Accepted Manuscript

Therapeutic journery of nitrogen mustard as alkylating anticancer agents: Historic to

future perspectives

Rajesh K. Singh, Sahil Kumar, D.N. Prasad, T.R. Bhardwaj

PII: S0223-5234(18)30328-3
DOI: 10.1016/j.ejmech.2018.04.001
Reference: EJMECH 10350

To appear in: European Journal of Medicinal Chemistry

Received Date: 6 February 2018

Revised Date: 30 March 2018
Accepted Date: 1 April 2018

Please cite this article as: R.K. Singh, S. Kumar, D.N. Prasad, T.R. Bhardwaj, Therapeutic journery of
nitrogen mustard as alkylating anticancer agents: Historic to future perspectives, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.04.001.

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 ACCEPTED MANUSCRIPT

THERAPEUTIC JOURNERY OF NITROGEN MUSTARD AS ALKYLATING ANTICANCER

AGENTS: HISTORIC TO FUTURE PERSPECTIVES

Rajesh K. Singh1*, Sahil Kumar2, D.N. Prasad1, T.R. Bhardwaj2

1
Department of Pharmaceutical Chemistry, Shivalik College of Pharmacy, Nangal, Dist. Rupnagar-

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140126 (Punjab), India

2
School of Pharmacy and Emerging Sciences, Baddi University of Emerging Sciences & Technology,

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Baddi, Distt. Solan (Himachal Pradesh), India

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*Corresponding author

Graphical Abstract

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THERAPEUTIC JOURNERY OF NITROGEN MUSTARD AS ALKYLATING ANTICANCER

AGENTS: HISTORIC TO FUTURE PERSPECTIVES
Rajesh K. Singh1*, Sahil Kumar2, D.N. Prasad1, T.R. Bhardwaj2
1
Department of Pharmaceutical Chemistry, Shivalik College of Pharmacy, Nangal, Dist. Rupnagar-
140126 (Punjab), India

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2
School of Pharmacy and Emerging Sciences, Baddi University of Emerging Sciences & Technology,
Baddi, Distt. Solan (Himachal Pradesh), India

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*Corresponding author
Abstract
Cancer is considered as one of the most serious health problems today. The discovery of nitrogen mustard

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as an alkylating agent in 1942, opened a new era in the cancer chemotherapy. This valuable class of
alkylating agent exerts its biological activity by binding to DNA, cross linking two strands, preventing

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DNA replication and ultimate cell death. At the molecular level, nitrogen lone pairs of nitrogen mustard
generate a strained intermediate “aziridinium ion” which is very reactive towards DNA of tumor cell as
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well as normal cell resulting in various adverse side effects alogwith therapeutic implications. Over the
last 75 years, due to its high reactivity and peripheral cytotoxicity, numerous modifications have been
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made in the area of nitrogen mustard to improve its efficacy as well as enhancing drug delivery
specifically to tumor cells. This review mainly discusses the medicinal chemistry aspects in the
development of various classes of nitrogen mustards (mechlorethamine, chlorambucil, melphalan,
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cyclophosphamide and steroidal based nitrogen mustards). The literature collection includes the historical
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and the latest developments in these areas. This comprehensive review also attempted to showcase the
recent progress in the targeted delivery of nitrogen mustards that includes DNA directed nitrogen
mustards, antibody directed enzyme prodrug therapy (ADEPT), gene directed enzyme prodrug therapy
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(GDEPT), nitrogen mustard activated by glutathione transferase and CNS targeted nitrogen mustards.
Keywords: Mechlorethamine; Chlorambucil; Melphalan; Cyclophosphamide; Steroidal mustards;
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ADEPT; GDEPT
*
Corresponding author E mail: rksingh244@gmail.com; Tel.: +919417513730; Fax: +911887221276
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1. Introduction
The scourge of cancer has afflicted mankind since the dawn of time. Cancer or neoplastic (Gr. neos-new
and plasma-formation) disease, is now regarded as a family of related disorders which may be defined as
a mass of new tissue, which grows independently, invades surrounding structures and which has no
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physiological use. This definition refers to the solid tumors, which are divided into sarcomas and
carcinomas. The leukemias are diseases of the blood forming organs, characterized by a marked increase
in the number of leukocytes and their precursors in the blood stream, together with the enlargement and
proliferation of lymphoid tissue, spleen, lymphatic glands and bone marrow. Cancer is the second largest
cause of deaths, next to cardiovascular worldwide and accounting for 8.8 million deaths in 2015.

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Globally, nearly 1 in 6 deaths is due to cancer [1].

1.1. Cancer Chemotherapy

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The cancer can be treated by chemotherapy, radiotherapy or surgical procedures. The youngest
approach for the treatment of cancer is chemotherapy which has been proved to be a boon for the

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improvement of the survival rates in last few decades. However, the major limitation of the
cancer chemotherapy is lack of site specificity of the drugs which leads to adverse effects on

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normal tissues as well. The administration of high doses of anticancer drugs during
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chemotherapy also impairs the immunity of patients by affecting biomolecular defence system of
the body.
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2. Nitrogen mustard agents

Although antineoplastic agents can be broadly classified into: alkylating agents; antimetabolites;
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antibiotics; plant products; hormones and various miscellaneous agents. Alkylating agents are further
classified into nitrogen mustard, ethylenimines, methanesulfonates, nitrosourea, triazene etc. Exploration
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in the area of tumor biology has led to the elucidation of the mechanism of action for antineoplastic
agents and also provides a basis for more rational design of new drugs. Since this review pertains to the
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nitrogen mustard, therefore we will restrict our discussion to the alkylating nitrogen mustards only.
Modern cancer chemotherapy is about seven decades old and was initiated from the discovery of the
antineoplastic activity of nitrogen mustard. During the World War II, a ship loaded with 100 tons of
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mustard gas, (bis-chloroethyl sulphide) exploded during an air raid in the harbor of Bari, Italy. A
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thousand men gave their lives and those rescued suffered from mustard gas poisoning and had leukopenia.
Therefore, sulphur mustards were tested in leukemic patients, but because of their vesicant action could
not be used therapeutically. The related nitrogen mustards were prepared and tested as anticancer agents.
However, a systemic study on nitrogen mustard was started before World War II, the above accident gave
an impetus.

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The nitrogen mustard based DNA alkylating agents were the first effective anticancer agents and remain
important drugs against many forms of cancer. More than 70 years of research on the nitrogen mustards
have yielded a broad range of therapeutically useful compounds. The figure 1 shows the long march
towards development of nitrogen mustard based molecules as anticancer agents. The orally
active molecules can be designed by reducing the electrophilicity of mustard agents, thereby

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yielding safer analogues. This approach has been employed for the development of clinically
useful anticancer agents such as chlorambucil, mechlorethamine, melphalan, cyclophosphamide

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and estramustine. Because of the detailed knowledge of the biochemical mechanism of alkylating
agents, now the goal of most efforts to discover nitrogen mustard anticancer drugs is to target molecular

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abnormality that are specific to tumor cells. This led to the development of prodrugs of nitrogen mustard
targeted to cancerous cells only using DNA intercalation, antibody directed enzyme prodrug therapy
(ADEPT), gene directed enzyme prodrug therapy (GDEPT), nitrogen mustard activated by glutathione

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transferase and CNS targeted nitrogen mustards.
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<FIG 1.>
2.1. Mode of action
Nitrogen mustards are commonly used as anticancer agents. These cytotoxic compounds are believed to
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exert their biological activity by binding to DNA, cross-linking the two strands and preventing cell
duplication. It binds to the N7 nitrogen on the DNA base guanine. This linkage represents the most toxic
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of all alkylation events. N-mustard alkylate DNA in two step process. First, bis(2-chloroethyl)amine
undergoes a first order SN2 intramolecular cyclization at neutral or alkaline pH by one step reaction, with
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the formation of highly reactive and unstable aziridinium cation. In the second step, the strained
aziridinium cation undergoes nucelophilic addition by DNA nucleophile to form a monoalkylation adduct
again through SN2 mechanism. These reactions can then be repeated with the other –CH2CH2Cl to give
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cross-link between two complementary strands of DNA [2] [Fig. 2].

<FIG 2>
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2.2. Mechlorethamine and its analogues

Mechlorethamine 1 (Fig. 3), also known as chlormethine is the prototype of alkylating nitrogen mustard
drugs. At physiological pH mechlorethamine is readily and unimolecularly converted by way of the free
base to a relatively stable aziridinium ion, which in turn reacts biomolecularly with available nucleophilic
centers. In vivo alkylation of guanine occurs mainly at the position 7. The drug is effective in Hodgkin’s
disease, other lymphomas and mycosis fungoides. The main toxicity is bone marrow depression. The

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original nitrogen mustard mechlorethamine has largely been supplanted by new congeners to enhance the
selectivity and specificity to target cancer cells by exploring various new strategies.

2.2.1. Hypoxia activated nitrogen mustard

Various previous investigations reported the presence of low oxygen levels, i.e. hypoxia in solid tumors.

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This distinctive feature has led to several strategies to utilize hypoxia as a basis for tumor selectivity,
especially for the synthesis of drugs that are toxic to hypoxic cells or activated under hypoxic conditions
[3-5].

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Using the above concept several nitrobenzyl quaternary salts as bioreductive prodrugs of
mechlorethamine, which have potential as hypoxia-selective cytotoxins, have been prepared [6]. The

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prototype member of the class, N-bis(2-chloroethyl)-N-(2-nitrobenzyl)ammonium chloride 2 has a
number of attractive features: very strong deactivation of the mustard and high water solubility by virtue

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of the permanent positive charge, and a reduction potential (-358 mV) within the range expected to be
suitable for metabolic activation. It was found to display extremely high hypoxia selectivities in vitro with
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the mechanism of action being established as reductive fragmentation to release mechlorethamine [7].
A number of benzyl-substituted, naphthyl and mustards analogues of 2 have been prepared and
investigated [8]. A series of heterocyclic analogues, including pyrrole, imidazoles, thiophene and pyrazole
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were synthesized and evaluated to cover a range of one-electron reduction potentials (from –277 to –511
mV) and substitution patterns. All quaternary salts were less toxic in vitro than mechlorethamine. As
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opposed to the multi-electron reduction required before fragmentation to mechlorethamine in 2, analogues

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3 and 4 required a clean one-electron reductive fragmentation, with high-yielding release of

mechlorethamine. Compounds 3 and 4 displayed significant hypoxia selectivity in vitro, although not to
the same extreme levels as observed in 2 (Fig. 3) [9-10].
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A series of diaryl and alkylaryl sulfoxide-containing nitrogen mustards were synthesized and evaluated
for their hypoxic-selective cytotoxicity against V-79 cells [11]. In general, the diaryl sulfoxide 5a-d
showed much greater hypoxia selectivity (11-27 fold) than the alkylaryl sulfoxide (∼3-fold) 5e-5f. The
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fused diphenyl sulfoxide 6a, 6b, on the other hand, show very slow selectivity (1.3-3-fold). Compound
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6a was highly cytotoxic under both aerobic and anaerobic conditions, while 6b showed low cytotoxicity
under both conditions. The bioreduction of NO2 group in 5c by the rat S-9 fraction under anaerobic
conditions was inhibited by menadione and enhanced by benzaldehyde, acetaldehyde, or 2-
hydroxypyrimidine suggesting the involvement of aldehyde oxidase in the reduction of sulfoxides.
Bioreductive metabolism studies on selected model sulfoxides suggested that diaryl sulfoxides are better
substrates for aldehyde oxidase than aryl alkylsulfoxides (Fig. 3) [12].

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Under physiological pH, the anionic nature of the carboxylate increased the electron density of aromatic
ring that favors the formation of the aziridinium species capable of alkylating DNA [12]. Benzoquinone
conjugate 7 of 4-aminophenyl nitrogen mustard was shown to release the 4-aminophenyl nitrogen
mustard upon reduction by DT-diaphorase under cell free conditions and exhibit selectivity toward both
the DT-diaphorase overexpressing cells under normoxic conditions and T47D cells that overexpress

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cytochrome P450 enzyme with a hypoxia selectivity (Fig. 3) [13].
Recently, dinitrobenzamide mustard (DNBM) pre-prodrugs (PR-104) 8 (Fig. 3) have been developed as a
hypoxia-activated prodrugs which is undergoing Phase I and II clinical trials in both single and

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combination therapies for solid tumours, lung cancer and leukemia. PR-104, consists of phosphate ester
promoiety to improve its aqueous solubility and nitro group as a hypoxia-selecive promoiety. After

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phosphate-catalyzed hydroxylation of PR-104 to dinitrobenzamide mustard (PR-104 A), containing latent
nitrogen mustard moiety is selectively bioreduced to hydroxylamine (PR-104H) or amine (PR-104M)

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under hypoxic condition, which are DNA-alkylating species (Scheme 1). This mechanism of “electron
switch” for activating nitrogen mustard (PR-104M) selectively in hypoxic cell results in hypoxia-selective
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cytotoxicity [14].
SCHEME 1
Novel Cu(II) complex with a mustard derivative of 1,4,7-tetraazacyclododecane (cyclen) 9 as the ligand
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exhibited 24-fold higher cytotoxicity under hypoxic conditions in vitro, and its cytotoxicity under
normoxic conditions was approximately 10-fold lower than SN24771, 10 (Fig. 3). Reversible redox
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behavior and stability of the cyclen-Cu (II) complex in aqueous solution correlated well with the hypoxia
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selectivity, making this complex a promising lead for the development of hypoxia-selective cytotoxic
agents [15].
The novel Cu(II) complexes of N-mustard derivatives of 1,4,7-triazacyclononane (tacn), 1,4,7,10-
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tetraazacyclododecane (cyclen), and 1,4,8,11-tetraazacyclotetradecane (cyclam) were assessed in vitro as

hypoxia-selective cytotoxins. The cyclen mustard complex showed 24-fold selectivity as a hypoxia-
selective bioreductive prodrug, with an IC50 value of 2 µM against the lung tumor cell line A549.
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In another study high reactivity and peripheral cytotoxicity of nitrogen mustard is reduced by making it
complex by co-ordinating its lone pair to an inert metal thereby attenuating its reactivity and cytotoxicity.
Such complexes have been synthesized by direct reaction of N-mustard with Cobalt (III) metal centre 11
bearing exchangeable ligands N-(2-hydroxyethyl) ethane-1,2-diamine (heen) can be co-ordinated to give
a single diastereomers of (oc-6-12’) [Co(heen)2NO2)2](NO3) (Fig. 3)[16].
FIG 3.

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2.2.2. Heterocyclic ring linked nitrogen mustard agent

Novel nitrogen mustard agents 12a-f acid (Fig. 4) involving 4-(N-bis(2-chloroethyl)-
aminophenyl)propylamine linked to 5-(4-N-alkylamidinophenyl)-2-furancarboxylic acid moiety by the
formation of an amide bond have been synthesized, characterized, and evaluated for their in vitro

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cytotoxic activity against MDA-MB-231 and MCF-7 human breast cancer cells. Compounds 12a and 12c,
which possess a cationic amidine and 4,5-dihydro-1H-imidazole functional moiety are approximately ten
times more potent than 4-[bis(2-chloroethyl)amino]benzenebutanoic [17].

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Quinazoline is an important pharmacophore having diverse pharmacological activities including
anticancer [18-19]. Recently, several anticancer drugs such as gefitinib, erlotinib, and lapatinib etc. have

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been approved by the FDA that are quinazoline derivatives. Based on the good performance of
quinazoline ring, thirteen quinazoline nitrogen mustard derivatives were designed and evaluated for

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anticancer activities. Compound 13 (Fig. 4) was found to be most potent with IC50 values of 3.06 mM to
HepG2 lower than sorafenib [20].
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The 1,4-dihydropyridine ring which is considered as “privileged structure” is frequently used as an
important building block to obtain compounds having diverse therapeutic activities including
antiproliferative and P-glycoprotein (P-gp) inhibitory inhibitory activities. Pgp is believed to play key role
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in access of anticancer drugs into the tumor cells. Based on this fact, Singh et al. has combined the 1,4-
dihydropyridine pharmacophore with nitrogen mustard to synthesize two series of twelve hybrids in the
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hope to produce the synergistic anticancer activity. The hybrids were tested against human cancer cell
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lines: A549 (lung), COLO 205 (colon), U 87 (primary glioblastoma cells), and IMR-32 (neuroblastoma
cell lines) using chlorambucil and docetaxel as the reference drugs. These hybrids exhibited moderate to
significant cytotoxic activity with IC50 ranging from 23.7 to 74.4 µM in comparison to 18.8 to 26.7 µM
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for chlorambucil. Among all compounds, 14 and 15 (Fig. 4) were found to be most active [21].
Pyrazolo[1,5-a]pyrimidine derivatives are potent inhibitor of cyclin-dependent protein kinases (CDKs)
which play a key role in regulating cell proliferation, apoptosis and gene expression. Based on this fact,
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several new compounds were formed by coupling the aniline N-mustard with the pyrazole [1,5-
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a]pyrimidine derivatives. Compound 16 (Fig. 4) exhibits excellent therapeutic efficacy at cell level
antitumor activity [22].
A recent study of synthesis of thiozol-2-yl hydrazide pharmacophore with nitrogen mustard moiety
demonstrated promising antiproliferative activity was published by Laczkowski et al. [23]. In a
continuing investigation, they reported thiazole-based nitrogen mustard containing benzenesulfonamide
pharmacophore and evaluation of their antiproliferative activity against human cancer cell lines. Among

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all the derivatives, 17 and 18 (Fig. 4) showed very strong activity against MCF-7 and HCT116 with IC50
of 3.02-4.13 mg/ml [24].
FIG 4.

2.2.3. Nitrogen mustard moiety linked to miscellaneous pharmacophores

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It has previously been investigated that mitochondrial DNA are over-expressed in tumor cells and
therefore this organelle could be a potential loci for selective tumor cell targeting [25-26]. L-carnitine
could be suitable vector to target mitochondria as it is a vital cofactor playing key role in transferring long

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chain fatty acids to the mitochondria for β-oxidation. Therefore, two series of nitrogen mustard
derivatives of L-carnitine were synthesized in which trimethylammonium moiety of L-carnitine was

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replaced by appropriate bis (2-chloroethyl)amine group of nitrogen mustard. The products 19a-d (“trans”
series), presenting the same relative configuration than L-carnitine, showed better cytotoxicities than

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compounds 20a-d (“cis” series) (Fig. 5) [27-28].
Among the growth factor receptor kinases, that have been identified as being important in cancer is
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epidermal growth factor receptor (EGFR) kinase (also known as erb-B1 or HER-1) and the related human
epidermal growth factor receptor HER-2 (also known as erbB- 2). They play a key role in signal
transduction pathway that regulates cell division and differentiation. Based on this fact, Zheng et al.,
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designed and synthesized some new amide-couple benzoic nitrogen mustard derivatives having EGFR
and HER-2 inhibitory compounds. Of all the study compounds, compound 21 & 22 (Fig. 5) exhibits most
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potent inhibitory activity which was comparable to the positive control erlotinib [29].
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Kapuria and coworkers, worked on series of novel water-soluble N-mustard-benzene conjugates bearing a
urea linker. The benzene moiety contains various hydrophilic side chains are linked to the m- or p-
position of the urea linker via a carboxamide or an ether linkage. The preliminary antitumor studies
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revealed that these agents exhibited potent cytotoxicity in vitro and therapeutic efficacy against human
tumor xenografts in vivo. The Anticancer activity of compound 23 (Fig. 5) has been shown to be most
cytotoxic against solid tumor cell lines tested [30].
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Formononetic is a type of isoflavanoids having many pharmacological activities including antiangiogenic

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and antiproliferative activities [31]. Based on this concept, a series of formononetin nitrogen mustard
derivatives were synthesized and evaluated in vitro for their cytotoxicity against five cancer cell lines
Compound 24 & 25 (Fig. 5) are the most potent drug among all the compounds [32].
Yadav et al. [33] combined the epipodopyllotoxin having DNA topoisomerase II isoenzyme inhibitory
activity and nitrogen mustard having DNA alkylation activity in a single compound to synthesize various
hybrids having various linkers. Amine or amide functionality was used to link nitrogen mustard to the

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epipodophyllotoxin, Optimal biological activity were obtained by varying the linking arm. Hybrids 26a
and 26b (Fig. 5) were found to be most active and displayed characteristics dual activity targeting both
topoisomerase II and DNA.
Chen et al. reported another approach to develop three novel aromatic nitrogen mustard prodrugs that are
activated by hydrogen peroxide (H2O2). These compounds 27, 28, 29 (Fig. 5) comprises of nitrogen

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mustard pharmacophore connected to H2O2-responsive trigger by different electron-withdrawing linkers,
so that they donot show cytotoxcity to DNA until and unless triggered by H2O2 to release active mustard
moiety. Among the three compounds, 28 was found to most potent anticancer prodrug that can serve as a

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model compound for further development [34].
Glutathione (GSH) is an important antioxidant and most abundant nonprotein thiol in the cell which has

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several vital metabolic functions such as protection of cell damage by reactive oxygen species like free
radicals, peroxides etc. Several studies revealed that the level of glutathione (GSH) is higher in cancer

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tissues than in normal tissues [35] and could be an attractive target for selective inhibition of tumor cell
multiplication. Some in vitro studies have shown that compounds which deplete GSH can increase the
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sensitivity of alkylating agents toward cancer cells. Sesqueterpene with α-methylene-β-lactone group has
proved to deplete GSH efficiently and also have a diverse range of biological activities including
anticancer [36-37]. Based on these facts, several sesqueterpene pharmacophore is combined with
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nitrogen mustard in the hope to obtain bifunctional antitumor agens with enhanced antiproliferative and
selectivity. Among these compounds, 30 and 31 (Fig. 5) showed the most potent antiproliferative
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activities with IC50 values ranging from 2.5 to 8.7 mM [38].

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Sophoridine has been widely used to treat various types of cancer and recently approved by FDA in 2005
to cure cancer patients. In order to assess the potential of sophoridine with the ability to inhibit DNA and
topoisomerase I (Topo I) and induce cell cycle arrest at G0/G1 phase, thereby causing apoptotic cell
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death, Li et al. designed and synthesized various nitrogen mustard sophoridinic acid derivatives and
evaluated for their cytotoxicity. Of the newly synthesized compounds, compound 32 (Fig. 5) exhibited a
potent effect against hepatocellular carcinoma in vitro and in vivo. SAR analysis indicated that the
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introduction of a nitrogen mustard group to the structure of sophoridinic acid significantly enhance the
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antitumor activity [39].

Chena et al. [40] reported on the design, synthesis and study of two novel mitochondria targeted nitrogen
mustard agent with fluorophores incorporated into the main structure to give 33 and 34 (Fig. 5). Its
results in direct reporting of flow cytometry and gel electrophoresis study without the use of fluorescent
tagging agents. Local accumulation of these compounds to the mitochondria is believed to increase their
selectivity and affinity for mitochondrial DNA.

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Brefeldin A, which was originally isolated from Eupenicillium brefeldianum with the hopes to obtain a
potent antiviral drug. Recently, Han et al., designed and synthesized a series of novel conjugates of
brefeldin A and nitrogen mustards at 4-OH or 7-OH position to explore anticancer agents having good
efficacy and less toxicity. Among them, 35 was the most potent derivative, although weak
antiproliferative potency but exhibited lower toxicity than the brefeldin A, when test against three cancer

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cell lones (HL-60, PC-3 and Bel-7402) [41].

FIG 5

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2.3. Chlorambucil and its analogues

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Chlorambucil 36 is a nitrogen mustard, which is used clinically against chronic lymphatic leukemia,
lymphomas and advanced ovarian and breast carcinomas. The clinical application of this drug is

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limited by its toxic side effects such as bone marrow suppression, anemia and weak immune system.
Besides, it also causes nausea, myelotoxicity and neurotoxicity [42]. One approach to overcome the
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toxicity of anticancer drugs to normal tissue is to attach cytotoxic drugs to a suitable carrier protein,
which accumulate in tumor tissue. Chlorambucil conjugates of the serum protein transferring the iron
(III) transport protein transferrin, exhibits a significant uptake in tumor tissue due to high amount of
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specific transferring receptors on the cell surface of tumor cells [43]. Thus, various maleimide
derivatives 37a-e (Fig. 6) of chlorambucil were bound to thiolate, human serum transferrin which
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differ in the stability of the chemical link between drug and spacer. Evaluation of the cytotoxicity of
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free chlorambucil and the respective transferring conjugates in the MCF 7 memmory carcinoma and
MOLT 4 leukemia cell line employing a propidium iodide fluorescence assay demonstrated that the
conjugates in which chlorambucil was bound to transferrin through acid-sensitive caroboxylic
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hydrazone bonds were as active as or more active than chlorambucil in both cell lines where as others
were not active [44].
To obtain potent antitumor agents with low toxicity, sulphonamide derivatives containing 5-
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fluorouracil and chlorambucil were designed, synthesized and evaluated. Compound 38 (Fig. 6)
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exhibited high antitumor activity and low toxicity with high therapeutic index [45].
Chlorambucil derivatives involving alkyl 2-aminodeoxy sugars have been synthesized by coupling the
chlorambucil moiety to positions C-2 or C-3 of the sugar, directly or via spacer. The final compounds
were tested for cytotoxicity and some of those that presented the best results were studied for
inhibition of cell proliferation [46-47]. The stereoselective synthesis of alkyl 2,3-diamino- and alkyl 2-
amino-3-thioglucopyranosides from alkyl 2-nitro sugar derivatives and amines or thiols has been

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described as intermediates in the synthesis of carriers of chlorambucil. The synthesis and antitumor
activity of three paclitaxel-chlorambucil hybrids has been studied and it was found to have significant
in vivo efficacy [48].
It was proposed that the AT-sequence specificity is derived from an avoidance of GC base pairs due to
the steric clash that would occur between the pyrrole hydrogens that protrude from the concave face of

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the molecules and the minor groove guianine-2-NH2 amino groups [49].
It was predicted that replacement of the pyrroles with imidazoles would remove the steric clash and
potentially introduce a hydrogen bond between the inward-facing imidazole nitrogen and guanine-2-

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NH2 hydrogen. A series of analogues relating to the NH2 terminal region of the fibrin α chain, i.e. Gly-
Pro-Arg-Pro were prepared by stepwise solid-phase synthesis and their abilities to inhibit fibrin

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polymerization and to prolong thrombin-initiated clotting time were evaluated. Replacement of the
NH2- terminal three residues of Gly-Pro-Arg-Pro by chlorambucil, p-nitrophenyl-L-alanine or p-

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aminophenyl-L-alanine gave inactive compounds in the thrombin time assay, whereas similar
substitution or extension of the COOH terminus produced the highly active analogue [50].
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The utility of the spermidine moiety as the homing device for the selective delivery of
chemotherapeutic and diagnostic agents into cancer cells has been explored [51-52]. The mechanism
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and kinetics of hydrolysis of chlorambucil and chlorambucil spermidine conjugates have also been
reported [53]. The structure and the biological significance of naturally occurring and synthetic
polyamine analogues and conjugates have also been described [54].
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Chlorambucil 36 has been reacted with sulphonamide with the aim to have potent antitumor agents
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with low toxicity. The compounds so obtained exhibited high antitumor activity with therapeutic index
twice as that of chlorambucil. 1,3-Dipalmitoylglycerol ester of chlorambucil has been synthesized and
tested. The results demonstrated that the esterification brings about considerably higher levels in the
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lymph and reduces plasma levels. Moreover, pharmacokinetic and biological data suggested that the
ester is most probably acting by itself rather than prodrug of chlorambucil [55].
The quaternary ammonium-chlorambucil conjugate 39 was synthesized which was found to be more
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stable than its parent compound, its hydrolysis rate being almost three times lower than that of 36.
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Compound 39 (Fig. 6) showed an enhanced efficiency against chondrosarcomas compared with

chlorambucil [56].
Previous studies investigated that prolidase is found in normal cells, substantially increased levels are
found in some neoplastic tissues. Because prolidase possess the ability to hydrolyse imido bonds of
various low molecular weight compounds coupled to L-proline, therefore, it was hypothesized that
coupling of L-proline through an imido bond to anticancer drugs might create prodrugs which would

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be locally activated by tumor-associated prolidase and consequently would be less toxic to normal
cells that evoke lower prolidase activity [57]. Lipidic prodrugs, also called drug-lipid conjugates, have
the drug covalently bound to a lipid moiety, such as a fatty acid, a diglyceride or a phosphoglyceride.
Drug-lipid conjugates have been prepared in order to take adavantage of the metabolic pathways of
lipid biochemistry, allowing organs to be target [58]. Albumin conjugates of the chlorambucil have

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been synthesized and evaluated to improve the selectivity and toxicity profile of antitumor agents [59].
Reactions of chlorambucil with 2’-deoxyguanosine have been reported [60]. Also a number of novel
cyclic amidine analogs of chlorambucil 40a-c (Fig. 6) were synthesized and examined for cytotoxicity

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in breast cancer cell cultures. In terms of reduction in cell viability, the compounds rank in both MCF-
7 and MDA-MB-231 cells in the order 40c > 40a > 40b > chlorambucil. Compound 40c was the most

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potent topoisomerase II inhibitors, with 50% inhibitory concentration (IC50) 5 µM [61].
Deoxyglucose preferentially accumulate in neoplastic cells and inhibit glycolysis process in cell. Based

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on this fact, a set of 19 new chlorambucil glycoconjugates were synthesized in which the alkylating drug
is attached to the C-1 position of 2-fluoro-2-deoxyglucose (FDG), directly or via different linkages 41
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(Fig. 6) and evaluated for in vitro cytotoxicity against different human normal and tumor cell lines. There
was a significant improvement in the in vitro cytotoxicity and four compounds were finally selected for
further in vivo studies owing to their lack of oxidative stress-inducing properties. Aromatic linker has a
M

positive impact on antiproliferative activity as compared to aliphatic linker. Compound 51 showed the
most active cytotoxicity among all the synthesized compounds [62-63].
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Combretastatin (inhibit tubulin polymerization) and nitrogen mustard (alkylating moiety) core is
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combined to form a series of chimeric compounds in an attempt to obtain potent antiproliferative

compounds having a dual mechanism of action. All the compounds were cytotoxic and having tubulin
polymerization inhibiting activity. Compound 42 (Fig. 6) was found to be most potent among them [64].
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In another novel approach to target cancerous cells expressing the estrogen receptor alpha (Era,)
Descoteaux et al. modified the natural amino acid i.e. L-para-tyrosine to mimic the steroid backbone. For
this, they first linked the tyrosine to various substituted aminoacid derivatives to convert into
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tyrosinamide molecule. The resulting tyrosinamides were then linked to chlorambucil either directly or
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via a 5 and 10 carbon atoms spacer chain. Interestingly, the meta-hydroxyphenyl-tyrosinamide-

chlorambucil 43 derivatives were more active than the ortho- and para- analogs 44 (Fig. 6) [65].
Glycosylated antitumor ether lipids (GAELS) kill cancer cells by a novel apoptosis independent
mechanism whereas chlorambucil kill cells by inducing apoptosis. Using this concept, Idour ed tal
designed and synthesized various molecules 45-47 (Fig. 6) combining both apoptosis dependent/
independent mode of actions in a single molecule thereby enhancing antitumor activity of chlorambucil

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by joint action. Hybrid 45 exhibited most potent activity DU 145 at CC50 of 6.0um, while hybrid 46
displayed the best activity on JIMT at 7.5um. Hybrid 47 exhibited no activity at the highest concentration
[66]
Based on the previous studies that nanoparticles have a high level of tumor cell uptake, Fan et al.
designed and synthesized chlorambucil nanomedicines by two step process. In the first step, hydrophilic

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gemcitabine (potent anticancer nucleoside analogue) covalently coupled to hydrophobic chlorambucil via
a hydrolysable ester linkage to synthesize 48 (Fig. 6). In the second step, the resulting amphiphilic
conjugate was self assembled into nanoparticles in water. This conjugate when tested on subcutaneous

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grafted SMMC-7721 hepatocellular carcinoma model, has found to have potential as nanomedicine for
efficient cancer therapy [67].

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Replacement of the aromatic ring of chlorambucil with N-methylbenzimidazole leads to the
development of more effective and safe water soluble anticancer agent, Bendamustine 49 (Scheme 2).

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It was was originally synthesized in the 1960 and approved for medical use in the United States in
2008 for treating chronic lymphocytic leukemia, multiple myeloma and non-Hodgkin’s lymphoma
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[68-71]. Aromatic ring is strategically replaced with purine like N-methylbenzimidazole moiety in the
hope to obtain an anticancer agent having synergistic activities i.e antimetabolite mechanism
alongwith DNA alkylation. Recenlty, Chen et al. developed a novel, efficient and scalable route for the
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synthesis of bendamustine hydrochloride by lowering the synthetic steps from eight to five and
increasing the yield from 12 % to 45 % [72].
D

SCHEME 2
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Bendamustine has also very good CNS penetration as reported by Li et al. using cheminformatic
screening and in a mouse model and therefore can be used for primary and secondary CNS tumors [73].
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However, the efficacy of bendamustine is limited by its poor drug potency. Therefore, Li et al.
synthesized a series of chemical derivatives of bendamustine. Among them, CY190602 50 was found to
very potent exhibiting 50- to 100- fold enhanced anticancer toxicity. They demonstrated that the enhanced
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potency of CY190602 was due to its ability to inhibit histone deacetylase (HDACs) which play a vital
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role in gene expression and DNA repair mechanism [74]. In the same way, Xie et al. combined
chlorambucil with aminobenzamide moiety as a DNA/HDAC dual targeting inhibitor to synthesized
compound namely chlordinaline 51 (Fig. 6) [75]. The chlordinaline exhibited both DNA and HDAC
inhibitory activities and showed potent antiproliferative activity against all the six test cancer cell lines
with IC50 values of as low as 3.1 -14.2 µM. It was concluded that combining nitrogen mustard and 2-

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aminobenzamide moieties into one molecule is an effective method to obtain promising candidate for
cancer therapy.

2.4. Melphalan and its analogues

The expectation that a nitrogen mustard derivative of a natural amino acid might be directed to a

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metabolic site critical to neoplastic cells led to the design of melphalan 52 (Fig. 7). It is often the drug of
choice in the treatment of metasatic melanoma, ovarian and breast cancer.
Studies have indicated that the transport of the L-isomer, L-phenylalanine mustard (L-PAM) is mediated

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equally by two separate amino acid transport systems [76]: (a) Corresponding to the system L, the
classical sodium-independent leucine-preferring transport system. Model: 2-

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aminobicyclo[2.2.1]heptanes-2-carboxylic acid (BCH) [77] (b) Monovalent cation-dependant transport
system, which exhibits highest affinity for leucine.

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The approach was used to utilize amino acids whose mode of entry are restricted to specific amino acid
transport systems in conjuction with the cytotoxicity produced by L-PAM to discern the mode of its entry
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into progenitor cells. The failure of the BCH to reduce L-PAM cytotoxicity to murine bone marrow
progenitor cells indicated that these cells lack system L or have a reduced affinity for BCH. In an attempt
to exploit this marked qualitative difference between tumor and normal host cells, a series of cyclic amino
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acids were examined to determine which were transported with the highest affinity by system L into the
tumor cells with the objective that they could be utilized selectively to introduce the cytotoxic bis(2-
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chloroethyl)amino group into the tumor cell. Results indicated that the aromatic bicyclicamino acid 2-
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amino-1,2,3,4-tetrahydronaphthoic acid was a potent competitive inhibitor of system L. These

observations promoted synthesis of the nitrogen mustard derivatives of 2-amino-1,2,3,4-
tetrahydronaphthoic acid [78].
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The two-target compounds, 53 and 54 (Fig. 7) were designed as tumor specific agents capable of being
selectively transported by the leucine-preferring transport system. Isomer 54 was an extremely potent
inhibitor of system L in murine L1210 leukemic cells. It possessed enhanced in vitro activity and reduced
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myelosuppression activity when compared to its prototype amino acid nitrogen mustard, L-PAM, and had
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a 2-3 fold shorter half-life.

In an attempt to improve the oral bioavailability and overcome some of the toxicity limitations of
melphalan 52, it was sought to synthesize and test the glycerol derivatives 55a-c (Fig. 7) [79]. From the
studies, it was concluded that esterification to the α-position of long-chain diacyl glycerols, a prodrug
approach, will not be therapeutically advantageous approach in the case of oral administration. An

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apparent in vivo instability of these esters in the intestinal tract need modification to an amide as evident
from compound 56 which shows some advantages over melphalan.
To enhance affinity for malignant cartilaginous tumors (chondrosarcomas), quaternary ammonium
conjugate of melphalan, 57 (Fig. 7) was prepared by linking the quaternary ammonium via an amide
bond. The conjugate displayed appreciable cytotoxicity [80]. Eight esters of 2-(1-hydroxyalkyl)-1,4-

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dihydroxy-9,10-anthraquinone with melphalan were prepared and tested for their antitumor activity (S
180) and cytotoxicity. 2-{1-[4-(p-bis(2-chloroethyl)aminophenyl)butanoyloxy]methyl}-1,4-dihydroxy-
9,10-anthraquinone and 2-{1-[4-(p-bis(2-chloroethyl)aminophenyl)butanoyloxy]ethyl}-1,4-dihydroxy-

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9,10-anthraquinone showed remarkable antitumor activity [81]. Peripheral benzodiazepine receptors
(PBRs) are located on the outer membrane of mitochondria, and their density is increased in brain tumors.

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Thus, they may serve as a unique intracellular and selective target for antineoplastic agents. A PBR
ligand-melphalan conjugate was synthesized and evaluated for cytotoxicity and affinity for PBRs [82].

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Synthesis of N-α-aminoacyl derivatives of melphalan for potential use in drug targeting has been done
[83]. Chikhale has reported that the reductive and bioreductive activation is controlled by electronic
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properties of substituents in conformationally-constrained anticancer drug delivery systems [84].
Benzoquinone based bioreductive prodrugs have been widely investigated as hypoxia selective
bioreductive anticancer agent. Benzoquinone derived prodrug 58 (Fig. 7) was shown to release the
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alkylating agent melphalan via lactonization following reduction of the compound 55 [85].
The amidine analogues of melphalan 59a-e differing by the nature of terminal basic side were synthesized
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and examined [86]. Evaluation of the cytotoxicity of these compounds was employing a MTT assay in
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both MDFA-MB-231 and MCF-7 human breast cancer cells. Although growth inhibition was
concentration-dependent in either cell line, it was more pronounced at shorter times, in MCF-7 than
MDA-MB-231. In terms of reduction in cell viability, the compounds rank in both MCF-7 than MDA-
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MB-231 cells in the order 59b > 59a > 59c > 59d > 59e > melphalan 52 (Fig. 7).
In order to increase their antitumor potency and tumor selectivity melphalan and bendamustine moiety
were integrated to form new bivalent bendamustine and melphalan derivatives 60a-d (Fig. 7). Two
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molecules of bendamustine esterify with N-(2-hydroxyethyl) maleamide were connected by diamines

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with various chains length. It was supposed that these conjugates cause cytotoxic effect preferred as
bivalent drug. The cytotoxicity of the new compounds increased compared to bendamustine and
melphalan as determined in concentration-dependent in vitro assays using the human MCF-7 and MDA-
MB-231 breast cancer cell lines [87].
The polymeric melphalan conjugate 61 (Fig. 7) was synthesized by Bogomilova et al. using
polyphosphoester as, biodegradable and water soluble polymer. Melphalan was chemically immobilized

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and conjugated to polyphosphoester through covalent bond. This polymeric conjugation resulted in
improved therapeutic effect without side effects when compared to the melphalan when evaluated in vivo
in the human hepatocellular carcinoma HuH7 xenograft mouse model [88].

Recently, novel prodrug of melphalan called melflufen 62 (Fig. 7) has been developed which is

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chemically ethyl ester of a dipeptide of melphalan and p-fluoro-L-phenylalanine. Melfufen is believed to
be activated by hydrolytic cleavage of the peptide bond by metalloproease aminopeptidase N (APN)
which has previously been reported to have a functional role in tumor angiogenesis and reported to be

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over expressed in human tumors. [89]. Melflufen has shown promising cytotoxic potential in both solid
tumor and in multiple myeloma and in under phase I/II clinical trial [90-91].

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Recently, Hu and coworkers, designed and synthesized hybrids of various nitrogen mustards with
quinolone alkalaoid evodiamine. Evodiamine was selected on the basis of previous studies that

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evodiamine alkaloid extracted from Evodiae fructus has various antitumor activities mainly by apoptosis
(cell cycle arrest) and topoisomerase I and II inhibition [92-93]. Among all the synthesized compounds,
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63 (Fig. 7) was found to be A most pormising cytotoxic activity against THP-1 and HL-60 cells with
IC50 value of4.04 µM and 0.5 µM respectively [94].
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2.5. Cyclophosphamide and its analogues

Cyclophosphamide 64(Fig. 8) remains one of the most booming and extensively utilized alkylating
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drugs. It is an oxazaphorphorine that contribute alkyl groups to DNA, forming covalent linkages.
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Cyclophosphamide was approved by FDA for clinical use in 1959 and is active against multiple
myeloma, chronic lymphocytic leukemia and acute leukemia of children (95-96). Moreover, it has
potent immunosuppressive action by depleting T and B cells and thereby suppressing antibody
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production. Therefore, it is the most commonly used drugs in blood and marrow transplantation
(BMT).
Cyclophosphamide is a prodrug and requires metabolic activation to elicit its activity. After initial
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hdroxylation by mixed-function oxidase, the resulting 4-hydroxycyclophosphamide 65 undergoes ring

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opening to its tautomer aldophosphamide 66 by liver cytochrome P450 enzyme followed by spontaneous
generation of the ultimate cytotoxic phosphoramide mustard and acrolein by β-elimination [97-99].
Tautomers are detoxified to inactive carboxycyclophosphamide by aldehyde dehydrogenase (ALDH)
Scheme 3. ALDH is present in relatively large concentration in rapidly proliferating tissues such as bone
marrow stem cells, liver and intestinal epithelium and and hence rapidly converted aldophosphamide to
inactive carboxycyclophosphamide thereby protecting these tissues. Therefore cyclophosphamide has

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relatively distinct cytotoxic properties as compared to other alkylating agents which also alkylate rapidly
proliferating normal cells [100].

SCHEME 3

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Many modifications have been carried out in the basic cyclophosphamide structure. Ifosfamide 67 (Fig.
8), an isomer of cyclophophamide, is indicated in combination therapy of germ cell testicular cancer, soft
tissue carcinoma and breast carcinomas and leukemias. Chemical stability and behavior of ifosafamide

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and its N-dechloroethylated metabolites have been investigated by 31P NMR [101]. Mafosphamide 68
(Fig. 8) has been introduced as a stable derivative of 4-hydroxycyclophosphamide. Its physicochemical

SC
characterization has been reported and its stability in aqueous buffer solution as been found to be highly
dependent on the pH [102-103]. It has undergone extensive preclinical evaluation, of toxicity and

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therapeutic efficacy. Predominant toxicity is myelosupression. Phase I clinical studies have shown that it
causes severe local toxicity presumably due to the high concentration of the 4-hydroxy compound and its
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subsequent metabolites [104].
A series of perhydrooxazine analogues of aldophosphamide 69a-d and 70a-d, (Fig. 8) were developed on
the basis of the idea that ring opening and tautomerization to an enamine intermediate might provide a
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mechanistic alternative to the β-elimination reaction for release of phosphoramide mustard [105-107].
The 4, 4, 6-trimethyltetrahydro-1, 3-oxazine moiety was selected on the basis of its rapid rate of iminium
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ion generation and relatively slow rate of hydrolysis. These analogues underwent phosphorodiamidate
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release by three distinct mechanisms: hydrolysis to aldophosphamide and subsequent β-eliminations;s

cyclization to produce 4-hydroxycyclophosphamides, which release phosphorodiamidate by ring opening
and elimination; and tautomerization to the enamine with rapid expulsion of phosphorodiamidate [108-
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109]. The results demonstrate that this approach can be used to deliver phosphorodiamidates that are non-
cross-resistant in cyclophosphamide resistant cell lines.
Although a series of ifosfamide analogues having modification in the N-(2-chloroethyl) group have been
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synthesized and evaluated, but only bromofosfamide 71 was found to possess activity exceeding that of
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racemic ifosfamide and cyclophosphamide [110]. Aldophosphamide-perhydrothiazine derivatives 72 and

73 (Fig. 8) are a new class of prodrugs, which spontaneously, with half lives of 2-12 h, hydrolyze to the
corresponding aldophosphamide in aqueous solution [111].
Glufosfamide (β-D-glucose-isophosphoramide mustard) 74 (Fig. 8), represents a member of a novel class
of compounds which contains the directly active alkylans isophosphoramide mustard linked to the carbon
atom I of glucose like a β-glycoside [112]. The development of this new drug was based on the rational

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that tumor cells display an increased uptake and utilization of glucose. It has been demonstrated that
tumor cells show an adaptive response to hypoxia, which leads to the inductioin of glucose transporters
and glycolytic enzymes. Furthermore, glucose uptake correlates with tumor aggressiveness and
prognosis. Glufosfamide represents an attractive new agent for cancer therapy. Its cytotoxic activities on
normal as well as tumor cells are still under investigations [113].

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A novel nitroheterocyclic bis(haloethyl)phosphoramidate prodrugs linked through lysine to a pteroic acid
75a-b (Fig. 8) has been prepared and evaluated as a potential alkylating agent. The data does not support
a contribution of the folate receptor to cytotoxicity [114].

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New methods for preparation of ifosfamide, cyclophosphamide and their side products have also been
reported [115]. Since the molecular structure of drug delivery system on the basis of segmented

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polyurethane has been determined to control the release of cyclophosphamide, and shown to reduce the
toxic action of the drug [116].

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The presence of hypoxia and hypoxic cells in many solid tumors is well established. Hypoxic cells
represent a therapeutic challenge that these cells are refractory to radiation therapy and resistant to many
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of the cytotoxic drugs used in chemotherapy. However, hypoxia can also be exploited via the design of
hypoxia-selective agents that undergo bioreductive activation to generate cytotoxic species under hypoxic
conditions. These hypoxia-selective agents should exhibit significantly greater toxicity to hypoxic
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compared to aerobic cells [117-118]. Many classes of hypoxia-selective agents have been synthesized
and evaluated preclinically for antitumor activity. Nitroaromatic compounds have been widely used in the
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development of hypoxia-selective agents because their favourable redox potentials and the marked
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reversal of electron affinity that results from a reduction to hydroxylamine or amine. Many of these
nitroaromatic compounds metabolized directly to cytotoxic intermediates under hypoxic conditions.
Nitroquinoline 76 (Fig. 8) was developed as a targeting moiety for bioreductive activated prodrug to
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achieve selective cytotoxicity towards hypoxic cells. Phosphoramide mustard attached to β-position was
released by nitro reduction under hypoxic conditions and subsequent β-elimination was facilitated by the
nearby basic quinoline nitrogen. Bioreduction of the nitro group in 76 gives the corresponding
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hydroxylamine intermediate, which then undergoes a spontaneous 1, 6-elimination to release the

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cytotoxic drug molecule. According to cytotoxic studies prodrug 76 was 11 fold more toxic toward HT-
29 human colon tumor cells under hypoxic conditions than under normoxic conditions [119]. Other nitro
heterocyclic compounds have been developed with latent alkylating moieties that are activated by
reduction of the nitro group [Scheme 4]. A series of novel nitroheterocyclic phosphoramidates 77a-c and
78a-c (Fig. 9) has been prepared and cytotoxicity of these compounds has been evaluated [120].
SCHEME 4

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All compounds showed a significant increase in life span compared to untreated controls in this modl, and
the efficacies were generally comparable to that of cyclophosphamide. In contrast to cyclophosphamide,
however, long-term survivors were observed in several of the treatment groups. Compound 78a appeared
to be most potent [120]. A series of 2- and 3-substituted indolquinone phosphormamidate prodrugs were
synthesized 79, 80 (Fig. 9) in the same laboratory to target DT-diaphorase enzymes (DTD) that will

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reduce quinone to quinine with the expulsion of the corresponding cytotoxic phosphoramide mustard
without formation of acrolein as by-product [121].
Several 1, 2-benzisoxazole phosphordiamidates 81a-c (Fig. 9) have been designed as prodrug of

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phosphoramide mustard requiring bio-reductive activation. Imine intermediate is produced by cleavage of
N-O bond after enzymatic reduction of 1,2-benzisoxazole moiety. The imine then hydrolyzed to a ketone

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metabolite, resulting in a base-catalyzed β-elimination of cytotoxic phosphoramide mustard [Scheme 5].
As expected, the proposed prodrug were at least 3-5 fold more potent cytotoxins than control compounds

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which lack phosphoramide mustard group [122]. AN
SCHEME 5
A series of sulfonyl-group containing aldophosphamide (Aldo) analogues were synthesized as potential
anticancer prodrugs 82a-c (Fig. 9) that liberate the cytotoxic phosphoramide mustard via β-elimination, a
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non enzymatic activation mechanism. The success of this method is based on the fact that β-elimination is
readily achieved under weakly alkaline conditions, provided the leaving group is situated in a position β
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to a sulfonyl group. The rate of β-elimination varies with different substituents in the sulfonyl activating
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group and also changes in the leaving group (Scheme 6).

SCHEME 6
Analogues with electron withdrawing substituents on the sulfonyl group showed faster β-elimination rates
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as compared to analogues containing electron donating substituents. All sulfonyl analogues demonstrated
in vivo antitumor activity equal to or better than cyclophosphamide against wild-type P388 cells [123].
The acyclic analogue 4-nitrobenzyl phosphoramide mustard LH7 83, (Fig. 9) which was originally
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synthesized as a control compound to explore the mechanism of activation of cyclic phosphoramide

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analogues, turned out to be most active. Prodrug 61 showed 170,000-fold selective cytotoxicity toward E.
coli nitroreductase expressing V79 cells and an IC50 as low as 0.4 nM [124]. Similar to the design of 4-
nitrobenzyl phosphoramide mustard, Evofosfamide formerly known as TH-302 84 (Fig. 9) is an
investigational hypoxia-activated prodrug that is in clinical development for cancer treatment. Compound
84 is achiral and requires reductive activation of methyl-2-nitroimidazol-5-yl-methyl to release the
corresponding bromo analogue of isophosphoramide mustard (Scheme 7). It was shown to be activated

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under hypoxic conditions with a 400-fold enhanced cytotoxicity toward H460 human non-small cell lung
cancer cells in culture under hypoxic versus aerobic conditions. In vivo studies using an orthotopic
xenograft model of pancreatic cancer demonstrated that compound 84 has antitumor efficacy both alone
and in combination with gemcitabine [125]. It is being evaluated in clinical trials for the treatment of
multiple tumor types as monotherapy as in combination with chemotherapeutic agents and other targeted

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cancer drugs [126].
SCHEME 7
A series of new prodrugs have been designed by Cystarska et al. [127] by reaction of isophosphoramide

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mustard with the corresponding acylcxymethyl halides in the hope to be catalyzed by carboxyesterase
with the release of cytotoxic isophoramide mustards 85a-c (Scheme 8). All compounds showed high

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antiproliferative activity against the human cancer cell lines. Compound 85b (Fig. 9) was found to be
most promising when tested for hydrolytic stability and enzymatic activation studies and could be used

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for further research.
SCHEME 8
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A novel sulfonylethyl phosphorodiamidate TLK58747 86 (Fig. 9) was synthesized and characterized
which releases an alkylating moiety upon cleavage at physiological pH. It was further selected based on
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its physicochemical properties, which facilitate its delivery both orally and parenterally. TLK58747 86
may offer several advantages over alkylating agents such as cyclophosphamide and ifosfamide as it does
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not produce acrolein and is its mild hematological toxicity, with little or no effect on red blood cells and
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platelets in animals [128].

Recently, Lin et al. incorporated phosphoramide mustard into the quinazoline scaffold as EGFR/HER2
inhibitors. These compounds were designed as multi-target-directed ligands against tumor cells.
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Activation of HER2 and EGFR stimulates intracellular pathway that regulate a variety of proteins
involved in cell differentiation, proliferation, motility and metastasis. In vitro assay result indicate the
tumor cells lines with high HER2 level of compounds are more sensitive than tumor cells with low HER2
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level. Compound 87 (Fig. 9) is the most potent inhibitor against EGFR and HER2 and found to be lead
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compound for the treatment of lung cancer [129].

Prostate-specific antigen (PSA) is an antigen with a serine protease activity and is reported to be over-
expressed in the prostate tissue and prostate carcinoma [130]. In an attempt to develop targeted
anticancer agent to prostate cancer cells, Wu and Hu designed a prodrug approach to specifically released
phosphoramide mustard in prostate tumor cells by the proteolytic action of PSA. They synthesized a
series of Glutaryl-Hyp-Ala-Ser-Chg-Gln-4-aminobenzyl phosphoramide mustard conjugates as potential

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prodrugs activated by PSA in prostate cancer cells. All conjugates were found to be substrates of PSA
with cleavage occurring between Gln and the para-aminobenzyl (PAB) linker. This cleavage of peptide
gives an intermediate that undergoes spontaneous decomposition to provide the cytotoxic phosphoramide
mustard inside the prostate tumor tissues (Scheme 9). The conjugate 88 (Fig. 9) with fluorine at ortho
position to benzylic phosphoramide was identified as a promising lead for further evaluation and

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optimization in the development of prostate cancer-targeted prodrugs [131].

SCHEME 9

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FIG 8
FIG 9

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2.6. Steroidal based nitrogen mustards

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Numerous studies previously explored the role of steroid hormones in preventing and treating various
hormone-sensitive cancers such as breast, ovarian, prostate and endometrium. It was later discovered that
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the mechanism of steroid hormones is based on binding to certain receptors called “steroid hormone
receptors” found mainly in breast cancer, prostate cancer, small lung cancer, meningiomas [132]. This led
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to the design and synthesis of various steroid-anticancer conjugates to target these cancer cells. Among
the various strategies used to decrease the reactivity and thus minimizing the side-effects of nitrogen
mustard, steroidal-mustard conjugates has been proved to be more efficacious, selective and less toxic
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antineoplastic treatment. The chemical linkage of alkylating agent to steroidal nuclei as the carrier
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molecule has been a promising approach to facilitate uptake of nitrogen mustard to these hormone
sensitive tumors [133]. The rationale behind selecting steroidal nuclei as it may provide the “biological
platform” on which the nitrogen mustard moiety could be transported easily through the cellular barrier to
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the target site. This approach gives several advantages such as reduced system toxicity, increased
bioavailability, reduced dose, synergistic activity and improved specificity of cancer therapy. This
C

concept has led to the successful generation of a various steroidal alkylating agents, which are being used
in clinical practice against several types of cancers (Fig. 10). Some of these agents are not marketed.
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Previous studies investigated that steroidal part in addition to act as “biological carrier” also plays an
important role in binding to the hormone receptors (binding site) present in the cell nucleus [134]. In
recent years, there has been increasing interest in development of steroidal based nitrogen mustards to
target hormone sensitive tumors of breast and prostate. Ongoing research in this field reviewed by Saha et
al. have shown that steroidal linked mustard increase the damaging effects on specific DNA sequence and
achieve better selectivity and reduced toxicity compared to nitrogen mustards themselves [135].

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FIG 10

2.6.1. Nitrogen mustard linked to unmodified steroid nucleus

Zhang et al. synthesized two novel steroidal nitrogen mustards 89a-b (Fig. 11) and evaluated against

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several cancer cells. The results showed that the 89a clearly demonstrated higher activities and, 89b did
not show any activities [136].

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Shervington et al. [137] synthesized a series of chlorambucil and 3-nitrochlorambucil esters of prasterone
and 90-93 (Fig. 11) and tested against several human cells. The prasterone esters are in general more
cytotoxic than the pregnenolone esters. The nitro functional group in esters 91 and 93 diminished the

SC
alkylating activity compared to esters 90 and 92. There was no correlation between the level of alkylation
and the IC50level in the tested cell lines. The overall results indicated that improved activity of 90 and 92

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are due to the increased lipophilicity of these compounds, thereby enhancing cell permeation.
A series of estradiol-chlorambucil hybrids with various chain lengths were synthesized for targeted
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chemotherapy of breast cancer. The novel compounds were synthesized by linking chlorambucil to the
modified estrone at position 16 alpha of the steroid nucleus which is close to the estrogenic binding site.
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The newly synthesized compounds were evaluated for their anticancer efficacy in different cancer cell
lines. The compound 94 (Fig. 11) bearing hydroxyl methyl group was found to be most potent [138].
Kharkar et al. synthesized the hybrids of nitrogen mustard in 16E and arylidene androstane steroidal ring
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95 (Fig. 11) with moderate antiproliferative activities. Glucocorticoid receptors were investigated as
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probable target for anntileukemic activity as indicated by insilico inverse screening and molecular
docking studies [139]. The same research group in continuing research explored the 17E position of
steroid nucleus and synthesized nitrogen-mustard conjugates of various steroidal oximes. The 17E-
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steroidal oxime-benzoic acid mustard ester 96 is the most potent conjugate having significant cytotoxicity
on most of the NCI 60-cell lines. D-ring derived steroidal oxime-nitrogen mustard conjugates represent a
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suitable lead to develop potent antineoplastic agents with better systemic stability and improved
antineoplastic activity [140].
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2.6.2. Nitrogen Mustards linked to modified steroidal ring

Arsenou et al., [141] synthesized a series of steroidal ester of chlorambucil’s active metabolite i.e. p-
[N,N-bis(2-chloroethyl)amino]phenylacetic acid and investigated the effect of 1. oxidized ∆5-steroids, 2.
presence of keto group at 7th position in B-ring and 3. Lactam function in D-ring on the antileukemic

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activity. It was concluded that the presence of the keto function in the oxidized ∆5-steroids esters and
lactam in D-ring increases antileukemic activity and decreased the toxicity. Compound 97 (Fig. 11)
exhibited most potent antiproliferative activity. In the continuing research, Kapou et al. [142] used 97 as a
prototype drug to design a series of analogs possessing diverse toxicities and antileukemic activities that
were investigated through QSAR studies. The study showed that small chemical modifications on the

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steroids can alter the overall conformation and have a major impact on the antileukemic activity. It was
deduced that the orientation of the nitrogen mustard moiety depends on the steroid overall structure and
that this orientation translates directly to the biological activity and the mechanism of action.

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After getting promising results, the same research group synthesized various steroidal derivatives of
chlorambucil and evaluated their antileukemic activity in vivo (P388 and L1210) and in vitro on normal

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human lymphocytes. It was demonstrated that the lactam ring on the B-steroidal ring lead to potent
hybrids but not as potent as the analogous 7-keto derivative 97.The lactam function on the D-steroidal

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ring was more beneficial for activity than the lactam on the B-steroidal ring. The compound 98 (Fig. 11)
was found to be most potent among all derivatives [106].
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In the same year, Fouseris et al. modified and synthesized steroidal skeleton with an acetamide substituent
and a B-lactam ring. It was linked to several nitrogen mustards by an ester function, and analyzed for their
ability to induce sister chromatid exchanges (SCEs) as well as to inhibit cell proliferation in normal
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human lymphocytes. It was observed that steroidal skeleton that carry a NHCO group are more powerful
than the analogous unmodified steroids bearing the same nitrogen mustard. Among the three hybrids
D

made, the steroidal derivative 99 (Fig. 11) was the most potent [143-144].
TE

From the previous results, it was observed that B-lactam ring decreased the antiproliferative activity, In
order to improve the antileukemic strength of these types of steroidal nitrogen mustard hybrids, it was
essential to modify the steroid portion itself. For this purpose, six novel 17β-acetamido-B-lactam steroidal
EP

ester derivatives of aromatic nitrogen mustards were synthesized and evaluated in vivo against leukemias
(P388, L1210) by Koutsourea and coworkers [145]. Hybrid 100 (Fig. 11) gave the best T/C% values in
P388 and L1210 leukemias and had low toxicity.
C

Based on the previous finding, Papageorgiou et al. synthesized three steroidal esters 101–103 (Fig. 11)
AC

and investigated their biological properties [146-147]. All the hybrids showed genotoxic effects
increasing the frequency of SCE and decreasing the PRI values in vitro in cultures lymphocytes and in
vivo in ascites cells of lymphocytic P388 leukemia [148]. Compound 103 was the most genotoxic and had
the highest cytostatic activity. The authors showed that the increasing SCE frequencies (in vitro and in
vivo) led to the cytostatic activity (decrease of PRI), the antileukemic activity and by the induction of

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apoptosis (via the activation of caspase-2and caspase-3). Their findings clearly demonstrate that the
hybrids have potential antineoplastic properties.
Trafalis et al. synthesized the hybrid 104 (Fig. 11) and tested its antitumor activity in vitro against several
breast cancer cell lines and, in vivo against murine tumors and a xenograft mammary tumor [149]. Hybrid
104 was quite active on breast cancer cells, inducing growth inhibition (GI50) ranging from 5 to 65 µM.

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The growth inhibitory activities were higher for hormone-dependentbreast cancers (MCF-7 and T47D).
The cytotoxic effect of hybrid 104 was low (IC50> 100 µM). The comparative study reported that hybrid
104 showed higher activity, low toxicity and high therapeutic ratio on human pancreatic adenocarcinoma

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celllines (Panc1, MiaPaCa2 and Hs766T) [150].
The homo-aza-steroidal ester called NSC 294859 (178) was synthesized by Catsoulascos et al., [151-152].

SC
It was shown that NSC 294859 gives a 100% and 383% ILS over controls during the treatment ofL1210
and P388 leukemia in mice. Hybrid 178 was found to be an interesting candidate for clinical trial.

U
Comparative in vitro and in vivo sstudy of homo-aza-steroidal alkylating ester NSC294859 105 with
dacarbazine (DTIC) [102], cisplatin (CP), carmustine (BCNU) and semustine (MeCCNU) was performed
AN
on malignant melanoma [153]. It was discovered that 105 (Fig. 11) had higher cytostatic and cyto-toxic
effect compared to DTIC, BCNU and MeCCNU, in all cases of in vitro screening, but was less activity
M

than CP.
It is observed, that the steroid nucleus and the attachment position of nitrogen mustard is crucial for
anticancer activity. The azasteroids and homo-aza-steroidal lactams derived from A- and D-rings are
D

important for improving the biological activity as compared to B- and C-ring substituted components. The
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steroidal skeleton has been linked with nitrogen mustard moieties such as chlorambucil and its metabolite,
phenylacetic acid and benzoic acid etc. The anticancer agents bearing phenylacetic acid and benzoic acid
mustards linked to aza-steroids and homo-aza-steroidal lactams at C-3 and C-17 positions of A and D-
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rings have been found to be promising analogues of utmost potential [5]. It was further investigated that
the steroidal skeletons bearing amide functionality possess potent biological activity as compared to
unsubstituted steroids. The ester based analoges of chlorambucil are found to be effective antileukemic
C

agents. The introduction of the amide group as d-lactam or as a 17-acetamido substituent also improves
AC

activity. The introduction of the keto group in the B ring also produced highly potent molecules. Thus, the
structure based drug design is highly crucial for the design of novel and potent steroids based nitrogen
mustards.

FIG 11

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2.7. Targeted delivery of nitrogen mustards

2.7.1. DNA directed nitrogen mustards

As discussed earlier, nitrogen mustard The majority of alkylating anticancer drugs alkylate DNA
primarily at the N-7 position of guanine, in a reaction dominated by the molecular electrostatic potential

PT
of the DNA site [154]. In terms of chemotherapy, there are major drawbacks with such a “bonding
dominated” [155] mechanism of alkylation. The sequence specificity of these drugs is limited.
There has been interest in overcoming these deficiencies by the use of DNA-targeted alkylating agents

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using DNA intercalating carriers to increase the binding component of the DNA interaction. However,
the binding selectivity of DNA intercalators is low and a more promising approach to highly potent

SC
sequence selective alkylators is the use of minor-groove binding structures as carriers.
Neoplastic cells can be distinguished from normal tissue by differences in their DNA. Since the

U
discovery of the structure of the DNA molecule, an enormous amount of research has been devoted to
producing high resolution X-ray crystallographic and NMR based structures of various short DNA
AN
molecules which variation in base sequence. These studies determined that in the B-DNA form (which
makes up the majority of cellular DNA) an antiparallel helix is right handed, the glycosidic bonds are in
the anti conformation and the ribose units are C2-endo puckered. The net effect of this is the creation of
M

two grooves, the major groove (≈12Ao wide) and the minor groove (≈ 6Ao wide).
In the major groove the C6 amino group of adenine or the C4 amino group of the cytosine can act as
D

hydrogen bond donating groups, while the adenine N7, thymine O4, guanine N7, O6 can act as hydrogen
TE

bond accepting groups, and the thymine methyl as a hydrophobic site. Similarly, in the minor groove the
adenine N3, thymine O2, guanine N3, and the cytosine O2 can act as H-bond acceptors and the C2 amino
group of guanine can act as donating group. Both grooves, therefore, carry base dependent sequence of
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potential H-bonding atoms that can be used as a target in the design of sequence specific DNA binding
compounds [156].
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2.7.1.1. Major groove alkylating agents

AC

Major groove alkylating agents have a long history in the treatment of cancer. The first clinically used
antitumor agent, the nitrogen mustard mechlorethamine, exerts its effects by alkylating cellular DNA,
preventing its use as a template for cellular proteins and preventing strand separation for replication. The
sequence selectivity of DNA alkylation is relatively poor, with most monoalkylating in the major groove
at the N7 position of guanine in runs of guanines, and cross-linking guanines at 5'-GMC sites (where N is

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any nucleotide). The antineoplastic activity of most DNA alkylating agents resides in their ability to kill
rapidly dividing cells, rather than any tumor cell selectivity. These compounds cause a high degree of
collateral damage to dividing cells in normal tissue, which is manifest as the side effects of
chemotherapy-toxicity to bone marrow and gut epithelia, hair loss and sterility.
DNA-directed alkylating agents comprising naphthalimide, nitrogen mustard and lexitropsin moieties

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have been designed, synthesized, characterized and evaluated. The results indicate that these alkylate
DNA at accessible guanine N7 sites within the major groove. Synthesis of 1-{ω-(9-acridinyl)amino)
alkyl}carbonyl-3-(chloromethyl)-6-hydroxyindolines 106a-c (Fig. 13) has been carried out. The

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sequence-specificity of DNA alkylation of these compounds was studied by gel electrophoresis cleavage
assay [157]. In contrast to the known trimethoxyindole-linked compounds, which alkylate exclusively at

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N3 of adenines in the minor groove, the acridine-linked analogues alkylate predominantly at the N7 of the
guanines in the major groove [158].

2.7.2.2. DNA intercalators

U
AN
Intercalators are the most vital group of compounds that have a reversible interaction with the DNA
double helix. They have major clinical significance and currently used for the treatment of ovarian and
M

breast cancer and acute leukemias. Some of them are in different phases of clinical significant, but they
generally show poor sequence selectivity. The presence of poly(hetero)cyclic system play an important
D

role in DNA intercalation which bind reversibly, but tightly to DNA by slotting the chromophore in
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between the stacked base pair [159]. These compounds work by inhibiting or poisoning the action of
topoisomerases, cellular enzymes, which relieve topological strain on the DNA molecule during
transcription and replication [160]. The sequence selectivity of these compounds is, however, generally
EP

very poor.
Acridine is one of the oldest classes of DNA and RNA binding bioactives with planar structure widely
studied as anticancer agents. Acridine ring is present in several antitumor agents that are in clinical use
C

such as actinomycin-D, daunomycin and adriamycin. Several recent researches have been focused on
AC

synthesis of acridine-derived DNA intercalating molecules because of its ability to intercalate DNA and
inhibit topoisomerase enzymes [161]. Using this approach several acridine linked nitrogen mustards have
been synthesized in an attempt to produce DNA directed alkylating agent with A dual mechanism of
action i.e. inhibition of DNA replication by both intercalation by acridine and alkylation by nitrogen
mustard (Fig. 12). It has been reported that acridine-carried mustards 107 are both more potent in vitro
and more active in vivo than the untargeted mustards themselves [162].

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FIG 12

Quinacrine mustards have greater sequence discriminating ability than any other mustard studied. It is a
DNA intercalator, which consists of a substituted acridine ring via a hydrocarbon chain to a bis-(2-
chloroethyl)amino moiety. A series of N-alkyl-N'-(2-chloroethyl)acridine mustards 108 (Fig. 13) with the

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hydrocarbon chain length varying from 2-6 methylene units have been prepared [163]. The degree of
sequence selectivity increased as the spacer chain length decreased below four methylene units. The 2-
methoxyacridine nucleus chosen for investigation was based on the quinacrine mustard, on the basis of

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results that indicated 2-methoxy substituent to be useful for increased antitumor activity. Length of the
spacer chain profoundly affected the reactivity at certain sites, whereas the nature of the alkylating group

SC
had relativity little effect.
Denny and coworkers have prepared and examined a series of aniline mustard derivatives employing 9-

U
anilinoacridine 109, 110, 111 (Fig. 13) as the carrier and studied their interaction with DNA, their
cytotoxic potencies and in vivo antileukemic activity [164-165].
AN
Prakash and co-workers have designed and synthesized a series of phenyl N-mustard link to a various
quinazoline and 9-aminoacridine via alkyl chain of variable length. The presence of these DNA-
intercalating binding rings improved the antitumor effectiveness of the nitrogen mustards [166-167].
M

Based on these findings, Bacherikov et al. synthesized a series of conjugates of N-mustard and 9-
anilinoacridine with linkers of various lengths such as methylene (CH2) or alkoxy such as O-ethylene (O–
D

C2), O-propylene, O-butylene (O–C4) spacer. There is little effect of length of spacer and location N-
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mustard group on antitumor activity. Of the entire synthesized compound 112 (Fig. 13), was found to be
most potent antitumor activity [168] but found to be unstable and low bioavailability. It was concluded
that instability may be due to the inductive effect of the alkoxy linker thereby increasing the reactivity of
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the N-mustard moiety.

To improve the chemical stability and poor bioavailability of 112, Kapuria and coworkers, synthesized a
series of phenyl N-mustard conjugates linked via urea, carbamate or carbonate linkers for antitumor
C

studies. The rational behind the selection of these linker was that these linkers were previously found to
AC

be stabilized reactive N-mustard in ADEPT and melenocyte directed enzyme prodrug therapy (MDEPT).
Among these derivatives, 113, 114, 115 (Fig. 13) were exhibited potent therapeutic efficacy and found to
be more stable than 112. It was found that urea linker 113 in phenyl N-mustard-9-anilinoacridine
conjugates plays a very important role in stabilizing the reactive N-mustard residue and the carbamate
114 or carbonate linkers 115 play an important role in enhancing the drug’s therapeutic effects (Fig. 13)
[169-170].

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 ACCEPTED MANUSCRIPT

Based on these promising results, same research group incorporated quinolines as DNA affinic carriers
for N-mustard moiety to form various mustard-quinoline conjugates having a urea or
hydrazinecarboxamide linker. N-mustard moiety is attached to C-4 position of quinoline nucleus. Both
linkers were able to lower the reactivity of the N-mustard thereby enhancing the half-life in rat plasma. Of
all the synthesized compound 116 and 117 (Fig. 13) were found to be most potent. In vitro cytotoxicity

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studies revealed that compounds with hydrazine carboxamide linker 117 were generally more cytotoxic
than the corresponding urea counterparts 116 in inhibiting human lymphoblastic leukemia and various
solid tumor cell growths in culture [171]. Similarly, N-mustard moiety is attached at the C-6 of the 4-

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anilinoquinazolines via a urea linker. Compound 118 was most potent but less potent than N-mustard-
quinoline conjugates 116, 117 [172]. Based on this finding that quinoline ring is more effective than

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quinazoline, Marvania et al. through bioisostere synthesized various quinoline linked N-mustard moiety
with urea linker. All the compounds were found to more cytotoxic than the corresponding N-mustard-4-

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anilinoquinazoline couterparts with improved solubility (Fig. 13) [173].
The hydroxyanthraquinones ring system was used by Zhao et al. to produce hybrid anthraquinone and
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nitrogen mustard conjugates via a bioisostere approach. Anthraquinone pharmacophore supposed to
provide a platform for DNA intercalation and bioreductive activation, whereas N-mustard imparts
cytotoxicity. Among them, compound 119 (Fig. 13) was the most cytotoxic with IC50 value of 0.263 nM
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and is more potent than DXR (IC50 = 0.294 nM) in inhibiting the growth of MCF-7 cells. The excellent
cytotoxicity and good selectivity of compound 119 suggest that it could be a promising lead for further
D

design and development of anticancer agents, especially for breast cancer [174].
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FIG 13
2.7.2.3. Minor groove binders
EP

A large number of compounds bind in the minor groove of DNA which includes: natural, synthetic, cell
permeable and sequence selective agents. A lot of research is going on in this area at a rapid pace [175-
176]. Minor groove binders have a number of characteristic features, these include: an overall annular
C

shape made up of aromatic rings which matches the curvature of the minor groove of DNA, cationic
AC

charges which provide affinity for the tunnel of negative molecular electrostatic potential in the groove,
and in addition many ligands possess H-bond donating or accepting atoms. These compounds tend to
bind in the minor groove with relatively little distortion of the phosphate backbone, and in fact stabilize
the regular B-DNA structure.
The molecular determinants of sequence specificity of minor groove binding ligands vary with ligand
structure. The vast majority of these compounds selectively bind to AT-rich DNA sequences and such

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sites possess several unique characteristics. The width of the minor groove is often considerably narrower
than mixed sequence DNA so that the planar aromatic rings of AT selective ligands can slot into the
groove and form stabilizing Van der walls contacts. The electrostatic potential of the minor groove varies
with base sequence and is most electronegative at AT rich sequences. Thus, positively charged with
minor groove binding ligands have a higher affinity for this tunnel of electronegativity on the floor of the

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groove. Additionally, AT base pairs have two H-bond accepting groups, the O2 of thymine and N3 of
adenine, which can further stabilize ligands with appropriately situated H-bond donating groups. In
addition, AT-rich sequences contain highly-structured networks of coordinated water molecules that add

RI
to the overall stability and the displacement of these by ligands provides a substantial entropy component
to the binding. Review on design of drugs that have specificity for modulation of gene expression and

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selectivity for target cells at the transcription level by targeting DNA [177].
DNA-targeted alkylating agents enhance electrophilicity and sequence specificity. A series of 4-

U
anilinoquinoline linked aniline mustards 120 and 121 (Fig. 14) of widely varying mustard reactivity have
been designed as minor groove binding agents, where aniline mustard ring itself is a part of the DNA
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binding ligand [178]. These compounds showed potent anticancer activity against human leukemia and
various solid tumor growth invitro, particularly compound 90 which was found to bind in the minor
groove and alkylate both adenines and guanines at the N3 position.
M

The use of the putative DNA minor groove binding 4-anilinoquinoline moieties resulted in aniline
mustards of enhanced potency. These compounds were active, but not found to be particularly dose-
D

potent against P388 leukemias in vivo. However, the modest dose potency and activity of these
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compounds may be related to their poor aqueous solubility [179].

Alkylation by 122 (Fig. 14) occurred almost exclusively at N-3 position of adenine in the minor groove.
These are very potent cytotoxins [180].
EP

The minor groove of DNA remains a site of interest for targeting of sequence-specific binding agents.
Netropsin and distamycin are two naturally occurring ligands that have been extensively studied for their
DNA binding properties and have been shown to bind in the minor groove of AT-rich sequences [181].
C

Another example, minor groove-binding targeted nitrogen mustards are a series of compounds based on
AC

antibiotic distamycin. Compound 123 (Fig. 14) has shown activity in vivo against a number of solid
tumor cell lines and given promising results in clinical trials [182-183].
The sequence specific of alkylation for a series of pyrrole and imidazoles containing analogues of
distamycin 124 and 125 (Fig. 14) that tether the nitrogen mustard chlorambucil was determined using
modified sequencing techniques.

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In this study, chlorambucil conjugates shown to alkylate DNA with similar sequence specificity to that
seen for the chlorambucil. The dose used to produce equivalent amounts of damage for the chlorambucil
conjugates was > 10 fold lower than that used for chlorambucil [184].
Synthesis, molecular modeling, antiproliferative and cytotoxic effects of carbohydrates derivatives of
distamycin, netropsin with chlorambucil moieties have been reported. All the compounds showed

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antiproliferative and cytotoxic effect [185].
Three different groups of analogues of the sequence specific minor groove alkylator, tallimustine such as
126 (Fig. 14) have been synthesized and investigated. The correlation between the cytotoxicity and

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alkylation pattern suggests that tallimustine exerts its cytotoxicity through DNA sequence-specific
alkylatioin of the adenine located in the sequence 5'-TTTTGA [186].

SC
The amino analogues with polymethylene (n = 3-6) chain of pentamidine as the DNA minor groove
binders and as a carrier for chlorambucil and their chlorambucil derivatives 127a-d (Fig. 14) were

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synthesized. The compounds have revealed the cytotoxic effect on MCF-7 human breast cancer cell line,
mainly by the induction of apoptosis [187].
AN
FIG 14

2.7.2. Nitrogen mustards in antibody-directed enzyme prodrug therapy (ADEPT).

M

One of the promising approaches to have selectivity for the treatment of cancer is to link an enzyme to an
antibody that catalyzes the conversion of a prodrug into its active form. According to this approach called
D

antibody-directed enzyme prodrug therapy or ADEPT a wide variety of drugs have been generated using
TE

monoclonal antibody-enzyme conjugates [188-193].

These alkylating agents in an ADEPT approach present many advantages: the activity of these drugs is
usually cell cycle-independent, dose dependent, and induce less acquired resistance than other
EP

chemotherapeutic agents.
FIG 15
ADEPT is a two-step approach for the treatment of cancer, which seeks to generate a potent cytotoxic
C

agent selectively at tumor sites. At the first step, the enzyme-antibody conjugates (mAb-enzyme) are
AC

delivered first which binds and accumulates at the cancer cells due to the specific antigen present only on
the surface of cancer cells. After the time needed to optimize localization and clearance of unbound
conjugates from the blood, a non-toxic drugs are administered in the form of the prodrug, which will be
activated by the enzyme in the conjugates, thus selectively liberating the cytotoxic agents at cancer cells
(Fig. 15). The general strategy is to conjugate the toxic component to the antibody in a single cytotoxic

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bifunctional agent, which has targeting potential. Cytotoxic agent is generated by the action of an enzyme
carboxypeptidase G2 (CPG2) on a relatively non-toxic prodrug [194].
A series of new prodrugs 128a and 128b have been synthesized where benzamide link has been replaced
by carbamate or ureido, respectively. The active drugs 129a and 129b derived from these prodrugs are
potent cytotoxic agents (Fig. 16) [195-196].

PT
Springer and coworkers have carried out a synthesis of six novel fluorinated potential prodrugs 130a-f
(Fig. 16) for ADEPT [197].

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The {2- and 3-fluoro-4-{(bis-2-chloroethyl)amino)benzoyl}-L-glutamic acid 130a and 130b, their
bis(mesyloxy)ethyl derivatives 130c and 130d and their chloroethyl(mesyloxy)-ethyl derivatives 130e
and 130f (Fig. 16) are bifunctional agents in which the activating effect of the ionized carboxyl function

SC
is masked through an amide bond to the glutamic acid residue. These diacids are converted by CPG2 in
vivo/in vitro to their corresponding active benzoic acid alkylating agents.

U
Prodrugs 131 and 132 (Fig. 16) of a phenol mustards include a glucuronide group which is connected to
the drug via an aromatic and/or aliphatic bis-carbamate spacer. The enzymatic catalyzed hydrolysis of the
AN
glucuronyl moiety of 131 by Escherichia coli β-glucuronidase resulted in the liberation of the parent
mustard drug. Surprisingly, prodrug 132 was not cleaved under the same conditions. According to in
M

vitro studies, prodrugs 131 and 132 were 50-and 80- fold less cytotoxic than the parent drug, but when
treated with β-glucuronidase, the level of cytotoxic activity of 131 became comparable to that of the drug
[198].
D

An antibody-directed enzyme prodrug therapy system under clinical trials (in Phase I/II clinical trials) is a
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prodrug, 4-[N-bis(2-iodoethyl)amino]phenoxycarbonyl L-glutamic acid (ZD2767P) 133 (Fig. 16) which

is a nitrogen mustard glutamate prodrug [199]. Due to its low yield, novel method has been described by
Niculescu-Duvaz et al., 2005 that overall increase the yield from 13% to 45% [200].
EP

The synthesis of cephalosporin derivatives of nitrogen mustard and mitomycin have been reported as
antibody-directed enzyme prodrug therapy [201].
A review highlighting evolving strategies in targeted prodrug design, including antibody-directed enzyme
C

prodrug therapy, and peptide transporter-associated prodrug therapy has been published. Potentiation of
AC

antitumor immunity by ADEPT has also been reported [202-203].

FIG 16

2.7.3. Nitrogen mustards in gene-directed enzyme prodrug therapy (GDEPT)

Gene-directed enzyme prodrug therapy (GDEPT) is a primary self immolative prodrug therapy related to
specific genes which is also known as suicide gene therapy. It is a promising strategy for cancer therapy

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similar to ADEPT, both guaranteeing selective killing of cancer cell by localizing activating enzymes
specifically into cancer sites before administration of prodrugs. In this method, specific genes encoded for
specific enzyme are introduced into cancer cells capable of catalyzing and activating the non-toxic
prodrug to cytotoxic drugs inside the cancer cells [204] (Fig. 17). Usually enzymes of viral or bacterial
enzymes are introduced which normally donot occur in mammals or human enzymes, which are absent

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from cancer cells or occur only at low concentration. For the prodrug in GDEPT, it must have high
affinity for the enzyme encoded by the suicide gene and low affinity for endogenous enzymes outside the
cancer mass [205]. Moreover, the expression of the exogenous enzyme does not occur in all cells of a

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targeted cancer, thus toxic form of the prodrug should be capable of intercellular diffusion to kill both the
cancer cells in which it is formed and neighboring cancer cells that do not express the enzyme. This

SC
process is called bystander effect or neighboring cell killing effect that will cause more widespread cell
death [206]. In ADEPT, prodrug activation occurs extracellularly, whereas intracellularly in GDEPT.

U
Therefore, the combination of enzyme and prodrug that is suitable for ADEPT may not be suitable for
GDEPT.
AN
FIG 17.

One example is a cytochrome P450s (CYPs) that can efficiently bioreduce the cytotoxic prodrug AQ4N
M

into cytotoxic AQ4 by CYPs under hypoxic condition. To enhance the metabolism of AQ4N for GDEPT
approach several investigations have been going on resulting in entering of AQ4N in phase II clinical
D

trials [207].
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An alternative candidate for GDEPT is carboxypeptidase G2 (CPG2) from Pseudomonas Sp. [208-209]
since it has no mammalian homologue. This enzyme catalyzes in scission of an amidic, urethanic, or
uridic bond between an aromatic nucleus and L-glutamic acid. Four new potential self-immolative
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prodrugs derived from phenol and aniline 134a and 134b (Fig. 16) nitrogen mustards, four model
compounds derived from their corresponding fluoroethyl analogues and two new self-immolative linkers
were designed and synthesized for use in GDEPT [210].
C
AC

As discussed earlier, clinical drug PR-104 (Fig. 3) also metabolically reduced into corresponding
hydroxylamine (PR-104H) and amine (PR-104M) in hypoxic cell, causing DNA interstrand cross-linking
(Scheme 1) [211].
No cancer suicide gene therapy has reached the desirable clinical importance despite of reaching various
enzyme/prodrug combinations in GDEPT inthe clinical trial stage [212-213].

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The virus-directed enzyme prodrug therapy (VDEPT) anti-cancer “gene therapy” strategy relies on the
use of viral vectors for the efficient delivery to tumor cells of a “suicide gene” encoding an enzyme which
converts a non-toxic prodrug to a cytotoxic agent [214]. The prodrug 5-(aziridin-1-y)-2,4-
dinitrobenzamide, CB1954, 135 was proposed for use in enzyme-prodrug gene therapy systems with the
E. coli enzyme nitroreductase (Ntr), which converts CB1954 to 2- and 4-hydroxylamino derivatives 136-

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137, whereupon the non-enzymatic reaction of the 4-hydroxylamino derivative 136 with cellular thio-
esters generates a potent cytotoxic bifunctional alkylating agent 138 capable of cross-linking DNA
[Scheme 10 ]. Nitroreductase delivery has been achieved in vitro using retroviral and adenoviral vectors

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and confirmed by immunocytochemical demonstration of Ntr expression [215-216].
SCHEME 10

SC
Cyclophosphamide analogues were developed through the incorporation of a trigger activation
mechanism by E. coli nitroreductase for gene therapy. They are based on the activation mechanism of

U
cyclophosphamide. The nitrobenzene fused cyclophosphamide analogue 139 (Fig. 18) exhibited good
substrate activity for E. coli nitroreductase with a half life of 13 min and a modest >33 fold enhanced
AN
cytotoxicity toward E. coli nitroreductase expressing cells (Scheme 11) [217].
Though not sufficiently potent, 140 (Fig. 18) represented a new structure type for reductive activation and
a new lead for further modification in the development of better analogue with much improved selective
M

toxicity to be used in gene directed enzyme prodrug therapy. The cytotoxicity and selectivity were
dependent not only on good substrate activity toward E. coli nitroreductase but also on the presence of a
D

benzylic oxygen para to the nitro group. Compared to the 4-nitrophenylcyclophosphamide 140 showed
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an over 100-fold increase in cytotoxicity and nearly 1,000-fold increase in selectivity toward E. coli
nitroreductase-expressing cells. The trans isomer was shown to be a better substrate of E. coli
nitroreductase than the corresponding cis isomer (Scheme 11) [218].
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Efforts have been focused on designing phosphoramide analogues incorporating site-specific activation
mechanisms such as nitro reduction by nitroreductase (E. coli) in order to move the site of activation from
the liver into the tumor sites. Cyclic (102, 103) and acyclic nitroaryl phosphorodiamidates 141 (Fig. 18)
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have been synthesized, activated by E. coli nitroreductase with a strategically placed nitrogroup on the
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benzene ring in the para position to the benzylic carbon [219]. The more active acyclic 4-nitrobenzyl
phosphoramide mustard 141 showed 1675000 X selective cytotoxicity towards nitroreductase expressing
V79 cells with an IC50 as 1000 as 0.4 nM (Scheme 11).
SCHEME 11

2.7.4. Latent alkylating agents activated by glutathione transferase

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A major cellular defense mechanism utilizes a tripeptide, glutathione (γ-glutarylcysteinylglycine, GSH),

which acts as a scavenger molecule able to couple to and neutralize many toxic electrophiles, including
chemotherapeutic agents. Such coupling is mediated by a family of enzymes named glutathione S-
transferases (GST’s) [220].
It has been shown that many types of cancer tissues often have elevated levels of GST’s compared to

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corresponding healthy tissue [221-222]. That results in enhanced detoxification of alkylating agents
leading to drug resistance. Ovarian carcinoma cells were found to be 10-fold resistant to chlorambucil and
had almost 5-fold higher glutathione S-transferase (GST) activity than the parental A2780 cells with 1-

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chloro-2,4-dinitrobenzene as substrate. The PI-class GST(s) was the major isoform(s) The latent prodrug
design was based on extensive literature showing that overexpression of GST in human tumors is

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associated with malignancy, poor prognosis and the development of drug resistance [223-225]. Thus,
selective targeting of susceptible tumors phenotypes is a strategy that should result in the release of more

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active drug in malignant cells compared with normal tissue, thereby achieving an improved therapeutic
index [226-228].
AN
Few reports demonstrated the association of GST’s with resistance to alkylating agents [229-231]. Some
recent studies revealed that microsomal GST play an important role in metabolism and adverse drug
reaction of chlorambucil [232] and melphalan [233]. A chemotherapeutic agent that takes advantage of
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this intrinsic property of many types of cancer cells may prove to be a valuable anticancer drug.
Interestingly, different cancers show different GST isozymes distributioin, but it is most often the P1-I
D

isozyme that is elevated to the greatest extent I many types of cancer tissue . As a result, an isozyme
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selective drug can be targeted to a specific cancer tissue on the basis of its isozyme distribution profile.
Targeting of GST’s can be accomplished in two ways. Firstly, to develop isozyme specific/selective GST
inhibitors and secondly by using an appropriate functionalized GST analogue that would serve both as a
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modified GSH substrate and a latent alkylating agent [234].

Satyam and coworkers [235] have designed and successfully demonstrated the glutathione-linked
nitrogen mustards 142a-e (Fig. 18). Two new analogues 142d and 142e of the latent alkylating agent
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142c have been designed, synthesized and evaluated. It was found that the compounds 142b and 142c are
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cleaved by physiological concentrations of GST’s at physiological temperature and pH to release toxic

phosphoramidate mustard. One of the diastereomers of 142e exhibited good selectivity for GST P1-I.
The tetrabromo analogue 142e of the tetrachloro compound 142c maintained its specificity and was found
to be more readily activated by GST than 142c.
Urethane mustard 143a and its diethyl ester 143b (Fig. 18) were also designed, synthesized and evaluated
following the same concept. Interestingly, 143a showed very good specificity for PI-1 GST [236].

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The most promising clinical candidate, that exploit high glutathione S-transferase PI-1 (GSTPI-1 or
GSTT I) levels in solid tumors and drug-resistant cell population is TLK 286 144 (Fig. 18) [237-238].
Chemically it is [γ-glutamyl-α-amino-β-(2-ethyl-N,N',N″-tetrakis(2-chloroethyl)phosphorodiamidate)-
sulphonyl-propionyl-(R)(-)phenylglycine. In TLK 286 144, the sulphydryl of a GSH conjugate has been
oxidized to a sulphone. The tyrosine-7 in GSTPI-1 promotes a β-elimination reaction that cleaves 144.

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The cleavage products are a GSH analogue and a phosphorodiamidate, which in turn spontaneously form
aziridinium species, the actual alkylating moieties [Scheme 12]. The cytotoxic moiety has tetra
functional alkylating properties, similar in concept to bifunctional nitrogen mustards that react with

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cellular nucleophiles with a short half-life [239-240].
SCHEME 12

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FIG 18
2.7.5. Peptides based nitrogen mustards

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Among many improvements in targeted and controlled delivery of therapeutics, cell targeting peptides
have emerged as the most valuable non-immunogenic approach to target cancer cells. In contrast to larger
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molecules, such as monoclonal antibodies, peptides have many advantages like small size, ease of
synthesis and modification, excellent tumor penetrating ability, and good biocompatibility, which make
them ideal carriers of anticancer drugs to the site of primary tumor and the distant metastatic sites [241]
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(Fig.19). Recently, peptides have been emerged as promising therapeutic agents in the treatment of
cancer. Peptides have also been utilized as biodegradable, non-toxic carrier for the targeted and site-
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specific delivery of anticancer drugs. This peptide research area is gaining momentum due to the
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possibility of improved drug potency and minimal side effects. Attachment of peptide to cytotoxic drugs
affords several advantages that include the specific delivery of drug to the target cell, regulated
cytotoxicity, by passing of drug resistance, reversal of drug resistance, delivery of higher payloads and
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simultaneous delivery of cytotoxic drugs with different mechanism of action [242-243].

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Fig. 19 Schematic representation of peptide based drug conjugate

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Utilizing this approach, Gellerman et al, designed and synthesized dendrimer-based peptide conjugates

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containing one, two, or four molecules of chlorambucil 145, 146, 147 respectively (Fig. 20). The murine
B-cell leukemic cells earlier resistant to a chlorambucil became sensitive to that drug when it is
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conjugated to a targeting peptide. There was also significant enhancement in the cytotoxic activity of
multidrug versus single-drug copy conjugates. The use of multifunctional dendrone linkers bearing
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several covalently bound cytotoxic agents allows the development of more effective targeted drug
systems and enhances the efficacy of currently approved drugs for B-cell leukemia [244]. Based on these
encouraging results, Gellerman and coworker, designed and synthesized various drug-carrier conjugates
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148, 149, 150 (Fig. 20) that consist of several different cytotoxic compounds linked via biodegradable
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linkages of Multifunctional Amino Acid Platforms (MAPP) using simple and convenient orthogonally
protective solid-phase organic synthesis (SPOS). Each arm of platform contains a different anticancer
agent having same or different functional group “switch off/switch on” regulation of drug cytotoxicity.
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The results presented here potentiate the application of amino acid platforms for targeted drug delivery
(TDD) [245]. In another work, Ragozin et al, using simple and convenient SPOS, synthesized various
oligopeptide compact carriers bearing several units of the same or different anticancer agents. Chemo-and
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biostability experiments revealed pH and liver homogenate dependent sequential release behavior. This
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approach may be beneficial in personalized medicine where multiple drug delivery is required in a
sequential and controlled manner [246]

Integrins, which is involved with tumor progression processes such as angiogenesis, invasion and
metastasis, are overexpressed in cancer cell and therefore could be exploited as a vital target for
therapeutic intervention [247] as well as for the selectively delivery of anticancer agents [248]. A recently

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identified peptidic ligand called iRGD selectively target integrins. Considering these facts, Gilad et al,
reported the synthesis of new cyclic peptide-anticancer agent conjugates 151a-b, (Fig. 20) based on the
cyclic (RGDfK) penta-peptide in which the methylated valine was mutated to either Lys or Ser enabling
primary amine or hydroxy group as a site for drug conjugation. Computational analysis suggested that all
conjugates occupy conformational spaces similar to that of the integrin bound bio-active parent peptide

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[249]. In continuing their research after getting encouraging results, a cyclic RG(DfK) penta-peptide
loaded with two anticancer drugs were synthesized to increase the drug loading and efficacy. The drug
release profiles of the new compounds were evaluated with the singly loaded analogs for cytotoxic

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activity against melanoma and non-small lung cancer cell lines. The peptide’s core was modified at the
side chain of its Lys residue that helped in two functional sites which allow the loading of two drugs onto

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a single targeting carrier. The conjugates were formed by coupling of chlorambucils 152a-b, and
camptothecins 153 (Fig. 20). Loading of two drugs enhanced cytotoxic efficacy of the conjugates. The

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compound occupied a conformational space similar to the bio-active conformation of an integrin-bound
cyclic RGD peptide reference peptide (c(RGDf(NMe)V). This lead to substantially increasing the efficacy
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of selective killing of tumor cells and while reducing the risk of the development of drug resistance [250].

Somatostatins are a five-membered family [251] of transmembrane G-protein coupled cell-surface

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receptors widely distributed in a variety of tumors [252-253], which also makes them an attractive
target for selective delivery of chemotherapeutics. In view of this, Redko and co-workers described the
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synthesis of novel peptide–drug conjugates based on the disulfide bridged backbone cyclic somatostatin
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peptide analog 3207-86 which is SSTR2 selective inhibitor [254]. Five chemotherapeutic molecules,
acting through different oncogenic mechanisms, were linked to the core peptide carrier, yielding SST–
chlorambucil conjugates 154 (Fig. 20). In that work chemo- and biostability of the peptide drug
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conjugates in various media were measured, representing release profiles for each drug. This information
is useful for further optimization of drug release capabilities from 3207-86 peptide–drug conjugates.
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2.7.6. Central Nervous system (CNS) targeted nitrogen mustards

Primary and secondary metastatic tumors of the Central Nervous System (CNS) represent a foremost
health problem globally. Male are affected more than females. It has been estimated that each year more
than 200,000 lakhs of population are diagnosed with a primary or metastatic brain tumor [255]. The major
obstruction to CNS drug delivery is the blood-brain barrier which limits the access of drugs to the brain.
Nitrogen mustard classes of drug are used extensively in the treatment of leukemia and solid tumors. But

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their inefficiency to cross the BBB due to their high polarity, poor physicochemical properties and
toxicity to normal tissues, remain challenges for the medicinal chemist and therefore lots of research have
been going on to make this class non-toxic to peripheral cells and permeable to cross the BBB by various
prodrugs approaches.
The combination of two pharmacological entities in a single compound has been utilized as a promising

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drug design strategy for site-specificity. In an attempt to obtain central nervous system (CNS) antitumor
agents, nitrogen mustard pharmacophore were designed to connect with the various lipophilic carrier.
The first attempt to make lipophile nitrogen mustard was performed by Peng et al. by linking nitrogen

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mustard moiety to lipophilic hydantoin ring. It was rationally designed as a lipophilic compound capable
of penetrating the CNS. The compound 145 (Fig. 22) was found to be most active active in the

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intraperitoneal leukemia L1210 and P388 systems as well as in B16 melanoma and Lewis lung carcinoma
[256-257]. It was further selected for phase I clinical trial with the name of spiromustine. Injection of

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spiromustine on a split-dose schedule decreased the acute neurological toxicity in mice and allowed a
larger total dosage to be delivered compared to single bolus dosage [258].
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Later, four phenothiazine derivatives 146a-d (Fig. 22) containing the bis(2-chloroethyl)aminopropyl side
chain were prepared and evaluated in the murine L-1210, P-388, and B-16 melanoma intraperitoneal
tumor systems. Moderate P-388 activity was observed. Aminoethyl phenothiazine mustard was compared
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with the aminopropyl analogs and was superior in all test systems. None of the compounds tested against
the murine ependymoblastoma brain tumor system were active [259].
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Bartzatt et al. combined the mustard pharmacophore to CNS active pyrimidine ring at 1 and 3 position to
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two nitrogen atoms. The various CNS active physcicochemical parameters analyzed were supporting the
contention that the N-mustard agent has potential as a potential CNS antitumor agent [260]. Vitamin
nicotinic acid has also been used to link with nitrogen mustard to obtain a CNS active anticancer agent
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147 (Fig. 22) and found to be stable at room temperature and sufficiently soluble in aqueous solution to
alkylate nucleophilic primary amine groups at 37◦C and pH 7.4 [261-262].
Singh et al., [263] designed two nitrogen mustard agents by conjugating it with a lipophilic
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benzodiazepine nucleus 148a-b (Fig. 22). The benzodiazepine part is aimed to serve as a CNS active
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carrier enabling the alkylating moiety to cross the BBB by altering its physicochemical properties. The
compounds were markedly active when subjected to in vitro biological evaluation using an MTT
colorimetric assay against four human cancer cell lines (A-549, COLO 205, U-87 MG and IMR-32). The
physicochemical ADME studies were also analyzed using Qikprop 2.5 tools of Schodinger software
which further indicates that both compounds can be potential candidates for the treatment of brain tumor.

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In another novel approach, Singh et al. designed and synthesized various 2-aminobenzophenone
derivatives of nitrogen mustard agents 149a-f (Fig. 22) as CNS active antitumor agents. Fist they study
the important pharamcophore of benzodiazepine nucleus responsible for the CNS activity resulting in
formation of open ring benzodiazepine nuclei. They assumed that incorporating these potent
pharmacophores of benzodiazepine nucleus responsible for CNS action together with N-mustard moiety

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may result in strong anticancer agent that may pass blood-brain barrier and effective against brain-tumor.
Most of the test compounds exhibited potent antitumor activity, especially compound 149f which
displayed the highest activity against CNS cancer cell line among the test compounds comparable to that

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of chlorambucil and doxorubicin. The 5-chloroaminobenzophenone-mustard 149a-c series exhibited
better antitumor activity than 5-nitroaminobenzophenone-mustard 149d-f series (Fig. 22) [264].

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These hybrids exhibited moderate to significant cytotoxic activity with IC50 ranging from 23.7 to 74.4
µM in comparison to 18.8 to 26.7 µM for chlorambucil. Among all compounds, 4 and 5a-d were found to

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be most active.
Bodor and coworker developed the most promising chemical delivery system (CDS) for brain delivery of
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therapeutic agents by the redox drug delivery approach analogous to the endogenous NADH↔NAD+
coenzyme system [265]. According to this, drug is linked to a lipophilic 1,4-dihydropyridine carrier
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which is easily permeable to blood-brain barrier (BBB). Inside the brain, the carrier part is oxidized to the
ionic pyridinium salt, whch is due to its hydrophilicity couldnot eliminate from the brain. Inside the brain,
the hydrophilic quaternary salt is subsequently cleaved into desired drug with facile elimination of the
D

carrier part (Fig 20). This system has been studied widely as a means to enhance the targeted of
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anticancer drugs to the brain [266].

FIG 20
Bodor et al., synthesized for the first time the CDS of chlorambucil as alkylating anticancer agent using
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either N-hydroethylnicotinamide or various aminoalcohols as linkers. A CDS 150 obtained with 2-

aminoethanol as a linker was found to most suitable and produce sustained level of drug in the brain after
i.v. administration [267].
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Similarly, El-Sherbeny et al. 2003 utilized the CDS approach for targeting nitrogen mustard across the
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brain. The synthesized redox derivatives of alkylating agent were subjected to various chemical and
biological investigations to evaluate their ability to cross the BBB. The in vitro and in vivo studies
showed that the compound 151 (Fig. 22) was able to cross the BBB at detectable concentrations [268].
Reversible redox drug delivery approach was thoroughly investigated by Singh et al. for improving the
targeting potential and sustained release of nitrogen mustard to the brain. Various redox derivatives CDS-
M 152a-e (Fig. 22) were synthesized incorporating different alkyl/aryl moiety at pyridine ring nitrogen

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and cytotoxic mustard moiety without any linker and subjected to in silico physicochemical parameters
determination required for CNS activity through computational, online and QikProp 3.2 software. The
results of stability study, in vitro chemical (silver nitrate) and biological oxidation studies in human
blood, rat blood and brain homogenate for all CDS-M 152a-e have been promising and suggest that brain
targeting could be possible with more stable CDS-M 152e. The in vivo study showed that CDS-M 152e

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was able to cross the BBB at detectable concentration and in vitro NBP alkylating activity of its
quaternary salt 3e was comparable to the known drug chlorambucil among all the synthesized derivatives.
The only drawback was that these compounds showed reasonable stability when stored at room

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temperature [269-271].
In order to improve the stability and retention ability, Singh et al. designed and synthesized various other

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CDS of nicotinic mustard using ethyl ester linker between dihydropyridine and mustard moiety 153a-d
(Fig. 22). The compound CDS-L-M 153d was found to be most promising for brain targeting as indicated

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by the storage stability study, in vitro chemical oxidation (silver nitrate) and pharmacokinetic studies in
human blood, rat blood and brain homogenate (Fig. 22) [272-273]
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FIG 22
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3. Conclusion
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Design and development of anticancer drugs are one of the most complicated tasks which can be achieved
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by the intelligent application of chemical principles. Apart from efficacy, toxic side effects,
pharmacokinetic compatibility would all be the major parameters in the ultimate development of
promising anticancer drugs. Nitrogen mustard that represents a major class of alkylating anticancer agent
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is suffering from limited therapeutic activity because of its narrow therapeutic window. This may be
attributed to their high reactivity, indiscriminate cytotoxicity and organ distribution. An understanding of
the mechanism underlying nitrogen mustard reactivity helps us to circumvent the toxic side-effects of new
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generation nitrogen mustard anticancer agents. This review emphasized on a potent anticancer activity
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profile of the nitrogen mustard and its analogs such as mechlorethamine, chlorambucil,
cyclophosphamide, melphalan etc. Recent progress in medicinal chemistry aspects of each analogues
were comprehensively described leading to the development of clinical and preclinical drugs. These
analogues need to be further explored and extensive research is required to find novel analogs suitable for
clinical applications. Numerous strategies have been explored to lower down nitrogen mustard reactivity
to convert it into efficacious and non-toxic anticancer drugs. The prodrug is one of the most promising

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strategies to improve the selectivity and efficacy of cytotoxic compounds. Development of prodrugs
strategies such as ADEPT, GDEPT, VDEPT etc. were based on the fact that cancer cells have unique
aberrant markers compared with normal tissues. Both ADEPT and GDEPT strategies are attractive
options for cancer treatment. Recent developments have led to various nitrogen mustard derivatives and
prodrugs with novel drug delivery that is highly specific to cancer cells. Noteworthy progresses of

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nitrogen mustards described herein will shed more light on it reactivity and its correlation with its
anticancer activity. However, there is evidently much yet to be investigated to fine-tune the chemistry of
nitrogen mustard to generate better anticancer drugs with improved selectivity as well as low toxicity.

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List of figure captions:

Fig. 1. Historical development of the mustard family of agents.

Fig. 2. Alkylating mechanism of nitrogen mustard agent with guanine base of DNA leading to cell
cytotoxicity.
Fig. 3. Chemical structures of compounds 1-11.

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Fig. 4. Chemical structures of compounds 20-18.
Fig. 5. Chemical structures of compounds 19-35.

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Fig. 6. Chemical structures of compounds 36-51.
Fig. 7. Chemical structures of compounds 52-63.

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Fig. 8. Chemical structures of compounds 64-76.
Fig. 9. Chemical structures of compounds 77-88.
Fig.10. Some typical representatives of steroidal linked nitrogen mustards used in clinical practice against

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several types of cancers.
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Fig. 11. Chemical structures of compounds 89-105.
Fig. 12. Mechanism of action of DNA intercalators
Fig. 13. Chemical structures of compounds 106-119.
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Fig. 14. Chemical structures of compounds 120-127.

Fig. 15. Mechanism of activation of nitrogen mustard at tumor site using antibody-directed enzyme
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prodrug therapy (ADEPT).

Fig. 16. Chemical structures of compounds 128-134.
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Fig. 17. Mechanism of activation of nitrogen mustard at tumor site using gene-directed enzyme prodrug
therapy (GDEPT).
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Fig. 18. Chemical structures of compounds 139-144.

Fig. 19. Schematic representation of peptide based drug conjugates

Fig. 20. Chemical structures of compounds 145-154.

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Fig. 21 Transport, activation and clearance of drug using redox delivery system.
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Fig. 22. Chemical structures of compounds 155-163.

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Fig. 1. Historical development of the mustard family of agents

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Fig. 2. Alkylating mechanism of nitrogen mustard agent with guanine base of DNA leading to cell
cytotoxicity.
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Fig. 3. Chemical structures of compounds 1-11.

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Fig. 4. Chemical structures of compounds 12-18.

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Fig. 5. Chemical structures of compounds 19-35.

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O
R
37a = -OCH2CH2-
R N
37b = O
Cl Cl O O
HOOCH2CH2CH2C N N
37c = -NH-N=CH--
R
Cl
36 37a-e 37d = -NH-N=C
Cl
CH3
37e = -NH-N=CH

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Cl
Cl
OO O N Cl
N
H2N S Cl O
O

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N N N N
H I
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39

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H OAc
N AcO
O N O O O
H O
O Cl AcO
R F N O Cl
H N
40a-c
NH Cl 41

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N N Cl
40a R= NH2 40b R= 40c R=
N HN
H
OH
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OH O
O Cl
O
N N N
O Cl H H O Cl
OMe N
Cl HN HN
MeO OMe HO 3 HO mN 3
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H
OMe O Cl O
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42 Cl 44
43
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NH2 N
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O O Cl N(C2H4Cl)2
HO OC16H33
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O
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O
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HO OC16H33 O O O
O O N
HN O O HO N NH2
HO N
O F F
HO O
N OC16H33
Cl
HO NH2 O
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Cl 48
45 46 47
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H
NHOH N
Cl Cl Cl
COOH O O
N N N N N H2N
Cl Cl
N N
Cl 51
49 50
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Fig. 6. Chemical structures of compounds 36-51.

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Fig. 7. Chemical structures of compounds 52-63.

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Fig. 8. Chemical structures of compounds 64-76.

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Fig. 9. Chemical structures of compounds 77-88.

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Fig.10. Some typical representatives of steroidal linked nitrogen mustards used in clinical practice against
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Fig. 11. Chemical structures of compounds 89-105.

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Fig. 12. Mechanism of action of DNA intercalators

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Fig. 13. Chemical structures of compounds 106-119.

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Fig. 14. Chemical structures of compounds 120-127.

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Fig. 15. Mechanism of activation of nitrogen mustard at tumor site using antibody-directed enzyme
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Fig. 16. Chemical structures of compounds 128-134.

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Fig. 17. Mechanism of activation of nitrogen mustard at tumor site using gene-directed enzyme prodrug

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therapy (GDEPT).
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Fig. 18. Chemical structures of compounds 139-144.

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Fig. 19. Schematic representation of peptide based drug conjugates

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Fig. 20. Chemical structures of compounds 145-154.

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Fig. 21: Transport, activation and clearance of drug using redox delivery system.

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Fig. 22. Chemical structures of compounds 156-163.

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List of schemes:

Scheme 1: Mechanism of activation of hypoxia activated dinitrobenzamide mustard (DNBM) pre-

prodrugs (PR-104).
Scheme 2: Strategically replaced aromatic ring of chlorambucil leading to development of bendamutine.

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Scheme 3: Biological activation of cyclophosphamide.
Scheme 4: Mechanism of activation of nitroheterocyclic phosphoramidates, 77 and 78 under hypoxic

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condition.
Scheme 5: Bioreductive activation of 1, 2-benzisoxazole phosphordiamidates under hypoxic condition.
Scheme 6: Bioreductive activation of sulfonyl-group containing aldophosphamide, 82 under hypoxic

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Scheme 7: Reductive activation of TH-302 84 into cytotoxic isophosphoramide mustard under hypoxic

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condition.
Scheme 8: Bioreductive activation of isophosphoramide mustard prodrug 85a-c
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Scheme 9: Proposed mechanism of activation of prodrug 88 by Prostate specific antigen (PSA)
proteolysis in prostate tumor tissues.
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Scheme 10: Mechanism of activation of prodrug CB1954 by E. coli nitroreductase (Ntr) using virus-
directed enzyme prodrug therapy (VDEPT).
Scheme 11: Proposed mechanism of bioreductive action of substituted nitrobenzyl phosphoramidates,
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Scheme 12: Biological activation of TLK 286 into cytotoxic alkylating mustard.
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Scheme 1: Mechanism of activation of hypoxia activated dinitrobenzamide mustard (DNBM) pre-
prodrugs (PR-104).
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Scheme 2: Strategically replaced aromatic ring of chlorambucil leading to development of bendamutine.

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Scheme 3: Biological activation of cyclophosphamide.

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Scheme 4: Mechanism of activation of nitroheterocyclic phosphoramidates, 77 and 78 under hypoxic

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Scheme 5: Bioreductive activation of 1, 2-benzisoxazole phosphordiamidates under hypoxic condition.

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Scheme 6: Bioreductive activation of sulfonyl-group containing aldophosphamide, 82 under hypoxic

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Scheme 7: Reductive activation of TH-302 84 into cytotoxic isophosphoramide mustard under hypoxic

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Scheme 8: Bioreductive activation of isophosphoramide mustard prodrug 85a-c

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Scheme 9: Proposed mechanism of activation of prodrug 88 by Prostate specific antigen (PSA)

proteolysis in prostate tumor tissues.
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Scheme 10: Mechanism of activation of prodrug CB1954 by E. coli nitroreductase (Ntr) using virus-

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directed enzyme prodrug therapy (VDEPT).
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Scheme 11: Proposed mechanism of bioreductive action of substituted nitrobenzyl phosphoramidates,

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Scheme 12: Biological activation of TLK 286 into cytotoxic alkylating mustard.

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Highlights

• Nitrogen mustards are an important class of alkylating drugs for cancer therapy.
• The linking of nitrogen mustards to various carriers has been a successful strategy to
reduce its side effect.
• The present review focus on historical as well as recent developments in nitrogen

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mustards.
• The development of clinical candidates of nitrogen mustards having reduced toxicities
and greater selectivity toward the target cancer cells has also been discussed.

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