
O
O
NH
OH
OH
HO
OH
OH
307
308
309
Kobayashi et al. isolated novel bromotyrosine alkaloids, maedamines A

(310) and B (311) from Okinawan marine sponge Suberea sp. [187].
Structures were elucidated on the basis of spectroscopic data and these
compounds were found containing a 2(1H)-pyrazinone moiety between two
bromotyrosine units. Maedamines A (310) and B (311) exhibited in vitro
cytotoxicity against murine leukemia L-1210 cells with IC50 values of 4.3 and
3.9 g/mL, respectively and epidermoid carcinoma KB cells with IC50 values
of 5.2 and 4.5 g/mL, respectively. Maedamine A (310) also demonstrated
inhibitory activity against c-erbB-2 kinase with IC50 value of 6.7 g/mL,
while compound 311 being inactive against c-erbB-2 kinase (IC50 >10
g/mL).

Br
H3CO
259
H
N
Br
310, R = CH3
311, R = H
N
R
Br
Two new bromotyrosine alkaloids, purealidin S (312) and purpuramine J

(313) were isolated from the Fijian marine sponge Druinella sp. [188].
Compound 313 contains a bromotyrosine N-oxide unit which is very rarerly
found in marine natural products. Both the compounds were tested for
cytotoxicity against A-2780 (Ovarian tumor) and K-562 (leukaemia) cell lines.
Compounds 312 and 313 showed mild cytotoxicity against these two cell
lines with IC50 values of 7.44 and 6.77 g/mL (A-2780) and values of 6.02 and
1.24 g/mL (K-562), respectively.
OMe
Br
HO
Br
O
N
H
N
O
Br
Br
H2
N
OH
H
N
Br
O
Br
312
NH
OH
313
Two new dimeric polysulfide alkaloids, lissoclinotoxins E (314) and F (315)

were isolated from the MeOH extract of a Philippine didemnid ascidian [189].
The polysulfide structures for compounds 314 and 315 were determined
by interpretation of spectroscopic data and chemical means. Computational
chemistry studies suggested the trans- and cis- orientations of N-alkyl chains
about the tricyclic systems of lissoclinotoxins E (314) and F (315), respectively.
Compounds 314 and 315 exhibited significant cytotoxicity against PTENdeficient human breast carcinoma, MDA-MB-468 cell lines with IC50 values of
2.3 and 1.5 g/mL, respectively.
Williams et al. isolated motuporamines A-C (316-318) from the marine
sponge Xextospongia exigua [190]. The crude mixtures of motuporamines A-C
could not readily be separated and they were obtained as a mixture of three (316318). The mixture of motuporamines A-C (316-318) showed significant
cytotoxicity against a panel of human solid tumor cancer cell lines with IC50
value of 0.6 g/mL.
260

N
OMe
OMe
MeO
SMe
MeO
S S
MeS
OMe
MeS
OMe
OMe
SMe
314
315
X
N
H
N
R
H2N
N
H
316, R = H, X = CH2
317, R = H, X = (CH2)2
318
Two novel alkaloids, pterocellins A (319) and B (320) were isolated from the
New Zealand marine bryozoans Pterocella vesiculosa [191]. The structures were
assigned by NMR and mass spectral data analysis and finally structure was
confirmed by single-crystal X-ray diffraction experiments. Pterocellins A (319)
and B (320) were evaluated for cytotoxicity against P-388 murine leukemia cell
lines and exhibited relatively potent activity with IC50 values of 477 and
323 ng/mL, respectively.
O
O
O
O
O
N
N
319
320
The cytotoxicity of pterocellins A (319) and B (320) was also evaluated by

the NCI in their 60 cell line panel, which represents a variety of human tumor cell
types such as leukemia, non-small cell lung, colon, central nervous system
(CNS), melanoma, ovarian, renal, prostate and breast cancers. Compounds 319
261
and 320 exhibited potent cytotoxicity with panel average values of GI50 = 1.4 M,
TGI = 4.8 M, LC50 = 17.0 M for pterocellin A (319) and GI50 = 0.7 M,
TGI = 2.1 M, LC50 = 6.9 M for pterocellin B (320). The leukemia cell line
(CCRF-CEM) was found to be the most sensitive cell line to pterocellin A (319)
with GI50 value of 0.05 M and TGI value of 0.8 M, although the high LC50
value of >100 M implied that pterocellin A (319) is cytostatic rather than
cytotoxic to this cell line. The most sensitive cell line to pterocellin B (320) was
the melanoma cell line MALME-3M with GI50 value of 0.03 M and TGI value
of 0.1 M, whereas cell lines such as NCI-H23, melanoma MALME-3M, M14,
SK-MEL-5, breast MDA-MB-435 and MDA-N were found to be sensitive to
both the compounds.
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Research Signpost
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ISBN: 978-81-308-0448-4
8. Microtubule binding natural substances

in cancer chemotherapy
Ram C. Mishra
Department of Biology, Georgia State University, P O Box 4010, Atlanta, Georgia
30302-4010, USA
Abstract. Microtubules constitute the major part of the

cytoskeleton and play active role in cell division. Their dynamic
instability and role in spindle formation during mitosis makes them
an interesting target for anti-cancer drug development. Natural
products are well known to be utilized for improving the human
health. There are many natural products currently in use for
providing cure to all kinds of diseases including cancer. Taxol and
Vinblastine are examples of such natural products which interact
with tubulin and are used in chemotherapy of cancer. This article
briefly describes the microtubule binding natural substances and
their use as anticancer agents.
1. Introduction
Natural products have shown to be the major source of anticancer drugs.
In the last 25 years more than 60 % of the anticancer drugs are either natural
products or have natural product origin [1]. Microtubules are one of the major
components of the cytoskeleton which are essential for many cellular
processes including maintenance of cell structure, protein transportation
and mitosis. These are also referred to as conveyer belts inside the cell [2].
The microtubules are composed of a group of cylindrical proteins
know as tubulins and perform many of their functions by binding to MAPs i.e.
Correspondence/Reprint request: Dr. Ram C. Mishra, Department of Biology, Georgia State University
P O Box 4010, Atlanta, Georgia, 30302-4010, USA. E-mail: mishra@rcmishra.in
270
Ram C. Mishra
microtubule associated proteins. Microtubules are directly involved in the

formation of mitotic spindle which helps in segregating the replicated
chromosomes towards two daughter nuclei at the end of mitosis. Involvement
of microtubules in this particular cell cycle event makes them an important
target in cancer chemotherapy [3]. The anticancer activity of taxanes and
vinca alkaloids is attributed to their affinity and binding ability to tubulin
units of the microtubules.
2. Microtubule
chemotherapy
structure
and
target
for
cancer
During the cell division i.e. mitosis, microtubules play an important role
in segregation of chromosomes via spindle formation. Microtubules as the
name suggests are the hollow tube like structures with a diameter of 15-25
nanometer and form the major part of the cytoskeleton. Their length may
vary from 200 nm to 25 micrometers. This hollow structure is formed by an
imperfect helix like arrangement of the protofilaments. The protofilaments in
turn, are the product of end to end polymerization of the tubulin heterodimers
namely alpha tubulin and beta tubulin. Polarity is another feature of the
microtubule structure as during the end to end polymerization process alpha
subunit of one tubulin dimer is attached to beta subunit of the other.
This leads to the formation of protofilaments with beta subunits exposed
at one end and alpha subunit at the other. These are designated as plus
(+) and minus () ends respectively. In a microtubule the protofilaments
bundle parallel to each other so that there is one end with beta tubulin
subunit (plus end) exposed and the other with alpha (minus end) unit
exposed. The minus end is capped, so that elongation occurs from the plus
end [4].
The mitotic spindles are formed by attachment of GTP-tubulin to the
growing end of the protofilament. The microtubules undergo rapid assembly
and disassembly leading to their dynamic instability [5,6]. This dynamic
instability along with their involvement in mitotic spindle formation helps
in the metaphase to anaphase transition of the mitosis. This continued
assembly and disassembly process in microtubules are crucial to the
normal cell division and any interference in this leads to cell death via
apoptosis. Usually the anti-mitotic agents arrest the metaphase to anaphase
transition in mitosis. The defective spindles formed due to disturbances in
dynamics of microtubules at low concentrations of the anti-mitotic agent
are unable to cross the mitotic spindle checkpoint and initiate the anaphase
stage. This leads to prolonged mitotic arrest and finally cell death by
apoptosis.
271
Figure 1. Microtubule architecture.

Table 1. Diverse origin of taxane-domain binding drugs.
ORIGIN
DRUG
SOURCE
PLANT
Paclitaxel
Docetaxel
Taxus brevifolia (Yew tree bark)

Taxus baccata (semi-synthetic)
10-deacetylbaccatin III
BACTERIAL Epothilones
Cyclostreptin
MARINE
Discodermolide
Dictyostatin
Laulimalide
CORAL
Peloruside
Eleutherobin
Sarcodictyins
Taxus brevifolia (Yew tree leaves)

Sporangium cellulosum
(myxobacterium)
Streptomyces sp.
Discoderma dissolute (marine
sponge)
Spongia (marine sponge)
Hyattella sp. and Fasciospongia
rimosa (marine sponges)
Mycale hentscheli (marine sponge)
Eleutherobia sp. (soft coral)
Sarcodictyon roseum (soft coral)
The beta unit of the tubulin heterodimer has the priority over the alpha
unit in interaction with the drugs. Its structure has been solved by electron
diffraction [7]. Beta-tubulin has the binding sites for both the taxane drugs
and the vinca alkaloids at different locations. The taxane drug, paclitaxel
binds on two sites of the beta subunit, the N-terminal unit and the region
between the amino acids 217-231 [8]. The vinca alkaloid drugs also bind to
same beta subunit but in the region bound by amino acids 175 and 213 [9].
272
Ram C. Mishra
The extensively studied natural ligand of the tubulin, colchicine, binds

between the two subunits and is not used clinically as anticancer drug.
Another group of the natural products known as epothilones also bind to
tubulin at its taxane binding site [10].
The natural products binding to the tubulin can affect its
dynamics either by promoting or by inhibiting the polymerization process.
Based on this general characteristic the tubulin binding natural products
have been classified as under inhibitors or promoters of the tubulin
polymerization.
3. Promoters of tubulin polymerization

The microtubule polymerization promotors can be broadly classified in
to the taxanes and the epothilones both of which bind to same domain of the
beta subunit of the tubulin heterodimer. Apart from these two classes, there
are few more compounds which are known for their tubulin polymerization
properties. In the following table the diverse origins of the drugs binding to
taxane domain and their source has been presented.
3.1. The taxanes

Paclitaxel has been the main chemotherapeutic agent for the various
types of cancers including breast, ovarian and the prostate cancer. This
compound was first isolated and reported from the pacific yew tree bark in
1960 and named as Taxol [11]. Its mechanism of action was discovered in
1980s. The new and currently used generic name Paclitaxel was given when
the drug was developed commercially by Bristol-Mayers Squibb and sold
under the trade name Taxol. The drug is also used in chemotherapy of
NSCLC in combination with Cisplatin [12].
273
The success of the paclitaxel led to the development of many of its

analogs which are currently in clinical trials. The only analog approved in
USA is the Docetaxel, which is a semi-synthetic analog and was developed in
France [13]. Apart from the paclitaxel and docetaxel which are the only
approved taxanes in therapeutic use there are many analogs in different
phases of in clinical trials which are mostly the semi-synthetic analogs
starting from 10-deacetylbaccatin III [14].
10-Deacetylbaccatin-III
3.2. Epothilones
Epothilones belong to macrolide class of the drugs and act as
microtubule stabilizers. They are produced by Myxobacterium Sorangium
cellulosum and initially found to have antifungal and cytotoxic activity [15].
Later, the cytotoxic activity of these epothilones A (R = H) and B (R = Methyl)
was found to be associated with mitotic arrest, which occurs via over
polymerization of microtubules. Patupilone which is a natural epithilone B
derivative is in phase III clinical trials by Novartis for the ovarian cancer.
It has been found to be many times more effective than paclitaxel and also
crosses the blood-brain barrier [16,17]. Initially another epothilone B
derivative, Ixabepilone [18] has shown to be of clinical use however later it
was dropped from further development.
3.3. Other compounds

Apart from the two major classes of the compounds described above with
tubulin polymerization promoter activity, there are some other recently
discovered compounds which have been shown to possess tubulin
polymerization properties. These include Discodermolide [19], Laulimalide
274
Ram C. Mishra
[20] and Eleutherobin [21,22]. Discodermolide along with Dictyostatins

was isolated from marine sponges. The sponges producing them use the
microtubule toxins as part of their self-defense mechanism. Although
development of the Discodermolide has been stopped there is a possibility of
its derivatives to become a clinical candidate in near future. Eleutherobon
was isolated [22] from corals and have similar binding properties as that of
paclitaxel. A total synthesis has been developed for this molecule, although it
is not yet in clinical trials [23]. Laulimalide which also stabilizes the
microtubules has a different binding site on tubulin in contrast to Paclitaxel.
It has potential to kill paclitaxel and epothilone resistant cells and a total
synthesis for this molecule has also been reported.
HO
O
OH
OH
OH
H
HO
O
O
OH
O
H
NH2
Discodermolide
O
Me
O
H
N
Me
O
O
O
Me
OMe
O
AcO
OH
Laulimalide
O
Eleutherobin
OH
275
4. Tubulin polymerization inhibitors

Vinca alkaloids constitute the major class of the compounds that inhibit
the polymerization of tubulins. Other important compounds in this category
include the Combretastatins, Dolastatins, Noscapine analogs, Hemisterlin and
Rhizoxins. The Table 2 gives a summary of the compounds with tubulin
destabilizing activity along with their natural origin and chemical nature.
Table 2. Vinca-domain binding drugs of diverse origin.
Name of the
drug
Source
Chemical nature
Plant origin
Vinca alkaloids
Alkaloids
Vinblastine
Vincristine
Vinorelbine
Catharanthus roseus
(Vinca rosea) and analogs
Vinflunine
Vindesine
Maytansinoids
Maytansine
Ansamitocins
Maytenus ovatus
Macrolide
Nocardia
Macrolide
Marine origin
Dolastatin 10
Dolastatin 15
Halichondrin
Spongistatin 1
Dolabella auricularia
Halichondira okadai Kadota
Hyrtios altum
Pseudo peptide
Lactone polyether
Macrocyclic lactone
Fungal origin
Rhizoxin
Rhizopus chinensis
Macrocyclic lactone
Phomopsin A
Phomopsis leptostomiformis
Peptide
Ustiloxin
Ustilaginoidea virens
Peptide
276
Ram C. Mishra
4.1. Vinca alkaloids

The vinca alkaloids vinblastine and vincristine were the first natural
products to enter in the clinical use for cancer chemotherapy. These
compounds were isolated by two different research groups in late 1950s and
early 1960s from Madagscar periwinkle known as Vinca rosea or
Catharanthus roseus [24].
One of the groups working on them was interested in finding a substance
affecting the blood glucose levels. However, at the same time they also
noticed the effect of the extract on the white blood cell counts. This lead to
the discovery of its antileukemic activity and finally the isolation and
structure elucidation of vincaleukoblastin which was later shortened to
vinblastine [25].
OH
N
H
N
H
O
O
O
N
H
N
O
O
Vinblastine
H
OH O
O
The vinca groups of alkaloids binding to the beta subunit of tubulin are
constituted by several closely related compounds. These include vincristine,
vindesine, vinorelbine and vinflumine, which are the semisynthetic vinca
alkaloids. Vinblastine and vincristine are in clinical use as anticancer drugs
since last 50 years. They are also used in combination therapy of acute
leukemias and lymphomas, bladder and breast cancers [26].
4.2. Combretastatins and derivatives

Although the vinca alkaloids are the only tubulin polymerization
inhibitor compounds which are in clinical use, there are several other groups
of compounds which bind to same domain of the tubulin and have similar
mechanism of action. Many of these analogs are in advanced stages of
clinical trials e.g. Combretastatins, which were isolated from the root bark of
Combretum caffrum [27].
277
OH
OMe
MeO
OMe
OMe
Combretastatin A4
They are well-known as antimitotic agents and Combretastatin A2 (CA2) &

Combretastatin A4 (CA4) are the most potent members of this family. CA4 is
highly cytotoxic than its tubulin destabilizing activity [28]. The phosphorylated
CA4 known as CA4P has anti-angiogenic properties via the disruption of the
endothelial cytoskeleton [29]. This compound is in phase III trials for treatment of
cervical, colorectal, NSCLC, prostate and ovarian cancers [30,31].
4.3. Dolastatins
Pettit isolated Dolastatin 10 from Dolabella auricularia, the most potent
member of a big family of dolastatins [32]. It has a distinct binding site on
tubulin where usual antimitotic peptides bind [33]. In 1990 it entered the
clinical trials by NCI for solid tumor treatments. Another peptide, Dolastatin
15 is also as potent as Dolastatin 10 but in contrast to the later, it is not
involved in the nucleotide exchange inhibition and aggregation induction.
One of the Dolastatin 15 derivatives is in phase II clinical trials [34].
H H
N
H
O
N
H3C
N
H
O
H3CO
N
O
Dolastatin 10
N
H
4.4. Noscapinoids
The phthalideisoquinoline alkaloid from Papaver somniferum, Noscapine
is in medicinal use since long for its antitussive activity [35]. Currently this
molecule is in phase I-II clinical trials for the treatment of multiple myeloma.
Noscapine and its derivatives are different from other microtubule binding
drugs in the fact that they keep the total polymer mass of the tubulin
unaltered [36].
278
Ram C. Mishra
NH2
O
N
O
OCH3
CH3
H
OCH3
O
O
OCH3
S,R-(Alpha)-Noscapine
Stoichiometric binding of Noscapine induces a conformational change in

tubulin and interrupts the cell cycle in mitosis. It inhibits the dynamic
instatbility of tubulins by extending the relaxation time. Several analogs of
the parent molecule have been prepared and evaluated against diverse cancer
cell lines. It has been concluded that noscapinoids are the gentlest molecules
involved in the microtubule dynamics creating mitosis checkpoints without
any significant toxicity profile [37].
4.5. Eribulin and halichondrins

These complex natural products of marine origin were isolated from
western pacific sponge Halichondria okadai and also from Axinell sp [38,39].
This class of molecules have shown to be highly cytotoxic specially the
Halichondrin B and homohalichondrin. These compounds were shown to
bind to tubulin and inhibit their polymerization. They have shown subnanomolar activity in NCIs 60 cell anti-cancer screening panel along with
promising activity in many animal models [39]. The attempts towards the
total synthesis of Halichondrin B resulted in the discovery of Eribulin [40].
Similar to its parent, it also inhibits tubulin polymerization and is currently in
phase III clinical trials for several cancer types [41].
OH MeO
H3N
H
O
O
O
Eribulin
OH
279
4.6. Hemiasterlin
Hemiasterlin is a tripeptide of marine origin. It was first isolated from
Hemiasterella minor and found to be active against murine leukemia cell
lines [42]. Later, its antitubulin and antimitotic activity was discovered by
Anderson [43]. The phenyl alanine derivative of the parent compound,
HTI-286 [44] has been found to be more potent and more synthetically
accessible. Both these molecules are in clinical trials [45].
N
Me
Me
NH
N
H
Me
N
O
OH
Hemiasterlin
4.7. Rhizoxin
Rhizoxin was isolated from a plant pathogenic fungus Rhizopus chinensis
and was discovered to be inhibitor of tubulin polymerization [46]. It is a
macrocyclic lactone, although very similar to maytansine [47], it is
comparatively more potent against human and murine tumor cells. It has been
synthesized and gone through the clinical trials. The molecule is yet to be
approved for clinical use.
HO
O
O
N
O
O
OMe
Rhizoxin
280
Ram C. Mishra
5. Challenges and future prospects

Since last two decades the importance of tubulin dynamics as a target for
anticancer drug development has been increased significantly. The
established tubulin interactive drugs include the two vinca alkaloids and the
taxens, paclitaxel and docetaxel. Epothilones have just been made available
clinically. There are many candidates in phase II-III trials as described
earlier. It is now well know that almost all tubulin interactive agents are the
natural products. The supplies of these compounds for clinical use in near
future should be guaranteed. In future apart from the discovery of the new
tubulin interactive agents, the development of the novel noscapinoids,
taxanes, epothilones and other compounds will continue towards finding the
new and improved drug candidates. Next, the targeted drug delivery approach
would augment the current situation e.g. the use of nanoparticles for the
targeted delivery of Noscapine is under investigation [49]. Design of simpler
synthetic compounds taking the clue from molecular modeling studies and
better understanding of the tubulin interactions with known molecules would
also play a major role in development of new and improved anticancer agents
in future [50].
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Research Signpost
37/661 (2), Fort P.O.
Trivandrum-695 023
Kerala, India
ISBN: 978-81-308-0448-4
9. Natural products: Anti-fungal agents

derived from plants
Tasleem Arif, T. K. Mandal and Rajesh Dabur
National Research Institue of Basic Ayurvedic Sciences
Nehru Garden, Kothrud, Pune-411038, India
Abstract. As new spectrums of human fungal infections are

increasing due to increased cancer and AIDS patients. The
increased use of antifungal agents also resulted in the development
of resistance to these drugs. It makes necessary to discover new
classes of antifungal compounds to treat fungal infections. The
research on natural products and natural products derived
compounds has accelerated in recent years due to their importance
in drug discovery. Plants are rich source of bioactive secondary
metabolites of wide variety such as tannins, terpenoids, alkaloids,
and flavonoids, reported to have in vitro antifungal properties.
A series of molecules with antifungal activity against different
strains of fungus have been found in plants, which are of great
importance to humans and plants. These molecules may be used
directly or considered as a model for developing better molecules.
This review attempts to summarize the current status of reported
antifungal compounds from plants.
1. Introduction
The prevalence of resistance to antifungal agents significantly increased
in the past decade. Resistance to antifungal agents has important implications
for morbidity, mortality and health care in the community. Until recently,
Correspondence/Reprint request: Dr. Rajesh Dabur, National Research Institue of Basic Ayurvedic Sciences
Nehru Garden, Kothrud, Pune, MH-411038, India. E-mail: rajeshdabur@yahoo.com
284
Tasleem Arif et al.
fungi were not recognized as important pathogens because the annual

death rate due to candidiasis was steady from 1950 to 1970 [1, 2]. Since
1970, this rate increased significantly due to more widespread use of
immunosuppressive therapies, indiscriminate use of broad-spectrum
antibacterial agents, the common use of indwelling intravenous devices and
immunosuppressive viral infections such as AIDS. These developments and
the associated increase in fungal infections [3] necessitated the search for
new, safer, and more potent agents to combat serious fungal infections. For
nearly 30 years, amphotericin B, which causes significant nephrotoxicity,
was the sole drug available to treat serious fungal infections. The imidazoles
and the triazoles in late 1980s and early 1990s were major advances in safe
and effective treatment of local and systemic fungal infections. The high
safety profile of triazoles, in particular fluconazole, has led to their extensive
use. Fluconazole has been used to treat in excess of 16 million patients,
including over 300,000 AIDS patients, in the United States alone since the
launch of this drug [4]. Due to selective pressure and widespread use of these
few antifungal drugs, there have been increasing reports of antifungal
resistance [5].
Medicinal plants have been a source of wide variety of biologically
active compounds for many centuries and used extensively as crude material
or as pure compounds for treating various disease conditions. Relatively 1-10 %
of plants are used by humans out of estimated 250,000 to 500,000 species of
plants on Earth [6]. The plants are relatively cheap source of biological
material having a vast variety of metabolites, primary or secondary, available
in them for selecting the molecule of desired biological activity. Mainstream
medicine is increasingly receptive to the use of antimicrobial and other drugs
derived from plants, as traditional antibiotics become ineffective. Another
driving factor for the renewed interest in plant antimicrobials in the past
20 years is due to the rapid extinction rate of (plant) species [7]. The scientific
discipline, ethno botany, is utilizing the impressive array of knowledge
assembled by indigenous peoples about the plant and animal products they
have used to maintain health [8,9]. Lastly, the ascendancy of the human
immunodeficiency virus (HIV) has spurred intensive investigation into the
plant derivatives, which may be effective, especially for use in
underdeveloped nations. Few of the compounds isolated from plants such as
2-decanone, hydroxydihydrocornin-aglycones [10], various indole derivatives
[11] and isoflavanone are reported to have antifungal activities. However,
development of useful antifungal drugs from these compounds has not yet
been possible.
Plants derived anti-fungal agents
285
2. Major groups of antifungal compounds from plants

Plants have an almost limitless ability to synthesize aromatic substances
of different functional groups, most of which are phenols or their oxygensubstituted derivatives [12]. Most are secondary metabolites, of which at least
13,000 have been isolated that is less than 10% of the total [13]. In many
cases, these substances serve as plant defense mechanisms against predation
by microorganisms, insects, and herbivores. Some plants used for their odors
(terpenoids), pigment, (quinones and tannins) and flavor (terpenoid capsaicin
from chili peppers) were found to be endowed with medicinal properties.
Some of the herbs and spices used by humans as season food yield useful
medicinal compounds.
2.1. Simple phenols and phenolic acids

In recent years, numbers of studies have been reported on the antifungal
activity of phenolic compounds from natural sources. Some of the simplest
bioactive phytochemicals consist of a single substituted phenolic ring (Fig 1).
The common herbs tarragon and thyme both contain caffeic acid, a
representative of a wide group of phenylpropane-derived compounds which is
effective against fungi [14]. The site(s) and number of hydroxyl groups on
the phenol group are thought to be related to their relative toxicity to
microorganisms, with evidence that increased hydroxylation results in
increased toxicity [12]. In addition, it was also reported that more highly
oxidized phenols are more inhibitory [15, 16]. The mechanisms thought to be
responsible for phenolic toxicity to microorganisms include enzyme inhibition
by the oxidized compounds, possibly through reaction with sulfhydryl groups
or through more nonspecific interactions with the proteins [17].
OH
OH
HO
HO
OH
1
HO
OH
O
HO
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Tasleem Arif et al.
Tannins and salicylic acid are polyphenol compounds extracted from

Gaullher procumbens, Rhammus purshiand and Anacardum pulsatilla
showed antifungal activity [18,19]. Piper crassinervium, Piper aduncum,
Piper hostmannianum and Piper gaudichaudianum, contain phenolic
acid derivatives crassinervic acid (1), aduncumene, hostmaniane and
gaudichaudanic acid, respectively, were reported for fungitoxic activity [20].
Phenolic compound Eriosemaones AD (2, MIC 20 mg/mL) are reported to
have good antifungal activities [21]. Phenolic compound from Croton
hutchinsonianus [22], and pinosylvin (3), a constituent of pine, showed growthinhibitory activity against C. albicans and Saccharomyces cerevisiae [23].
Four
phenolic
amides,
dihydro-N-caffeoyltyramine,
trans-Nferuloyloctopamine, trans-N-caffeoyltyramine, and cis-N-caffeoyltyramine
isolated from Lycium chinense reported to have anti-fungal activity in a range of
5-10 g/mL [24a]. Three phenolic compounds, 1-galloyl--D-glucopyranosyl-(1->4)--D-galactopyranoside, 2-methoxy-5-(1,2,3'-trihydroxypropyl)-phenyl-1O-(6"-galloyl)--D-glucopyranoside and 2-methoxy-5-hydroxymethyl-phenyl-1O-(6"-galloyl)--D-glucopyranoside together with the known compounds from
the leaves of Baseonema acuminatum were reported for antifungal activity
against Candida albican strains with inhibitory concentration to 50% microorganism (IC50) values in the range of 25-100 g/mL [24b].
2.2. Flavonoids
Flavones are phenolic structures containing one carbonyl group and the
addition of a 3-hydroxyl group yields a flavonol (Fig 2). Flavonoids are
hydroxylated phenolic substances synthesized by plants in response to
microbial infection. They have been found to be effective antimicrobial
substances against a wide array of microorganisms. Their activity is probably
due to their ability to complex with extracellular and soluble proteins and to
complex with fungal cell walls. More lipophilic nature of flavonoids may also
disrupt fungal membranes [25].
Flavonoids isolated from the stem bark of Erythrina burtii [26] were
reported for antifungal activity. 4-methoxy-5,7-dihydroxyflavone 6-Cglucoside (isocytisoside) from the leaves and stems of Aquilegia vulgaris
showed activity against the mould A. niger [27]. Pelalostemumol (4) from
Pelalostemium had strong antifungal activity against many pathogenic fungi
[28]. Galangin (5), derived from the perennial herb Helichrysum aureonitens,
seems to be a particularly useful compound, since it has shown activity
against wide range of fungi [29]. A flavonoid from rhizome of Alpinia
officinarum had strong antifungal activity against variety of pathogenic fungi.
Minimum inhibitory concentration (MIC) against the fungi varied from
287
3 g/mL [30,31]. A flavon 3,4',5,7-tetraacetyl quercetin isolated from

heartwood of Adina cordifolia exihibited moderate antifungal activity against
A. fumigatus and Cryptococcus neoformans [32]. Flavonoid derivative
phloretin from Malus sylvestris have antifungal properties [33].
Isopiscerythrone (6), allolicoisoflavone A (7), piscisoflavones A (8) and B (9)
from different plants were reported to be endowed with antifungal activity [34].
HO
HO
OH
O
HO
O
OH
5
OH
O
HO
OH
OH
OH
4
HO
OH
OH
8
HO
OH
O
O
O
O
OH
HO
O
HO
OHO
OH
7
Heartwood extracts of Acasia auriculiformis and Acasia mangium

were reported to have antifungal activity due to the compounds 3,4',7,8tetrahydroxyflavanone and tetrracidin [35]. The four compounds
eupomatenoid-3, eupomatenoid-5, conocarpan and orientin, from Piper
solmsianum exhibited antifungal action against all the dermatophytes tested,
with MIC values in range of 2 to 60 g/mL and with a potency as high as
the standard antifungal drug ketoconazole [36]. Flavonoids, azulenes,
sesquiterpenes and essential oils from Inula viscosa were proved to have a
significant antifungal activity against dermatophytes even at low
concentrations (0.01 mg/mL). The high concentration of the sesquiterpene
288
Tasleem Arif et al.
(carboxyeudesmadiene), occurring in the leaf extracts, is reported to have

greater antifungal activity [37].
The 95% ethanol extract of the bark of Swartzia polyphylla afforded the
flavonoids biochanin A and dihydrobiochanin A as antifungal constituent and
another plant from the Fabaceae family Teramnus labialis was also reported
for antifungal flavonoid [38]. The antifungal activity of a series of prenylated
flavonoids purified from five different medicinal plants of moraceae family
was reported antifungal against C. albicans and S. cerevisiae [39]. These
results also support the use of prenylated flavonoids in traditional medicine to
treat fungal infections. A. nobilis furnished several flavonoids which showed
good fungicidal activity against C. cladosporioides [40]. Amentoflavone
from Selaginella tamariscina exhibited potent antifungal activity against
several pathogenic fungal strains but had a very low hemolytic effect on
human erythrocytes [41].
2.3. Coumarins
Coumarins have been reported to stimulate macrophages which could
have an indirect negative effect on infections. Coumarins are phenolic
substances made of fused benzene and -pyrone rings (Fig 3). Their fame has
come mainly from their antithrombotic50, anti-inflammatory [42] and
vasodilatory [43] activities and their use to prevent recurrences of cold sores
caused by HSV-1 in humans 48. Hydroxycoumarin scopoletin (10) was
isolated from seed kernels of Melia azedarach [44] reported to be antifungal
against Fusarium verticillioides.
O
HO
HO
O
O
O
OH
11
10
O
HOOC
OH
O
OCH3
H3CO
O
12
N
O
13
H
14
289
Tithoniamarin is a new isocoumarin dimer isolated from Tithonia

diversifolia [45] showed antifungal and herbicidal activities. Deng and
Nicholson [46] reported the antifungal properties of surangin B (11), a
coumarin from Mammea longifolia. Phytoalexins, which are hydroxylated
derivatives of coumarins, are produced in carrots in response to fungal
infection and can be presumed to have antifungal activity [47]. A coumarin
namely, 6,7-dimethoxycoumarin (12), isolated from P. digitatum-infected
Valencia fruit confers resistance against the mycotoxigenic fungi A. parasiticus
[48]. Clausenidin (13), dentatin, nor-dentatin, and carbazole alkaloid
clauszoline J (14) isolated from Clausena excavata showed antimycotic
activity (MIC 50 g/mL). Methylated clausenidin (MIC 50 g/mL), a
synthetic coumarin, also exhibited moderate antimycotic activity [50]. Data
about specific antibiotic properties of coumarins are scarce, although many
reports give reason to believe that some utility may reside in these
phytochemicals [51].
2.4. Quinones
Quinones are aromatic rings with two ketone substitutions and
characteristically highly reactive. Fig 4 shows some of the important
antifungal quinones. They can switch between diphenol (or hydroquinone)
and diketone (or quinone) easily through oxidation and reduction reactions.
These compounds, being colored, are responsible for the browning reaction in
cut or injured fruits and vegetables [52]. In addition to providing a source of
stable free radicals, quinones are known to complex irreversibly with
nucleophilic amino acids in proteins [53]. Therefore the quinone inactivate
the protein and impair there function. Quinones bind with surface-exposed
adhesins, cell wall polypeptides, membrane-bound enzymes and form
complex which inactivate the enzymes.
In the anthraquinone group, there are only a few reports concerning their
antifungal activity. Schmidt et al. [54] reported the antifungal activity of the
major anthraquinone aglycones, alizarin (15) and emodin (16) of Rubia
tinctorum and Rhamnus frangula. Hypericin (17), from Hypericum
perforatum, known as an antidepressant and Duke reported in 1985 that it had
general antimicrobial properties [14]. Examples of other antifungal
anthraquinones from medicinal species also included a new 1,3-dihydroxy-2methyl-5,6-dimethoxyanthraquinone (18) from the roots of Prismatomeris
fragrans [55]. The naphthoquinones kigelinone (19), isopinnatal, dehydroalpha-lapachone, and lapachol from Kigelia pinnata were reported for
antifungal activity [56].
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Tasleem Arif et al.

OH
O
OH
OH
OH
CH3
O
15
16
O
OH
18
CH3
OH
CH3
OH
17
O
OH
OH
O
OH
OH
HO
OH
OH
OH
HO
O
19
O
20
A novel compound 11-hydroxy-16-hentriacontanone isolated from

Annona squamosa was reported for its antifungal potential [57]. Hypericum
an anthraquinone extracted from Hypericum perforatum showed antifungal
activity [58]. The 2-hydroxy-1,4-naphthoquinone (Lawsone) (20) from
Lawsonia inermis were found to exhibit strong fungitoxicity [59]. Emodin,
physcion and rheins were isolated from Cassia tora showed strong fungicidal
activity against the microorganism tested [60]. Hopeanolin MIC value range
0.1-22.5 g/mL, an unusual resveratral trimer with an O-quinone nucleus, from
the stem bark of Hopea exalata is reported to have antifungal activity [61].
2.5. Saponins
Saponins are secondary metabolites that occur in wide range of plant
species (Fig 5). They are stored in plant cells as inactive precursors but are
readily converted into biological active antibiotics by enzymes in response to
pathogen attack. Saponins are glycosylated compounds widely distributed in
plant kindom and can be divided into three major groups, a triterpenoid, a
steroid or a steroidal glycoalkaloid. CAY-1, a triterpene saponin from the
Capsicum frutescens was found to be active against sixteen different fungal
strains, including Candida spp, A. fumigatus and C. neoformans [62].
Importantly, CAY-1 appears to act by disrupting the membrane integrity of
fungal cells.
Recently, steroidal saponins ypsilandroside B, ypsilandroside A, iso
ypsilandroside A, iso ypsilandroside B and isoypsilandrogaine isolated from
Ypsilandra thebetica were reported for antimicrobial activities by [63]. Two
new spirostanol saponins were isolated from the roots of Smilax medica,
291
together with the known smilagenin 3-O--D-glucopyranoside (21)

exhibited antifungal activity against the human pathogenic yeasts C. albicans,
C. glabrata and C. tropicalis in a range of 6.25-50 g/mL [64]. Mollugo
pentaphylla, a tropical herb, contains an antifungal saponin, mollugogenol-A
(22) [65]. Phytolaccosides B (23) and E (24) from Phytolacca tetramera
showed antifungal activities against a panel of human pathogenic
opportunistic fungi [66]. Novel spirostanol saponins together with three
known saponins were reported for antimycotic activity. The most active
compound was found to be 6-O-[-D-xylopyranosyl-(13)--Dquinovopyranosyl]-(25,S)-5-spirostan-3-ol, with IC50 values of 25 g/mL
against T. mentagrophytes and T. rubrum [67]. From Solanum species,
Solanum chrysotrichum five new spirostan saponins showed antimycotic
activity against T. mentagrophytes, T. rubrum, A. niger and C. albicans.
Another compound isolated from same plant, 6--O--D-xylopyranosyl(1-->3)--D-quinovopyranosyl-(25R)-5-spirostan-3,23-ol was reported
to be active in a rage of 12.5 to 200 g/mL against T. mentagrophytes,
T. rubrum, A. niger and C. albicans [68]. Recently, saponins isolated from
Alternanthera tenella is reported to have strong antifungal in the range varied
from (MIC) 50-500 g/mL [69].
Bioassay-guided fractionation of the ethanol extracts of the aerial parts of
the Tibetan medicinal herb Clematides tangutica led to the isolation of two
new antifungal triterpene saponins 3-O--L-arabinopyranosyl hederagenin
28- O-- L-rhamnopyranosyl ester and 3-O--D-glucopyranosyl-(1-->4)-L-arabinopyranosyl hederagenin 28-O--L-rhamnopyranosyl ester (MIA
2.5 g/disc) [70]. Two dammarane saponins from the stems of
Anomospermum grandifolium jujubogenin 3-O--l-arabinofuranosyl(1-->2)[-D-glucopyranosyl(1-->6)-D-glucopyranosyl(1-->3)]--l-arabinopyranoside,
ujubogenin 3-O--l-arabinofuranosyl(1-->2)-[6-O-[3-hydroxy-3-methylglutaryl]-D-glucopyranosyl(1-->3)]--l-arabinopyranoside and a new lupane saponin,
3-hydroxylup-20(29)-en-27,28-dioic acid 28-O--D-glucopyranosyl(1-->2)-[D-xylopyranosyl(1-->3)]--D-xylopyranosyl(1-->2)--D-glucopyranoside ester,
jujubogenin 3-O--l-arabinofuranosyl(1-->2)-[-D-glucopyranosyl(1-->3)]--larabinopyranoside and 3-hydroxylup-20(29)-ene-27,28-dioic acid revealed
antifungal properties against C. albicans [71].
From the rhizomes of Dioscorea cayenensis, the dioscin (25) exhibited
antifungal activity against the human pathogenic yeasts C. albicans, C. glabrata
and C. tropicalis [72]. Three antifungal steroidal saponins were isolated from the
root of Smilax medica [73, 74]. Saponins, named minutoside A, minutoside B,
minutoside C, sapogenins, alliogenin and neoagigenin, were isolated from the
bulbs of Allium minutiflorum showed promised antifungal activity [75].
292
Tasleem Arif et al.
H
O
OH
HO
HO
OH
H
O
OH
OH
OH
OH 22
21
HO
HO
O HO
H
HO
HO
OH
HO
HO
OH
HO
23
O
O
HO
HO
HO
O
OH
HO
O
24
Nineteen saponins from Medicago sativa, M. murex, M. arabica and

M. hybrida, were were reported to be active against three dermatophytic
fungi Microsporum gypseum, T. interdigitale and T. tonsurans [76].
OH
HO
OH
O
HO
HO
O
HO
O
OH
O
O
25
O
OH
O
293
Two saponins from Tribulus terrestris were reported for promised

antifungal activity against fluconazole resistant Candida strains
(MIC 0.15 mg/mL) [77,78]. Tigogenin-3-O--D-xylopyranosyl (1-->2)-[-Dxylopyranosyl (1-->3)]--D-glucopyranosyl (1-->4)-[-L-rhamnopyranosyl
(1-->2)]--D-galactopyranoside was reported to have in vivo activity in
C. albicans vaginal infection model. Another saponin from same plant,
tigogenin-3-O--D-glucopyranosyl (1-->2)-[-D-xylopyranosyl (1-->3)]-D-glucopyranosyl (1-->4)--D-galactopyranoside was found to be in vitro
very effective against several pathogenic Candida species (MIC80 = 4.4, 9.4
g/mL), C. neoformans (MIC80 =10.7, 18.7 g/mL) and inherently resistant
C. krusei (MIC80 = 8.8, 18.4 g/mL [79]. The Avenacin obtained from the
Avena sativa showed varying degree of in vitro antifungal activity [80]. Other
antifungal saponins from medicinal plants also included Astragalus verrucosus
[81], A. suberi [82], A. auriculiformis [83] and Hedera taurica which possessed
in vitro antifungal activity against C. albicans, C. krusei and C. tropicalis [84].
Saponins from several plants i.e Hedera colchica [85], Kalopanax pictus [86],
Dracaena mannii and D. arborea [87], Trillium grandiflorum [88] and
Solidago virgaurea [89] were reported for antifungal activity.
2.6. Xanthones
Xanthones are a restricted group of plant polyphenols, biosynthetically
related to the flavonoids. These are planar-six carbon molecules in a
conjugated ring system consisting of a backbone molecule and various
chemical groups attached to it. Xanthone backbone consists of two benzene
rings attached through a carbonyl group and oxygen not allowing free
rotation about the carbonZcarbon bonds. The unique backbone along with
type and position of the attached chemical groups defines specific properties
of xanthones. Xanthones possess numerous bioactive capability including
antifungal properties (Figure 6).
Caledonixanthone E (26) isolated from the stem bark of Calophyllum
caledonicum was reported for strong antifungal activity (MIC80 8 mg/mL)
[90]. Isoprenylated xanthones, toxyloxanthone C (27), and wighteone (28)
showed antifungal activity against C. albicans with MIC values of 25 and
12.5 mg/mL, respectively [91]. The dichloromethane extract of Securidaca
longepedunculata yielded 1,7-dihydroxy- 4-methoxyxanthone (29) which
exhibited antibacterial activity against Staphylococcus aureus and antifungal
activity against A. niger, A. fumigatus, and a Penicillum species [92]. 1,3,6Trihydroxy-2,5-dimethoxyxanthone (30) isolated from the aerial part of
Monnina obtusifolia was reported to have antifungal potential [93]. Seven
xanthanolides from Xanthium macrocarpum were reported to be effective
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Tasleem Arif et al.
OH
OH
O
OH
OH
OH
27
26
HO
OH
OH
28
HO
HO
O
O
HO
O
HO
OH
O
29
30
against C. albicans, C. glabrata, and A. fumigatus [94,95]. Two new

2-hydroxy-3-methylbut-3-enyl-substituted xanthones, -caledol and -dicaledol,
were isolated from a dichloromethane extract of the leaves of Calophyllum
caledonicum and have been reported for antifungal activity against
A. fumigatus [96]. Xanthones from the green fruits of Garcinia mangostana
were reported to have strong antifungal activities [97]. Cudrania fruticosa
yielded an isoprenylated xanthone, cudrafrutixanthone which showed
antifungal activity against C. albicans [98, 98]. Xanthone analogues bearing
the basic chain of butenafine were reported for significant activity against
C. neoformans (1.5 mg/mL) [100].
2.7. Terpenoids and essential oils

A large number of studies have been done in recent years on the
antifungal activity of terpenoids of natural origin (Fig 7). These reports
concern mainly sesquiterpenes and sesquiterpene lactones. The fragrance of
plants is carried in essential oil fraction. These oils are secondary metabolites
that are highly enriched in compounds based on an isoprene structure. They
are called terpenes, their general chemical structure is C10H16, and they occur
295
as diterpenes, triterpenes, and tetraterpenes (C20, C30, and C40), as well as

hemiterpenes (C5) and sesquiterpenes (C15). The mechanism of action of
terpenes is not fully understood but is speculated to involve membrane
disruption by the lipophilic nature. Mendoza et al. [101] found that increasing
the hydrophilicity of kaurene diterpenoids by addition of a methyl group
drastically reduced their antimicrobial activity.
A number of terpenes or terpenoids are reported active against fungi
[102-104]. In 1977, it was reported that 60% of essential oil derivatives
examined to date were inhibitory to fungi while 30% inhibited bacteria [105].
The antifungal activities of the essential oil from Agastache rugosa and its
main component, estragole (31), combined with ketoconazole, were reported
to show significant synergistic effects [106]. A known sesquiterpene lactone,
encelin (32), isolated from the Mexican species Montanoa speciosa
has a determining action on growth and the morphogenetic process of
fungal cells [107]. The roots of Delphinium denudatum have yielded
8-acetylheterophyllisine, panicutine, and 3-hydroxy-2-methyl-4H-pyran-4one (33) has shown antifungal activity against a number of human pathogenic
fungi [108]. Khaya ivorensis afforded methyl angolensate and 1,3,7trideacetylkhivorin displayed antifungal activity, with 62.8 and 64% mycelial
growth inhibition at 1000 mg/L, respectively [109]. Estragole and the
essential oil of A. rugosa exhibited strong activities against the tested fungi
and showed synergism with ketoconazole against B. capitatus [110]. From
other Centaurea species, C. thessala and C. attica, two eudesmanolides,
4-epi-sonchucarpolide, 8-(3-hydroxy-4-acetoxy-2-methylene-butanoyloxy)
derivative and eudesmane derivative named atticin (35) showed a
considerable antifungal effect against nine fungal species [111]. Two new
dammarane type triterepenes, ailexcelone and ailexcelol from Ailantus
excelsa were reported to be endowed with antifungal activities [112].
Amesterol, isolated from Amaranthus viridis strongly inhibited a growth of
pathogenic fungus [113,]. Two diterpenes isolated by Batista et al. [114]
were found to be active against Candida spp. Terpenoid isolated from Atrus
sinenisis, Armoracia rusticana, Metha peperita [115] and grapefruit showed
antifungal activity [116]. A. sativum oil exhibited the strongest inhibition of
growth of T. rubrum, T. erinacei, and T. soudanense with MIC of 4.0 g/mL,
while the activities of A. cepa and A. fistulosum were relatively mild [117].
The bark extract of Drimys brasiliensis led to the isolation of the
sesquiterpene polygodial, 1--(p-methoxycinnamoyl)-polygodial (36),
drimanial and 1--(p-cumaroyloxy)-polygodial which were selectively active
against fungi [118]. An antimicrobial diterpene 817-epoxylabd-12-ene-15,
16-dial from Alpinia galanga synergistically enhanced the antifungal activity
296
Tasleem Arif et al.
of quercetin and chalcone against C. albicans [119]. Triterpenoid glycosides

obtained from Solidago virgaurea and Bellis perennis inhibit the growth of
human-pathogenic yeasts (Candida and Cryptococcus species) [120].
O
O
O
O HO
O
31
O
33
32
O
O
O
O
O
O
34
HO
O
O
O
O
OH
H
OH
HO
36
35
The oil from leaves of J. oxycedrus spp. oxycedrus was reported

antifungal with MIC and MLC values ranging from 0.08-0.16 M/L and
0.08-0.32 M/L, respectively. The chemical constituents of the essential oil
extracted from the fruits of Lindera glauca have epishyobunol acetate,
caryophyllene oxide and 3,6,6-trimethyl-2-norpinene exhibited more
manifest antifungal properties with MIC between 0.03-0.5 mL/L for
pathogenic fungi species [121]. The essential oil from the leaves of
Litsea cubeba have cis-ocimene,3,7-dimethyl-1,6-octadien-3-ol and ntransnerolidol had manifest antifungal activities with MIC between 0.03-0.4
L/mL for utilized pathogenic fungi and 1.0-2.0 L/mL for moulds [122,].
The oil of the leaves, clemateol, and the alcohol from Calea clematidea
showed a moderate antifungal activity [123]. Daucus carota (Apiaceae),
afforded four sesquiterpene daucane esters [124] found to contain a range of
low antifungal activity against Fusarium oxysporum and A. niger. Carotol,
which was observed to be the main constituent of carrot seed, inhibited the
radial growth of fungi by 65%. The Vernonanthura tweedieana afforded one
297
antifungal active sesquiterpene, 6-cinnamoyloxy-1-hydroxyeudesm-4-en-3one [125]. Barrero et al. [126] investigated six Centaurea species:
C. bombycina, C. granatensis, C. monticola, C. incana, C. maroccana and
C. sulphurea of Astraceae family. The sesquiterpene lactones costunolide and
dehydrocostunolide showed noticeable IC50 values. Other antifungal
sesquiterpene lactones from the Asteraceae family also included those
isolated from Ajania fruticulosa [127].
A fruit pulp extract of Detarium microcarpum endowed with four
new clerodane diterpenes which showed antifungal activity [128]. The
diterpenoids 16-hydroxy-cleroda-3,13-(14)-Z-diene-15,16-olide and 16oxo-cleroda-3,13-(14)-E-diene-15-oic acid isolated from the hexane
extract of the seeds of Polyalthia longifolia demonstrated significant
antifungal activity [129]. Five new diterpenoids from Casimirella namely,
humirianthone, 1-hydroxy-humirianthone, 15R-humirianthol, patagonol and
patagonal showed activity against pathogenic fungi [130]. Antifungal
activity of oxygenated pimarane diterpenes from Kaempferia marginata
was reported by Thongnest et al. [131]. The triterpenoids pristimerin and
celastrol isolated from the roots of Celastrus hypoleucus exhibited
inhibitory effects against diverse pathogenic fungi [132]. Oleanane
triterpenoid, triterpenetetrol isolated from the chloroform extract of the
aerial parts of Leontodon filii were reported to have antifungal prop [133].
Carvone, dinydrocarvone, limonene, dillapiole and dillapional from
Anethum sowa revealed antifungal activity at a concentration of 1:100 and
1:250 [134,135,136]. A derivative of dillapiole, isodilapiole tribromide
found to more active [137]. The steam distillate of fresh mature
leaves containing odorous oil rich in cyclic tri-and tetra-sulphides of C3,
C5 and C9 units exihibited antifungal activity at 125 g/mL in vitro [139].
The active compounds, 1'-acetoxychavicol acetate from Alpinia galanga
strong inhibitory affects at a MIC 0.024 g/mL against several fungal
pathogens [140 a,b]. Most of the species of Oscimum showed in vitro
antifungal activities against a broad range of fungi as well as bacteria.
An Indian chemotype Ocimum gratissimum, with a high level of ethyl
cinnamate, presents, in vitro, an interesting spectrum of antifungal
properties [141].
2.8. Alkaloids
Heterocyclic nitrogen compounds are called alkaloids (Fig 8 A-B). The
first medically useful example of an alkaloid was morphine; isolated in
1805 from the opium poppy Papaver somniferum [142,], Codeine and heroin
are both derivatives of morphine. Diterpenoid alkaloids, commonly isolated
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Tasleem Arif et al.
from the plants of the Ranunculaceae [143, 144] are found to have
antimicrobial properties [145]. While alkaloids have been found to have
microbiocidal effects including against Giardia and Entamoeba species [146],
the major antidiarrheal effect is probably due to their effects on transit time in
the small intestine.
Recently, a novel alkaloid, 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)1-methylethyl pentanoate (38) was isolated from the plant Datura metel
showed in vitro as well as in vivo activity against Aspergillus and Candida
species [147]. Another novel alkaloid, 6,8-didec-(1Z)-enyl-5,7-dimethyl-2,3dihydro-1H-indolizinium (37) from Aniba panurensis demonstrated the
activity against a drug-resistant strain of C. albicans [148]. The antifungal
alkaloids -carboline, a tryptamine- and two phenylethylamine-derived
alkaloids and N-methyl-N-formyl-4-hydroxy-beta-phenylethylamine (39)
from Cyathobasis fruticulosa [149] and haloxylines A and B, new piperidine
from Haloxylon salicornium displayed antifungal potentials [150].
Jatrorrhizine (40) from Mahonia aquifolium was found to be the most
effective against all fungal species tested (MIC ranges from 62.5 to
125 g/mL), while the crude extract, berberine , and palmatine exhibited only
marginal activity (MIC 500 to >/= 1000 g/mL) [151].
Cocsoline (43), a bisbenzylisoquinoline alkaloid from the Epinetrum
villosum displayed antifungal activities [152]. The alkaloids N-methylhydrasteine
hydroxylactam and 1-methoxyberberine chloride from Corydalis longipes
showed high efficacy individually [153]. Four alkaloids, dicentrine (41), glaucine
(42), protopine, and alpha-allocryptopin (46) from Glaucium oxylobum
exhibited good activity against Microsporum gypseum, Microsporum canis,
T. mentagrophytes and Epidermophyton floccosum [154].
C8H17
O
N
O
C8H17
HN
OH
39
38
37
O
O
O
O
O
O
OH
N
O
O
O
40
41
42
299
Flindersine (45) and haplopine from Haplophyllum sieversii were

growth-inhibitory compounds against various fungi [155]. Canthin-6-one and
5-methoxy-canthin-6-one of Zanthoxylum chiloperone var. angustifolium
exhibited antifungal activity against C. albicans, A. fumigatus and
T. mentagrophytes [156]. Frangulanine, a cyclic peptide alkaloid and
waltherione A, a quinolinone alkaloid from leaves of Melochia odorata were
reported to exhibit antifungal activities against a broad spectrum of
pathogenic fungi [157].
The indole alkaloid venenatine exhibited antifungal activity against all
the 10 tested fungi, showed an especially high sensitivity towards this
compound, exhibiting germination levels below 10% [158]. From the root
bark of Dictamnus dasycarpus two antifungal furoquinoline alkaloids
were isolated. 3-methoxysampangine from Cleistopholis patens exhibited
significant antifungal activity against C. albicans, A. fumigatus, and
C. neoformans [159].
O
NH
O
O
O
O
HO
43
44 O
N
O
O
N
H
45
46
2.9. Lectins and polypeptides

Peptides, which are inhibitory to microorganisms, were first reported in
1942 [160]. They are often positively charged and contain disulfide bonds
[161]. Their mechanism of action may be the formation of ion channels in the
microbial membrane [162] or competitive inhibition of adhesion of microbial
300
Tasleem Arif et al.
proteins to host polysaccharide receptors [163]. Recent interest has been

focused mostly on studying fungi by these macromolecules, such as that from
the herbaceous Amaranthus, has long been known [164]. Thionins are
peptides commonly found in barley and wheat and consist of 47 amino acid
residues. Thionins AX1 and AX2 from sugar beet were active against fungi
but not bacteria [165, 166].
Raw Allium Sativum extract showed antifungal activity beyond 48h in
extract stored at room temperature. Antifungal activity was stable upto 8h
under these conditions [167]. A novel lectin was isolated from the roots of
Astragalus mongholicus [168] and a protein with a novel N-terminal
sequence from ginger rhizomes exerted antifungal activity towards various
fungi [169]. An antifungal peptide was reported from fresh fruiting bodies of
the mushroom Agrocybe cylindracea [170]. Two antifungal peptides from
seeds of Pharbitis nil exhibited potent antifungal activity against both chitincontaining and non-chitin-containing fungi in the cell wall. Concentrations
required for 50% inhibition of fungal growth were ranged from 3 to 26
micrograms/mL and from 0.6 to 75 g/mL [171].
From Phaseolus species, mung bean (Phaseolus mungo) seed, chitinase
with antifungal activity was isolated [172]. This species also yielded a novel
lysozyme exhibiting antifungal activity toward Botrytis cinerea [173].
Another Fabaceae species, Trigonella foenum-graecum, yielded defensins,
small cysteine rich peptides, which exhibited antifungal activity against the
broad host range fungi [174]. A novel antifungal peptide, cucurmoschin, was
isolated from the seeds of the black pumpkin inhibited mycelial growth in the
fungi [175]. A peptide designated cicerarin from seeds of the green chickpea
Cicer arietinum showed antifungal activity. The antifungal activity was
preserved after exposure to 100 degrees C for 15 min [176]. Two other
antifungal peptides cicerin and arietin were reported from seeds of the
chickpea (Cicer arietinum). Arietin manifested a higher antifungal potency
toward Mycosphaerella arachidicola, Fusarium oxysporum and Botrytis
cinerea [177]. An antifungal peptide designated angularin was isolated from
the adzuki bean exhibited antifungal activity against a variety of fungal
species [178]. Two novel antifungal peptides, designated alpha- and betabasrubrins, respectively, were isolated from seeds of the Ceylon spinach
Basella rubra [179].
An antifungal protein, AFP-J, was purified from potato tubers, Solanum
tuberosum strongly inhibited yeast fungal strains, including C. albicans,
Trichosporon beigelii and S. cerevisiae [180]. Pineapple leaf chitinase-B
from Ananas comosus exhibits strong antifungal activity toward Trichoderma
virida [181]. Another chitinase with antifungal activity was also purified
from the bulbs of the plant Urginea indica, known as Indian squill.
301
The protein was an active growth inhibitor of the fungal pathogens in an

in vitro assay [182]. A novel protein was isolated from the Chinese herb
Astragalus mongholicus Bunge. It exerted selective antifungal activity
against various fungi [183]. Recently, a new protein from E. coli, having
anti-Aspergillus activity was reported by Yadav et al. [184]. Antifungal
peptides and proteins from medicinal species also included two chitin-binding
proteins from spindle tree Evonymus europaeus [185], a thaumatin-like protein
from banana Musa acuminate [186], and a protein from ginger rhizomes
Zingiber officinalis (Zingiberaceae), which exerted antifungal activity toward
various fungi [187].
2.10. Other compounds

Many phytochemicals not mentioned above have been found to exert
antifungal properties. This review has attempted to focus on reports of
chemicals, which are found in multiple instances to be active. There are
reports of chemical having antifungal properties associated with several
different classes not covered above (Figure 9).
OH
O
HO
O
HO
HO
N
H
47
48
O
49
OH
OH
HO
HO
50
51
O
O
O
52
53
302
Tasleem Arif et al.
N-trans-feruloyl-4-methyldopamine (47) recently isolated from

Achranthes ferruginea was reported to be active against a broad range of
fungi [188]. Leaves of Piper aduncum accumulate the anti-fungal
chromenes, methyl 2,2-dimethyl-2H-1-chromene-6-carboxylate and methyl
2,2-dimethyl-8-(3'-methyl-2'-butenyl)-2H-1-chromene-6-carboxylate [189].
Iridodial -monoenol acetate, from Nepeta leucophylla, and actinidine from
N. clarkei were found to be endowed with antifungal activities [190].
Anofinic acid and fomannoxin acid (48) from Gentiana algida were found
to be active against fungi [191]. The antifungal activity of Artemisia
herba-alba was found to be associated with two major volatile compounds
as carvone and piperitone [192]. The phenylpropanoids p-coumaric acid
and ferulic acid from Kigelia pinnata were observed to have antifungal
activity [193]. Piper cubeba afforded the compounds (8R,8'R,9'S)-5methoxyclusin, (-)-clusin, (-)-yatein, ethoxyclusin, and (-)-dihydroclusin
showed very potent and selective inhibitory activity against CYP3A4 with
IC50 values (0.44-1.0 M) identical to that of the positive control,
ketoconazole (IC50, 0.72 M) [194].
Demethoxyageratochromene (49) from Ageratum conyzoides showed
antifungal activity [195,196] at a concentration of 2000 ppm. The leaf
extract was found to be active and showed strong toxicity against the fungi
causing ringworm [197]. 2,4-Dihydroxy-3-methoxychalcone (50) and
2,4-dihydroxychalcone (51) in the dichloromethane extracts of Zuccagnia
punetata was found to have antifungal properties [198]. Plumbagin from
Plumbago zeylanica was also reported to inhibit the fungel species viz.
Trichophyton, Epidermophyton, Microsporum in a range of 30-40 g/mL
[199]. Hexane extracts of the stem bark as well as (-)-kaur-16-en-19-oic
acid of Annona glabra revealed antifungal activity [200]. Isoalantolactone
(52) and alantolactone (53), the major constituents of the roots of Inula
racemosa showed in vitro antifungal activity againest T. mentagrophytes
and Microsporum canis [201]. Withafarin A and amemonine isolated from
Withania somniferm and Anemone pulsatillea, salicin a phenolic glucoside
compound from Salix alba showed antifungal activity [202]. The psoralen,
8-methoxypsoralen and imperatorin in extracts of leaf, fruit, stem, bark and
root of Zanthoxylum americanum demonstrated a broad spectrum of
antifungal activity and inhibited fungal species in a disk diffusion assay
[203]. Myrothecium roridum contains macrocyclic trichothecenes of the
verrucarin type, 16-hydroxyverrucarin A and verrucarin X exhibited
moderate antifungal activity. Both compounds were reported to
preferentially inhibit in vivo protein biosynthesis [204].
303
3. Conclusion
Phytomedicines are major component of traditional system of healing in
developing countries, which have been an integral part of their history and
culture. Besides wide spread use of botanicals as medicinal products in
developing countries, such products are becoming part of the integrative
healthcare system of industrialized nations, known as complementary and
alternative system of medicines (CAM).
Existing costly therapy is not affordable well for the millions of
individuals particularly in the developing world. Plant extracts are the cheap
and easily available source to poor people. Plants are great source of
thousands new useful phytochemicals of great diversity, which have
inhibitory effects on all types of microorganisms in vitro. Till date more than
600 plants have been reported for their antifungal properties, however a few
of them were explored for the active components. The current pharmaceutical
armoury of antifungal is a clear cause for satisfaction, not from gloom.
However, we still do not have agents that fulfil every one of the criteria that a
physician would set as desiterata for antifungal drugs. They need to be active
against those fungai causing infections which we cant yet depend on
eradacting. They need to be formaluted for both oral and parenteral
administration, they need to be extremly safe and as cheep as possible. The
search for new antifungal agents therefore must go on.
Identification of new chemotypes for drug development remains an
urgent need in antifungal therapeutics. Simultaneously, a number of
antifungal compounds reported till date, are tested for their in vitro activities
not for in vivo acitivities. In vivo and in vitro activities of a compound may be
different and a very small number of plants extracts or components have been
studied for their in vivo activity. Therefore these should be subjected to
animal and human studies to determine their effectiveness in whole-organism
systems. Also in vitro testing and method of extraction should be
standardized so that the search could be more systematic.
The current set of clinically available antifungal agents includes three
classes of natural product and four classes of synthetic chemicals. We
therefor cant abandon intrest in biodiversity as a source of natural antifungal
products. Furthermore, the inactive plant extracts may be subjected to
chemical diversification of their components to increase the activity. The
transformation of chemical groups in natural products into rare chemical
groups is possible which are rarely produced by secondary metabolism.
Therefore, biosynthesis machinery can be complemented to produce a whole
range of new semisynthetic compounds in one step which may become an
alternative source of compounds to feed the discovery process for new
intresting compounds.
304
Tasleem Arif et al.
The study of alternative mechanisms of infection prevention and

treatment is essential. Plant products furthermore may be structurally
modified to increase their in vivo activity. For example isodilapiole
tribromide, a derivative of dillapiole was found to more active. Another
example include echnicandin type peptide FR901379, chemical modification
of which lead toward more active FK463 compound. Therefore, attention
toward the plant derived principles, their chemical modification and
chemotherapeuitic potential is needed.
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Research Signpost
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ISBN: 978-81-308-0448-4
10. Sesquiterpene lactones: Structural

diversity and their biological activities
Devdutt Chaturvedi
Natural Products Chemistry Division, North-East Institute of Science and Technology (CSIR)
Jorhat-785006, Assam, India
Abstract. Sesquiterpenes lactones (SLs) have been isolated from

numerous genera of the family Asteraceae (compositae) and can
also be found in other angiosperm families. They are described as
the active constituents of a variety of medicinal plants used in
traditional medicine for the treatment of inflammatory diseases.
They are known to possess wide variety of biological and
pharmacological activities such as antimicrobial, cytotoxic, antiinflammatory, antiviral, antibacterial, antifungal activities, effects
on the central nervous and cardiovascular systems as well as
allergenic potency. Their wide structural diversity and potential
biological activities have made further interest among the chemists.
The present chapter will be highlighted on the recent developments
on the SLs and their diverse biological activities.
1. Introduction
Sesquiterpene lactones (SLs) constitute a large and diverse group of
biologically active plant chemicals that have been identified in several
plant families such as Acanthaceae, Anacardiaceae, Apiaceae, Euphorbiaceae,
Lauraceae, Magnoliaceae, Menispermaceae, Rutaceae, Winteraceae and
Hepatideae etc [1]. However, the greatest numbers are found in the Compositae
Correspondence/Reprint request: Dr. Devdutt Chaturvedi, Natural Products Chemistry Division, North-East
Institute of Science and Technology (CSIR), Jorhat-785006, Assam, India
E-mail: ddchaturvedi@rrljorhat.res.in
314
Devdutt Chaturvedi
(Asteraceae) family with over 3000 reported different structures [2].

Sesquiterpene lactones are a class of naturally occurring plant terpenoids that
represent a diverse and unique class of natural products and are important
constituent of essential oils, which are formed from head-to-tail condensation
of three isoprene units and subsequent cyclization and oxidative
transformation to produce a cis or trans-fused lactone. These secondary
compounds are primarily classified on the basis of their carbocyclic skeletons
into pseudoguainolides, guaianalides, germanocranolides, eudesmanolides,
heliangolides and hyptocretenolides etc (Figure 1).
The suffix "olide" refers to the lactone function and is based on
costunolide, a germanacranoride which is related to the ten-membered
carbocyclic sesquiterpene, germacrone. However, SLs exhibit variety of other
skeletal arrangements. An individual plant species generally produces one
skeletal type of SLs concentrated primarily in the leaves and flower heads.
The percentage of SLs per dry weight may vary from 0.01% to 8%. Losses of
livestock intoxicated by plants containing SLs are well known. In fact, they
have been shown to exhibit a wide range of biological activities.
O
O
Guaianolides
Pseudoguaianolides
O
O
O
O
O
Germacronolides
O
O
O
O
O
Heliangolides
Eudesmanolides
O
Hypocretenolides
Figure 1. Basic skeletons of sesquiterpenes lactones.
315
Biologically active sesquiterpene lactones
An important usual feature of the SLs is the presence of a -lactone ring

(closed towards either C-6 or C-8) containing in many cases, an -methylene
group. Among other modifications, the incorporation of hydroxyls or
esterified hydroxyls and epoxide ring are common. A few SLs occur in
glycoside form and some contain halogen or sulfur atoms [3].
Majority of SLs have shown cytotoxic activity (KB and P388 leukemia
in vitro) and activity against in vivo P388 leukemia. Structure activity
relationship studies showed that various cytotoxic SLs react with thiols, such
as cystiene residues in the protien, by rapid Michael type of addition. These
reactions are mediated chemically by ,-unsaturated carbonyl system
present in the SLs. These studies support the view that SLs inhibit
tumor growth by selective alkylation of growth regulatory biological
macromolecules such as key enzymes, which controls cell division, thereby
inhibiting a variety of cellular functions, which directs the cell into apoptosis.
Differences in activity between individual SLs may be explained by different
number of alkylating structural elements. However, other factors, such as
lipophilicity, molecular geometry, and chemical environment or the target
sulfhydryl may also influence the activity of sesquitepenes lactones.
OH
O
O
OH
O
O
HO
HO
O
Costunolide (1)
Tagitinin C (3)
Tagitinin A (2)
OH
O
HO
CH2OH
O
O
HO
CH2OH
O
O
O
O
Cynaropicrin (4)
O
Parthenin (6)
Eupatoriopicrin (5)
HO
H
O
O
O
O
O
O
H
O
O
CH2
O
O
O
OH
Helenalin (7)
O
Artemisinin (8)
Vernodalin (9)
Figure 2. Structurally diverse sesquiterpenes lactones (SLs 1-9).
316
Devdutt Chaturvedi
Some of structurally diverse sesquiterpenes lactones have been shown in

Figure 2 and 3. Distribution of different structural classes of sesquiterpene
lactones have been depicted in Table 1.
2. Biological activity of sesquiterpene lactones

[A] Anticancer activity
In recent years, many researchers over the world have reported that
sesquiterpenes lactones possess potential anticancer activity. Some of the
important compounds of this class have been discussed below:
O
H
O
O
H
O
O
OH
HO
O
Parthenolide (10)
Dehydrocostus lactone (11)
Vernolide (12)
Hanphyllin (13)
O
O
O
O
Epoxy(4,5)- 4,5-dihydrosantonin (15)
Alantolactone (14)
O
OCOCH3
OH
O
OCOCH3
OiVal
HO
O
O
HO
O
OH
OAc
H
HO
O
O
11,13-dihydrovernodalin (19)
Vernodal (18)
OH
CH2
OH
MeO
O
Tagitinin C (3)
O
Neurolenin B (17)
CH2
O
O
OH
O
4,(5) Epoxy-4,5-dihydrosantonin (16)
Rupicolin A 8-Acetate (20)
Ridentin (21)
Figure 3. Structurally diverse sesquiterpenes lactones (10-21).
317
Table 1. Distribution of different structural classes of sesquiterpene lactones in the

family-Compositae.
Tribes
(No. of genera)
No. of genera with

sesquiterpene
lactones
Eupatorieae (50)
Vernonieae (50)
Astereceae (100)
Inuleae (100)
Heliantheae (250)
24
Type of lactones
present
Germacranolides
Elemanolides
Guaianolides
Ambrosanolides
Seco-Ambrosanolides
Germacranolides
Elemanolides
Guaianolides
Germacranolides
Guaianolides
Elemanolides
Guaianolides
Xanthanolides
Ambrosanolides
Helenanolides
Seco-Eudesmanolides
Seco-Ambrosanolides
Germacranolides
Elemanolides
Guaianolides
Eudesmanolides
Xanthanolides
Ambrosanolides
Helenanolides
Seco-Eudesmanolides
Seco-Ambrosanolides
Seco-Helenanolides
Germacranolides
Xanthanolides
Eremophilanolides
Helenanolides
Bakkenolides
Anthemideae (50)
10
Germacranolides
Elemanolides
Guaianolides
Helenanolides
Cadinanolides
Chrymoranolides
ArcototeaeCalenduleae (50)
Guaianolides
Senecioneae (50)
318
Devdutt Chaturvedi
Table 1. Continued
Cynareae (50)
Mutisieae (55)
Lactucae (75)
Germacranolides
Elemanolides
Guaianolides
Eudesmanolides
Eudesmanolides
Germanocranolides
Eudesmanolides
Guaianolides
I. Costunolide
Costunolide (1, Figure 2) is an active component from the crude extract of
Saussurea lappa roots, a traditional Chinese medicinal herb [3]. The anticancer
property of costunolide was first reported in a rat intestinal carcinogenesis model
induced by azoxymethane and supported by a subsequent study using a DMBA
induced hamster buccal pouch carcinogenesis model [4]. Following these two
in vivo experiments, considerable efforts have been devoted to understad the
mechanism responsible for the anti-cancer activity of costunolide. First,
costunolide is a potent apoptotic inducer in cancer cells, via multiple
pathways. It has been reported that costunolide readily depletes intracellular
GSH and disrupts the cellular redox balance [5]. It triggers an intracellular
reactive oxygen species (ROS) burst which leads to mitochondrial
dysfunction: loss of mitochondrial membrane potential, onset of mitochondrial
membrane transition, and release of mitochondrial pro-apoptotic proteins [6].
The apoptosis-inducing activity of costunolide was found to be closely
associated with Bcl-2, based on observations that costunolide treatment
decreased the anti-apoptotic Bcl-2 protein expression [7], while over expression
of Bcl-2 protein attenuated costunolide-induced apoptosis [12]. Second,
costunolide suppresses NF-kB activation via prevention of IkB phosphorylation
[8], a process also responsible for the strong anti-inflammatory activity of
costunolide [9]. Third, costunolide is capable of promoting leukemia cell
differentiation [10], inhibiting endothelial cells angiogenesis [11], and
disrupting nuclear microtubule architecture in cancer cells [12].
II. Parthenolide
Parthenolide (10, Figure 3), is the major SL responsible for bioactivity of
feverfew (Tanacetum parthenium), a traditional herb plant which has been
used for the treatment of fever, migraine and arthritis for centuries [13]. One
well-explored bioactivity of parthenolide is its potent anti-inflammatory
319
effect, which is mainly achieved through its strong inhibitory effect on

NF-kB activation. It has been well established that parthenolide acts on
multiple steps along the NF-kB signaling pathway [14]. By suppressing
NF-kB parthenolide inhibits a group of NF-kB regulated pro-inflammatory
cytokines, such as interleukins and prostaglandins [15]. The anticancer
activity of parthenolide has been pursued in a number of laboratories. A large
number of studies have been undertaken to investigate the mechanism of
action of parthenolide at molecular levels in the different phases of
carcinogenesis. The data were obtained using different tumor cell systems.
Parthenolide induced apoptosis in pre-B acute lymphoblastic leukemia lines,
including cells carrying chromosomal translocations [16]. Parthenolide
induced rapid apoptotic cell death distinguished by loss of nuclear DNA,
externalization of cell membrane phosphatidyl-serine, and depolarization of
mitochondrial membranes at concentrations ranging from 5 to 100 M. Steele
et al. investigated the in vitro actions of parthenolide on cells isolated from
patients with chronic lymphocytic leukemia. Brief exposure to the
sesquiterpene lactone (one to three hours) was sufficient to induce caspase
activation and commitment to cell death. The mechanism of cell killing was
via parthenolide induced generation of ROS, resulting in turn in a proapoptotic Bax conformational change, release of mitochondrial cytochrome C
and caspase activation. Other studies also demonstrated that parthenolidemediated apoptosis correlated well with ROS generation. Parthenolide
strongly induced apoptosis in four multiple myeloma cell lines, although
there are considerable differences in susceptibility to the sesquiterpene
lactone. KMM-1 and MM1S sensitive to parthenolide possess less catalase
activity than the less sensitive KMS-5 and NCI-H929 cells. These findings
indicate that parthenolide-induced apoptosis in multiple myeloma cells
depend on increased ROS and that intracellular catalase activity is a crucial
determinant of their sensitivity to parthenolide. Chen et al. also reported the
anti-proliferative and apoptosis-inducing effects of parthenolide on
human multiple myeloma cells, mediated by an enhancement of caspase-3
activity [17].
III. Helenalin
Helenalin (7, Figure 2) is another SL, from Arnica species, which has
been reported to possess cytotoxicity and anti-cancer activity [18]. Earlier
studies demonstrated its potent activity to inhibit nucleic acid and protein
synthesis [19]. Similiar to other anticancer SLs, mechanism of action mainly
involve: (i) thiol depletion, (ii) inhibition of NF-kB, and (iii) induction
of apoptosis [20]. These prominent bioactivities make helenalin another
potential anti-cancer agent.
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Devdutt Chaturvedi
IV. Artemisinin and its derivatives

Given the high accumulation of iron in cancer cells, researchers Henry
Lai and Narendra Singh became interested in possible artemisinin (8, Figure 2)
activity against malignant cells and have used artemisinin against numerous
cancer cells in vitro [21]. There are a number of properties shared by cancer
cells that favor the selective toxicity of artemisinin against cancer cell lines
and against cancer in vivo. In addition to their high rates of iron flux via
transferrin receptors when compared to normal cells, cancer cells are also
particularly sensitive to oxygen radicals. Artemisinin becomes cytotoxic in
the presence of ferrous ion. Since iron influx is naturally high in cancer cells,
artemisinin and its analogs can selectively kill cancer cells in vivo [22].
Furthermore, it is possible to increase or enhance iron flux in cancer cells by
supplying conditions that lead to increased intracellular iron concentrations.
However, intact in vivo systems do not need holotransferrin, since the body
provides all the necessary iron transport proteins. In recent years, in order to
search for potential anticancer agents many researchers have directed their
efforts in synthesizing various kinds of artemisinin dimers, trimers, tetramers
wherein several of which have shown potential anticancer activity and are in
the various phases of clinical trials [23].
[B] Anti-inflammatory activity
Sesquiterpenes lactones have displayed potential anti-inflammatory
activity through NF-kB pathway. Since NF-kB plays a central role in most
disease processes, and since it can regulate the expression of many key genes
involved in inflammatory as well as in a variety of human cancers [24],
NF-kB represents a relevant and promising target for the development of new
chemopreventive and chemotherapeutic agents. Some of the important SLs
have displayed anti-inflammatory activity are as follows:
I. Costunolide
Costunolide (1, Fig. 2) is a closely related sesquiterpene lactone analogue
of parthenolide present in several plants such as Magnolia grandiflora,
Tanacetum parthenium. Koo et al. showed that costunolide also dosedependently inhibited LPS-induced NF-kB activation. In this assay system,
costunolide even exhibited more potent inhibitory activity than parthenolide.
Detailed mechanism studies revealed that, similar to parthenolide,
costunolide also significantly inhibited the degradation of IkB- and IkB-.
In addition, costunolide also inhibited the phosphorylation of IkB-. These
321
accumulative results indicate that costunolide inhibits NF-kB activation by

preventing the phosphorylation of IkB, and therefore, sequestering the
complex in an inactive form [8].
II. Parthenolide
Parthenolide (10, Figure 3) is a sesquiterpene lactone present in several
medicinal plants that have been used in folk medicine for their antiinflammatory and analgesic properties. Several in vitro studies have shown
that a great part of the anti-inflammatory action of this compound appears
to be related to its ability to inhibit the NF-kB pathway. In vitro studies
have proven that the sesquiterpene lactone parthenolide does not interfere
with the generation of oxygen radicals [25], whereas it specifically inhibits
activation of the NF-kB pathway by targeting IKK [26] and/or preventing
the degradation of IkB- and IkB- [25]. Furthermore, parthenolide has
recently been reported to exert beneficial effects during endotoxic shock in
rats through inhibition of NF-kB DNA binding in the lung [27]. These
effects of parthenolide may also accounts for its inhibition of proinflammatory mediator genes, such as the gene for the inducible nitric
oxide synthase after endotoxin stimulation in rat smooth muscle cells [28]
and the gene for IL-8 in immune-stimulated human respiratory epithelial
cells [29]. In addition, parthenolide has also been demonstrated to protect
against myocardial ischemia and reperfusion injury in the rat by selective
inhibition of IKK activation and IkB degradation [30].
III. Helenalin
Since different types of sesquiterpene lactones showed inhibition of
NF-kB activation at similar concentrations, this effect seems to be
characteristic for many of the sesquiterpene lactones with an exomethylene
group like parthenolide and costunolide. Exomethylene groups of
,-unsaturated carbonyl compounds can react by Michael type addition to
sulfhydryl groups of cysteine residues in the DNA binding domain of the NF-kB
subunit [31]. Recently, Lyu et al. provided evidence that a sesquiterpene
lactone, helenalin (7, Fig. 2), containing two functional groups, namely
,-unsaturated carbonyl group and -methylene--lactone ring, exerts its
effect by direct alkylation of the p65 subunit of NF-kB without inhibition of
IkB degradation [32]. In vitro studies also demonstrated that helenalin
selectively modifies the p-65 subunit of NF-kB at the nuclear level, therefore
inhibiting its DNA binding [33]. However, costunolide differs from helenalin
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Devdutt Chaturvedi
in a number of functional groups and inhibits degradation of IkB by

inhibiting phosphorylation of IkB. Therefore, another functional group other
than the exomethylene group and the molecular geometry of sesquiterpene
lactone compounds appear to be important factors to determine the mode of
NF-kB inhibition. However, the epoxide group in parthenolide is not likely
important because parthenolide is at least less effective to inhibit both NF-kB
activation and NO production.
[C] Anti-malarial activity

I. Artemisinin and its derivatives
In 1972, a group of Chinese researchers isolated a new anti-malarial drug
(+)- artemisinin (8, Figure 4), a sesquiterpene lactone of the amorphene
sub-group of cadinene from the hexane extract of a traditional Chinese
medicinal plant Artemesia annua (Asteraceae) - a plant which has been used
for the treatment of fever and malaria since ancient times [23]. Artemisinin is
a sesquiterpene lactone containing an endoperoxide linkage in it. This highly
oxygenated sesquiterpene lactone peroxide, unlike most other anti-malarials,
lacks nitrogen containing heterocyclic ring systems and was found to be
superior plasmocidal and blood schizontocidal agent to conventional antimalarial drugs, such as chloroquine, quinine etc against malaria strains,
without obvious adverse effects in patients.
Artemisinin is active at nanomolar concentrations in vitro both against
chloroquine sensitive and resistant P. falciparum strains. However, the practical
values of artemisinin, nevertheless, are impaired (i) poor solubility either in oil or
water; (ii) high rate of parasite recrudescence after treatment; (iii) short-plasma
half life (3-5h) and poor oral activity. However, a low level of resistance has
O
O
22
23
24
25
26
O
O
O 12
11
13
R = H (Dihydroartemisinin)
R = Me (Artemether)
R = Et (Arteether)
R = COCH2CH2COONa (Sodium artesunate)
R = COCH2C6H4COONa (Sodium artelinate)
OR
8 (Artemisinin)
Figure 4. Structure of artemisinin and its analogs.
323
recently been observed using artemisinin, which disappeared as soon as the

drug-selection pressure has been withdrawn. However, artemisinin with an
endoperoxide linkage is a sensitive molecule for large scale derivatization.
Fortunately, it was found that the carbonyl group of artemisinin 8, can be easily
reduced to dihydroartemisinin 22 in high yields using sodium borohydride, which
has in turn led to the preparation of a series of semi-synthetic first-generation
analogues included oil soluble artemether 23, arteether 24, water soluble sodium
artesunate 25, and sodium artelinate 26.
These three analogs become very potent anti-malarial drugs effective against
chloroquine-resistant strains of P. falciparum. Artemether 23 has been included
in the WHO lists of Essential Drugs for the treatment of severe MDR malaria.
In this family, the Walter Reed Institute of research has patented a stable, watersoluble derivative called artelinic acid 26 which is now being tested in animals.
A key advantage of these endoperoxides containing anti-malarial agents, which
have been used for nearly two decades, is the absence of drug resistance. It has
been realized through the structure-activity relationship (SAR) of artemisinins
that mainly endoperoxide affects the antimalarial activity. In order to increase
antimalarial potency of these molecules, researchers around the world become
interested to synthesize artemisinin dimers, trimers and tetramers in recent years.
Many of them have shown promising antimalarial activity than artemisinin and
their first generation analogs.
II. Miscellaneous antimalarials

Antimalarial activity of sesquiterpenes lactones from Neurolena lobata has
been documented (Figure 5) [34]. Germacranolide sesquiterpenes lactones like
neurolenin B (17, IC50 = 0.62 M) more potent than furanoheliangolides lobatin
B (IC50 = 16.51 M). Among the germacranolides, the shift of the double bond
from the 2,3-position (neurolenin B) into the 3,4-position (lobatin A) led to
dramatic decrease in the activity suggesting that one of the structural
requirements is the presence of /-unsaturated keto function. Additionally, a
free hydroxyl group at C-8 increased the antiplasmodial activity, while a free
hydroxyl group at C-9 decreased the activity.
Goffin et al. investigated the antiplasmodial properties of Tithonia
diversifolia against three strains of P. falciparum, and sesquiterpene lactone (Fig.
5/Fig 3) Tagitinin C (3) was found to be active against FCA strain (IC50 = 0.33
g/mL) [35]. Jenett-Siems et al. reported four sesquiterpenes, vernodalol (18),
11,13-dihydrovernodalin 11,13-dihydrovernolide (19) and 11,13,17,18tetrahydrovernolide from Vernonia colorata. Among these, vernodalol (18) and
11,13-dihydrovernodalin (19) exhibited the strongest antiplasmodial activity
(IC50 = 4.8 and 1.1 g/mL) respectively). Among the sesquiterpene lactones
obtained from Artemisia afra, 1-desoxy-1-peroxy-rupicolin A-8-O-acetate
324
Devdutt Chaturvedi
OCOCH3
OH
O
OCOCH3
OiVal
HO
OH
MeO
O
Tagitinin C (3)
O
Neurolenin B (17)
HO
O
Vernodalol (18)
OH
OAc
CH2
O
H
CH2
O
O
OH
OH
O
O
Helenalin (7)
Rupicolin A 8-Acetate (20)
11,13-dihydrovernodalin (19)
O
OH
R
O
O
HO
HO
O
O
Ridentin (21)
O
Hanphyllin (13)
OH
27, 28
R = H, CH3
O
Figure 5. Structures of some of anti-malarials sesquiterpene lactones.
(20), 1,4-dihydroxy-bishopsolicepolide and rupicolin A-8-O-acetate (20)

possessed in-vitro antiplasmodial activity (IC50 = 10.8-17.5 g/mL) [36].
Passreiter et al. have isolated sesquiterpene lactones of the pseudoguaianolide
type from Arnica Montana, helenalin (7), dihydrohelenalin and their acetates
showing activities against P. falciparum in vitro (IC50 = 0.23 to 7.41 M) [37].
Inhibitory effect upon the growth of P. falciparum has been reported
for sesquiterpene lactones (27) and (28) isolated from Camchaya calcarea
(IC50 = 1.2 and 0.3 g/mL) respectively [38].
[D] Antiviral activity
In spite of an effective and safe vaccine therapy against hepatitis B virus
(HBV), viral infection by HBV caused a global health problem in the world,
especially the third world. Moreover, because direct antiviral therapy against
HBV infection is not yet perfectly developed, it is important to discover the
lead compounds for novel anti-HBV agents from the potential library.
Recently, there was a report about anti-HBV activity of artemisinin (8)
and artesunate (25) based on the screening by using HBV-transferred
HepG2 2.2.15 cell [39], which is derived from hepatoblastoma HepG2 cell
325
[40]. This screening method is a useful in vitro model for evaluation of novel
anti-HBV drugs, as well as to study several steps of the HBV biology [41].
Artemisinin (8), artesunate (25), and a variety of purified compounds from
traditional Chinese medicine remedy were investigated by measuring the
release of surface protein (HBsAg) and HBV-DNA after drug exposure
(0.01-100 M) for 21 days [39]. As a result, artesunate (25) strongly inhibited
the HAsAg secretion with an IC50 of 2.3 M and IC90 of 16 M, respectively,
whereas artemisinin (8) had a mild inhibition activity. To evaluate an
enhancement in viron production, the amount of the HBV-DNA release to the
HepG2 2.2.15 culture medium during different treatments was measured, and
it was significantly reduced. In addition, it was discovered that, for artesunate
(25), toxicity in host cell was shown in drug concentration of 20 M and
therapeutical index (TI) calculated from IC50 of HBV-DNA release was 40.
When comparing to TI value (500) of lamivudine as positive control, the
value of artesunate (25) is quite low, but reasonable value for further
investigation. Finally, artesunate (25) was tried in combination treatment with
lamivudine. When both compounds were administered together in
concentration of 20 nM each, no toxicity was observed, but a synergic
inhibitory effect in HBsAg release was found. It means that it is possible to
be potential antiviral agent against infection of lamivudine-resistance
HBV strains, frequent problem in clinical treatment [42]. This result was
quite similar to potency previously reported for human cytomegaloviruses
[39].
Anti-viral activity of various sesquiterpene lactones was reported by
Hsieh and their coworkers against hepatitis C virus (Fig. 6) [43]. They have
tested a series of 10 compounds such as parthenolide (10, EC50 = 2.21 M),
costunolide (1, EC50 = 2.69 M), dehydrocostus lactone (11, EC50 = 3.08
M), Helenalin (7, EC50 = 1.25 M), alantolactone (14, EC50 = 2.03 M),
Epoxy-dihydrosantonin (15, EC50 = >10 M), artemisinin, and two other
conjugated lactones. Wherein they found the best anti-HCV activity was
shown by helenalin. They have further derivatized a series of parthenolide
analogs 29 wherein they found that best activity was realized while putting a
piperidine moiety (R = piperidine, EC50 = 1.64 M).
[E] Antibacterial activity
There has been an overwhelming amount of evidence indicating that
certain SLs are effective in exerting antibacterial activity. Rabe et al. showed
that Vernonia colorata, a member of the Compositae found in west,
central and South Africa possess SLs with antibacterial activity primarily
against Gram-positive species and lower activity towards Gram-negative
species [44]. The SLs vernodalin (30), vernolide (12) (Fig. 7) and 11,13dihydroovernolide were isolated and screened against Staphylococcu aureus
326
Devdutt Chaturvedi
H
O
parthenolide (10)
HO
Costunolide (1)
O
Dehydrocostus lactone (11)
OH
Helenalin (7)
O
O
O
O
O
O
O
Epoxy(4,5 ) - 4,5-dihydrosantonin (15) 4,(5) Epoxy-4,5-dihydrosantonin (16)
Alantolactone (14)
R = N(CH3)2, N(Et)2, Pyrrolidine, Piperidine, Morpholine
O
29
Parthenin derivatives
Figure 6. Antiviral sesquiterpene lactones.
and Bacillus subtilis (Gram-positive species) and Escherichia coli and

Klebsiella pneumoniae (Gram-negative species).
11,13-Dihydroovernolide is a novel SLs in that it has never been isolated
from a Vernonia species before. All three of the compounds screened had very
low inhibitory action against the Gram-negative bacteria. However, S. aureus and
B. subtilis showed the most sensitivity towards all of the SLs screened. It needs to
be noted, however, that although 11,13-dihydrovernolide is a novel SL, it had
the lowest activity against the Gram-positive species compared to vernolide and
vernodalin which had MIC values of 0.1-0.5 mg/mL.
Taylor and Towers isolated, characterised and screened three SLs
belonging to the pseudoguaianolides class of SLs from Centipeda minima, a
member of the Compositae [45]. This plant is used throughout Southeast Asia
to treat colds, coughs, and sinus infections. Three SLs, 6-O-methylacrylylplenolin
(31), 6-O-angeloylplenolin (32), and 6-O-isobutyroylplenolin (33) (Figure 8)
were isolated, with 6-O-methylacrylylplenolin being novel, and were then
screened for antibacterial activity against B. subtilis and S. aureus. All three of the
SLs screened had significant activity against the bacteria with 6-Oisobutyroylplenolin being the most bioactive. Both 6-O-isobutyroylplenolin and
6-O-methylacrylylplenolin exhibited MIC value of 150 g/mL against B. subtilis.
6-O-Angeloylplenolin was less active with a MIC of 300 g/mL. All three
327

O
HO
O
O
CH2
O
O
OH
O
O
Vernodalin (30)
Vernolide (12)
Figure 7. Antibacterial sesquiterpenes lactones (30, 12).
SLs showed activity against both methicillin-resistant and methicillinsensitive strains of S. aureus. Both 6-O-isobutyroylplenolin and
6-O-methylacrylylplenolin had a MIC of 300 g/mL against methicillinresistant S. aureus, while 6-O-angeloylplenolin was less active with a MIC of
600 g/mL against this strain. With respect to the methicillin-sensitive strain
of S. aureus, 6-O-methylacrylylplenolin and 6-O-angeloylplenolin had MIC
values of 75 g/mL while 6-O-isobutyroylplenolin had a MIC of 38 g/mL
indicating that this SL is more bioactive against methicillin-sensitive
S. aureus than the other SLs screened.
Further amplifying the possibility for the use of SLs found in plant oils,
Wang and coworkers recently discovered four new SLs in a plant species known
as Ligulariopsis shichuana, which is a new genus of the Compositae [46]. The
four SLs isolated and characterised were: (a) 3-acetoxy-9-angeloyloxy-1,
10-epoxy-8-hydroxyeremophil-7(11)-en-8-(12)-olide (34); (b) 3-senecioyloxy-1,10-epoxy-8-hydroxyeremophil-7(11)-en-8-(12)-olide (35); (c) 6angeloyloxy-8-hydroxyeremophil-1(10),7(11)-dien-8-(12)-olide (36); and (d)
1-oxo-6-senecioyloxy-8-hydroxyeremophil-7(11),9(10)-dien--(12)-olide (37)
O
O
O
O
6-O-Methylacrylylphenolin (31) 6-O-Angeloyphenolin (32) 6-O-Isobutyroylphenolin (33)
Figure 8. Antibacterial sesquiterpene lactones (31-33).
328
Devdutt Chaturvedi
O-Ang
OH
H
OH
O
O
O
O
AcO
SenO
34
35
OH
OH
OSen
OAng
37
36
Figure 9. Antibacterial sesquiterpenes lactones (34-37).
(Fig. 9). Only compounds 34 and 35 were screened for their antibacterial activity.
Compound 34 showed moderate activity towards both the Gram-positive and
Gram-negative species B. subtilis and E. coli, respectively. Compound 35, while
exhibiting moderate activity towards E. coli, showed much stronger activity
against B. subtilis at MIC concentrations up to 100 g/mL.
Finally, there have been reports that other SLs, such as helenalin 7, showed
inhibitory action against Mycobacterium tuberculosis as well as activity against
Corynebacterium diptheriae [47]. Helenalin 7, a mixture of alantolactone 14 and
isoalantolactone 38, is derived from the plant species Inula helenium (Fig. 10).
Helenalin 7 has primarily been utilised as an antiseptic for the urinary tract [47].
However, helenalin 7 was also shown to inhibit both Gram-positive and Gramnegative bacterial growth, with the former showing more sensitivity [48]. As one
can see, there is certain hope for those essential oils containing SLs in
therapeutics. The preclinical data implicates that SLs are effective in reducing
bacterial growth which gives strength to the idea that SLs could be potentially
used in the medical treatment of both Grampositive and Gram-negative bacterial
infections.
O
O
O
Alantolactone (14)
Isoalantolactone (38)
Figure 10. Antibacterial sesquiterpene lactones (14, 38).
329
[F] Antifungal activity

There certainly exists a vast amount of empirical data supporting that
certain SLs found in essential oils have the potential to act as antibacterial
agents. It also needs to be shown that certain SLs also possess antifungal
activities. The following section will focus on studies implicating the SLs for
probable use as antifungal agents.
Calera et al. isolated, characterised, and screened two bioactive SLs from
the roots of yellow flowered perennial herb Ratibida mexicana. This plant is
found primarily along the Sierra Madre Occidental in the northwestern part of
Mexico [49]. Indian tribes find that the roots are useful in alleviating
headaches, colds and rheumatism. The two SLs isolated from this plant are
isoallolantolactone (38) and elema-1,3,11-trien-8,12-olide (39) (Fig. 11).
The in vitro antifungal screen revealed that both SLs inhibited the radial
growth of Helminthosporium with the MIC being 650 g/mL for both SLs.
Pythium growth was far more sensitive to isoallolantolactone (38) with a
MIC of 125 g/mL. Fusarium was also screened for sensitivity against
isoallolantolactone (38) and elema-1,3,11-trien-8,12-olide, with again
isoallolantolactone (39) showing the most bioactivity by inhibiting 45% of
radial growth at 200 g/mL for this particular fungus.
O
O
O
Isoalantolactone (38)
Elema-1, 3,11-trien-8,12-olide(39)
Figure 11. Antifungal activity of sesquiterpene lactones (38, 39).
Two new eudesmanolides were isolated from the aerial parts of Centaurea
thessala spp. drakiensis and C. attica spp. attica, plants which are primarily used
in folk medicine in the Mediterranean region [50]. The two novel eudesmanolides
isolated were 8-hydroxy-4-epi-sonchucarpolide (40) and the 8-(4-acetoxy-3hydroxy-2-methylenebutanoyloxy) derivative (41) of the 8-hydroxy-4-episonchucarpolide, also known as 40-acetoxymalacitanolide (Fig. 12).
A variety of fungal species showed sensitivity towards 8-hydroxy-4epi-sonchucarpolide (40) and 40-acetoxymalacitanolide (41) [50].
8-Hydroxy-4-epi-sonchucarpolide 40, when compared to 40acetoxymalacitanolide 41, showed higher activity against all the fungal
330
Devdutt Chaturvedi
OH
OH
OH
OAc
O
O
H
CHO
40
H
CHO
O
O
OH
O
O
41
Figure 12. Antifungal activity of sesquiterpene lactones (40-41).
species screened, with the exception of one species. The MIC values for
8-hydroxy-4-epi-sonchucarpolide 40 were considerably lower indicating
that sensitivity is much higher for this compound. The exception was for
Cladosporium cladosporioides; this species showed higher sensitivity
towards 40-acetoxymalacitanolide, with a MIC value of 0.06 g/mL while
the MIC value for 8-hydroxy-4-epi-sonchucarpolide was 0.5 g/mL.
In addition, both SLs had indentical MICs against Penicillium funiculosum,
showing no disparity between these two SLs in their inhibitory action against
this particular species. The authors of this paper speculated that the
differences in activity between these two SLs could be attributed to the
different skeletal types and functional groups present on the compounds.
Finally, it needs to be mentioned that both SLs, possessed greater antifungal
activity that miconazole, a commercial fungicide used as the positive control.
3. Structural-activity relationships (sar) of sesquiterpene

lactones
It is generally believed that the bioactivity of SLs is mediated
by alkylation of nucleophiles through their , - or , , -unsaturated
carbonyl structures, such as -methylene--lactones or ,-unsaturated
cyclopentenones. These structure elements react with nucleophiles, especially
the cysteine sulfhydryl groups by Michael-type addition. Therefore, it is
widely accepted that thiol groups such as cysteine residues in proteins, as
well as the free intracellular GSH, serve as the major targets of SLs.
In essence, the interaction between SLs and protein thiol groups or GSH
leads to reduction of enzyme activity or causes the disruption of GSH
metabolism and vitally important intracellular cell redox balance.
The relationship between chemical structure and bioactivity of SLs
has been studied in several systems, especially with regards to cytotoxicity,
331
anti-inflammatory and antitumor activity. It is believed that the exomethylene group on the lactone is essential for cytotoxicity because structural
modifications such as saturation or addition to the methylene group resulted
in the loss of cytotoxicity and tumor inhibition. However, it has also been
shown that the factor responsible for the cytotoxicity of SLs might be the
presence of the O=C-C=CH2 system, regardless of lactone or cyclopentenone.
It was latter demonstrated that the presence of additional alkylating groups
greatly enhanced the cytotoxicity of SLs. Furthermore, it was established that
the -methylene--lactones and ,-unsaturated cyclopentenone ring (or epoxycyclopentenone) present in SLs essential for their in vivo anti-tumor
activity. It has been confirmed through various published reports that the various
kinds of biological activities displayed by SLs is due to presence of either methylene--lactones and ,-unsaturated cyclopentenone ring. In summary, the
differences in activity among individual SLs may be explained by differences in
the number of alkylating elements, lipophilicity, molecular geometry, and the
chemical environment of the target sulfhydryl group.
n
O
O
O
Figure 13. General structure of sesquiterpene lactones.
4. Conclusions
Sesquiterpene lactones are an important group of natural products
obtained from many species of medicinal plants. Their structural diversity
and diverse potential biological activities such as anticancer, antiinflammatory, anti-tumor, anti-malarial, antiviral, antibacterial, antifungal
etc. have made further interest among the chemists to the drug discovery
research. Although, the exact mechanism of action of SLs are not well
known but it has been documented through the various published reports
that the biological activity displayed by majority of sesquiterpene lactones
is due to the presence of -methylene--lactones and ,-unsaturated
cyclopentenone ring. The present chapter deals an overview on the various
kinds of biologically activity of structurally diverse sesquiterpene lactones
332
Devdutt Chaturvedi
which may be useful for the chemists/pharmacologists working in the area of

drug discovery of the relevant subject.
Acknowledgements
Author is thankful to the Director, North-East Institute of Science and
Technology (CSIR), Jorhat, Assam, for providing the necessary facilities
during the preparation of this book chapter.
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Research Signpost
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ISBN: 978-81-308-0448-4
11. A review on natural products with

mosquitosidal potentials
Navneet Kishore, Bhuwan B. Mishra, Vinod K. Tiwari and Vyasji Tripathi
Abstract. Mosquito, a flying insect of family Culicidae, serves as

crucial vector for a number of arboviruses (arthropod-borne
viruses) and parasites that are maintained in nature through
biological transmission between susceptible vertebrate hosts by
blood feeding arthropods (mosquitoes, psychodids, ceratopogonids,
and ticks) responsible for inflammation/encephalitis, dengue,
malaria, rift valley fever, yellow fever and others. Despite of a
direct human affliction, they are also known to transmit several
diseases and parasites that are lethal to dogs and horses, i.e., dog
heartworm (Dirofilaria immitis), West Nile virus (WNV) and
Eastern equine encephalitis (EEE) with ability to affect the central
nervous system and cause severe complications and death. Vector
control is by far the most successful method for reducing the
incidences of diseases, but the emergence of widespread insecticide
resistance and the potential environmental issues associated with
some synthetic insecticides (such as DDT) has indicated that
additional approaches to control the proliferation of mosquito
population would be an urgent priority research. In concern to
quality & safety of life on controlling mosquito vectors has shifted
steadily from the use of conventional chemicals toward alternative
insecticides that are target-specific, biodegradable, environmentally
safe, and botanicals in origin. In present article, we have discussed
Correspondence/Reprint request: Dr. Vyasji Tripathi, Department of Chemistry, Faculty of Science, Banaras
Hindu University, Varanasi-221005, India. E-mail: vyas_45@rediffmail.com
336
Navneet Kishore et al.
the local and traditional uses of plants in mosquito control and have reviewed 185
phytochemicals of paramount importance for the development of efficient chemical
entities to control mosquito population by direct as well as indirect inhibitions. In
order to highlight any possible mechanism based action for promising mosquitosides,
the review has been organized according to chemical structural classes.
1. Introduction
Mosquito serves as crucial vector for a number of arboviruses
(arthropod-borne viruses) and parasites that are maintained in nature through
biological transmission between susceptible vertebrate hosts by blood feeding
arthropods responsible for inflammation/encephalitis, dengue, malaria, rift
valley fever, yellow fever and others. The Word Health Organization (WHO)
estimates that each year 300-500 million cases of malaria occur and more
than 1 million people die of malaria. About 1,300 cases of malaria are
diagnosed in the United States each year. In addition, some 2500 million
people (two fifth of the world's population) are now at risk from dengue [1].
One can imagine the dangers of these mosquitoes with all the other diseases
that it can transmit.
Vector control is by far the most successful method for reducing incidences
of mosquito born diseases, but the emergence of widespread insecticide
resistance and the potential environmental issues associated with some
synthetic insecticides (such as DDT) has indicated that additional approaches to
control the proliferation of mosquito population would be an urgent priority
research. Currently, numerous products of botanical origin, especially the
secondary metabolites, have received considerable renewed attention as
potentially bioactive agents used in insect vector management. However, there
is a little other than anecdotal, traditional or cultural evidence on this topic [2].
The Greek natural philosopher Pliny the Elder (1st century AD)
recorded all the known pest control methods in Natural History. The use
of powdered chrysanthemum as an insecticide comes from Chinese record.
The other natural products like pyrethrum, derris, quassia, nicotine, hellebore,
anabasine, azadirachtin, d-limonene, camphor and turpentine were among
some important phytochemical insecticides widely used in developed
countries [3]. The discovery of DDTs and the subsequent development of
organochlorines, organophosphates and pyrethroids suppressed natural
product research as the problem for insect control were thought be solved.
However, high cost of synthetic pyrethroids, environment and food safety
concerns, the unacceptability and toxicity of many organophosphates and
organochlorines, and increasing insecticide resistance on a global scale
argued for stimulated research towards potential botanicals [4].
Mosquitosidal natural products
337
Mosquitoes in the larval stage are attractive targets for pesticides because
they breed in water and, thus, are easy to deal with them in this habitat. Some
of new significant larvicidal insect growth regulators such as methoprene,
pyriproxyfen, diflubenzuron and endotoxins obtained from Bacillus
thuringiensis israelensis and B. sphaericus have been developed. The plant
Azardichita indica has gained wide acceptance in some countries as an
antifeedant [5] while many essential oils from plant origin such as citronella,
calamus, thymus, and eucalyptus are reportedly promising mosquito
larvicides [6-10].
The use of herbal products is one of the best alternatives for mosquito
control. The search for herbal preparations that do not produce any adverse
effects in the non-target organisms and are easily biodegradable remains a top
research issue for scientists associated with alternative vector control [11].
Many plant species are known to possess biological activity that is frequently
assigned to the secondary metabolites. Among these, essential oils and their
constituents have received considerable attention in the search for new
biopesticides. Many of them have been found to possess an array of
properties, including insecticidal activity, repellency, feeding deterrence,
reproduction retardation and insect growth regulation against various
mosquito species [12-16].
2. Traditional mosquito repellents and usage custom

There are several reports particularly in Africa describing about the
burned plant materials effective to drive away mosquitoes. Thirteen percent
of rural Zimbabweans use plants and 15% using coils [17] while 39% of
Malawians burn wood dung or leaves [18]. Up to 100% of Kenyans burned
plants to repel mosquitoes [19], and in Guinea Bissau 55% of people burned
plants or hung them in the home to repel mosquitoes [20].
The local communities adapt various methods to repel the insects/
mosquitoes. Application of smoke by burning the plant parts is one of the
most common practices among the local inhabitants. Other types of
applications are spraying the extracts by crushing and grinding the repellent
plant parts, hanging and sprinkling the repellent plant leaves on the floor etc.
The leaf of repellent plant is one of the commonly and extensively used plant
parts to repel the insects and mosquitoes, followed by root, flower and
remaining parts of repellent plants [21]. Various traditional repellent plants
used by the local inhabitants in order to avoid mosquito bites have been listed
in Table 1.
338
Table 1. Traditional plants as mosquito repellents [21].

Traditional Names
Tinjut
Woira
Neem
Wogert
Kebercho
Waginos
Eucalyptus
Ades
Gemmero
Tej-sar
Ats-faris
Endode
Azo-hareg
Berberra
Gullo
Scientific Names
Ostostegia integrifolia
Olea europaea
Azadirachta indica
Silene macroserene
Echinops sp.
Brucea antidysenterica
Eucalyptus camaldulensis
Myrtus communis
Capparis tomentosa
Cymbopogen citrates
Datura stramonium
Phytolacca dodecandra
Clematis hirsuta
Millettia ferruginea
Ricinus communis
Family
Lamiaceae
Oleaceae
Meliaceae
Caryophyllaceae
Asteraceae
Simaroubaceae
Myrtaceae
Myrtaceae
Capparidaceae
Rutaceae
Solanaceae
Phytolaccaceae
Ranunculaceae
Fabaceae
Euphorbiaceae
3. Natural products as potential antimosquito agents

The plant world comprises a rich untapped pool of phytochemicals that
may be widely used in the place of synthetic insecticides. Plant-based
products have been used to control domestic pests for a very long time. The
search for and investigation of natural and environmentally friendly
insecticidal substances are ongoing worldwide [22-24]. Insecticidal effects of
plant extracts vary not only according to plant species, mosquito species and
plant parts, but also to extraction methodology [25]. A brief delve into the
literature reveals many laboratory and applied investigations [26-28] into the
biological activity of many plant derived components against a large number
of pathogens and arthropods but the lack of reviews in this area is somewhat
surprising since much effort been invested in locating mosquitocidal
phytochemicals from edible crops, ornamental plants, herbs, grasses, tropical
and subtropical trees and marine angiosperms.
A review by Roark, 1947 [29] highlights about 1200 plant species with a
wide spectrum of bioactive insecticides. A relevant effort to present context
comes from a review by Sukumar et al., 1991 [30] who listed 344 insecticidal
botanical agents. Reviews by Schmutterer, 1990 [31] and Mulla & Su, 1999
[32] did not cover significant topics such as structure-activity relationship
based activity, mode and site of action and joint action of botanical extracts
with other phytochemicals and synthetic insecticides. This review is focused
to cover the entire formal and constant research on mosquitocidal natural
339
products from the 1947 to early 2010 with special attention on structureactivity relationship (SAR) based activity and mechanism of action for most
of natural products, in addition to a number of bioassay procedures and
toxicities of crude plant extracts on different species of mosquitoes reported
in literature Table 2.
Table 2. Mosquitocidal activity of crude plant extracts against different mosquito
larvae as well as adults.
Plant Family
Plant species
Parts
Mosquitoes
Acoraceae
Acorus calamus [91,92]
Rhizome
Agavaceae
Alliaceae
Annonaceae
Agave sisalana [93]

Allium sativa [94]
Annona squamosa [95]
Fiber
Bulb
Leaf
Apiaceae
Mkilua fragrans [96]

Xylopia caudata [96]
Xylopia ferruginea [96]
Calotropis procera [97]
Catharanthus roseus [98]
Daucus carota [92]
Aerial Part
Leaf
Leaf
Root
Whole
Seed
Acanthaceae
Rhinocanthus nasutus [99]
Leaf
Hygrophila auriculata [98]
Shoot
Cx. quinquefasciatus
Ae. aegypti
Ae. albopictus
An. tessellates
An. subpictus
Cx. fatigans
Cx. pipiens
Cx. pipiens
Ae. aegypti
An. gambiae
Ae. aegypti
Ae. aegypti
An. labranchiae
Ae. Aegypti,
Cx. fatigans
Ae. aegypti
An. Stephensi
Justicia adhatoda [98]

Anthemis nobilis [100]
Baccharis spartioides [101]
Cotula cinerea [97]
Sassurea lappa [92]
Leaf
Flower
Aerial Part
Whole Plant
Leaf
Tagetes minuta [102]
Whole Plant
Araceae
Betulaceae
Homalomena propinqua [92]

Alnus glutinosa [103]
Rhizome
Old Litter
Cucurbiataceae
Caesalpinaceae
Bryonopsis laciniosa [104]

Cassia tora [105]
Whole Plant
Seed
Apocynaceae
Asteraceae
Cx. pipiens
Ae. aegypti
An. labranchiae
Ae. Aegypti
Cx. fatigans
Ae. aegypti
An. stephensi
Aedes aegypti
Cx. pipiens
Ae. rusticus
Ae. albopictus
Ae. aegypti
Ae. aegypti,
Cx. pipiens pallens
340
Table 2. Continued
Cupressaceae
Callitris glaucophylla [106]
Wood
Clusiaceae
Calophyllum inophyllum [99]
Leaf and Seed
Cannabaceae
Cannabis sativa [107]
Leaf
Caulerpaceae
Caulerpa scalpelliformis
[108]
Cleome viscosa [109]
Dictyota caryophyllum [110]
Dictyota dichotoma [109]
Codiaeum variegatum [95]
Whole Plant
Whole Plant
Flower
Whole Plant
Leaf
Jatropha curcus [111]

Ricinus communis [112]
Abrus precatorius [98]
Cassia obtusifolia [105]
Leaf
Whole Plant
Shoot
Seed
Croton bonplandianum [98]

Vicia tetrasperma [105]
Pelargonium citrosum [96]
Endostemon tereticaulis [113]
Lavandula afficinalis [112]
Leucas aspera [98]
Mentha arvensis [112]
Mentha piperita [114]
Shoot
Seed
Whole Plant
Aerial Parts
Whole Plant
Whole
Whole Plant
Aerial Parts
Minthostachys setosa [115]
Whole Plant
Moschosma polystachyum
[96]
Ocimum basilicum [116]
Leaf
Ae. aegypti
Ae. aegypti
Ae. Aegypti,
An. stephensi
Ae. aegypti,
Cx. pipiens pallens
Ae. Aegypti,
Ae. aegypti
An. gambiae
An. stephensi
An. stephensi
Ae. aegypti
An. Tessellatus
Ae. Aegypti
An. Tessellatus
Aerial Parts
An. stephensi
Origanum majoranal [100]

Plectranthus longipes [116]
Pogostemon cablin [117]
Rosmarinus officinalis [118]
Thymus capitatus [119]
Cinnamomum iners [96]
Cinnamomum kuntsleri [96]
Leaf
Aerial Parts
Leaf
Shoot
Whole Plant
Leaf
Leaf
Cx. pipiens
An. gambiae
Ae. aegypti
An. stephensi
Cx. Pipiens
Ae. aegypti
Ae. aegypti
Cinnamomum pubescens [96]
Leaf, Bark and

Twig
Bark
Ae. aegypti
Ae. aegypti
Bark and Leaf
Ae. aegypti
Capparidaceae
Caryophyllaceae
Dictyotaceae
Euphorbiaceae
Fabaceae
Geraniaceae
Labiatae
Lauraceae
Cinnamomum scortechinii
[96]
Cinnamomum sintoc [96]
Ae. aegypti,
Cx. annulirostris
Cx. quinquefasciatus,
An. Stephensi
Ae. aegypti
An. Stephensi,
Ae. aegypti
Ae. aegypti
341
Table 2. Continued
Cinnamomum zeylanicum
[118]
Bark and Leaf
Liliaceae
Lythraceae
Gloriosa superb [98]

Pemphis acidula [120]
Whole
leaf
Menispermaceae
Meliaceae
Abuta grandifolia [115]

Azadirachta indica [95]
Fruit
Leaf and Seed
Khaya senegalensis [106]

Lansium domesticum [95]
Seed
Leaf
Melia azadirachta [112]
Whole Plant
Melia volkensii [121]
Seed and Fruit
Eucalyptus camaldulensis
[122]
Eugenia caryophyllus [112]
Eucalyptus globules [100]
Fruit
Syzygium aromaticum [117]
Leaf
Oleaceae
Papaveraceae
Pinaceae
Piperaceae
Jasminum fructicans [100]

Argemone mexicana [111]
Cedrus deodara [112]
Piper longum [123]
Piper nigrum [124]
Leaf
Leaf
Whole Plant
Fruit
Fruit
Plumbaginaceae
Plumbago dawei [125]
Root
An.gambiae
Plumbago stenophylla [125]

Plumbago zeylanica [125]
Cymbopogon citratus [96]
Cymbopogon flexuosus [112]
Cymbopogon martini [112]
Sorghum bicolour [126]
Vetiveria zizanioides [100]
Root
Root
Whole Plant
Whole Plant
Whole Plant
Seedling
Rhizome
An.gambiae
An.gambiae
An. stephensi
An. stephensi
Cx. pipiens
Cx. pipiens
Spermacoce hispida [98]

Citrus limon [94]
Zanthoxyllum acanthopodium
[96]
Whole
Peel
Stem
Cx.quinquefasciatus
Cx. pipiens
Ae. aegypti
Myrtaceae
Poaceae
Rubiaceae
Rutaceae
Whole Plant
Whole Plant
An. stephensi
Ae. aegypti
Ae. aegypti
Ae. aegypti
Ae. aegypti
Cx. annulirostris
Ae. Aegypti
An. stephensi
Cx. pipiens molestus
Cx. pipiens molestus
Ae. aegypti
An. arabiensis
Cx. pipiens
An. stephensi
An. stephensi
Cx. pipiens
Ae. albopictus
Ae. aegypti
An. dirus
Cx. pipiens
An. stephensi
Cx. pipiens pallens
Cx. pipiens pallens
Ae. aegypti
Ae. togoi
342
Table 2. Continued
Solanaceae
Simaroubaceae
Thymelaeaceae
Umbelliferae
Valerianaceae
Verbenaceae
Zingiberaceae
Solanum elaeagnifolium [97]

Solanum indicum [98]
Solanum sodomaeum [97]
Withania somnifera [111]
Solanum xanthocarpum [127]
Quassia amara [128]
Aquilaria malaccensis [96]
Dirca palustris [129]
Angelico glauca [92]
Berry
Shoot
Seed
Leaf
Leaf
Whole Plant
Wood
Seed
Aerial Parts
Pimpinella anisum [122]

Valarian wallichii [92]
Aloysia citriodora [101]
Clerodendrun inerme [98]
Stachytarpheta jamaicensis
[98]
Vitex nequrdo [109]
Curcuma domestica [91]
Kaempferia galangal [98]
Zingiber officinalis [130]
Seed
Rhizome
Whole Plant
Leaf
Shoot
An. labranchiae
An. labranchiae
Ae. aegypti
Ae. aegypti
Ae. aegypti
Cx. fatigans
Cx. pipiens
Ae. Aegypti
Ae. aegypti
Whole Plant
Rhizome
Whole
Tubers
An. culicifacies
4. Alkanes, alkenes, alkynes and simple aromatics

The hydrocarbon, octacosane (1) isolated from Moschosma polystachyum
shows significant larvicidal activity against Culex quinquefasciatus mosquito
with LC50 value of 7.21.7 mg/L [33]. The (E)-6-hydroxy-4,6-dimethyl-3heptene-2-one (2) isolated from Ocimum sanctum exhibit toxicity against fourthinstar larvae of Aedes aegyptii with LD100 value of 6.25 g/mL in 24 h [34].
Among the acetylenic compounds, falcarinol (3) and falcarindiol (4) isolated
from Cryptotaenia canadensis display strong activity against Culex pipiens larvae
[35,36]. The more lipophilic 3 with LC50 values of 3.5 and 2.9 ppm in 24 h and
48 h, respectively exert strong toxicity than the more polar acetylene 4 with LC50
values of 6.5 and 4.5 ppm in 24 and 48 h, respectively [37].
The volatile aromatics, 4-ethoxymethylphenol (5), 4-butoxymethylphenol
(6), vanillin (7), 4-hydroxy-2-methoxycinnamaldehyde (8), and 3,4dihydroxyphenylacetic acid (9) isolated from Vanilla fragrans show very
efficient mortality against mosquito larvae. The compunds 5-8 display 100%
larval mortality at 0.5, 0.4, 2.0 and 1.0 mg/mL concentrations, respectively while
9 shows 17% larval mortality at a concentration of 1.0 mg/mL [38].
The hexane extract of Delphinium cultorum shows significant
mosquitosidal activity (100% mortality at a concentration of 10 mg/mL)
against Ae. aegyptii larvae at 2 h. A literature report comprising GC-EIMS
343
analysis of hexane extract of D. cultorum resulted into isolation of six

volatiles, ethylmethylbenzene (10), 1-isopentyl-2,4,5-trimethylbenzene (11),
2-(hex-3-ene-2-one)phenyl methyl ketone (12), E and Z isomers of
3-butylidene-3H-isobenzofuran-1-one (13 and 14) and 2-penten-1-ylbenzoic
acid (15) [39]. The trans-asarone (16) isolated from seeds of Daucus
carota shows 100% mortality at a concentration of 200 g/mL against fourthinstar larvae of Ae. Aegyptii [40]. The compound (17) isolated from rhizomes
of Curcuma longa display 100% mortality against Ae. aegyptii larvae with
LD100 value of 50 g/mL in 18 h [27]. Similarly, 18 isolated from leaf and
stem of Ocimum sanctum display mosquitocidal activity against fourth-instar
larvae of Ae. aegyptii with LD100 value of 200 g/mL in 24 h, respectively
[34].
The 5-allyl-2-methoxyphenol (19) isolated from seeds of Apium graveolens
exhibit 100% mortality on fourth-instar Ae. aegyptii larvae at 200 g/mL
concentration [41]. The trans-anethole (20), methyl eugenol (21) and iso-methyl
eugenol (22) isolated from Myrica salicifolia display 100% mortality with LD100
value of 20, 60 and 80 ppm in 24 h against 4th instar larvae of Ae. aegypti [42].
The stilbenes (23-29), isolated from the root bark of Lonchocarpus chiricanus
possess larvicidal activities Ae. aegypti mosquito larvae. Among these, 27 at a
concentration of 3.0 ppm exhibits highest activity while 24 and 25 with minimal
concentration of 6.0 ppm each, display pronounced affect by kill all the larvae in
24 h. The compounds 23, 26, 28 and 29 with 50 ppm concentrations show
moderate activity against larvae of Ae. aegypti [43].
5. Lactones
The lactones 30 and 31, isolated from Hortonia floribunda,
H. angustifolia and H. ovalifolia, exhibit potent larvicidal activity against the
second instar larvae of Ae. aegypti with LC50 values of 0.41 and 0.47 ppm,
respectively [44]. The 3-n-butyl-4,5- dihydrophthalide (32) isolated from
seeds of Apium graveolens show 100% mortality on fourth-instar Ae. aegyptii
larvae at a concentration of 25 g/mL [41]. The sedanolide (33) isolated from
seeds of same species exhibits 100% mortality at 50 g/mL concentrations
against fourth-instar larvae of Ae. aegyptii [45].
6. Essential oils and fatty acids

The essential oils, -phellandrene (34), limonene (35), p-cymene (36),
-terpinene (37), terpinolene (38) and -terpinene (39) isolated from leaves
of Eucalyptus camaldulensis possess significant larvicidal activity against
344
CH3
H2C
OH
H 3C
CH3
OH
CH3
R
CH3
CH3
OH
OH
3R=H
4 R = OH
COOH
CHO
CHO
OCH3
CH3
OCH3
C2 H5
C4H9
OH
OH
OH
CH3
CH3
CH3
OH
10
CH3
CH3
O
CH3
H3 C
O
CH3
CH3
H 3C
11
12
CH3
13
CH 3
CH3
OCH3
H3CO
14
OH
OCH3
H 3C
H3 C
H3 C
O
CH2
CH2
15
17
16
18
OH
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
CH 3
CH2
CH2
H 3C
19
20
22
21
CH3
OH
OH
H3C
OH
CH3
CH3
OCH3
OCH3
OH
H3C
H3C
CH3
CH3
24
23
H3C
O
25
CH3
OH
OCH3
OCH3
OH
27
26
H3C
HO
CH3
H3C
CH3
O
CH3
OCH3
CH3
OH
OH
H3C
28
29
CH3

H3C
345
O
O
O
9
O
H
H3C
30
C4H9
C4H9
32
31
33
fourth-instar larvae of Ae. aegypti and Ae. albopictus. The compound 39

exert the strongest activity against Ae. aegypti larvae with LC50 value of 14.7
g/mL (LC90 = 39.3 g/mL) in 24 h, following the compounds 34 (LC50 =
16.6 g/mL, LC90 = 36.9 g/mL), 35 (LC50 = 18.1 g/mL, LC90 = 41.0
g/mL), 36 (LC50 = 19.2 g/mL, LC90 = 41.3 g/ mL), 38 (LC50 = 28.4
g/mL, LC90 = 46.0 g/mL) and 37 (LC50 = 30.7 g/mL, LC90 > 50.0 g/mL)
[46]. Similarly, 40-49 isolated from leaves of different Cinnamomum
osmophloeum exhibit strong activity against Ae. aegypti larvae.
CH3
H3C
CH3
CH3
H3C
34
CH2
H3C
35
CH3
CH3
CH3
CH3
H3C
CH3
H3C
37
36
CH3
H3C
CH3
38
CH3
39
CHO
CHO
OH
CHO
COOH
HO
41
40
43
42
O
O
44
OCOCH3
CH3
H2C
H3C
H
CH3
CH3
49
CH3
CH3
CH3
45
H3C
H
H2C
47
46
O
HO
CH3
H2C
H2C
CH3
CH3
H2C
CH3
CH3
O
48
CH3
50
OH
CH3
51
346
Among these volatiles, benzaldehyde (40) 4-hydroxybenzaldehyde (41),

benzenepropanal (42), cinnamic acid (43), cinnamyl alcohol (44), bornyl
acetate (45), -caryophyllene (46), caryophyllene oxide (47) and linalool (48)
possess strong activities with LD50 value of 50 g/mL while 49 with LD50
value of 33 g/mL produce significant larvicidal effect [47]. Likewise,
among the 2,2-dimethyl-6-vinylchroman-4-one (50) and 2-senecioyl-4vinylphenol (51) isolated from the roots of Eupatorium betonicaeforme, 50
shows efficient larvicidal potential, causing 84% larval mortality at a
concentration of 12.5 g/mL in compared to 51 exhibiting 40-100% mortality
at 5-100 g/mL concentrations [48]. The fatty acid constituents, linoleic acid
(52) and oleic acid (53) isolated from Dirca palustris exhibit mosquitocidal
activity against fourth instar Ae. aegyptii larvae with LD50 values of
100 g/mL at 24 h, each [49].
H3C(H2C)6H2C
H3C(H2C)4(HC
CHCH2)2(CH2)5CH2COOH
52
CH2(CH2)5CH2COOH
C
H
53
7. Terpenes
7.1. Monoterpenes
The monoterpenoids, thymol (54), cholorothymol (55), carvacrol (56),
-citronellol (57), cinnamaldehyde (58) and eugenol (59) isolated from a
number of plant species possess mosquitocidal activity against forth instar
larvae of Culex pipiens with LC50 values of 37.95, 14.77, 44.38, 89.75, 58.97
and 86.22 g/mL, respectively. The N-methyl carbamate derivatives of
54-57, i.e. 60-63 display high toxicities against forth instar larvae of Cx.
pipiens with LC50 values of 7.83, 11.78, 4.54, 15.90 g/mL, respectively.
Moreover, the N-methyl carbamate derivatives of geraniol (64) and borneol
(65) also exhibit significant activity against forth instar larvae of Cx. pipiens
with LC50 values of 24.08 and 33.00 g/mL, respectively [50].
Likewise, 1,8-cineole (66) isolated from leaves of Hyptis martiusii
display pronounced insecticidal effect against Ae. aegypti larvae at
concentrations 25 (10%), 50 (53%), 100 (100%) mg/mL [51]. Other
monoterpenoids, geranial (67) and neral (68) isolated from Magnolia
salicifolia show 100% mortality with LD100 value of 100 ppm in 24 h against
4th instar Ae. aegypti [42].

CH3
347
CH3
CH3
Cl
OH
CH3
OH
OH
OCH3
CHO
OH
OH
CH2
H3C
CH3
H3C
CH3 H3C
CH3 H3C
55
54
CH3
56
CH3
CH3
CH3
Cl
Cl
H3C
OCNHCH3
OCNHCH3
H3C
CH3
H3C
61
60
CH3
O
OCNHCH3
H3C
CH3
CH3
CH3
CH3
CHO
O
CHO
H3C
65
CH3
63
CH3
CH3
64
O
OCNHCH3
62
OCNHCH3
H3C
CH3
O
OCNHCH3
CH3
59
58
57
CH3
66
CH3
H3C
67
H3C
CH3
68
7.2. Sesquiterpenes
The -selinene (69) isolated from seeds of Apium graveolens show 100%
mortality against fourth-instar larvae of Ae. aegyptii at a concentration of
50 g/mL [41]. The pregeijerene (70), geijerene (71), and germacrene D (72)
isolated from leaves of Chloroxylon swietenia possess activity against An.
gambiae, Cx. quinquefasciatus and Ae. aegypti. The results of SAR indicate
that 72 with LD50 values of 1.8, 2.1 and 2.810-3 exert highest activity
followed by 70 with LD50 values of 3.0, 3.9 and 5.110-3 while 71 with LD50
values of 4.2, 5.4 and 6.810-3 display lowest activity against An. gambiae,
Cx. quinquefasciatus and Ae. aegypti, respectively [52]. The sesquiterpene
lactones, 73 and 74 isolated from leaves, stem bark, flowers and fruits
of Magnolia salicifolia exhibit significant toxicity against Ae. aegypti
larvae. The lactone 73 with LD100 value of 15 ppm kills all the mosquito
larvae of Ae. aegypti in 24 h while 74 possess 100% mortality with LD100
value > 50 ppm in 24h [53]. The sesquiterpene, 74 does not show
mosquitocidal activity at 50 ppm, thus suggesting the presence of a double
bond rather than an epoxide at C-4 and C-5 in 73 is essential for
mosquitocidal activity [42].
348

CH3
CH3
CH3
CH2
CH2
CH2
CH3
CH2
CH3
69
CH3
71
70
CH3
CH3
CH3
H3C
CH3
CH2
CH3
72
CH2
H3C
73
CH2
O
O
74
7.3. Diterpenes
Among the diterpenes, 75-77 isolated from Pterodon polygalaeflorus
exhibit significant larvicidal activity against fourth-instar larvae of Ae.
aegypti with LC50 values of 50.08, 14.69 and 21.76 g/mL, respectively [54].
Similarly, hugorosenone (78) isolated from the Hugonia castaneifolia display
larvicidal activity against mosquito larvae An. gambiae with LC50 values of
0.3028 and 0.0986 mg/mL at 24 and 48 h, respectively [55].
O
CH3
CH3
O
O
H3C
H
CH3 OH
75
H3C
H
CH3 OH
76
CH2
CH3
OH
CH3
OH
H3C
H
CH3 OH
77
OH
CH3
OCH3 HO
H3C
H
CH3
78
7.4. Triterpenes
The
triterpenes,
3,24,25-trihydroxycycloartane
(79)
and
beddomeilactone (80) isolated from Dysoxylum malabaricum and
D. beddomei possess strong larvicidal, pupicidal and adulticidal activity and
also affect the reproductive potential of adults by acting as oviposition
deterrents. Among these, the 79 at a concentration of 10 ppm kills more than
90% of pupae and 85% of adults. Similarly, 80 at the same concentration
results in more than 95% of pupal and larval mortality and more than 90%
mortality in case of adult An. Stephensi [56].
349
OH
H3C
CH3
H2C
H
CH3
HO
H3C
H
CH3
CH3
OH
CH3
O
H2C
CH3
H
CH3
H3C
79
CH3
H3C
CH3
H
COOH
80
7.5. Tetranortriterpenoids
The limonin (81), nomilin (82) and obacunone (83) isolated from the
seeds of Citrus reticulate [57] exhibit mosquitocidal activity against
fourth instar larvae of Cx. quinquefasciatus at 59.57, 26.61 and 6.31 ppm
concentrations, respectively [58]. The limonoids 84-86, isolated from the
root bark of Turraea wakefieldii exhibit activity against late third or early
fourth-instar larvae of An. gambiae. In SAR, the strong larvicidal activities
of 84, 85 and 86 with LD50 values of 7.83, 7.07 and 7.05 ppm, respectively
indicate that the epoxidation of the C-14, C-15 double bond or deacetylation of the 11-acetate group does not alter the larvicidal activity
[59]. Other limonoids, azadirachtin (87), salannin (88), deacetylgedunin
(89), 17-hydroxyazadiradione (90), gedunin (91) and deacetylnimbin (92)
isolated from Azadirachta indica possess significant activity against An.
stephensi larvae. Among these, 87 with EC50 value of 0.014, 0.021, 0.028
and 0.034 ppm, 88 with EC50 value of 0.023, 0.036, 0.047 and 0.061 ppm,
89 with EC50 value of 0.028, 0.041, 0.0614 and 0.078 ppm, 90 with EC50
value of 0.047, 0.054, 0.076 and 0.0104 ppm, 91 with EC50 value of 0.058,
0.073, 0.095 and 0.0117 ppm and 92 with EC50 value of 0.055, 0.067, 0.091
and 0.0113 ppm, show activity against first, second, third and fourth instar
larvae of An. stephensi, respectively. The metabolite 87 exerts 100% larval
mortality at 1 ppm concentration thus demonstrates that the A. indica
(Neem) products may have benefits in mosquito control programs [60].
Likewise, 93-95 isolated from Turraea wakefieldii and T. floribunda
exhibit toxicity against An. gambiae larvae with LD50 values of 7.1, 4.0,
and 3.6 ppm, respectively and display more potency than azadirachtin (87;
LD50 value of 57.1 ppm), a commonly used positive control [61].
350

O
O
CH3
O
OAc
CH3
H3C
81
OAc
OAc CH
3
CH3
H3C
H
CH3
H3C
H3C
84
O
H3C
CH3
OH
86
H3C
O
CH3
OH
H3C
O
CH3
CH3
AcO
CH3
88
CH3
CH3
CH3OH
CH3
O
H3C
CH3
CH3
OAc
CH3
92
H3C
AcO
OAc
CH3
CH3
93
HO
H3C
H
O
(H3C)2HCOCO
CH3
AcO
OAc
CH3 CH3
CH3
CH3
OAc
CH3
94
H
OH
O
(H3C)2HCOCO
H
O
CH3
91
OAc
H3C
90
CH2
CH3
O
OAc
CH3
CH3
CH3
O
OH
H3C
89
CH3
CH3
OH
CH3
O
HO
CH3
O
H 3C
H3C
CO
CH3
87
CH3
CH3
H3C
OCH3
OAc
H
CH3
H 3C
H3C
H3C
AcO
AcO
83
OAc
OH
CH3
H
CH3
CH3
85
OAc
CH3
OH O
O O
O
H3C
CH3
CH3
H
CH3
OAc
OAc CH
3
82
CH3
H
CH3
O
O
CH3
CH3
O
O
O
O
CH3
CH3
CH3
HO
H3C
H
O
OH
CH3
95
351
8. Alkaloids
8.1. Alkamides
The alkamides, undeca-2E-4Zdien- 8,10-diynoic acid isobutylamide
(96), undeca-2Z,4E-dien-8,10-diynoic acid isobutylamide (97), dodeca2E,4Z-dien-8,10-diynoic acid isobutylamide (98), undeca-2E,4Z-dien8,10-diynoic acid 2-methylbutylamide (99), dodeca-2E,4Z-dien-8,10diynoic acid 2-ethylbutylamide (100), and a mixture of dodeca2E,4E,8Z,10E-tetraenoic acid isobutylamide (101) and dodeca2E,4Z,8Z,10Z-tetraenoic acid isobutylamide (102) isolated from the dried
roots of Echinacea purpurea and other plant species of family Asteraceae
[62] display significant mosquitocidal activity against Ae. aegyptii. The
mixture of 101 and 102 exert most effective mosquitocidal activity at
100 g/mL concentration with 87.5% mortality of mosquito larvae in
15 min while 96 display 100% mortality at same concentration in 2 h.
The alkamides, 97 and 98 exhibit 50% mortality at the end of 9 h with
100 g/mL while 99 and 100 show least activity with 10% mortality at a
concentration of 100 g/mL in 24 h [63].
Among isobutyl amides, pellitorine (103), guineensine (104), pipercide
(105), and retrofractamide-A (106) isolated from fruits of Piper nigrum
exhibit toxicity against Cx. Pipiens larvae. The toxicities against Cx. pipiens
larvae falls in the order: 105 (0.004 ppm)> 106 (0.028 ppm) > 104 (0.17
ppm) > 103 (0.86 ppm). These compounds also display larvicidal activity
against Ae. aegypti larvae in which 106 exerts pronounced activity at a
concentration of 0.039 ppm than 105 (0.1 ppm), 104 (0.89 ppm) and
103 (0.92 ppm). Also, the amides 105, 106 and 104 exhibit 255, 31 and
5 times more toxicity than 103, respectively. The SAR indicates that the
N-isobutyl amine moiety might play a crucial role in the larvicidal
activity, but the methylenedioxyphenyl moiety does not appear essential for
toxicity [64].
8.2. Carbazole alkaloids

Among carbazoles, mahanimbine (107), murrayanol (108) and
mahanine (109) isolated from leaves of Murraya koenigii display
promising mosquitocidal activity against Ae. Aegyptii [65]. The alkaloid
107 exhibits 100% mortality at a concentration of 100 g/mL while
108 and 109 at 12.5 g/mL concentration display 100% mortality
[66,67].
352

O
96
H2
C CH2
C NH
C
CH3
H
H
C
C
CH3
O
H3C
H
H
O
C
C
C
H2
C
N
H
H2
C CH2
101
H
H
C
102
C
H
CH3
CH3
C
H2C CH2
H
H
C
C
C
C
103
H2
C CH
H
C
H3C
H
N
H
H
CH3
H2C
H2
C CH
CH2
H2C
CH
H
C
N
H
H
H3C
C
C
H
N
H3C
O
CH3
CH3
O
H3C
H2C
100
CH3
CH3
H2
C CH2
H2
C CH
H
99
H
N
C
C
CH3
H2
C CH2
H2
C CH
H
N
98
CH3
H2
C CH2
H
97
H2
C CH
H
N
H2
C CH
CH3
CH3
O
CH3
CH3
CH3
353
O
104
CH3
H
N
CH3
CH3
H
N
105
O
CH3
O
O
CH3
H
N
106
O
CH3
O
H3C
CH3
R
N
H
H3CO
CH3
CH3
OH
CH3
N
H
CH3
CH3
CH3
108
107: R = H
109: R = OH
8.3. Naphthylisoquinoline alkaloid

The alkaloid, dioncophylline-A (110) isolated from Triphyophyllum
peltatum [68] possess promising activity against different larval stages of An.
stephensi with LD50 values of 0.5, 1.0 and 2.0 mg/L concentrations at 3.33,
2.66 and 1.92 h, respectively. In each instar larval stage, the LC50 values
decrease as a function of time indicating that 110 continues to exert its action
during at least 48 h [69].
CH3
CH3
N
OH
CH3
H3CO
H3CO
110
8.4. Piperidine alkaloids

The alkaloid, pipernonaline (111) isolated from fruits of Piper longum
exhibits activity against the fourth-instar larvae of Ae. aegypti [70] and Cx.
Pipiens [71] with LC50 values of 0.25 and 0.21 mg/L, respectively in 24 h.
354
The larvicidal potential of 111 against the Ae. aegypti, is comparable to that
of pirimiphos-methyl, a commonly used insecticide and may be useful for
development of new mosquito larvicides [70]. Similarly, N-methyl-6-(decal',3',5'-trienyl)-3--methoxy-2--methylpiperidine (112) isolated from stem
bark of Microcos paniculata shows significant insecticidal activity against
second instar larvae of Ae. aegypti with MC50 value of 1.0 ppm and LC50
value of 2.1 ppm at 24 h against second instar larvae of Ae. aegypti [72].
O
O
111
O
O
CH3
112
H3C
CH3
CH3
Insecticidal activity evaluation of piperidine derivatives (113-145)

against female adults of Ae. aegypti, along with the structure-activity
relationships (SAR) using piperine (E,E)-1-piperoyl-piperidine as standard
insecticide (LD50 value of 8.13 g per mosquito) reveals that different
moieties (ethyl-, methyl-, and benzyl-) attached to the piperidine ring are
responsible for different toxicities (i.e. 113, 1.77; 114, 2.74; 115, 8.76; 116,
1.20; 117, 1.09; 118, 1.13; 119, 4.14; 120, 1.92; 121, 2.07; 122, 1.80; 123,
4.90; 124, 4.25; 125, 2.63; 126, 6.71; 127, 1.22; 128, 1.67; 129, 0.94; 130,
1.56; 131, 1.83; 132, 0.84; 133, 29.20; 134, 14.72; 135, 19.22; 136, 12.89;
137, 0.80; 138, 1.38; 139, 3.59; 140, 1.32; 141, 2.07; 142, 7.43; 143, 1.54;
144, 2.72 and 145, 14.72 g) against Ae. aegypti.
The 3-methylpiperidines (119-122) exhibit slightly lower toxicities than
that of 2-methyl-piperidines (113-118) with LD50 values ranging from 1.80 to
4.14 g. However, there is no significant difference found between the
toxicities of 3-methyl piperidines (119-122) and 4-methyl piperidines (123127), whose LD50 values range from 1.22 to 6.71 g while the saturated long
chain derivatives of 4-methyl-piperidine (123 and 126) show lower toxicity
than others with LD50 values of 4.90 and 6.71 g, respectively [73]. Further,
SAR among the piperidines with two different moieties (ethyl- and benzyl-)
attached to the carbons of the piperidine ring against Ae. aegypti establishes
that 2-ethyl-piperidines (128-132) show higher toxicity than the benzylpiperidines (133-136) with LD50 values ranging from 0.84-1.83 and 12.8929.20 g, respectively. The results of SAR suggest that ethyl-piperidines
355
O
O
R
H3C
H3C
CH3
C6H13
CH3
120
CH3
122
CH3
CH3
125
O
N
CH3
CH3
H3C
127
O
N
CH3
129
130
O
C8H17
CH3
132
H3C
N
134
H2C
135
136
O
H2C
O
H2C
N
137 R = CH3
139 R = C6H5
N
138
H3C
O
O
H2C
H2C
N
141
140
CH3
O
H2C
N
CH3
143
O
N
144
CH3
H2C
142
H2C
CH3
131
133
CH3
124
CH3
O
(H2C)2
CH3
128
O
N
118
121
O
N
123 R = C9H19
126 R = C11H23
H3C
O
N
119
H3C
117
O
CH3
H3C
114 R = C9H19
115 R = C11H23
116 R = C6H13
113
O
N
H2C
CH3
N
145
356
generally exhibit higher toxicities than methyl-piperidines, followed by

benzyl-piperidines whose toxicities are lowest.
Among the three 1-undec-10-enoyl-piperidines (134-139) with the three
different moieties at the second carbon of the piperidine ring, the 137
displays highest toxicity with LD50 value of 0.80 g, in compared to 138
(LD50 value of 1.38 g) and 139 (LD50 value of 3.59 g). Similarly, among
the three 1-undec-10-enoyl-piperidines (140-142) with the three different
moieties attached to the third carbon of the piperidine ring, the 140 exhibit
highest toxicity (LD50 value of 1.32 g), followed by the 141 and 142 with
LD50 values of 2.07 and 7.43 g, respectively. Likewise, among the three
1-undec-10-enoyl-piperidines (143-145) with the three different moieties
attached to the fourth carbon of the piperidine ring, the 143 shows highest
toxicity (LD50 value of 1.54 g), following 144 (LD50 value of 2.72 g) and
145 (LD50 value of 14.72 g).
8.5. Stemona alkaloids

The Stemona alkaloids, stemocurtisine (146), stemocurtisinol (147) and
oxyprotostemonine (148) isolated from roots of Stemona curtisii exhibit
potency against mosquito larvae An. minimus with LC50 values of 18, 39 and
4 ppm, respectively. Among these, 148 display highest potency with LC50
value of 4 ppm [74].
H
H
H3C
H H
OCH3
H3C
H
N
H3C
O
147
146
CH3
H
HC
OCH3 3
O
N
H3C
O
HH
OCH3H3C
O
148
O
N
CH3
OH
357
9. Phenolic derivatives
9.1. Naphthoquinones
The cordiaquinones (149-152), isolated from the roots of Cordia curassavica
show toxic properties against larvae of the yellow fever-transmitting Ae. aegypti.
The quinones 149 and 151 with 25.0 g/mL concentration result in 100% larval
mortality while 150 and 152 with 12.5 g/mL concentrations kill all the Ae.
aegypti larvae in 24 h [75]. Likewise, the alkaloids 153-155 isolated from the
roots of Cordia linnaei exhibit larvicidal potency against Ae. aegypti at 12.5, 50.0
and 25.0 g/mL concentrations, respectively [76]. The naphthoquinone,
plumbagin (156) isolated from Plumbago zeylanica [77] and other plant species
[78,79] exhibit mosquito larvicidal activity against An. gambiae with LC50 value
of 1.9 g/mL [80,81]. Some of natural and synthetic naphthoquinones e.g.
lapachol (157) and its synthetic derivatives (158-160) possess toxicity against
fourth instar larvae of Ae. aegypti. The quinone 159 with LC50 value of 15.24 M
exerts higher activity in compared to 160 (19.45 M), 158 (33.94 M) and 157
(108.7 M). Likewise, juglone (161) and its synthetic derivatives (162-170)
display significant toxicity against fourth instar larvae of Ae. aegypti. The bromonaphthoquinone 167 with LC50 value of 3.46 M exhibits the best larval toxicity
in compared to 164 (4.64 M), 165 (3.98 M), 166 (36.48 M), 167 (3.46 M),
168 (24.79 M) and 169 (21.62 M) while 161 and derivatives 162, 163 and 170
display relatively weak toxicity with LC50 values of 20.61, 21.08, 42.12 and
86.93 M, respectively [82].
The shikonin (171), alkannin (172) and shikalkin (173) isolated from root of
Lithospermum erythrorhizon [83], Alkanna tinctoria [84] and young leaves and
stems of L. officinale [85] exhibit toxicities against mosquito larvae. The quinone
171 at a concentration of 3.9 mg/L show high toxicity against mosquito larvae
followed by 173 and 172 with 8.73 and 12.35 mg/L concentrations, respectively.
Results of SAR indicate that naphthoquinones, compared with other natural
compounds with larvicidal activity, are very toxic against mosquito larvae and
would be a potential source of natural larvicidal substances [86].
9.2. Coumarins
Coumarin, pachyrrhizine (174), isolated from Neorautanenia mitis
exhibits activity against An. gambiae adults with LC50 value 0.007 mg/mL.
The marmesin (175), isolated from Aegle marmelos exhibits toxicity against
An. gambiae adults with LC50 and LC90 values of 0.082 and 0.152 mg/L,
respectively [87].
358

CH3
H3C
CH3
O O
CH3
OH
H3C
O
CH3
O
149
O CH3
H3C
150
O
CH3
CH3
CH3
H2C
O
151
CH3
153
CH3 CHOH
CH3
H3C
O
O
152
H3C
CH3
154
CH3
H3C
OH
H3C
OH
OH
O
O
O
155
OH
OAc
CH3
CH3
CH3
159
OH
CH3
158
157
OLi
CH3
CH3
O
OH
O
156
O
Br
CH3
CH3
OH O
OCH3 O
OH
166
165
O
Br
OAc
164
Br
Br
OH O
163
162
OCH3O
OAc O
161
160
OH
OAc
167
OCH3 O
169
168
OH
Br
Br
O
OH
CH3
CH3
CH3
O
170
OH
O
171
OH
CH3
OH
172
OH
CH3
CH3
OH
O
173
OH
CH3
359
O
O
H3C
O
H3C
OCH3
HO
O
175
174
9.3. Isoflavonoids
The isoflavonoids neotenone (176), neorautanone (177) isolated from
Neorautanenia mitis display activity against adult An. gambiae mosquitoes
with LD50 values of 0.008 and 0.009 mg/mL, respectively [88].
O
OCH3
OCH3
OCH3
OCH3
O
177
176
9.4. Pterocarpans
The pterocarpans, neoduline (178), 4-methoxyneoduline (179), and
nepseudin (180) isolated from tubers of Neorautanenia mitis exhibit
mosquitocidal activity against An. gambiae and Cx. quinquefaciatus larvae
with LD50 values 0.005, 0.011 and 0.003 mg/mL, respectively [87,89].
O
O
O
O
H
H
O
178
H3CO
OCH3
OCH3
179
180
360
9.5. Lignans
The lignans, conocarpan (181), eupomatenoid-5 (182), eupomatenoid-6
(183) and decurrenal (184) isolated from Piper decurrens possess significant
mortality at 10 g/mL concentrations against mosquito larvae [90].
O
OCH3
O
OH
H3C
181
O
H
OH
H3C
CH3
182
CH3
O
OH
OH
C
O
H
183
CH3
H3C
CH3
184
10. Conclusive remarks

Our ancestors exclusively depended on the use of plant-derived products
to repel or kill mosquitoes and other blood sucking insects. Modern synthetic
chemicals could provide immediate results for the control of
insects/mosquitoes; on the contrary they bring irreversible environmental
hazard, severe side effects and pernicious toxicity to human being and
beneficial organisms. In concern to the quality and safety of life and the
environment, the emphasis on controlling mosquito vectors has shifted
steadily from the use of conventional chemicals toward alternative
insecticides that are target-specific, biodegradable, and environmentally
safe, and these are generally botanicals in origin. Therefore, right now use
of eco-friendly and cost-free plant based products for the control of
insects/mosquitoes is inevitable. Efforts should be made to promote the
use of easy accessible and affordable traditional insect/mosquito repellent
plants.
Acknowledgement
The author sincerely acknowledged Department of Chemistry, Faculty of
Science, Banaras Hindu University, Varanasi, India, for infrastructural
facilities.
361
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1289.
Research Signpost
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ISBN: 978-81-308-0448-4
12. Soybean constituents and their

functional benefits
Ajay K. Dixit1, J. I. X. Antony1, Navin K. Sharma1 and Rakesh K. Tiwari2
1
Biosciences, ITC R & D Center, Peenya Industrial Area, Peenya, Banglore-560058, India
Dept. Biomed. & Pharma. Res., University of Rhode Island, Kingston, RI 02881 0809, USA
Abstract. Soybean [Glycine max (L.)] is in use for more than 5000
years in China and South East Asia as food. Epidemiological studies
show its importance in prevention of several diseases. Recently, an
upsurge of consumer interest in the health benefits of soybean and
soy products is not only due to its high protein (38%) and high oil
(18%) content, but also due to the presence of physiologically
beneficial phytochemicals. Past several years of clinical and
scientific evidences have revealed the medicinal benefits of the soy
components against metabolic disorders (cardio-vascular diseases,
diabetes and obesity etc.) as well as other chronic diseases (cancer,
osteoporosis, menopausal syndrome and aneamia etc.). Many of the
health benefits of soy are derived from its secondary metabolites,
such as, isoflavones, phyto-sterols, lecithins, saponins etc. In this
review we discuss the bioactive components of soybean and their
role in prevention, maintenance, and/or curing of diseases.
1. Introduction
The term functional foods was first introduced in Japan in the mid1980s and refers to processed foods containing ingredients that aid specific
bodily functions in addition to being nutritive [1]. The soybean [Glycine
max(L.) Merrill] a native of China, have been extensively used as important
Correspondence/Reprint request: Dr. Ajay K. Dixit, Biosciences, ITC R & D Center, Peenya Industrial Area
Peenya, Banglore-560058, India. E-mail: ajay.dixit@itc.in
368
Ajay K. Dixit et al.
source of dietary protein and oil throughout the world. Though, soybean is a
widely cultivated crop, most of it is used as the raw material for oil milling,
and the residue (soy meal) is mainly used as feedstuff for domestic animals
[2]. Dry soybean contain 36% protein, 19% oil, 35% carbohydrate (17% of
which dietary fiber), 5% minerals and several other components including
vitamins [2].
Several years of rigorous scientific and clinical research has established
that most of the components of soybean have beneficial health effects as
characterized by its preventive potential for the so-called life-style-related
diseases. The impact of most of the nutritionally and physiologically
functional components of soybean [3] have been summarized in Table 1.
Table 1. Functional components of soy and their impact [3].
-Linolenic acid
Isoflavones
Lecithins
Lectins
Linoleic acid
Peptides
Phytosterols
Protein
Saponin
Essential fatty acid, hypotriglyceridemic, improves heart

health
Estrogenic, hypocholesterolemic, improves digestive
tract function, prevents breast, prostate,
and colon cancer, bone health,
improve lipid metabolism
Improve lipid metabolism, improve memory and learning
abilities
Anti-carcinogenic, immunostimulator
Essential fatty acid, hypocholesterolemic
Readily absorbed, reduce body fat, anticancer
Hypocholesterolemic, improves prostate cancer
Hypocholesterolemic, antiatherogenic, reduces body fat
Regulates lipid metabolism, antioxidant
2. Constituents of soybean
2.1. Proteins
Soybean contains 3540% protein on a dry-weight basis, of which, 90%
is comprised of two storage globulins, 11S glycinin and 7S -conglycinin
[2,4]. These proteins contain all amino acids essential to human nutrition,
which makes soy products almost equivalent to animal sources in protein
quality but with less saturated fat and no cholesterol. Soybean also contains
the biologically active protein components hemagglutinin, trypsin inhibitors,
-amylase and lipoxygenases [2]. As per the FDAs Protein Digestibility
Corrected Amino Acid source method, soybean is not only high quality
protein, but it is now thought to play preventive and therapeutic roles for
several diseases [5].
369
2.2. Oil
Soybean contains roughly ~19% oil, of which the triglycerides are the
major component. Soy oil is characterized by relatively large amounts of
the polyunsaturated fatty acids (PUFA), i.e., ~55% linoleic acid and ~8%
-linolenic acid, of total fatty acids [6] (Fig. 1). Linoleic acid in soy oil is an
essential fatty acid (EFA) belonging to the -6 family of PUFAs, which
exerts important nutritional and physiological functions. Even the -linolenic
acid is also an EFA belonging to -3 fatty acid family, and plays an
important role in the regulation of a number of metabolic pathways.
However, due to the presence of lipoxygenases in soybean, linoleic acid
renders the soybean oil prone to rancidification [2]. The minor components of
crude soybean oil are phospholipids, collectively called lecithin, as well as
phytosterols, and tocopherols.
O
HO
Linoleic acid
O
HO
-Linolenic acid
Figure 1. Two EFAs present in soy oil.
2.3. Carbohydrates
Soybean contains ~35% carbohydrates, most of which is nonstarch
polysaccharides. It also contains oligosaccharides [5] such as, stachyose
(4%), and raffinose (1.1%). Stachyose is a tetraose with a galactosegalactose-glucose-fructose structure, while raffinose is a triose with a
structure of galactose-glucose-fructose. Polysaccharides are composed
mainly of insoluble dietary fiber. Soybean curd refuse (Okara) contains
soluble polysaccharides with galacturonic acid as its underlying
structure. In addition to use as a dietary fiber supplement, soluble
polysaccharides have been used to modify the physical properties of various
foods [7].
370
2.4. Vitamins and minerals

Soybean is a better source of B-vitamins [2] compared to cereals,
although it lacks B12 and vitamin C. Soybean oil also contains tocopherols
[2,3], which are excellent natural antioxidants. Soybean oil contains
-tocopherol, -tocopherol, -tocopherol, and -tocopherol in trace
amount (mg/kg). Soybean also contains ~5% minerals [3]. It is relatively rich
in K, P, Ca, Mg, and Fe. Soy ferritin can supplement reasonable quantities of
iron.
2.5. Isoflavones
Isoflavones is a sub-group of heterocyclic plant phenolic category called
flavonoids. Besides isoflavones, the other subclasses of flavonoids include
flavones, flavonols, flavanols, aurones, red and blue anthocynin pigments,
and chalcones. In isoflavones the phenyl ring B is connected at position 3 of
1,4-benzopyrone ring (Fig. 2).
The soybean is most abundant source [8] of isoflavones (up to 3 mg/g
dry weight) in the nature. Soybean contain three types of isoflavone aglycone
viz., daidzein, genistein and glycitein; each of them present in three
glycosidic forms in addition to their aglycone form (Fig. 3). Daidzein,
genistein and their glycosides contribute to >90% of total isoflavone; whereas
glycetein and its glycoside are present as minor component (<10%), only.
Isoflavones are structurally similar [6] to mammalian estradiol as shown
in Fig. 4, and can bind to both and isoforms of estrogen receptor (ER),
thus called phytoestrogens. Though, the isoflavones are not essential nutrients
that are required to support life, still they exert many beneficial health effects,
therefore, are of immense help for maintaining healthy life.
8
7
A
6
2'
1'
3'
6'
4'
5'
Figure 2. Basic structure of isoflavones.
371
Figure 3. Chemical Structures of 12 isoflavones found in soybean.

OH
OH
HO
O
Isoflavone
(Equol)
HO
O
Estrogen
(Estradiol)
Figure 4. Comparison of equol (isoflavone metabolite) and estradiol structure.
2.6. Phytosterols
Soybean oil contains about 300 to 400 mg of plant sterols per 100 g. The
major components of soy sterols are -sitosterol (53 to 56%), campesterol (20
to 23%), and stigmasterol (17 to 21%) [9]. These phytosterols differ from
cholesterol only in the structure (Fig. 5) of their side chains; sterols differ
from stanols in being unsaturated versus saturated at the C5-C6 double bond
in their B ring. These sterols are proven to have cholesterol-lowering activity,
though the mechanism is not completely understood [10].
2.7. Phospholipids
Soybean oil contains 1-3% phospholipids [2,3], of which ~35%
phosphatidyl choline, ~25% phosphatidyl ethanolamine, ~15% phosphatidyl
inositol, ~5-10% phosphatidic acid. The phospholipids are removed from the
372
HO
HO
-sitosterol
Campesterol
HO
HO
Stigmasterol
Cholesterol
Figure 5. Soy phytosterols and their structural similarity with cholesterol.

Table 2. Soy phospholipids and their structure.
O
RO
R'O
O
O
O
O P OR''
R and R' O
same or different fatty acids
O
Name
Phosphatidyl
choline
Phosphatidyl
ethanolamine
Phosphatidyl
inositol
Phosphatidic acid
R
-CH2CH2NH3
-CH2CH2N(CH3)3
(CH(OH))6
H
oil mainly during the degumming process and are used as a natural food
emulsifier. They are polar lipids and contribute to the structure of cell
membrane. The structure of soy phospholipids are given in the Table 2.
2.8. Saponins
Soybean also contains ~2% soy saponins (triterpene glycosides) which
are currently attracting lot of scientific attention. Soybean saponins have
unique chemical structures and physiological functions. Soy saponins are
oleonane type triterpene glycosides. They can be classified according to type
373
of aglycon, the moiety attached at the C-22 position on the aglycon, and
the carbohydrate sequences at the C-3 position on the aglycon [11,12].
A representative structure is shown in Fig. 6. So far, total 30 soy saponins are
reported, but their presence and quantity differ from genetic and agronomical
variation. Soy saponins are found to have several biological activities [13]
such as hepatoprotective, anti-hyperlipidemic, anti-cancer, anti-oxidative, and
anti-HIV etc.
R1
21
22
R2
HOOC
O
O
OH
CH2OH
R1
R2
HO
OH
O
O
OH
Group A saponin
OH
Disaccharide
Group B saponin
OH
Group E saponin
=O
DDMP saponin
Maltol
CH3
OH OH
Figure 6. The chemical structure and types of soy saponins.
2.9. Ferritins
Soybean contains ferritin, a multimeric iron storage protein [14]. It is
now well proven that the iron from soybean ferritin is as much absorbed and
bio-available as much it is from the animal products [14]. Therefore, soybean
is recommended to be incorporated in the diet of people suffering from
anemia.
3. Health benefits of soybean

The health effects of soy components have been extensively studied
through human clinical trials, experimental animal studies, and in vitro cell
culture studies. Going further, we will confine our discussion to various
374
scientifically validated/supported heath benefits of soybean consumption to

ameliorate various human health issues, though there are few studies which
present less promising and unfavorable role of soybean in human health.
3.1. Soybean and cardiovascular disease

Cardiovascular disease (CVD) includes all diseases that affect the heart
and blood vessels, such as coronary heart disease (CHD), coronary artery
disease, dyslipidemia, and hypertension [15]. Looking at present scenario
CVD has become one of the major health problems around the world
including developing countries.
The role of soy in the prevention of CVD, particularly LDL cholesterol
lowering effects, has been the subject of numerous controlled clinical studies
[15]. In 2006, a study [16] reported findings from a 1-year trial in which 66
individuals who adhered well to the portfolio diet (31.8% of participants)
experienced reduced serum LDL cholesterol levels by 29.7%. In response to
increased interest and the expanding body of knowledge in soy and health
[17], the U.S. Food and Drug Administration (FDA) approved in 1999 a
health claim for use on food labels [18] which stated that daily diet
containing 25 g/day of soy protein, which is also low in saturated fat and
cholesterol, may reduce the risk of heart disease. Modest reductions in serum
LDL cholesterol levels have been achieved with soy intake, especially for
subjects with hypercholesterolemia [15]. Soy protein consumption in several
human controlled clinical trials ranged between 14 and 113 g/day (with a
median of 36 g/day). The beneficial effects which have been documented
include decreased low-density lipoprotein (LDL) concentrations,
triglycerides, lipoprotein, C-reactive protein, homocysteine, oxidized LDL,
and blood pressure, and increased high-density lipoprotein (HDL)
concentrations [17,19-24]. In these studies, the amount of isoflavones
prescribed was up to 185 mg/day with a median of 80 mg/day.
The lack of understanding of the mechanism remains an obstacle for a
better acceptance of soybean protein by clinical community. There are
different hypothesis to explain these mechanisms. One of these hypothesis is
that amino acid composition or distribution in soybean change the cholesterol
metabolism, possibly, due to changes in endocrine status, because there are
alterations in insulin: glucagon ratio and thyroid hormone concentrations
[25], as well as an increase in plasma thyroxin concentrations which is related
to reduced plasma cholesterol [26]. Another hypothesis proposes that nonprotein components such as saponins, fibre, phytic acid, minerals and
isoflavones associated with soybean protein affect cholesterol metabolism.
Several in vitro and in vivo studies have shown favourable effects of
375
isoflavones in a variety of atherosclerosis models [27]. These include the

reduction of homocysteine levels in plasma [28], prevention of LDL oxidation
[29], improvement of vascular reactivity [30], inhibition of proinflammatory
cytokines, or cell adhesion proteins [31,32], inhibition of reactive nitrogen species
[33], as well as reduction of platelet aggregation [34].
Legumes are relatively rich in soluble fiber, which may play an important
role in the prevention of heart disease [35]. The major effects of soybean
soluble fibers on serum lipoproteins appear to be related with bile acid
binding and with a decrease in the reabsorption of bile acid [36]. Therefore,
there is an increase in the cholesterol used to synthesize bile acids [37]. Also
the fermentation of soluble fibers in the colon produces short-chain fatty
acids that contribute to reduce hepatic cholesterol synthesis [38]. It has been
shown that propionic acid, one of the short-chain fatty acids, decreases the
hepatic cholesterol [39]. Moreover, the attenuation in the synthesis of
cholesterol in the liver leads to reduction in serum insulin concentrations,
which in turn, reduces the activation of an enzyme that participates in
cholesterol synthesis. Perhaps, on the other hand, it might also be due to an
alteration of the bile acid profile in the liver [37].
There is also a hypothesis that isoflavones may inhibit atherosclerotic
development, because they have antioxidant properties against LDL
oxidation, which generates a cascade of events producing atherosclerotic
plaques. Additionally, isoflavones possess a hypocholesterolemic effect, due
to the interaction of isoflavones with estrogenic receptors. Serum cholesterol
concentrations may decrease by this mechanism, also.
Another component of soybean is phytosterols. The mechanism
underlying the capability of plant sterols/stanols to reduce plasma LDL
cholesterol levels relates to their structural similarity to cholesterol (Fig. 5).
This enables them to compete with cholesterol for incorporation into
micelles, which are translocated over the brush border membrane, from the
gut into the plasma, via intestinal cholesterol transporters, known as NPC1L1
and SR-BI [40,41]. Moreover, free phytosterols are assumed to be taken up
by enterocytes directly and to prevent cholesterol from being esterified by
blocking the ACAT system and subsequent transport to the mesenteric
lymph. Free sterols/stanols are hypothesized to stimulate the ABC-ATP
binding cassette mediated enterocytic cholesterol transfer back to the
intestinal lumen for excretion from the body. Consequently, sterols/stanols
will significantly down regulate the cholesterol influx and stimulate the
efflux over the brush border membrane [42].
In brief, various components of soybean might be contributing
independently towards its overall cardiac health benefits. To overemphasize
the importance of one and/or another component may not be able to justify
the total effect of soybean on cardiovascular system.
376
3.2. Soybean and cancer

In the last two decades, many groups of researchers have suggested that
the regular consumption of soybean is associated with the relatively lesser
risk of different cancers in countries that include soybean in their diets
[6,43,44]. Researchers have evaluated dietary differences between Japan
and the Western nations to try to explain variations in death rates from
cancer [45]. A number of soy components have been investigated for
potential anticancer activity. Soybean contains several components with
anticancer activity, such as, isoflavones, protease inhibitors, phytosterols,
saponins, phenolic acids, and phytates. Most of the data support that
predominantly isoflavones are responsible for the anticancer effects of
soybean [6,43,45].
Based on the estrogenic activity of isoflavones, they can possibly be
used for prevention and treatment of hormone dependent cancers [47].
Prevailing hypothesis is that isoflavones may act like antiestrogen when
they are in a high estrogen concentration, and like estrogen when they are
in a low estrogen environment. Genistein, one of the two primary
isoflavones, may be contributing its anticancer effects due to its very good
antioxidant properties. The anticancer effects of genistein are also due to
the fact that it is a specific inhibitor of protein tyrosine kinase, MAP kinase,
ribosomal S6 kinase, topoisomerase II, which form part of growth factorstimulated signal transduction cascades in normal and transformed cancer
cells. It has also been proved in vitro, that genistein increases
concentrations of TGF-, which may inhibit the growth of cancer cells.
Moreover, genistein has an important role as a potent inhibitor of
angiogenesis in vitro [43]. Japanese people with high phytoestrogens
(isoflavone) plasma levels, have low incidence of breast, prostate, and
colon cancer. This also indicates the safety aspect of isoflavones and
soybean consumption [47].
3.2.1. Breast cancer
Several studies suggest that consumption of soy foods during childhood
and adolescence in women reduces the risk of breast cancer in later part of
life. The growth of both estrogen-dependent and estrogen independent breast
cancer cells in vitro has been inhibited by genistein, but it is not clear if the
concentrations reached in vitro could be reached in vivo. Studies have shown
that soybean intake may help in preventing the initiation of breast cancer
cells [6,43].
377
3.2.2. Prostate cancer

It is known that estrogens cause programmed cell death of prostate
cancer cells and inhibit enzymes associated with different processes in the
development of cancer [47]. Soybean foods may be a factor contributing to
the diminution of prostate cancer mortality of the soy consuming populations.
Genistein has been shown to reduce DNA synthesis in human prostate
cells in vitro and inhibit testosterone effect in prostate cancer development in
rats [48]. Prostate cancer is also reported to be associated with increased
levels of dihydrotestosterone, and soybean isoflavones are known to
inhibit 5-reductase, which is involved in the conversion of testosterone to
dihydrotestosterone [49]. However, the mechanism by which soybean may
prevent prostate cancer still remains unclear and controversial [50].
3.2.3. Colon cancer
Epidemiological evidences show protective effects of soybean products
on colon cancer. In vitro studies on soybean products have shown an
antiproliferative effect even on cells of gastrointestinal tract among the
various other cell types [51]. An important role in colon cancer is attributed
to dietary fiber from soy, which also reduces the risk of other chronic
diseases in digestive system. The fermentation of soy fiber in colon produces
an increase of short-chain fatty acids that present a potential protective effect
against colon cancer and bowel infections through inhibition of putrefactive
and pathogenic bacteria, respectively [52]. Nonetheless, scientific evidence in
support of the protective effect of soybean isoflavones on colon cancer is
limited [47].
3.3. Soybean and menopause

Menopause is a natural stage of life all women experience as they age.
Thermoregulatory disturbances like hot flashes (HF), night sweats, mood
swings and lack of energy can make menopause one of the most physically
and emotionally miserable times in a woman's life [53]. HF arises as a sudden
feeling of heat in the face, neck, and chest [54]. Menopausal hormone therapy
(MHT) is the most effective therapy for vasomotor symptoms [55]. However,
current data have indicated adverse effects of MHT by increasing the risk of
e.g. stroke, breast cancer and gallbladder disease [56].
Dietary soy has gained much attention since reports of reduced
menopausal discomfort and reduced morbidity incidence of several hormone-
378
dependent diseases in soy consuming Asian compared with non-soy

consuming Western populations. Epidemiological studies in Japanese women
suggest that consumption of soy products has a protective effect against
menopausal symptoms [57-59]. In Japan, the total isoflavone intake from soy
food averages from 25 to 50 mg per day. A small proportion of the Asian
population (<10%) seems to consume more than 100 mg isoflavones per day
[60].
Isoflavones bind to the estrogen receptors in certain cells in the body and
produce weak estrogenic effects, especially when an inadequate amount of
estrogen is present in the body [61]. Over 20 human studies have tested the
hypothesis that soy products alleviate post menopausal symptoms [62,63].
In these studies, perimenopausal women as well as postmenopausal women
who suffered menopausal symptoms consumed soy proteins for 4 weeks or
longer. These studies show that the soy isoflavone do have the beneficial
health effects on menopausal symptoms.
In summary, though the available human studies seem to show a few
conflicting results in terms of the consistent efficacy of soy isoflavones in
alleviating post menopausal symptoms, from the epidemiological studies it
can be stated that isoflavone do help the regular soybean consumer to better
manage their post menopausal symptoms.
3.4. Soybean and osteoporosis

The loss of estrogen during menopause puts women at greater risk to
develop weaker bones and joints as they age. Menopause leads to rapid
decrease in estrogen levels, which causes bone breakdown and loss of more
calcium via urine. Over time, bones may become weak and brittle with tiny
holes inside, this condition is called osteoporosis.
Phytoestrogens help prevent osteoporosis in the presence of
subnormal endogenous estrogen [64]. Studies in Asia reveal that women
in Shanghai, China, who ate sumptuous amount of soy foods, were onethird less likely to experience a fracture than Chinese women who
consumed lower amount of soy [65]. This observation has led to the
hypothesis that soybean or soybean isoflavones are a possible alternative
option for the prevention of osteoporosis. Randomized controlled studies
[66,67] that used isoflavone extracts or pure genistein reported that soy
isoflavones have a mild but significant and independent effect on the
maintenance of bone mineral density at doses ranging between 35 to 54 mg
of aglycone equivalent. The mechanism of isoflavones on bone health is
yet to be understood.
379
3.5. Soybean and diabetes

Soybean diet may be a good option in type 2 diabetes individuals due to
its effect on hypertension, hypercholesterolemia, atherosclerosis and obesity,
which are very common diseases in diabetic patients [68].
Furthermore, substituting animal protein for soybean or other vegetable
protein may also decrease renal hyperfiltration, proteinuria, and renal acid
load and therefore reduces the risk of renal disease in type-2 diabetes [69].
Soluble fiber from soybean may be useful because of its insulin-moderating
effect. It is generally accepted that a high fiber diet, particularly soluble fiber,
is useful to control plasma glucose concentration in diabetics. Soybean fiber
intake has also been implicated for the improvement of the blood glucose
levels of diabetics [43]. It also increases fecal excretion of bile acid and
therefore may cause a low absorption of fat [20,69].
Though further research is needed, it can be suggested that diabetic
patients with soybean diets show several potential advantages, such as,
reduced insulin resistance, renal damage, and fatty liver, thereby improving
their quality of life.
3.6. Soybean and obesity

Obesity poses a major public health challenge since it is a well
recognized independent predictor of premature mortality [70]. The dramatic
increase in the occurrence of overweight and obesity over the past several
decades is attributed in part to changes in dietary and lifestyle habits, such as
rapidly changing diets, increased availability of high-energy foods, and
reduced physical activity of peoples in both developed and developing
countries [71]. Ingestion of foods with high protein content is well known to
suppress appetite and food intake in humans [72].
Several nutritional intervention studies in animals and humans indicate
that consumption of soy protein reduces body weight and fat mass in addition
to lowering plasma cholesterol and triglycerides. In obese humans, dietary
soy protein also reduces body weight and body fat mass in addition to
reducing plasma lipids [73].
Several lines of evidence suggest that soy protein may favorably affect
lipid absorption, insulin resistance, fatty acid metabolism, and other
hormonal, cellular, or molecular changes associated with adiposity. It is well
established that soy protein consumption reduces serum total cholesterol,
LDL cholesterol, and triglycerides as well as hepatic cholesterol and
triglycerides. Studies in animals indicate that soy protein ingestion exerts its
lipid-lowering effect by reducing intestinal cholesterol absorption and
380
increasing fecal bile acid excretion, thereby reducing hepatic cholesterol

content and enhancing removal of LDL [74,75]. Dietary soy protein has also
been shown to directly affect hepatic cholesterol metabolism and LDL
receptor activity [76].
4. Conclusion
Several nutritional advantages could be obtained by incorporating
soybean based foods in the diet. Soybean represents an excellent source of
high quality protein with a low content in saturated fat, with no cholesterol,
and a great amount of dietary fiber. Therefore, the possible use of soybean in
functional food design is very promising, since the consumption of soybean
protein and dietary fibre seems to reduce the risk of cardiovascular diseases
and to improve glycemic control. Furthermore, soybean and several of its
components have shown in various in vitro, in vivo, and human clinical
studies their effectiveness and potential role in the prevention and treatment
of different diseases. The use of soybean in food form for several centuries
assures us of its safety and nutritive value for human health. Consequently, it
is imperative for all the conscious societies to incorporate this abundantly
available treasure of functionality in their daily diet and harness the
complete benefit of this yellow miracle seed.
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Research Signpost
37/661 (2), Fort P.O.
Trivandrum-695 023
Kerala, India
ISBN: 978-81-308-0448-4
13. Mutasynthesis of medicinally important

natural products through manipulation of
gene governing starter unit
Deepak Sharma, Syed Khalid Yousuf and Debaraj Mukherjee
Natural Product Chemistry (Microbes), Indian Institute of Integrative Medicine
Jammu-180001, India
Abstract. Many of our most valuable drugs are natural products or

are, in some way, inspired by their structures. However, the
complex molecular architecture that lies behind the biological
activity makes it extremely difficult and time consuming to modify
these metabolites by semisynthesis, or to assemble analogues in
stereospecific way from scratch. One way to circumvent this problem
is mutasynthesis, which couples the power of chemical synthesis
with molecular biology to generate derivatives of medicinally
important natural products. Mutasynthesis grew from precursordirected biosynthesis (PDB). Some recent developments in this area
citing literature from 2001 to 2009 will be covered in this review.
1. Introduction
Since the dawn of medicine, natural products have played an important
role throughout the world in treating and preventing human diseases. In fact
history of natural product based medicine dates back practically to the
existence of human civilization. Plants, animals and microorganisms are the
different sources of these important materials. Nearly 28% of all new
chemical entities (NCEs) launched onto the market are natural products, and
Correspondence/Reprint request: Dr. Debaraj Mukherjee, Natural Product Chemistry (Microbes), Indian
Institute of Integrative Medicine, Jammu-180001, India. E-mail: dmukherjee@iiim.ac.in
386
Deepak Sharma et al.
24% of the NCEs launched during 1981-2002 were synthetic or natural

mimic compounds. The data (52% of all NCEs are natural products or are
derived from them) suggests that natural products are important sources
for new drugs and are also good lead compounds suitable for further
modification during drug development. This can be attributed to diverse
structures and the intricate carbon skeletons of natural products. As
secondary metabolites from natural sources have been elaborated within
living systems, they are often perceived as showing more drug-likeness and
biological friendliness than totally synthetic molecules, making them good
candidates for further drug development. Recently Newman and Cragg have
reviewed the continuing value of natural products as sources of potential
chemotherapeutic agents [1].
2. Different approaches used to obtain bioactive natural

compounds
Natural compounds are commercially developed and modified to
generate a collection of chemically related structures to fulfill the needs of
high throughput screening (HTS). The newly developed strategies in modern
organic chemistry has played a crucial role in optimization of the drug leads,
but this approach is limited in applicability for complex, high molecular
weight natural products that contain a great number of reactive groups, some
of which require multistep orthogonal protection/deprotection steps during
the reaction process. Even though Nicolaou and others have convincingly
demonstrated that highly complicated molecules such as vancomycin,
everninomycin, or epothilone can be synthesized de novo [2], still there is a
pressing need for alternative techniques for the production of large amounts
of the compounds needed for pharmaceutical purposes.
Some of the recently devised techniques being practiced for achieving
the growing needs of HTS and the development of new chemical entities
include the utilization of genes from different natural product-producing
microbes to create novel hybrid compounds [3]. This combinatorial
biosynthetic approach was adapted by Madduri and coworkers for the
synthesis of epirubicin (4-epidoxorubicin) by introducing a desoxysugar
biosynthesis gene into the doxorubicin producer S. peucetius [4]. Over last
few years, combinatorial biosynthesis has been shown to be a powerful tool
for modifying several natural compounds with highly diverse structure [5].
Another important technique for the generation of novel secondary
metabolites is precursor-directed biosynthesis. This requires the basic
knowledge concerning the cultivation of the producer organisms and
deep understanding of biosynthetic pathway of the target natural product.
Mutasynthesis of medicinally important natural products
387
Again this is the earliest example of combining chemical and biological

approaches for the generation of novel natural products [8]. However, one
drawback of this approach is that, under normal circumstances, the
alternative precursors must compete with the natural precursors, so the yield
of novel derivatives is rather low. This disadvantage can be overcome by
blocking the synthesis of the natural precursor, either by mutating key genes
[6] in the respective synthetic pathway or by adding specific inhibitors of
biosynthetic enzymes.
3. Precursor-directed biosynthesis (PDB)

In PDB, synthetic chemistry is used to construct analogues of the early
intermediates in the pathway, leaving the producing organism to accomplish
the remaining, more difficult, transformations. In its simplest form, this
technique involves feeding the producing organisms with precursor
analogues, which get incorporated into a new product [7] (Fig. 1). This
approach can be effective at generating new analogues of complex
natural products; for example the orally active -lactam antibiotic
penicillin V (phenoxymethylpenicillin) is produced by adding
phenoxyacetic acid to fermentation broths of Penicillium chrysogenum [9].
A comprehensive example of PDB is that recently applied by Zeeck, de
Meijere and coworkers who generated new analogues of hormaomycin, a
peptide lactone from Streptomyces griseoflavus with a broad spectrum of
biological activities, including antibacterial as well as antimalarial activities.
Preliminary feeding experiments [10] with standard deuterium-labelled
building blocks pointed to 2-nitrocyclopropylalanine as a suitable precursor
for the cyclopropane units. Interestingly, 2-aminocyclopropylalanine is
neither an intermediate nor an acceptable substrate for the multienzyme
complex. Subsequent feeding experiments [11] with a variety of building
blocks resulted in the incorporation of unnatural amino acids, thus
leading to novel hormaomycins. However, the authors noted difficulties in
separation of the analogues from the competitively produced hormaomycin,
a principal problem for PDB.
Nonetheless, structureactivity relationship (SAR) studies were
conducted and this revealed the unexpected antibacterial activity for one
derivative against the opportunistic fungal pathogen Candida albicans by an
order of magnitude equivalent to the antimycotic agent nystatine. Other
remarkable studies exemplifying the potential of PDB include the
generation of soraphen-derivatives employing its natural producer
Sorangium cellulosum [12] and formation of novel rhamnopyranosides
exploiting Streptomyces griseoviridis [13].
388
However, there are drawbacks to precursor-directed biosynthesis; a

mixture of natural and unnatural products with similar physical properties are
often produced, leading to complex downstream purification procedures; high
concentrations of synthetic precursor are often required to compete with the
preferred natural precursor and a limited range of intermediates are efficiently
incorporated into the final product [7]. Once a suitable type of precursor has
been chosen for supplementation experiments, several uncertainty factors
remain, like whether these analogues will (a) be assimilated by the organism,
(b) exhibit non-predictable toxic side-effects, (c) be accepted by the
biosynthetic machinery in the presence of the natural building block, and
finally (d) be possible separable from the natural variants with a reasonable
amount of work.
4. Mutational biosynthesis (MBS)

An important technique that combines chemical synthesis with
metabolic engineering is mutasynthesis (mutational biosynthesis; MBS),
which developed from precursor-directed biosynthesis (PDB) (Fig. 1). Both
techniques are based on the cellular uptake of modified biosynthetic
intermediates and their incorporation into complex secondary metabolites.
Mutasynthesis utilises genetically engineered organisms in conjunction with
feeding of chemically modified intermediates. From a synthetic chemists
point of view the concept of mutasynthesis is highly attractive, as the method
combines chemical expertise with natures synthetic machinery and thus can
be exploited to rapidly create small libraries of secondary metabolites. MBS
was first demonstrated 40 years ago using mutants of the neomycin producer
Streptomyces fradiae [14].
Mutasynthesis, a term coined by Rinehart (1977) and originally proposed
as an alternative method to PDB, is a means of obtaining new structural
diversity (Birch 1963). According to Rinehart, the mutasynthesis approach
consists of five central steps: (1) generation of the biosynthetic block mutant;
(2) generation of the mutasynthon; (3) integration of the mutasynthon;
(4) isolation of the target compound (metabolite); and (5) evaluation of
biological activity [15]. Two advances have recently converged to make
MBS an even more attractive prospect. Although the original PDB
experiments relied on laborious chemical synthesis to generate suitable
precursor compounds, tens of thousands of complex small molecules are now
available from commercial suppliers not only as racemic mixtures but also in
stereochemically pure forms. When the desired analogues cannot be
purchased, commercial compounds can still be elaborated into the target
389
Figure 1. Diagrammatic representation of precursor-directed biosynthesis (PDB) and

mutational biosynthesis (MBS). (a) Biosynthesis of natural product by the wild type
strain. (b) Precursor-directed biosynthesis: the culture medium is supplemented with
an analogue of the natural building block (mutasynthon), which competes for
incorporation into the natural product. Both original and required (modified) products
are formed. (c) Mutasynthesis: biosynthesis of only the required product by mutant
strain by inactivating gene which regulate synthesis of a precursor. Only novel
analogues are produced.
molecules using state-of-the-art chemical synthesis. The second important

development is the immense growth in sequence information for many
natural product pathways. In silico analysis of the proteins encoded by the
gene clusters, coupled with gene inactivation in vivo, often enables each
gene product to be assigned, unambiguously, to a particular role in the
biosynthesis. Together, these experiments can identify suitable candidate
genes for constructing metabolic nonproducers, a much more efficient
alternative to screening vast numbers of randomly generated mutants for the
desired phenotypes [15]. Some early examples of precursor mutational
biosynthesis have been summarized in table 1 and schematically described in
trailing diagrams (Fig. 2-6).
390
Table 1. Some early examples of precursor mutational biosynthesis.

Year Mutants
1969
1977
1984
1991
Streptomyces
fradiae
S. fradiae
Streptomyces
tendae Tu901
1997
Pseudomonas
aeruginosa
S. coelicolor
CH999
1998
S. avermitilis
1999
S. tendae
Tu901
Active
mutasynthons
Streptamine and
epistreptamine
Streptamines
Pyrimidines
Salicylic acid
analogues
N-acetylcysteamine
thioesters
Cyclohexanecarb
oxylic acid
Benzoic acid
derivatives
Product
Activity
Fig.
Hybrimycins Aminoglycoside antibiotics

Neomycins
Antibiotics
Nikkomycin Antifungal,
X and Z
insecticidal,
acaricidal
Pyochelins
Iron
transporter
6Precursor for
Deoxyerythr erythromycin
onolide
antibiotics
Doramectin Antiparasitic
Antibiotic
Nikkomycin
Bx/Bz
2
3
4
5
6
5. Antiproliferative and immunosuppressant

Streptomyces species produce three anticoumarins (aminocoumarin
antibiotics) which inhibit DNA gyrase - novobiocin, clorobiocin and
coumermycin. Among them novobiocin is the best-known and most
important. However, aminocoumarins possess considerable toxicity towards
eukaryotic cells, so that less toxic derivatives of this class of antibiotics are
required.
H 2N
O
HO
HO
H2 N
H2 N
S. fradiae mutant
HO
R
NH2
O
HO
H2N
R
NH2
HO
HO
HO
R=H, deoxystreptamine
R=OH, streptamine
HO
HO
H2N
NH2
OH
R=H, neomycin B/C

R=OH, neomycin analog
Figure 2. Mutasynthesis of neomycins usng streptamine mutasynthone in S. fradiae

mutant cuture [16].
391
OH
Streptomyces tendae Tu901
NH2
HO
N
R=
COOH
H
N
O
N
OHC
OH
N
H
N
H
Nikkomycin Z
OH
Nikkomycin X
Figure 3. Mutasynthesis of nikkomycin Z and nikkomycin X from Streptomyces tendae

Tu901 supplemented with substituents uracil and 4-formyl-imidazolin-2-one [17].
Ar
COOH
P. aeruginosa mutan A602

S
Ar-COOH
OH
OH
OH
Ar =
F
Figure 4. Mutasynthesis of siderophore analogs 5-fluoropyochelin, 4-methyl pyochelin

and 6-azapyochelin using mutant P. aeruginosa IA 602 [18].
O
O
OH
O
O
O
S. avermitilis mutant
O
O
R=
O
H
H
Doramectin
Avermectin A1
Ivermectin
unsaturated
O
H
Figure 5. Mutasynthesis of doramectin by using a mutant strain of the avermectin

producing organism Streptomyces avermitilis supplemented with cyclohexanecaroxylic
acid [19].
S. tendae mutant
nikC::aphII
OH
HO
COOH
NH2
H
N
nikkomycin Bx/Bz
COOH
O
O
OH
OH
Figure 6. Mutasynthesis of nikkomycin Bx and Bz using mutant S. tendae supplemented

with benzoic acid [20].
392
The aminocoumarin antibiotic chlorobiocin consists of three

components: an aminocoumarin moiety, a noviose sugar, and a
3-dimethylallyl-4-hydroxybenzoic acid (DMAHB) moiety. Mutasynthesis of
this class of antibiotics was achieved by employing a mutant deficient in the
biosynthesis of the 3-dimethylallyl-4-hydroxybenzoyl moiety (DMAHB, ring
A), which is coupled via an amide synthetase to the aminocoumarin ring (ring
B, Galm et al. 2004). In a biochemical study, the amide transferases of all
three coumarin antibiotics (CloL, NovL and CouL) were heterologously
expressed in Escherichia coli and screened for promiscuity in substrate
conversion. Among these three enzymes the amide synthetase CloL was
found to be most promising and therefore subsequent mutasynthesis
experiments were preferentially performed with the prenyltransferasedeficient cloQ-mutant unable to synthesize the DMAHB precursor (Fig. 7).
A key step in the biosynthesis of DMAHB is catalysed by CloQ, a
dimethylallyl transferase. In order to produce analogues of chloroiocin, a
strain of the producing organism Streptomyces roseochromogenes was
constructed in which the cloQ gene was inactivated [21].
Streptomyces hygroscopicus produces polypeptide-polyketide nature
compound rapamycin. Rapamycin inhibits the signals required for cell cycle
progression, growth and proliferation by binding protein mTOR (the
mammalian target of rapamycin), a downstream protein kinase within the
phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) signalling
pathway [22]. Thus, rapamycin is useful as a lead structure for development
of anti-cancer, anti-inflammatory and immunosuppressive agents. All
rapamycin analogues currently in clinical trials have been altered by
semi-synthesis in the starter unit, 4,5-dihydroxycyclohex-1-enecarboxylic
acid (DHCHC) [23]. In rapamycin biosynthesis, DHCHC acts as a starter unit
for polyketide chain extension on a modular polyketide synthase (PKS) [24],
resulting in the release of pre-rapamycin. Finally, pre-rapamycin is
modified through regio and stereospecific oxidation and methylation by a
series of post-PKS enzymes. As recently reviewed by Weissman, researchers
at Biotica were able to generate a strain of Streptomyces hygroscopicus that
was unable to make the DHCHC starter unit. Supplementing the strain
with different analogues of DHCHC resulted in novel rapamycin analogues
(Fig. 8).
Borrelidin is a polyketide derivative inhibiting angiogenesis [25]. Natural
starter unit in borrelidin is trans-cyclopentane-(1R,2R)-dicarboxylic acid. As
recently reviewed by Weissman, researchers at Biotica and their collaborators
at the University of Oviedo cloned and sequenced the borrelidin gene
cluster from Streptomyces parvulus Tu4055. This advance enabled them to
mutate a key gene involved in starter unit production, using insertional gene
393
OH
HO
O
3-dimethyllyl-4-hydroxybenzoic acid
OH
OH
O
H
N
Streptomyces roseochromogenes
inactivated gene cloQ
H
N
O
Cl
Chlorobiocin
NH2
OH
HO
HO
OH
O
HO
O
OH
HO
3-dimethyllyl-4-hydroxybenzoic acid analogues supplemented in

feed
Figure 7. Mutasynthesis of chlorobiocin analogues using supplement of DMAHB

(Galm et al. 2004).
HO
HO
HO
O
COOH
HO
COOH
COOH
COOH
COOH
COOH
COOH
DHCHC analogues supplemened in feed
HO
H3CO
S. hygroscopicus MG210
gene inactivated rapK
OH
N
O
O
O
O
H3CO
HO
O
OCH3
Rapamycin
Figure 8. Mutasynthesis of rapamycin analogues using supplement of DHCHC.
394
OH
OH
Streptomyces parvulus Tu4055

O
NC
COOH
Feed supplemented with cyclobutane analogues

Borreldine
COOH
COOH
COOH
COOH
COOH
H
COOH
H3CS
Figure 9. Mutasynthesis of borrelidin analogues using supplement of different

dicarboxylic acid.
inactivation. More than 40 commercially available mono and dicarboxylic

acids were fed to BIOT-1302 cultures to evaluate their ability to initiate the
biosynthesis of modified borrelidins [26].
Although the monocarboxylic acids were not incorporated, use of
cyclobutane-trans-1,2-dicarboxylic acid resulted in a novel compound at 50%
of the normal borrelidin titre. Because the trans isomer of the cyclobutane
dicarboxylic acid was accepted, and not the cis (meso) form, the researchers
surmised that the particular geometrical arrangement of carboxylic acids in
the precursor was vital for substrate recognition (Fig. 9).
Kalaitzis et al. (2003) reported the generation by MBS of wailupemycin
and enterocin analogs from Streptomyces maritimus, which is the first
example of the use of MBS for type-II PKS (Fig. 10) [27].
Another unusual starter unit, 3-amino-5-hydroxybenzoic acid (AHBA), is
required for the biosynthesis of ansamycin antibiotics, a distinctive class of
polyketide-based metabolites. Ansamitocins are cytotoxic compounds known
to inhibit different leukemia cell lines as well as human solid tumors at very
low concentrations. The natural producer is Actinosynnema pretiosum.

OH
395
O
OH
Streptomyces maritimus
encP-mutant
OH R
OH
HO
O
O
R-COOH
HO
wild type
HO
O
Enterocin
Waliupemycin
S
R=
S
Figure 10. Mutasynthesis of waliupemycin and enterocin analogues by feeding

culture with carboxylic acid derivatives.
The different members of this group differ in the nature of the acyl sidechain, with ansamitocin P-3 (AP-3) being an important example [29].
Ansamitocins are highly potent and are currently being evaluated in phase I
studies for target-directed antibody conjugates [29]. Total synthesis has not
provided new AP-3 derivatives, while semisynthesis has mainly addressed
ester side chain modifications and dehalogenation [28]. Meanwhile, work
towards generation of novel ansamitocin analogues employing a mutant
blocked in the biosynthesis of the unique starter unit AHBA has been
conducted [30]. By using different benzoic acid derivatives as a supplement
in culture of A. pretiosum mutant, novel AP-3 could be generated in amounts
suitable for structural identification and activity analysis (Fig. 11) [31]. The
analogues exhibited strong antiproliferative activity against several tumor cell
lines (IC50 values in pg mL1 range).
MeO
NH2
wild type
O
R
O
O
COOH
Actinosynnema pretiosum
mutant
Feed supplemented with benzoic acid analogues
N
OH H
OMe
R
NH2
R= F, Cl, Br
COOH
Figure 11. Mutasynthesis of ansamitocin analogues using AHBA supplement.
396
The soil bacterium Streptomyces thioluteus produces auerothin. Starer

unit for this is p-nitro benzoate (PNBA) [32,33]. Aureothin exhibits a
variety of pharmacological properties, which include weak cytotoxic,
antifungal, and antiviral activities [34]. Expression of the mutated gene
clusters in the S. lividans host appeared suitable for mutasynthesis, since
the Aur pathway could only be restored with exogenously supplied PABA
or PNBA. To explore the substrate specificity of the Aur PKS, at first p-,
m-, and o-PNBA were administered to a growing culture of S. lividans
ZX1::pHJ79. Not surprisingly, only the p-substituted acid was used as
substrate, while the regioisomers were not incorporated. In a series of
further feeding experiments a variety of p-substituted PNBA surrogates
were tested, either as free acids or as the corresponding N-acetyl
cysteamine (NAC) thioesters, which were synthesized using the
dicyclohexyl
carbodiimide/4-dimethylaminopyridine
(DCC/DMAP)
method [35,36] The NAC adducts serve as activated acyl CoA mimics
[37] that may diffuse into the bacterial cells and bypass a potential
bottleneck, the putative acyl CoA ligase AurE. Of the various
p-substituted benzoic acids, p-iodo, p-bromo, p-chloro, and p-fluoro
benzoate, as well as p-N-acetamido anthranilic acid and p-dimethylamino
benzoate were probed on a 100-mL fermentation scale. Unfortunately,
novel aureothin derivatives could not be detected in the crude extracts of
these feeding experiments. Also, toluic acid and terephthalic acid
monomethyl ester failed to incorporate. Strikingly though, addition of
p-cyano benzoic acid to a culture of the aurF null mutant yielded a novel
metabolite, which was detected by ESI-MS in the positive mode (m/z
378). As only relatively low quantities of the new compound were
produced, p-cyano benzoyl-SNAC was tested to find if it would provide
higher yields, as it does not need to be activated as an acyl CoA adduct by
the ligase. However, the yield did not exceed that obtained with the free
acid, which reveals that in this case acid activation by the ligase AurE is
not a bottleneck (Fig. 12) [38].
COSCoA
COOH
CoASH
NAC
DCC
DMAP
PCBA-CoA
O
S
AurABCHI
AurE
H
N
PCBA-SNAC
O
OMe
Aureonirile
O
Figure 12. Mutasynthesis of aureonitrile with PCBA and PCBA-SNAC.
397
Salinosporamide A, a chlorinated natural product from the marine

bacterium Salinispora tropica, is a potent proteasome inhibitor currently in
development for the treatment of multiple myeloma and other cancers
[39-41]. Besides the major analogue 2, S. tropica also accumulates the
deschloro compound salinosporamide B. Administration of synthetic 5-FDA
[42] to a salL-knockout mutant of S. tropica [43] devoid of 2 led to the
production of a new salinosporamide derivative (Fig. 13).
NH2
N
O
N
Cl
L-Met
Cl-
N
OOC
NH
N
N
se
na
i
r
lo
Ch lL NH
2
Sa
NH3
-
HO
OH
Cl
5'-CIDA
N
N
Salinosporamide A(2)
NH2
N
N
S
OH
L-Met
N
F
N
HO
Fluorinase
OH
SAM
HO
OH
5'-FDA
O
NH
-
salL mutant of S. tropica
OH
O
Fluoriosalinosporamide (1)
Figure 13. Fluorosalinosporamide was generated from 5-FDA in a salL-mutant of

S. tropica. SAM: S-adenosyl-l-methionine; 5-ClDA: 5-chloro-5-deoxyadenosine;
5-FDA: 5-fluoro-5-deoxyadenosine [44].
Geldanamycin is a potential antitumor drug [45] that binds to the

N-terminal ATP-binding domain of heat shock protein 90 (Hsp90) and
inhibits its ATP-dependent chaperone activities [46]. Most geldanamycin
derivatives reported to date are 17-aminated compounds and were obtained
by semisynthesis [47]. Related to geldanamycin is reblastatin, which is
saturated across C4-C5 and has a benzene chromophore instead of a
benzoquinone or a hydroquinone moiety [46]. Importantly, reblastatin
shows lower cytotoxicity than geldanamycin but has a higher affinity for
Hsp90 [48].
398
The producing microorganism Streptomyces hygroscopicus var. geldanus

NRRL 3602 creates geldanamycin through biosynthetic machinery based on
a polyketide synthase (PKS) and additional post-PKS enzymes. The starter
unit for geldanamycin is 3-amino-5-hydroxybenzoic acid (AHBA), which
originates from a shikimate-type biosynthetic pathway. New geldanamycin
derivatives were obtained by using mutational biosynthesis with an AHBAblocked mutant of the geldanamycin producer, Streptomyces hygroscopicus
K390-61-1 [49]. Twenty different 3-aminobenzoic acids were chosen and
individually added to cultures of strain K390-61-1. Mutasynthons 1, 2, 3
(fig. 14) and aminonicotinic acid turned out to be the most promising
candidates with respect to yields [50].
O
OH
MeO
MeO
1
7
21
O
N
H
N
H
21
4
OH
MeO
1
7
OH
MeO
O
Geldanamycin NH2
O
Reblastatin
Reblastatin analogues
S. hygroscopicus K390-61-1
Successfull precursor added

OMe
F
O
OH
NH2
O
OH
NH2
Br
N
O
O
OH
NH2
NH2
OH
Aminonicotinic acid
Figure 14. Mutasynthesis of reblastatin analogues.
O
NH2
399
5.1. Antimicrobial
A series of triketide analogues were synthesized and shown to be
processed by supplemented culture of S. venezuelae BB138 strain with
N-acetyl cysteamine thioester of the triketide (Fig. 15). Four of them were
shown to be processed into new biologically active 14-membered macrolide
products. The levels of production of these new macrolides varied, but in all
cases were at least tenfold lower than seen for pikromycin production from
the natural triketide. Preliminary analysis of one new product, 15,16dehydropikromycin, indicated slightly improved antibacterial activity [51].
Studies integrating sophisticated methods of molecular biology and
chemical synthesis were carried out with the aim of elucidating the
acceptance of advanced intermediates by the 6-deoxyerythronolide B
synthase (DEBS) of Saccharopolyspora erythraea, the producer of the broad
spectrum antibiotic erythromycin B [54]. The DEBS system represents the
most extensively characterised modular polyketide synthase and for a more
detailed description of the insights gained into its utilization of advanced
intermediates, the reader is directed to the work of Ward et al. [52].
Studies were carried out with mutants of the natural producers, due to the
selectivity of the DEBS loading module for standard biosynthetic building
blocks such as propionyl-CoA, the elimination of internal precursor
competition is not possible by blocking the respective starter unit
biosynthesis. To make the DEBS PKS suitable for a mutasynthesis approach,
a different strategy was employed. A point mutation was introduced into the
active site of the first PKS ketosynthase (KS1) domain; thereby blocking
diketide formation based on the available internal starter units [53]. These
engineered KS10 DEBS systems were shown to convert the natural diketide
as well as modified diketides and triketides into analogues of 6-deoxyerythronolide B [54,55]. These advanced precursors could be further modified
O
S. venezuelae (BB138)
PikAl mutated gene
SNAC
a
OH
Synthetic triketide SNAC
+ R1
R2
N
R1
R2
R1
R2
HO
OH
N
OH
1a R1=CH3, R2=CH2CH3,pikromycin
2a R1=CH3, R2=CH=CH2, 15,16-dehydropikromycin
1b R1=CH3, R2=CH2CH3,narbomycin
2b R1=CH3, R2=CH=CH2, 15,16-dehydronarbomycin
Figure 15. Mutasynthesis of pikromycin, dihydropikromycin, nabromycin and

dihydronabromycin using synthetic triketide SNAC.
400

O
Streptomyces coelicolor mutant
OH
OH
O
R
R= Bu,Bn,Et ,
56-58%
Diketide feeded
6%
25%
OH
SNAC
OH
Streptomyces coelicolor mutant
OH
O
O
O
SNAC
R1
OH
OH
R2
R1=H R2=Me
R1=Me R2=H
R1=H R2=H
Triketide incorporated
HO
HO
O
O
O
O
OH
OH
O
O
O
Post-PKS modification
OH
OH
OH
6-Deoxy-erythronolide B
NMe2
OH
O
O
OMe
Erythromycin B
Figure 16. Mutasynthesis of 6-deoxy-erythronolide and formation of erythromycin B

in post PKS modification.
into novel erythromycins by application of a S. erythrea mutant unable to

synthesise the core polyketide, but equipped with the full set of post-PKS
tailoring enzymes. Remarkably, when SNAC-esters of 2,3-unsaturated
triketide derivatives were administered, polyketide elongation and
macrolactonisation yielded 16-membered lactones which spontaneously
formed the corresponding lactols (fig. 16) [56].
401
The triketide analogues were apparently accepted as surrogates for the

absent diketide precursors and treated accordingly by the biosynthetic
machinery, finally resulting in an increased ring size. In a more recent study
it was demonstrated that removal of the DEBS loading domain and the first
module, rather than catalytic inactivation of the latter, resulted in an increased
utilisation of supplemented diketide precursors by the engineered PKS
system [52].
Vancomycin is a glycopeptidal antibiotic used for treatment of lifethreatening infections with methicillin-resistant Staphylococcus aureus
(MRSA) [57]. Due to resistance development in enterococcal and
staphylococcal against vancomycin, treatments require the novel vancomycin
analogues. Amycolatopsis balhimycina is the producer of the vancomycintype glycopeptide antibiotic balhimycin [58]. The first series of
mutasynthesis experiments were performed with an in-frame deletion mutant
of the bhp perhydrolase gene (Puk et al. 2002) inactivated in Hty
biosynthesis. Other mutasynthetically generated fluoro-balhimycines were
obtained by feeding 2-fluoro- or 3,5-difluoro--hydroxytyrosine, respectively
(Fig. 17). All novel fluorobalhimycines were found to be active and showed
antibiotic activity against the strain Bacillus subtilis. The DL-Tyrosine or
phenyl serines lacking the 4-OH group were not accepted as substrates [59].
The lantibiotics are a class of polycyclic peptide natural products
that possess antimicrobial activity [60, 61]. They are also used in food
preservation [62]. The major problem associated with lantibiotics is solubility
and stability. Lantibiotics are ribosomally synthesized as linear prepeptides
and are extensively post-translationally modified to their active forms. These
modifications introduce thioether containing cross linked amino acids called
lanthionine (Lan) and methyllanthionine (MeLan) as well as dehydroalanine
(Dha) and dehydrobutyrine (Dhb) residues. The latter motifs result from
enzymatic dehydration of Ser or Thr residues, respectively. (Me)Lan
crosslinks are then installed through enzymatic intramolecular Michael type
addition of Cys thiols onto the unsaturated amino acids [63].
Lacticin 481 is a lantibiotic produced by Lactococcus lactis CNRZ 481
that contains three Me(Lan) rings and one Dhb residue [64]. Lacticin 481
synthetase (LctM) introduces these post-translational modifications by
catalyzing both dehydration and cyclization of its substrate prepeptide, LctA
[65]. LctA is a 51 amino acid peptide consisting of a C-terminal structural
peptide that undergoes posttranslational modification and an N-terminal
leader peptide that is required for efficient processing by LctM [66,67].
Several recent reports have characterized the promiscuous substrate
specificity of LctM toward truncated LctA mutant substrates [68-70]. A
significant challenge for the introduction of nonproteinogenic amino acids
402
A.balhimycin
Fluoro balhmycin
bhp deleted mutant
NH2
HO
COOH
OH
NH2
NH2
HO
HO
COOH
NH2
NH2
HO
HO
COOH
NH2
COOH
COOH
OH
HO
COOH
F
OH
OH
Accepted mutasynthone
Non-accepted mutasynthone
OH
OH
HO
CH2OH
O
H2 N
H3C
CH3
O F
O
N
H
OH
H
N
H
N
H
N
NH
O
NH2
NH
NH
H
HOOC
OH
OH
HO
Fluoro balhimycin
Figure 17. Mutational biosynthesis of fluorobalhimycin analogues by using mutated

strain of A. balhimycin.
into lacticin 481 is the preparation of full-length LctA prepeptides. For this
purpose, a triazole-linked LctA peptide analogue (3) has been synthesized via
Cu(I)-catalyzed 1,3-dipolar cycloaddition of an alkyne functionalized LctA leader
peptide (1) and an azide modified LctA structural region (2) [71]. This strategy
403
was utilized to prepare triazole-linked LctA substrates containing several

nonproteinogenic amino acids in the structural peptide, including -amino acids,
D-amino acids, and N-alkylglycine (peptoid) residues. Additional two mutations,
Asn15Arg and Phe21His, were included in substrate 3 to improve solubility [71].
Amino acids that are tolerated by LctM,14 substrates including the following
nonproteinogenic amino acid mutations were prepared: sarcosine (Sar) and
aminocyclopropanoic acid (Acpc) in place of Gly5, D-valine at position 6, 4cyanoaminobutyric acid (Cba) in place of Glu13, 3- homoarginine (3-Arg) at
position 15, N-butylglycine (N-Nle) and -Ala replacing Met16, naphthylalanine
(Nal) at Trp19, 4-pyridynylalanine (Pal) at position 21, and homophenylalanine
(hPhe) in place of Phe23. A final analogue contained three concomitant
replacements of Gly2, Gly3, and Gly5 with -Ala (Fig. 18). The triazole linked
Figure 18. In vitro mutasynthesis of lacticin 481 analogues. Synthetic substrate

analogues were prepared using copper-catalyzed [2+3] cycloaddition of two fragment
peptides. The resulting LctA analogues were treated with LctM, which dehydrated the
underlined Ser and Thr residues and incorporated the thioether rings shown. Mutant
analogues generated are indicated with arrows. Lan residues are shown in red, and the
MeLan crosslink is shown in blue [72].
404
LctA substrate analogues (0.5-1.5 mg) are incubated with 0.5 M LctM in the
presence of 10 mM Mg2+ and 1 mM ATP. Assay progress was monitored by
MALDI-TOF MS. Each of the unnatural LctA substrates was dehydrated four
times by LctM. To produce bioactive lacticin 481 analogues, the leader
peptide and the triazole linker are removed by proteolysis using
commercially available endoproteinase LysC, which efficiently cleaved the
modified substrates C-terminal to Lys1. Thus, lacticin 481 analogues are
produced in high purity. To ensure that complete cyclization had occurred,
the analogues were incubated with a thiol modifying reagent, demonstrating
that no free thiols remained in the LctM-treated peptides [72].
5.2. Insecticidal
The insect pathogen Beauveria bassiana produces several secondary
metabolites, including the cyclooligomer nonribosomal depsipeptides
beauvericin and bassianolide, the diketomorpholine bassiatin, the cyclic
peptides beauverolides, the 2-pyridone tenellin, and the dibenzoquinone
oosporein [73-77]. The cyclooligomer depsipeptides beauvericin
and bassianolide represent rich pharmacophores with diverse biological
activities.
Bassianolide is a tetramer of the dipeptidol monomer d-Hiv-N-methyl-lleucine. Bassianolide causes smooth muscle contraction by inhibiting
acetylcholine activity [78]. It is toxic to insect larvae [75], and exerts
antimycobacterial, antiplasmodial, and cytotoxic activity [79]. Beauvericin is
a cyclic trimer assembled from three d-Hiv-N-methyl-l-phenylalanine
dipeptidol monomers the main cyclooligomer depsipeptide product of B.
bassiana, transports monovalent ions across membranes; this uncouples
oxidative phosphorylation [80].
Beauvericin is insecticidal [81], displays moderate antifungal and
antibiotic activity [73], reverses multidrug resistance in Candida albicans
[82,83] possesses broad spectrum antiproliferative activity (activating
calcium-sensitive cell apoptotic pathways) [84] and is a potent inhibitor of
haptotactic motility [85].
As a consequence of the oligomeric nature of beauvericin, precursordirected biosynthesis with the wild-type strain and an appropriate d-Hiv
analogue yields beauvericin and a series of three beauvericin analogues in
which one, two, or all three d-Hiv moieties are replaced by the externally
supplied precursor [86,87] (Fig. 19). In contrast, the kivr mutant strain is
unable to produce any beauvericin-like compounds unless the fermentations
are supplemented with an appropriate d-Hiv analogue. Moreover, upon
d-Hiv analogue supplementation, this strain biosynthesizes only a single
405
beauvericin compound, in which all the d-Hiv positions are fully substituted
by the externally supplied precursor. For mutasynthetic approach, kivr
mutant B. bassiana strain was supplemented with d-2-hydroxybutyrate
(d-Hbu) instead of d-Hiv and the major product obtained is beauvericin G3.
To produce a larger variety of beauvericin analogues, combinatorial
simultaneous feeding of pairs of precursor analogues was also used during
mutasynthesis.
O
O
OH
D-2-Hydroxyisovaleric acid (D-Hiv)
R
O
N
OH
OH
Dipeptidol monomer
4x
3x
N
O
O
O
N
O
O
O
O
O
O
Beauvericin R= CH2-C6H5
Enniatins R= iPr, sBu or iBu
Bassianolide
Figure 19. Mutational biosynthesis of beauvericin analogues by using kivr mutant

B. bassiana strain supplemented with d-2-hydroxybutyrate (d-Hbu).
6. Summary
Mutasynthesis seems to have a healthy future in lead optimization and
drug discovery. In the current review we have demonstrated with example
that using mutasynthesis one can not only synthesize a very complex
bioactive natural product but also construct subset analogues obviating
406
several multistep laborious synthetic protocols. Despite these, mutasynthesis

has clear limitation that only the portion of structure can be modified.
However, such a minor modification in structure can result a drug e.g.
Doramectin is commercially available insecticidal differ from avermectin
only in starter unit [88].
Acknowledgements
Authors are thankful to Dr Ram. A. Vishwakarma, Director IIIM Jammu
for his keen interest and support.
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Research Signpost
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Kerala, India
ISBN: 978-81-308-0448-4
14. Carbohydrate-containing natural

products in medicinal chemistry
1
Hongzhi Cao1, Joel Hwang2 and Xi Chen2
National Glycoengineering Research Center, Shandong University, Jinan, Shandong 250012

P. R. China; 2Department of Chemistry, University of California-Davis
One Shields Avenue, CA 95616, USA
Abstract. Carbohydrates are essential components of many natural

products known for great medicinal importance. The carbohydrate
moieties can increase drug water solubility, decrease toxicity,
and/or contribute to the bioactivity of the natural products. This
review provides a short summary of diverse carbohydratecontaining natural products, recent advances in introducing glycan
diversity to natural products, and their potential application in
medicinal chemistry.
1. Introduction
Carbohydrates are the most abundant biomolecules. They are presented as
free monosaccharides, oligosaccharides, polysaccharides, and as essential
components of glycoconjugates, including glycolipids, glycoproteins or
glycopeptides, and glycosylated natural products. Glycosylated natural
products have been commonly used as antimicrobial drugs and now as
emerging anti-cancer drug candidates. The sugar moieties in many bioactive
natural products do not only increase water solubility thus the bioavailability
of the compounds, but also decrease toxicity. Some glycans are also the
essential components for the bioactivity of the natural products. This review
Correspondence/Reprint request: Dr. Hongzhi Cao, National Glycoengineering Research Center, Shandong
University, Jinan, Shandong 250012, P. R. China. E-mail: hzcao@sdu.edu.cn; Prof. Xi Chen, Department of
Chemistry, University of California-Davis, One Shields Avenue, CA 95616, USA
E-mail: chen@chem.ucdavis.edu
412
Hongzhi Cao et al.
summarizes the diverse carbohydrate-based and glycosylated natural

products that have been used as drugs and those have great drug potential.
Recent advance in developing novel glycosylated natural products by
glycorandomization/glycodiversification is also included in the review.
2. Iminosugars, aminoglycosides and carbohydrate mimics

Iminosugars, also known as azasugars or polyhydroxylated alkaloids, are
a family of naturally occurring carbohydrate mimics. These sugar mimics in
which the ring oxygen is replaced by nitrogen are classified into five
structural classes: polyhydroxylated piperidines, pyrrolidines, indolizidines,
pyrrolizidines, and nortropanes [1]. Some representative iminosugar
structures are shown in Figure 1. Nojirimycin (1, Figure 1) [2] was the first
natural iminosugar isolated from Streptomyces roseochromogenes R-468 and
S. lavendulae SF-425 and was shown to be a potent inhibitor of - and
-glucosidases from various sources [3]. The chemically more stable 1-deoxy
nojirimycin (DNJ, 2) was also isolated shortly after the discovery of
nojirimycin. So far, many iminosugars have been isolated from natural source
and most of them exhibit specific potent inhibition against different
glycosidases, a class of carbohydrate catabolic enzymes [1-4].
Aminoglycosides are an important group of carbohydrate-based
antibiotics typically containing one aminocyclitol linked to two or more
uncommon monosaccharides. Streptomycin (9, Figure 2) from Streptomyces
griseus was the first aminoglycoside natural product discovered [5].
Streptomycin and many other aminoglycosides (Figure 2) isolated from
natural sources have been widely used as antibacterial agents, especially
against mycobacterium tuberculosis. Recently, aminoglycosides have been
demonstrated to inhibit catalytic RNAs in vitro as well as to interfere with
HIV replication by disruption of essential protein-RNA contacts [6-8].
Figure 1. Structures of representative iminosugars.
413
H2N
NH2
NH
HN
OH
OH
HO
NH
H2N
NH CHO
O
H3C
HO
HO
HO
HO
H2N
NH2
HO
OH
O OH
NHMe
H2N
O
H2N
NH2
HO
OH
O OH
NH2
H2N
HO
OH
H2N
OH
Streptomycin, 9
H2N
NH2
O
O
H2N
H2N
O
HO
NH2
O
Kanamycin B, 12
H2N
O
HO
H2N
NH2
O
H2N
O
HO
NH2
O
OH
O
HO
OH
Paromomycin, 11
NHCH3
NH2
O
OH
Neomycin B, 10
NH2
HO
HO
H2N
HO
HO
HO
OH
OH
NH2
O
HO
CH3HN
Gentamicin C1, 13
CH3
OH
O
HO
CH3HN
CH3
OH
Sisomicin, 14
Figure 2. Structures of representative aminoglycosides.
Many natural occurring and synthetic carbohydrate mimics including

iminosugar- and aminoglycoside-based glycosidase inhibitors have been used
as drugs to treat diabetes, viral infections, cancers, and Gaucher disease
(Figure 3) [1-4]. For example, acarbose (15), a pseudotetrasaccharide isolated
from the fermentation broth of the Actinoplanes strain SE 50, was the first
marketed -glucosidase inhibitor. It was introduced in early 1990s under the
name of GlucobayTM [3]. Two other synthetic carbohydrate mimics, miglitol
(16) and voglibiose (17), have also been introduced to the market as
-glucosidase inhibitors for the treatment of type II diabetes [4,9].
As -glucosidase inhibitors, acarbose (15), miglitol (16) and voglibiose (17)
can decrease the carbohydrate digestion rate and reduce postprandial
hyperglycaemia (PPHG).
Neu5Ac2en (20), the dehydrated Neu5Ac, is a transition state analog
inhibitor of sialidases (also known as neuraminidases). To overcome the low
efficiency and poor selectivity of Neu5Ac2en in inhibiting influenza virus
neuraminidases, Relenza (18) and Tamiflu (19) were developed in recent
years as competitive inhibitors against influenza viral neuraminidase [1,4].
These two blockbuster flu drugs have played important roles in combating
the recent flu pandemic and epidemics.
414
Hongzhi Cao et al.
Figure 3. Some glycosidase inhibitor-based drugs.
The iminosugar Miglustat (22) was the first market azasugar anticancer
drug. It was also used to treat Gaucher disease by inhibiting the
glycosyltransferase involved in the biosynthesis of glucosylceramide [4,9]. Other
iminosugars, such as naturally occurring swainsonine (5), castanospermine (6),
and a DNJ synthetic derivative (NMDNJ, 21), are current anticancer drug
candidates in ongoing clinic trails. These iminosugars are inhibitors against
catabolic glycosidasees associated with cancer progresses [1,4].
During the course of synthesizing novel inhibitors against glycosidases
and glycosyltransferases, many new synthetic approaches and methods for
iminosugars have been developed. For example, Wong and co-workers
developed a one-pot chemoenzyamtic approach for the synthesis of a
library of iminocyclitols using fructose-6-phosphate aldolase (FSA) [10].
Most recently, the Wong group also developed a two-step chemical synthesis
of iminocyclitols using Petasis-type aminocyclization as the key step [11].
The organocatalytic aldo reaction was also intensively investigated in recent
years for the synthesis of iminosugars [12,13].
415
3. Saponins
Saponins are a class of glycosylated secondary metabolites that have
been found in various plant species and some marine organisms. Thousands
of saponins have been characterized and they usually can be classified into
steroidal glycosides and triterpenoid glycosides according to their aglycones.
As natural surfactants, saponins have not only been used as detergents or
foaming agents for many years, they have also been used in Africa to kill
infected snails and prevent the transmission of schistosomiasis. Plant saponin
extracts from ginseng, liquorice, horse chestnuts, ivy leaves, quillaia barks,
primula roots, senega roots, sarsaparilla roots and others have been used as
folk medicines [14-16]. The cardiac glycosides include well-known drugs
such as digoxin (23) has been used for many years to treat congestive
heart failure.
O
OH
HO
OH
O
OH
OH
O
OH
Digoxin, 23
Some recent studies showed that digoxin also has anti-cancer activity and
can be used as a novel cancer therapeutic agent [17,18]. Antimicrobial,
especially antifungal, activities of many steroidal saponins (e.g. 24-29 in
Figure 4) have also been reported [19-25].
Ginseng (Panax genus) is a family of slow-growing perennial plants
belonging to the family Araliaceae. Its root has been used to increase the
quality of life in China and East Asia since ancient time. So far, more than 30
different ginsenosides (Figure 5) have been isolated, and these triterpene
saponins are considered to be the main active compounds in the ginseng
products [26,27]. Accumulated evidences have shown that ginsenosides also
have anti-inflammation [28], anticancer [29-31] anti-diabetic [32,33]
activities, and can prevent neurodegeneration [34,35].
OSW-1 (Figure 6, 34) is a high potent anticancer cholestane glycoside.
OSW-1 and its four natural analogues (35-38) have been isolated from the
bulbs of Ornithogalum saundersiae, a perennial grown in southern Africa
where it is cultivated as a cut flower and garden plant [36]. These cholestane
416
Hongzhi Cao et al.

O
O
HO
O HO
O
HO
HO
OH
OH
HO
O
HO
HO
OH
HO
HO
OH
TTS-12, 24
HO
O HO
O
HO
HO
HO
OH
OH
HO
O
HO
HO
OH
HO
HO
OH
TTS-15, 25
O
O
OH
O
OH
O
HO
HO
HO
O
HO
HO
Dioscin, 26
HO
OH
29
O
OH
O
HO
O HO
O
HO
HO
OH
HO HO
HO
HO
OH
O
OH
HO
O
HO
O
O
OH
OH
OH
O
Aginoside, 27
HO
HO
HO
HO
O HO
O
OH
HO
HO
O
O
OH
HO
HO
OH
O
OH
O
HO
O
HO
O
OH
OH
CAY-1, 28
Figure 4. Structures of representative antimicrobial saponins.
417

OH
O
HO
HO
O
HO
HO
OH
OH
OH
O O
HO
HO
HO
O
RO
O
HO
HO
OR
30, R = Rha, Ginsenoside Re,

HO 31, R = Glc, Ginsenosied Rg1
32, R = H, Ginsenoside Rh2

33, R = Glc, Ginsenoside Rg3
Figure 5. Structures of some representative ginsenosides (3033).
glycosides exhibited extremely potent cytotoxicity against human

promyelocytic leukemia HL-60 cells with IC50 between 0.1 and 0.3 nM.
OSW-1, the major constituent, exhibited high potent activity against
various malignant tumor cells, including leukemia, mastrocarcinoma, lung
adenocarcinoma, pulmonary large cell carcinoma and pulmonary squamous
cell carcinoma. The cytotoxicity is 10-100 fold more potent than some wellknown anticancer agents in clinical use, such as mitomycin C, cisplatin,
camptothecin, adriamycin and taxol [37].
Due to its unique structure and exceptional highly potent anticancer
activity, OSW-1 has been an attractive synthetic target for organic chemists.
Three groups have reported the total synthesis of OSW-1. All of these
synthetic approaches utilized a convergent strategy that glycosylated the
aglycone acceptor with disaccharide donor to realize the coupling (Figure 7).
The most challenging part of the total synthesis was the synthesis of aglycone
O
OH
O
AcO
R1O
HO
HO
34, R1 = H, R2 = p-methoxybenzoyl, OSW-1

35, R1 = H, R2 = 3,4-dimethoxybenzoyl
O
36, R1 = H, R2 = (E)-cinnamoyl
O O OH 37, R1 = Glc,R2 = p-methoxybenzoyl
38, R1 = Glc,R2 = (E)-cinnamoyl
OR2
Figure 6. OSW-1 and its analogs.
418
Hongzhi Cao et al.

(a) Hui and Yu's synthesis (1999)
O
35%
39
O
OH
OH
3 steps
9 steps
HO
54%
30%
40
TBSO
2 steps
41
TBSO
OSW-1
(b) Jin's synthesis (2001)

O
66%
39
TBSO
O
OH
OH 3 steps
OAc 2 steps
7 steps
HO
58%
74%
42
41
TBSO
OSW-1
(c) Guo's gram scale synthesis (2008)

O
HO
39
4 steps
3 steps
37%
41%
TBSO
40
TBSO
O
OH
OH
3 steps
41%
41
OSW-1
Figure 7. Total synthesis of OSW-1 (34).
acceptor. It was achieved from commercially available 5-androsten-3-ol-17one (39) in all three reports. The Hui and the Yu groups reported the first
total synthesis of OSW-1 (34) in 1999 (Figure 7, path a) [38]. In their
synthesis, the side chain elongation was realized by employing sequentially
Wittig olefination, Ene reaction, Dess-Martin oxidation, Grignard addition,
PDC oxidation, and protection of keto with ethylene glycol to give the key
diene intermediate 40. The diene 40 was subjected to OsO4 to afford the
corresponding 16,17 diol intermediate in moderate yield, which was
converted to the natural aglycone as the acceptor for the next glycosylation
step by reversing the 16-OH to 16-OH through an oxidation-reduction
process. Finally, the OSW-1 (34) was constructed from commercially
available dehydroisoandro-sterone, L-arabinose, and D-xylose in 14 linear
steps with a total yield of 6%.
Jin and co-workers developed a new strategy for steroselective
introduction of the aglycone side chain via 1,4-addition of -alkoxy vinyl
cuprate to 17(20)-en-16-one steroid to give the intermediate 42 (Figure 7,
pathway b) [39,40]. In Jins synthesis, a new strategy was developed to
introduce the 16,17 diol to avoid using toxic OsO4. The total synthesis was
finished in 10 linear steps with a 28% overall yield.
419
Recently, Guo and co-workers reported gram-scale synthesis of OSW-1

(34) (Figure 7, pathway c) [41]. In their synthesis, an efficient new approach
was developed to elongate the steroid side chain to give the key diene
intermediate 40 in 4 steps with a 37% total yield. Then, following the similar
approach as described by Hui and Yu, the OSW-1 was synthesized in 10 linear
steps in a 6% overall yield. Structure-activity relationship studies of OSW-1
analogues revealed that a new 23-oxa-analogue of OSW-1 had much more
potent antitumor activity than its parent OSW-1 [42]. Recently, a biotinylated
OSW-1 was successfully synthesized for cell targeting studies [43].
Other than the anti-cancer activity of some saponins such as OSW-1 and
its analogs described above, some saponins such as QS-21Aapi and QS-21Axyl
has been used as the potent immunoadjuvants for vaccine. The saponin
extracts of South American tree Quillaja saponaria Molina have long been
used as foaming agents in beverages. Recently, the extracted saponins attract
tremendous attention for its immunological adjuvant activities and have been
used as a critical adjuvant component in many vaccine therapy trials [44].
QS-21A (the 21st fraction from Reverse Phase-HPLC) was the minor
constituent which comprised two isomeric triterpene glycoside saponins
QS-21Aapi (43) and QS-21Axyl (44) (Figure 8) [45].
These
two
complex
triterpene-oligosaccharide-normonoterpene
conjugates consist of a quillaic acid as a central lipophilic core, a branched
trisaccharide, a linear tetrasaccharide, and an extended glycosylated diester
side chain (Figure 8). QS-21Aapi (43) and QS-21Axyl (44) have a -D-apiose
and a -D-xylose, respectively, as the terminal saccharide residue on the
linear tetrasaccharide substructure. Unfortunately, obtaining sufficient
quantities of these natural products in pure form is a daunting project due to
their low abundance.
OH
O
HO
O
HO
HO
O HO
O
OH
HO OH
O
HO
CO2-
OH
O
HO
O
CHO
OH HO
O
HOO
HO
OR
O
OH
OH
OH
OH
43, QS-21Aapi R=
HOOOH
44, QS-21Axyl R= HO
O
OH
OH
Figure 8. Structures of QS-21Aapi (43) and QS-21Axyl (44).
OH
420
Hongzhi Cao et al.
The synthesis of the fully protected branched trisaccharide and the linear
tetrasaccharide components of QS-21Aapi were reported by Zhu et al. [46].
The total synthesis of QS-21 Aapi (43) [47], QS-21Axyl (44) [48], QS-7-Api
[49] was successfully accomplished by Gin and his co-workers. The total
synthesis of QS-21 Aapi (43) was achieved in 2006 by judicious choice of the
coupling protocols and protecting patterns (Figure 9) [47]. A convergent
coupling strategy was used for conjugating four building blocks including a
branched trisaccharide (45), a quillaic acid acceptor (46), a tetrasaccharide
fragment (47), and an acyl chain (48) (Figure 9). The glycosyl acceptor, a
30-carbon triterpene quillaic acid ester (46), was prepared by acid-mediated
hydrolysis of natural semipurified QS saponins followed by selective
protection.
In Gins total synthesis, most of the glycosidic linkages were constructed
with sulphoxide-mediated dehydrative glycosylation (Ph2SO-Tf2O) method
using hemiacetal donors. The steroselective coupling between branched
trisaccharide -imidate (45) and the quillaic allyl ester (46) was achieved
using a less common B(C6F5)3 Lewis acid as the promoter. The coupling of
linear tetrasaccharide (47) and acyl chain (48) under Yamaguchi conditions
provided the complex sugar ester in 90% yield. This sugar ester was then
converted to its -imidate and coupled with the acid of glycosylated quillaic
acid triterpene to give the fully protected QS-21Aapi. QS-21Aapi was finally
achieved after global deprotection [47].
BnO
BnO
O
BnO
BnO
CO2Me
AcO
O
OBn
O
OO
CO2All
OH
NH
BnO
CCl3
OBz
HO
46
CHO
45
H
OH
BnO
TIPSO
O
O
O
O
O BnO
O
Ph
OO
HO
OBn
OBn
TBSO
O
TBSO
O
O
O
TBSOO
TBSO
47
OTBS
48
Figure 9. Structures of four building blocks for the synthesis of QS-21Aapi (43).
421
The total synthesis of the other two QS saponins, QS-21Axyl (44) [48]
and QS-7-Api [49], were also accomplished by the same group using a
similar approach. Most recently, Gin and his co-workers designed and
synthesized several amide-modified, non-natural QS-21 analogs [50]. These
synthetic saponins were chemically stable and exhibited similar or even
better immunopotentiating effects in in vivo assays with GD3-KLH
melanoma conjugate vaccine. The highly convergent synthesis of these novel
non-natural saponins provides new avenues for searching and identifying
improved molecular adjuvants for specifically tailored vaccine therapies.
Some other saponins have been isolated and characterized with significant
biological activities such as anticancer, anti-infection, anti-fungi etc. Because of
the lengthy steps of protection/deprotection and sometimes low yields and low
stereoselectivity in the glycosidic coupling processes suffered in saponin
synthesis, only a small portion of these natural products have been synthesized.
For example, two complex triterpene saponins, Lobatoside E (49) [51] and
Candicanoside A (50) [52] both exhibited potent anticancer activity, have been
recently synthesized by Yus group (Figure 10). However, many others, such as
Avicin D (Figure 10, 51) [53] which also exhibited potent anticancer activity, are
still attractive total synthetic targets that have not been synthesized.
O
HO
O
HO
HO
HO
O
O
O HO
O
OH
OH
OH
O
HO
O
HO
O
O
OH O
O
O
HO
HO OH
OH
HO
HO
HO
OH
O
HO
HO
OH
Anticancer
Total Synthesized in 2008
Lobatoside E (49)
Anticancer
Total Synthesized in 2007
Candicanoside A (50)
O
O
HO
O
HO
HO
OH
O
O HO
HO
OH
OH
HO
O
O
NHAc
OH
O
O
O
HOO
OH
OH
HO
O
HO
HO OH
HO
OH
OH
O
OH
OH
OH
OH
Anticancer
(To be synthesized)
Avicin D (51)
Figure 10. Structures of three bioactive complex triterpene saponins.
422
Hongzhi Cao et al.
4. Glycosylated macrolides
Many microlides produced by bacteria have sugar moieties and have
excellent activity against Gram-positive bacteria. Many of them such as
erythromycin A (52), oleandomycin, spiramycin, josamycin, tylosin, and
midecamycin have been successfully used in clinic for years [54,55].
In addtion, cytotoxic tetraene macrolide CE-108 (53) [20-56], a secondary
metabolite of Streptomyces diastaticus 108, and amphotericin B (54) [57] are
good candidates for broad-spectrum antifungal drugs. Most recently, a new
18-membered macrolide glycoside, biselyngbyaside (55) from marine
cyanobacterium Lyngbya sp., has been reported to exhibit uncommon broadspectrum antitumor activity in a human tumor cell line panel [58].
5. Glycosylated cyclic peptides

Cyclic polypeptides have been considered as potential antimicrobial
agents for pharmaceutical, preservation, cleaning, and disinfection uses [59].
Most of these cyclic polypeptides are of microbial origin and many have
carbohydrate moieties. Many of these glycosylated cyclic peptides, such as
O
HO
HO
HO
OH
NMe2
HO
O
O
O
OH
HO
OH
OH
HO
(antibacterial)
Erythromycin A, 52
OH
NH2
(Antifungal)
CE-108, 53
OH
OH
O
OH
OMe
HO
HO
OH
OH
OH
OH
OH
O
O
HO
(Antifungal)
Amphotericin B, 54
O
NH2
OH
HO
HO
MeO
OMe
OH
(antitumor)
Biselyngbyaside, 55
Figure 11. Structures of representative glycosylated macrolides.
423

OH
H2N
HO
OH HO
Cl
O
O
HO
HO
HO
O
O
Cl
HO
HO
Cl
O
H
N
N
H
HN
O
O
OH
OH
HO
NH2
H H
N
H2N
HO
O
O
H
N
HN
H
N
N
H
NH
O
HO
NH2
O
O
HO
OH
HO
OH
O
H
N
S+
H
N
N
H
Vancomycin, 56
NH2
OH
OH
OH
O
OH
Teicoplanin, 57
OH
NH2
H
N
H
Cl
O
AcHN
O
NH2
H
N
N
H
O
HO
OH
O
O
O
HO
O
O
H
N
O
HO
O
H
H H
N
O HO
H O
H O
HO
N
H
H H
N
O
H
OH
O O
N
H
OH
OH
O
OH
OH
O
O
NH2
HO
H
N
S
H
N
NH N
Bleomycin, 58
HO
H
N
O
N
H
NH
H2N
O
HO
HO
HO
N
O
O
R
OH
O
NH2
HN
H
N
O HO
N
H
O
O
R = OH, Hassallidin A, 59a

R = L-Rhamnose, Hassallidin B, 59b
Figure 12. Structures of some glycosylated cyclic peptides.
vancomycin (56), teicoplanin (57), bleomycin (58) and ristocetin etc., are
very important antibiotics and some of them have been considered as the last
resort for treating multiple resistant bacteria infections (Figure 12) [60].
Hassallidins A (59a) [61] and B (59b) [62] isolated from a cyanobacterium
Hassallia sp., have shown broad-spectrum antifungal activity. Compared to
hassallidin A, hassallidin B has an extra rhamnose attached to the 3-hydroxyl
group of the acyl chain and was shown to have increased water solubility
without decreasing its potent antifungal activity [20,62].
6. Cyanogenic glycosides
Cyanogenic glycosides are secondary metabolites widely distributed
in more than 2500 plant species. They comprise a sugar moiety, mostly
D-glucose, beta-linked to an alpha-hydroxynitrile type aglycone. The sugar in
some cases can also be gentibiose, primeverose or others, and the aglycones
can be aliphatic or aromatic compounds (Figure 13) [63,64]. Cyanogenic
glycosides can release hydrocyanic acid (HCN) upon hydrolysis. They are
believed to participate in defense mechanisms of many plants against
different phytopathogens [63,65].
424
Hongzhi Cao et al.

HO
HO
HO
CN
CH3
O
OH
HO
HO
HO
O
OH
CO2H
HO
HO
HO
OH
Cynocardin, 62
NC
HO
HO
HO
O
O
NC
O
O
OH
OH
CO2H
HO
HO
OH
Lithosperm, 64
Tryglochinin, 63
NC
O
O
OH
Aciapetalin, 61
CN
O
OH
CH3
Linamarin, 60
HO
HO
HO
HO
HO
HO
NC
OH
65
Figure 13. Structures of some cyanogenic glycosides.
7. Glucosinolates
Glucosinolates are sulfur-rich secondary metabolites of plants
which contain beta-D-thioglucose and sulpholated oxime moieties (Figure
14) [66-68]. The glucosinolates share some common features with
cyanogenic glycosides, such as similar biosynthetic pathway at the early
stages and both can be hydrolyzed to generate toxic degradation products in
plant defense. The biosynthesis of glucosinolates comprised three steps, sidechain elongation of precursor amino acids, formation of the core
glucosinolate structure, and side-chain decoration. The biological activity of
glucosinolates is not limited to protection against various pathogens and
weeds in case of plants, and recently studies demonstrated it has antifungal,
antibacterial, antioxidant, antimutagenic and anticarcinogenic effects [69-73].
HO
HO
HO
N
O
S
OH
OSO3-
HO
HO
OH
HO
OSO3-
O
S
H
N
HO
HO
HO
Glucobrassicin, 67
OH
OH
Sinalbin, 66
OSO3-
Sinigrin, 68
Figure 14. Structures of some glucosinolates.
8. Other carbohydrate-containing natural products

Other carbohydrate-containing major secondary metabolites which have
recently been identified as potent antimicrobial agents or dietary supplements
include antifungal glycosylated flavonoid 69, antimicrobial glycosylated
iridoids (monoterpenoids) 71 and 73, antimicrobial glycosylated lignan 74,
and antibacterial glycosylated terpenoid 72 (Figure 15) [20,74-76]. In
addition, flavonol glycoside rutin (70, from buckwheat and rue) and the
flavanone glycoside hesperidin from Citrus peels have been used as vitamin P
in dietary supplements [5].
425

MeO2C
O
HO
HO
OH
HO
OH
HO
OH
HO
HO
HO
OH
O
73
O
H
O
AcO O
O
71
H
CO2H
OH
72
O
O
OH
OMe HO
OH
O
HO
H OH
AcO
OMe
HO
O
H OH
HO
OH HO
HO
HO
rutin, 70
69
O
OH
O-Glc-Rha
OH
OH
HO
HO
HO
OH
MeO
OMe
OH
74
OH
OMe
HO
HO
OH
OH
OH
hesperidin, 75
Figure 15. Some glycosylated flavonoids, iridoids, lignans & terpenoids.
9. Current advances in glycosylated natural products:

Glycorandomization/glycodiversification
Many glycosylated naturally occurring antibiotics and its synthetic
analogs are widely used in the clinic for the treatment of various human
diseases such as antibacterial, anticancer, antifungal, antiparasite drugs,
etc [77,78]. These antibiotics can be classified to macrolides, enediynes,
anthracyclines, coumarins, non-ribosomal peptides, aminoglycoside, polyenes,
aureolic acids, and others, according to their specific architectures.
The emergence of pathogenic bacteria that are resistant to multiple
antibiotics represents a growing threat to human health and has given
additional driving force for the search for novel antibiotic drugs [79-81].
Fewer and fewer new drugs have been found in target screening programs
during the past two decades, and scientists have started to look for new
technologies to generate new compounds. Accumulating evidence has shown
that the sugar moiety of many antibiotics play pivotal roles in drug targeting
and activity. Alteration of the carbohydrate structures of drugs, therefore, will
have profound effect to their molecular targeting and organism specificity.
Recently, Thorsons group reported a promising new glycoengineering
(glycorandomization or glycodiversification) strategy for drug development by
quick accessing a library of diverse natural product analogs [82,83]. One example
is glycoengineering of vancomycin using a promiscuous glucosyltransferase GtfE
and an expanded pool of NDP-sugars (Figure 16) [84,85]. Vancomycin (56), a
glycosylated natural product from Amycolatopsis orientalis, is considered the last
defense against infections caused by methicillin-resistant Gram-positive bacteria.
Two glycosyltransferases, GtfE and GtfD, are involved in the vancomycin
biosynthetic pathway to stepwisely add L-vancosaminyl-1,2-D-glucosyl
disaccharide to the 4-hydroxyphenylglycine of the heptapeptide vancomycin
aglycone (Figure 16a) [86].
426
Hongzhi Cao et al.

OH
H 2N
a) Glycosylation steps in the vancomycin biosynthetic pathway
O
HO
R
OH
vancomycin
aglycone
dTDP-Glc
dTDP-vancosamine
GtfE
HO
HO
GtfD
vancomycin
aglycone
O
O
vancomycin
aglycone
b) Glycoengineering and futher chemical diversification of vancomycin (Thorson, 2003)

R1
OH
NDP-sugars
vancomycin
aglycone
R2
alkynes
vancomycin
aglycone
GtfE
OH
O
vancomycin
aglycone
HN
N
H
vancomycin
aglycone
OH
O
N
H
O
H
N
O
N
H
H
N
NH2
O
HO
H
N
HO
OH
O
Cl
HO
O
Click-chemistry
Cl
OH
HO
HO
N
OH
OH
Figure 16. Glycoengineering of vancomycin.
Previous studies have shown that GtfD and GtfE have flexible substrate
specificity [86,87]. Thorson and his co-worker further exploited these
properties and found that 21 of the 23 TDP-sugars generated through
chemoenzymatic synthesis were utilized by GtfE to give a library of novel
vancomycin analogs (Figure 16, Path way B). The vancomycin analog which
has an azidosugar moiety (6-azido-6-deoxy-glucopyranose) can be further
modified in the presence of alkynes via Click-Chemistry to generate 39
additional vancomycin derivatives. One of the new compounds displayed
improved antibiotic activity against Staphylococcus aureus and Enterococcus
faecium (Figure 16) [84,85].
The glycodiversification strategy has been recently employed by the
same group in generating calicheamicin analogs. A new reversible reaction
mechanism catalyzed by the glycosyltransferases (GTs) was discovered
during the course of their studies (Figure 17) [88]. Calicheamicin (Figure 17,
77) is a member of the enediyne family of antitumor antibiotics isolated from
Micromonospora echinospora. Thorson and his co-workers demonstrated
427

A) Glycosylation step catalyzed by CalG1 in the Calicheamicin biosynthetic pathway
O
HO
O
HO
MeSSS
O
I
H
O N
HO
OMe OH
HO
OMe
NHAc HO
MeO
OTDP
CalG1
B) Modification Calicheamicin by in vitro glycodiversification
S
OMe OH
HO
NHAc
MeSSS
O H
N
HO
76
OMe
O
HO
MeO
OH
OMe OH
OMe
O H
N
HO
OH
Calicheamicin, 77
OTDP
O
CalG1
OH
MeSSS
O
I
NHAc
HO
O
O
OH
HO
OH
76
MeSSS
OMe OH
O H
N
HO
NHAc
H
O
OH
OMe
Calicheamicin derivatives with non-natural sugar moiety
C) Modification Calicheamicin by reverse glycosyltransferase reaction
S
OMe OH
HO
NHAc
MeSSS
O H
N
HO
O
HO
O
HO
O
I
OTDP
O
CalG1
OH
MeSSS
OMe OH
O H
N
HO
NHAc
H
O
OH
OMe
OMe
Calicheamicin derivatives with non-natural sugar moiety
O
OTDP
HO
MeO
OH
CalG1
TDP
O
HO
MeSSS
O
I
OMe OH
O
O
HO
MeO
OH
OMe
O H
N
HO
NHAc
H
O
OH
77
Figure 17. Glycoengineering of calicheamicin by in vitro glycodiversifation and

reverse glycosyltransferase reaction.
that ten different TDP-sugars can be utilized by calicheamicin

glycosyltransferase CalG1 to give calicheamicin derivatives with different
sugars (Figure 17, Pathway B). Quite interestingly, when TDP-3-deoxy--Dglucose was incubated with CalG1 in the presence of calicheamicin (77), a
calicheamicin derivative with the sugar moiety being replaced by 3-deoxy-D-glucose was identified (Figure 17, Pathway C). Close investigation
revealed that CalG1 catalyzed a reverse glycosyltransferase reaction in the
presence of TDP to generate TDP-sugar and deglycosylated calicheamicin as
a glycosyltransferase acceptor for producing the final derivative (76) [88].
More than 70 different calicheamicin derivatives were generated by this
CalG1 catalyzed reverse reaction from 8 calicheamicin derivatives and 10
CalG1 recognized TDP-sugars. This GTs catalyzed reverse reaction was
demonstrated to be a novel approach for the aglycone exchange reaction
and can be applied for the synthesis of NDP-sugars [88]. Calicheamicin
aminopentosyltransferase (CalG4) and vancomycin GTs (GtfD and GtfE)
were also shown to catalyze reversible reactions in this study, suggesting that
reversibility may be a general property of GTs involved in glycosylation of
natural products in vitro [88].
428
Hongzhi Cao et al.
10. Prospective and conclusion

Finding drugable carbohydrate-containing natural products remains an
ongoing process. With the increasing interests in the field of carbohydrates
and the rapid advance of the powerful tools including chemical synthetic
strategies, cheomenzymatic methods, and glycodiversification strategies, it is
now possible to expand the existing repertoire of carbohydrate-containing
natural products to find new drugs that can be used to protect human health
and to combat and treat diseases. Nevertheless, developing more efficient and
more economic synthetic approaches for synthesizing carbohydratecontaining natural products remains to be a great challenge and thus an active
area of research for years to come.
Acknowledgements
We are grateful for financial supports from Shandong University (to
H.C.), the National Science Foundation of China (No. 20902087 to H.C.), the
University of California-Davis (to X.C.), the National Institutes of Health
(R01GM076360 and U01CA128442 to X.C.), the National Science
Foundation (CAREER Award 0548235 to X.C.), Alfred P. Sloan Foundation
(to X.C.), and the Camille & Henry Dreyfus Foundation (to X.C.). X.C. is an
Alfred P. Sloan Research Fellow, a Camille Dreyfus Teacher-Scholar, and a
UC-Davis Chancellors Fellow.
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Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry

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Opportunity, Challenge and Scope of

Natural Products in Medicinal

Research Signpost, T.C. 37/661 (2), Fort P.O., Trivandrum-695 023

Published by Research Signpost

development, while chapters 2-12 are focused on natural product drug

Editorial Advisory Board

Prof. M. Shibasaki, Japan

1. Natural products in drug discovery:

Bhuwan B. Mishra & Vinod K. Tiwari

organisms that enhance their survival and competitiveness. Today, R&D

Natural products in drug discovery

immunological, cardiovascular, neurological, inflammatory and related

Bhuwan B. Mishra & Vinod K. Tiwari

2. Drug approval processes

Natural products in drug discovery

3. NP based drugs approved during 1998-2004

3.1. NP based drugs approved during 2005-2010

Bhuwan B. Mishra & Vinod K. Tiwari

Natural products in drug discovery

Zotarolimus 9, a derivative of sirolimus 10, is an active principle of

Bhuwan B. Mishra & Vinod K. Tiwari

Trabectedin (Yondelis, ecteinascidin-743, ET-743) 19, an alkaloid

Natural products in drug discovery

Ixabepilone (IxempraTM, BMS-247550) 20, a semi-synthetic derivative of

Bhuwan B. Mishra & Vinod K. Tiwari

thus is significant in management of alcohol and opioid dependence [27].

Everolimus (LuveniqTM or LX211) 24, an mTOR inhibiting derivative of

Natural products in drug discovery

Telavancin (VibativTM, TD-6424) 25, a semisynthetic derivative of

Bhuwan B. Mishra & Vinod K. Tiwari

Natural products in drug discovery

sinusitis (BS), chronic bronchitis (CB), CAP and uncomplicated (SSSIs).

Bhuwan B. Mishra & Vinod K. Tiwari

Dalbavancin (Zeven, BI-397) 38, a semi-synthetic derivative of the

Natural products in drug discovery

Bhuwan B. Mishra & Vinod K. Tiwari

Moli1901 (duramycin, 2262U90) 48, obtained from Streptomyces

Natural products in drug discovery

Erythromycin 51, macrolide antibiotic produced by actinomycetes, exerts

Bhuwan B. Mishra & Vinod K. Tiwari

by Novartis form Paratek for collaborative Phase III clinical development.

Natural products in drug discovery

CBR-2092 60, a hybrid antibiotic inhibiting RNA and DNA synthesis is

CH3 CH3 CH3

Bhuwan B. Mishra & Vinod K. Tiwari

Natural products in drug discovery

Bhuwan B. Mishra & Vinod K. Tiwari

Betulinic acid 68, a topoisomerase I inhibitor isolated from bark of

Natural products in drug discovery

caffeoylquinic acid) 78, a HIV-1 integrase inhibitor extracted from Inula

Bhuwan B. Mishra & Vinod K. Tiwari

Natural products in drug discovery

formulation bridging study of 86 as Investigational New Drug (IND) to treat

Bhuwan B. Mishra & Vinod K. Tiwari

The University of Florida licensed 89 to Osprey Pharmaceuticals whose

Natural products in drug discovery

Phlorizin 92, a flavonoid that belongs to the group of dihydrochalcones

Resveratrol 94, a triphenolic stilbene occurring in many plants is

Bhuwan B. Mishra & Vinod K. Tiwari

6. Cardiovascular and metabolic diseases

Natural products in drug discovery

Pharmaceuticals against Fabry disease. Isofagomine (PliceraTM, AT2101) 99,

Bhuwan B. Mishra & Vinod K. Tiwari