Kadric-Master Thesis PDF

PALACKÝ UNIVERSITY OLOMOUC
Faculty of Science
Department of Organic Chemistry
Master Thesis
Synthesis of Novel Derivatives of 3-Hydroxyquinolin-4(1H)-on-

carboxamides via Solid-Phase Synthesis and Evaluation of Their
Biological Properties
Student: Jasna Kadric

Supervisor: RNDr. Miroslav Soural, Ph.D.
Field of Study: Organic Chemistry
Olomouc, 2012
I declear that the present Thesis is written by myself under the supervision of
RNDr. Miroslav Soural, Ph. D., using sources which are listed in the literature.
In Olomouc Jasna Kadric

I sincerely thank the Palacký University and Head of the Department of
organic chemistry doc. RNDr. Jan Hlaváč, Ph.D. giving me the
opportunity for studying and for all the support provided during the study.
I refer special and greatest gratitude to my supervisor RNDr. Miroslav Soural,
Ph.D. for his patience, support and appreciating advices that followed me through the
studies and my staying in Czech Republic.
Thank to all workers, doctorates and colleagues at the department who
have accepted me and made that three-year absence from home easier, less painful
and help me to achieve my goal.
In particular I would like to thank to esteemed Professors who with the
understanding and patience enabled me to overcome the difficult study program and
passed on to me the knowledge which is priceless.
I want to dedicate this work to my family and partner without whose support and
assistance this work would not been possible.
Content
Glossary and Abbreviations ............................................................................................... 5
1. Introduction ..................................................................................................................... 7
2. Target of Thesis ............................................................................................................... 7
3. Theoretical part ............................................................................................................... 11
3.1. Solid Phase Synthesis – how it all began..................................................................... 11
3.2. Concept of Solid Phase Synthesis – basic terms......................................................... 13
3.2.1. Solid supports – types of Resins .................................................................................. 14
3.2.1.1. Polystyrene – Divynilbenzene Copolymers ............................................................. 15
3.2.1.2. Poly (ethylene glycol) – Polystyrene Graft Polymers .............................................. 16
3.2.1.3. Polyacrylamide based Resin ..................................................................................... 17
3.2.1.4. Other Polymer Supports ........................................................................................... 17
3.3. Linkers – types, properties and uses ........................................................................... 19
4. Solid Phase Synthesis and its application ...................................................................... 27
5. Biological part .................................................................................................................. 30
5.1. Some Routes to the 3-Hydroxyquinolin-4(1H)-ones...................................................... 30
5.2. Biological properties of 3-Hydroxyquinolin-4(1H)-ones ............................................... 33
6. Results and Discussion .................................................................................................... 38
6.1. SP Synthesis of Novel Derivatives of
3-Hydroxyquinolin-4(1H)-on-carboxamides .............................................................. 38
6.2. Optimalization of conditions regarding the SP Synthesis Chemistry and the
occurred problems during Synthesis .......................................................................... 45
a) Synthesis of the Diethanolamine Derivative ..................................................................... 45
b) Reduction of the Nitro group ............................................................................................ 47
c) Attempt for the Synthesis of the Benzimidazole Derivative ............................................. 49
6.3. Biological Properties of the synthesized Compounds ................................................ 50
7. Experimental part............................................................................................................ 53
7.1. Analytical data of Hydroxyquinolinones ........................................................................ 61
8. Conclusion ........................................................................................................................ 63
9. Literature ......................................................................................................................... 64
4
Glossary and Abbreviations
AcOH Acetic acid

Bn Benzyl
Cbz Carboxybenzyl protecting group
CAN Cerric Ammonium Nitrate
DBU 1,8-Diazabicyclo[5.4.0]undec-5-ene
DCM Dichloromethane
DIC Diisopropylcarbodiimide
DIEA N,N-Diisopropylethylamine
DMAP 4-Dimethylaminopyridine
DMSO Dimethyl sulfoxide
DMF Dimethylformamide
et al. and others
Et3N Triethylamine
Fmoc Fluorenylmethyloxycarbonyl
Fmoc Ala N-(9-Fluorenylmethoxycarbonyl)-L-alanine
Fmoc-OSu Fmoc N-hydroxysuccinimide ester
GI50 Concentrations required to inhibit growth by 50%
g gram
h Hour
HBr Hydrobromic acid
HF Hydrogen fluoride
HCl Hydrochloric acid
HPLC High Performance Liquid Chromatography
HOBt 1-Hydroxybenzotriazole
In vitro Latin: in glass or within glass
In situ Latin: in place
IC50 Value of the drug concentration which is lethal to 50% of the tumor
cells
K+ -OtBu Pottasium tert-butoxide
KOH Potassium hydroxide
KOTMS Potassium trimethylsilanolate
5
LC-MS Liquid chromatography coupled with Mass spectrometry
LDA Lithium diisopropylamide
LAH Lithium aluminium hydride
µM micromolar
MeOH Methanol
min. minutes
MS Mass spectrometry
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide
NMR Nuclear Magnetic Resonance spectroscopy
NaBH(OAc)3 Sodium triacetoxyborohydride
NaOH Sodium hydroxide
NaOEt Sodium ethoxide
NMP N-methylpyrrolidone
Nvoc Nitroveratryloxycarbonyl protecting group
o- ortho
p- para
PPA Polyphosphoric acid
PIP Piperidine
PS Polymer support
ppm parts per million
rt room temperature
Ref. Reference
THF Tetrahydrofuran
TFA Trifluoroacetic acid
TFMSA Trifluoromethane sulfonic acid
t-Boc Di-tert-Butyl dicarbonate protecting group
SPPS Solid Phase Peptide Synthesis
SAR Structure-activity relationship
SnCl2*2H2O Stannous Chloride dihydrate
6
1. Introduction
The target of the diploma work was the preparation of novel derivatives of 3-
hydroxyquinolin-4(1H)-on-carboxamides via solid-phase synthesis and evaulation of their
biological activity.
As already confirmed, through the exhaustively research of the Department of
organic chemistry, Faculty of Natural Science, Palacky University, derivatives of 3-
hydroxy-4(1H)-quinolinone-7-carboxamides (3-hydroxyquinolinones hereafter) showed a
significant in-vitro cytotoxicity. In connection with this we decided to focus on a
preparation of novel 3-hydroxyquinolinones derivatives which would be a result of
combination of previous reported structures and their cytotoxic screening. At the same
time we tried to draw attention on importance of structure-activity relationship (SAR)
studies and to get the best values, for biological activity, as possible.
To efficient preparation of series of novel derivatives we used solid-phase
chemistry to which we dedicate the most of our interest in this diploma work.
2. Target of Thesis
As already mentioned in the introduction, we tried to prepare set of novel
derivatives of 3-hydroxyqiunolinones with phenyl in position 2 substituted with nitro group
and various primary and secondary amines, and N-substituted carboxamide group in
position 7.
The selected amines were chosen on the basis of previous studies and published
work1, 2
and we can say that the obtained compounds were result as a combination of
following structures 1 and 2:
7
O O
R1 OH OH
HN Cl NO2
N N
H H
O R2
NH2 N
Cl
1 R3
2
O
R1 OH
HN NO2
N
H
O
R2
Target compounds
As our previous experience showed a different variations of structure can have

significantly different impact on the resulting biological activity of the corresponding
hydroxyquinolinones. From this reason we decided to combine in the target molecules R1-
R3 ligands from the most active compounds of the structure 1 and 2.
Overview of the used amines:
R1:
H H
H N CH 3 N CH 3
N CH 3
HN N
H
R2:
OH CH3
NH CH3 N N N
N N
H
H3C CH3
OH CH3
8
All of the target derivatives were prepared with use of solid-phase chemistry about
which we will write in detail in the theoretical part of this diploma work.
Efficient preparation, quick and easy isolation of intermediates and final products
are the most important features of the solid-phase chemistry and are worth mentioning
even now. The synthetic route leading to target derivatives was developed on a basis of the
previously reported method 3, (Scheme 1).
OMe
R1NH2
O 10 % AcOH/DMF, rt
PS NH = PS L NH
L O NaBH(OAc)3, rt PS R1
3O
COOH
COOMe
COOMe
TMSOK, THF, rt O
HO NH2
NH2 O
NH2 L N
O PS R1
L N
DIC, HOBT, DMF, DCM PS R1
O
Br NO2
TEA, DMF
Cl
NO2
NO2
Cl
R2 O
O
O
O
O O O
O NH2
NH2 amine, DMSO, rt
L N L N
PS PS R1
R1
1. TFA/DCM, rt
2. AcOH, reflux
O
OH
O NO2
N
H
NH
R1 R2
PS-polymer support L-linker
Scheme 1. Synthesis of 3- hydroxyquinolinones with SP synthesis
9
Our next goal was also attempt to prepare a bis-heterocyclic derivative, more
precisely, bis-heterocyclic derivative with benzimidazole scaffold (Scheme 2, structure 3).
As a natural occurring compound we can find a benzimidazole heterocyclic in vitamin B12
known as cobalamine, isolated from the fungus Streptomyces griseus. The importance of
benzimidazole is not only narrowed as a natural product compound, synthetic
benzimidazole derivatives are also used in medicine as drugs. One of the world’s best-
selling drug in the late 1990s4 with the benzimidazole nucleus, is Omeprazole, known as a
proton pump inhibitor.
The suggested synthetic strategy leading to derivatives 3 is based on the preparation
of appropriate o-phenylendiamine intermediates and their conversion to desired derivative
of benzimidazole (Scheme 2).
NO2 NO2
H
Cl N
O O R2
O O
O O O O
NH2 NH2
L N L N
PS R1 PS R1
NH2
H
N
O R2
O
OH O
R1
O O
HN N NH2
N L N
H R3
O PS R1
N
R2
Scheme 2
As a last goal of a diploma work was the isolation, if possible, of the synthesized
compounds and their evaluation of biological activity with use of cytotoxic screening on
the human cancer cell lines. More details about that and obtained results will be given in a
part dedicated to the biological activity of prepared derivatives.
10
3. Theoretical part
3.1. Solid Phase Synthesis - how it all began
Crucial year for the discovery of the synthesis on the solid supports was the year
1963, where the name of Nobel prize winner, Merrifield B. R. will be engrave for the
future generations and for all times. The beginnings of the novel type of synthesis were
modest and, if we can say so, reduced to the synthesis of peptides, in Merrifield’s case
synthesis of tetrapeptides5 (Scheme 3). The huge need to solve technical problems which
appears while dealing with peptides, as solubility or purification, resulted with innovative
method which promises simplicity, speed, effectiveness. To date, this visions and
characteristics did not change, only improved, and represent the major advances of solid
phase synthesis.
As far as the principle of the synthesis more or less it stayed the same. The main
changes are noticeable at the type of amine protecting groups, carboxyl group activating
agents, the variety of linkers6 and maybe some novel types of solid supports which we can
be used today in laboratories.
The Merrifield synthesis was conducted on the chloromethylpolymer
(copolystyrene-2% divinylbenzene) to which the first protected amino acid was attached
through benzyl ester linkage. The used protecting group, carbobenzoxy group was cleaved
with diluted 10% HBr and the ester bond with concentrated 30% HBr. The former
protecting group is known already from Max Bergmann, which first reported its use for
synthesis of oligopeptides at 19327. After the cleavage of the N-terminal protecting group,
the free amino acid was acylated with the next protected amino acid. The activation of the
carboxyl group proceeded with carbodiimide. The desired peptides were cleaved from the
polymer support either by saponification or with treatment with HBr. The former steps
represent one cycle through which the peptide bond was lengthened by one amino acid.
This cycle, containing bonding, deprotection and washing steps, represent the strategy of
solid phase synthesis.
11
Scheme 3. Basic approach to synthesis of peptides by Merrifeld5
From the famous 1963 till to date, solid phase synthesis has to justify her existence.
Various reviews and critics, dealing with innumerable reactions done on solid supports,
were directed to this new method of synthetic chemistry8,9,10,11. The first mentioned solid-
phase synthesis was developed for the synthesis of peptides5,12 and short after that the first
report an automated peptides synthesis was already lounched13. Through time and
tremendous effort it developed to synthesis which can be used for synthesis of
oligonucleotides14,15, oligosaccharides16,17,18 and various small organic molecules whose
3,19-26
examples can be found in literature . The main effort is still in optimization,
adjustment and implementation of traditional solution-phase chemistry into chemistry on
solid supports.
Obviously is clear that Merrifield, in that time by himself, had a vision and
anticipation that solid-phase chemistry could be a new leader in synthetic organic
chemistry. To date, in combination with combinatorial chemistry, we clearly can see that
he was right. Solid phase combinatorial chemistry became a standard for the most of
pharmaceuticals companies, as a new tool for discovering novel drugs and unavoidable
tool for basic research. These methods are often complemented and connected with each
other but we cannot consider them as a whole. Therefore we will through this diploma
work approach them individually.
12
3.2. Concept of Solid Phase Synthesis – basic terms
As mentioned earlier in the text, the concept of solid-phase (only “SP” hereafter)
synthesis did not change during the last years of development and research. The target
molecule as well as all intermediates stay connected on a polymer support, through linker,
during the whole synthesis. The beauty of SP lies in her simplicity. This unique method
does not require any experience in advance, which is for solution chemistry hardly to
claim. The typical routine steps are shaking a support containing solvents and reagents,
washing with suitable solvents, filtering. Cleavage proceeds in dependence of the used
linker and often yields product of high purity, which is usually used for analysis and
reaction monitoring. If necessary the isolated product can be used immediately or further
purified by various methods.
Few other aspects for solid-phase synthesis are important when one is planning
such synthesis:
1. The route to a library synthesis requires development of efficient synthesis that
means that reaction time, temperature, reagents and equipment should be determined in
advance
2. Reactions must be driven to completion, because during the synthesis it is not
possible to purify the intermediates
3. If the reaction is not completed or it does not continue smoothly it is possible to
increase yields by repeating the reaction, increasing temperature or to use reagents in
excess or higher concentrations.
4. Reaction monitoring is usually done by cleavage of the intermediate from the
polymer support by using of appropriate cleavage cocktails and analyzing compounds in
solution by HPLC, MS, NMR.
5. As for the equipment for SP synthesis it can be performed with standard
equipment for organic synthesis while some adjustment will be required. For typical SP
synthesis fritted syringes, shaker for mixing the reagents in the syringe (which can be
featured with heater), and domino blocks (www.torviq.com) designed for “washing” the
resin can be used. Shaker or tumbler can be used for temperatures up to 100°C6.
13
Literature27 present commercially available low budget SP synthesis equipment
under $30.000 while literature9 present commercially available equipment for automated
SP synthesis in the high price category between $125.000-300.000. Recent developments
ensured newest equipment for efficient preparation of small to medium size libraries, with
possibilities to use various reaction conditions, with no need for robotic or expensive
automation28.
3.2.1. Solid Supports – type of Resins

The crucial step of successful SP synthesis is choosing the appropriate solid support
and the linker. Various reactions on different solid supports have already been
reported3,8,9,29. As it is not possible to discuss all existing polymer supports we will focus
only on those which are mostly or commonly used in SP synthesis as also in the presented
diploma work.
Before we move on the specific types of resins here are some general requirements
which are common to all:
1. Resins should be mechanical stable during the synthesis – by all means to
prevent formation of clusters by breaking down of support into smaller particles
2. Resins should be chemical inert and stable under the conditions which are used
for the synthesis
3. Mostly they are compatible with wide range of polar and no polar solvents
4. Mechanical stirring should be avoid and replaced with alternatives
5. Resins should be chemically functionalized providing a suitable group to connect
the linker.
14
3.2.1.1. Polystyrene - Divynilbenzene Copolymers
Most commonly used polymer supports for SP are cross-linked polystyrenes. The
ones with 1-2% divinylbenzen are the most common (Scheme 4). As they represent so
called gelatinous solid supports they are not recommended to use for continuous-flow SP
peptide synthesis although some recent report claims the opposite30. These types of resins
are commercially available with broad choice of functional groups and linkers (Scheme 5).
First condition for successful synthesis relies on the properties and quality of chosen resin.
Literature compares performance of those, commonly used supports in SP synthesis,
through synthetic sequence developed for traceless SP synthesis of nitrogen-containing
heterocycles31. Attention was also given to the site of functionalization, where the amino
functional-group was analyzed by using optical analysis which provided a clear, concise
and accurate picture of the spatial distribution of those sites within the bead32.
Hydrophobic polystyrenes generally swell well in nonpolar solvents causing the
size of the beads to increase. Loading of the resin is expressed in mmol/g and is usually
about 1 mmol/g. The loading decreases with the increasing weight of compound attached
to the support33. A broad range of various reagents used in the synthesis is compatible with
polystyrene resin as for example strong bases (DBU, LDA), acids (HBr, TFA), mild
oxidizing agents, reducing agents (LAH, NaBH(OAc)3), Lewis acids and many others.
Scheme 4. Polystyrene cross-linked with divinylbenzene
Cl NH2 Br
Merrifield PS Aminomethylated Bromomethylated PS

resin PS resin resin
Scheme 5
15
3.2.1.2. Poly (ethylene glycol) - Polystyrene Graft Polymers
Poly (ethylene glycol)-polystyrene graft polymers (PEG-PS) are prepared by
grafting or linking suitable functional PEG chains to the polystyrene resin. While the cross-
linked PS resins do not show good swelling properties in polar protic solvents the PEG-PS
resins swell equally well in protic and aprotic solvents. The two representatives of these PS
are TentaGel (TG) and Argogel (AG). Some examples can be seen on scheme 6 and 7.
O O O
O
TentaGel N CHO TentaGel N N
H H
O
O
Scheme 6. TentaGel Resins
O O
PS Cl PS NH2
Scheme 7. ArgoGel Resins
Both types of polymers are the most favored and became the preferred resins when
it comes to solid phase chemistry due to the positions of functionalized groups compared to
the polystyrene matrix. While TG has it on the end of PEG chains with loading from 0.2-6
mmol/g, AG differs from it by branching of the PEG chain at the attachment point to the
PS core which doubles the loading level to 0.4 mmol/g34. In comparison to the cross-
linked PS the TG and AG polystyrene resins are more expensive and do not allow use of
aggressive reagents such as Lewis acids.
The PEGA35 resin, as hydrophilic resin, is a poly (ethylene glycol)

dimethylacrylamine copolymer which shows good swelling in water and allows inner
penetration of macromolecular reagents such as enzymes, also for preparation
combinatorial libraries8, but has a limited use due low mechanical stability and reactivity
of the amide bonds36, 37.
16
3.2.1.3. Polyacrylamide based Resin
These so called macroporous non-swelling resins contain a rigid structure which
does not allow any swelling of the matrix. Usually cross-linked polyacrylamide resins are
not suitable for use of continuous-flow solid phase peptide synthesis but generated on
Kieselguhr their rigid structure becomes more mechanically stable allowing the use for
continuous-flow solid phase synthesis38. Other resins from the same family are POLYHIPE
resin containing 10-50% cross-linked PS, and controlled pore glass (CPG) 39 resins made of
highly porous pure silica. Characteristics of those resins, beside their use for continuous-
flow synthesis, are that they are washed easily then classical PS resins and the transfer
from solution chemistry to solid phase chemistry is not difficult and do not require some
special conditions.
3.2.1.4. Other Polymer Supports

The next few supports are maybe not really interesting for routine solid-phase
organic synthesis but when talking about solid-phase peptide synthesis they are
unavoidable. Peptide synthesis on so called “pins” support provides over a quarter of
million peptides yearly and the method was commercialized as PepSets by Multipin
technology (Picture 1). Pin support is made by a radiation-grafted polyethylene support40.
These are hydrophilic, chemical reactive matrix providing amino groups for peptide
coupling.
Cellulose paper was developed for automated multiple peptide synthesis in a form
of cotton, paper and beaded support. Synthesis on paper proceeds by attaching to the
surface spots of amino acids used for peptide synthesis. Coupling is encouraged by typical
activating agents as carbodiimide and the protecting groups are removed with a standard
TFA/scavenger cocktail.
17
http://www.mimotopes.com/
Picture 1. Type of PepSets

http://www.mimotopes.com/
18
3.3. Linkers – types, properties and uses
We can consider linkers as “bridges” which connect the target component to the
polymer support. The connected compound stays anchored on the polymer, through the
linker, until it is cleaved under proper conditions. So, when speaking about linker it is not
possible to divide the process of linking and cleavage and it should be taken as an
inseparable feature when deciding which linker to choose for specific SP synthesis.
The SP synthesis can be proud to present a hundreds of linkers developed over the
years starting with Merrifield’s peptide synthesis till synthesis of small to medium size
organic libraries. It seems almost impossible to note all linkers but some general features
can be applied to all:
• Linker should be chemically inert to all conditions used during the
synthesis procedure
• The target compound must be cleaved form the support without
problem and the cleavage procedure must be as quantitative as
possible
Literature34 states two others features which are important when speaking about
linkers:
• The cleavage conditions should not degrade the library compounds
• Cleavage conditions should be user friendly and provide released
compounds ready for screening
As for the great numbers of linkers it is difficult to name all of them or at least not
to omit the most frequently used once. Different authors offer a variety of division for
linkers. For instance literature41 classified it as integral linkers – part of the solid support
forms a part or whole linker and nonintegral linkers – linker is attached to the resin
scaffold. Solution prepared linkers are defined as unloaded linkers. Literature34, 37 deals
with the division by summarizing the structures into the commercially and noncommercial
available and the most used in the SP synthesis of small organic compounds.
This diploma work will briefly describe the most used linkers in the SP synthesis in
general. Perhaps the division of such linkers on weak acid and strong acid cleavable
linkers is the only appropriate division, as those differences are just the most visible.
How the structure and substituted groups can have influence on the sensitivity of
the linker toward acidic cleavage is obvious from the following types.
19
A) Wang linker – belongs to weak - mild acid cleavable linker which means that
cleavage usually occurs with 50% TFA in DCM. Literature42 cites apparatus and method
for cleavage with gaseous reagents as HF or HCl. Usually carboxylic acids are anchored on
to the Wang linker using activating groups as DIC. Literature43 cites that the same
anchoring process can be achieved under Mitsunobu conditions. Wang linker is the
standard and common support for the synthesis of peptide acids using Fmoc protecting
strategy (Scheme 8).
O NHFmoc
O
OH H
PS N O
HO R L O
O
DIC R O
PS Fmoc protection
Wang resin linker = PS L OH
Deprotection piperidine
O
NH2
O
O R
Cleavage O
H2N
OH
TFA/DCM PS
R
Scheme 8. Wang resin linker, Fmoc protecting strategy and cleavage
As mentioned earlier in the text various substitutions can have different impact on
the properties of the linkers as it is visible on the SASRIN and HAL.
B) SASRIN – super sensitive acid linker is a modification of the previous Wang

linker where the sensitivity against acids increases due to the introduced o-methoxy group.
Carboxylic acids can be cleaved now by using 1-3% TFA/DCM. Sasrin resin was also used
for the synthesis of highly substituted pyridines44 which are representing one the most
important core for drugs and novel drug discovery (Scheme 9).
20
OMe
HO
= HO PS
O
SASRIN
PS
HO PS O O O O
O R1CHO
O Piperidine
Wang or H3C O PS
H3 C O PS PrOH-C6/H6
Sasrin DMAP, DCM
R1
DMF R2
H 2N
O R1 O R3 O
1. CAN
Me2NCOMe O R1 O
R2 OH
2. TFA/DCM R2 O
R3 N CH3 PS
H
R3 N CH3
H
Scheme 9. Preparation of highly substituted pyridines44 with Sasrin or Wang linker
C) Additon of the second methoxy group increases the sensitivity in such matter
that 0.1% TFA/DCM, for 5 min at rt or 10% AcOH/DCM for cleavage is enough. The
HAL (Scheme 10) or the hyper sensitive acid-labile linker justifies its name.
OMe
HO O
MeO O OH
Scheme 10. HAL – hypersensitive acid unloaded linker
D) RINK linker – was first introduced 1938 demanding 10% AcOH/DCM or 0.2%
TFA/DCM as cleavage conditions41. Today various modification of the Rink linker exists
as the –NH2 version, the Fmoc amine version, the RAM – Rink amide linker which is used
for formation of amides or sulfonamides45. Various analogues derived from the Rink linker
can be found. RAM linker is obtained by coupling the unloaded Knorr linker on an
aminomethylated polystyrene resin41 and have been widely used for nonpeptide synthesis
21
under cleavage with TFA to release primary amides46. Formerly mentioned linkers can be
seen in Scheme 11.
OMe OH
OMe NH2
MeO O
MeO O
Rink linker PS
Rink amine PS
OMe NHFmoc
OMe NHFmoc
MeO O O
MeO O
OH
PS
Knorr
Rink Fmoc amine
OMe NHFmoc
O
MeO O
NH
RAM linker PS
Scheme 11
E) PAL – peptide amide linker (Scheme 12). As the name already says it is the most
widely used linker for the peptide amide synthesis, bearing two methoxy groups and Fmoc-
protected group instead of the hydroxymethyl.
OMe
FmocHN O
MeO O OH
Scheme 12. PAL – peptide amide-unloaded linker
F) BAL – backbone amide linker allows immobilization of amines by reductive

amination and synthesizing amides, in our case carboxamides, after acylation and cleavage
steps (Scheme 13.)
22
OMe
PS R1NH2
O 10 % AcOH/DMF, rt
= L O H
HN PS overnight L N
3O PS R1
O then NaBH(OAc)3,rt
BAL
O DIC,
HOBt,
OMe DMF,
HO DCM
NH2
O
O O
R1 R1
OMe OMe
TFA/DCM
HN L N
NH2 PS NH2
O O
Scheme 13
The BAL linker strategy was first directed to the synthesis of the C-terminally
modified peptides as well as cyclic peptides which are both naturally occurring and also
important synthetic targets, among others e.g. cyclosporine A, rapamycin (widely
successful immunosuppressant agents) and valinomycine (antibiotic). Jensen et al. reported
novel BAL approach for the synthesis of C-terminal modified and cyclic peptides as well47.
Synthesis of the original p-BAL was reported by Jensen et al. and relied on the synthesis
develop by Albericio and Barany48,49 (Scheme 14). Boas et al. also reported efficient and
easy preparation of the o-BAL50.
O H
MeO OMe
+ Br OC2H5
O
OH
K+ -OtBu DMF
O H
O H
NaOH MeO OMe
MeO OMe
O OH
O OC2H5
O
PAL
O
Scheme 14. Preparation of PALdehayde 49
23
First use of BAL for the synthesis of small organic heterocyclic molecules was
51,52
reported by Boojamra et al. by preparing library of diverse 1,4-Benzodiazepine-2,5-
diones. To date myriad reactions especially of small organic molecules justifies the uses of
BAL strategy. Commonly used acids, for the cleavage cocktails and releasing the final
products from the linker, are TFA, HF and TFMSA. Cleavage is likely to proceed by
protonation of the amide carbonyl oxygen releasing imidate, which tautomerizes to the
desired amide48 (Scheme 15).
R' OH
+
R' O
R' OH +
N
N R
R N
H+ R MeO OMe
MeO OMe
MeO OMe
O
O PS
PS O
PS
R' OH
N +
R
MeO OMe
+
R' O O
PS
HN R = H (PAL)
R - CHR'COR'' (BAL)
Scheme 15. Proposed mechanism for the cleavage step48
Previously mentioned linkers are the common used ones and are commercially
available. Due to the broadness of the literature and existing linkers Scheme 16 and 17
shows others type of linkers belonging to the group weak -mild acid cleavable linkers
which were not mentioned previously.
OR O X PS
O
PS O O
THP - tetrahydropyran linker R1 R2
for alcohol immobilization
Ketal linkers - for carbonyl group immobilization
X = O, S
Scheme 16
24
O
S
CH3
FmocHN
O
NHFmoc
O
NH
O
O O OH
S
H 3C O PS
XAL
SCAL - for SPPS
CHO
Cl PS
O
N
N
H
Indole Linker X
PS
Various Trityl linkers
X= Me, OMe, Cl
Scheme 17
The above listed linkers require weak to mild acids in contrast to the following
types of linkers that require some stronger acids:
A) Merrifield resin – benzyl type linkage used for the synthesis of tetrapeptides5
requires harsh conditions as 10% HBr for the deprotection and alkaline conditions for the
release of the peptide. Novel Merrifield synthesis of the bradykinin53 used improved
conditions for both deprotection and cleavage steps. In that synthesis previous Cbz group
was replaced for the t-Boc group which could be deprotected by the use of 1M HCl.
Chloromethylated PS was not nitrated or brominated anymore due to the change of the
protecting group. Nitration and brominating was introduced to reduce loss of the peptide
upon cleavage with the HBr acid and in addition it requires the alkaline conditions for
cleavage. Due to the new used protecting group cleavage could proceed further with
HBr/TFA and deprotection, as mentioned earlier in the text, with 1N HCl/AcOH while the
loss of the peptide was completely avoided.
Benzyl type linkers require mostly harsh cleavage conditions, usually liquid HF, but
various substitutions can have significant effect on the acid lability of the linkers. Some
represents are shown in Scheme 18.
25
O O O
O O N O Cl
PS O PS
Disuccinimidyl carbonate linker Chloroformate linker
O NO2
O
O NRR1 O O
PS PS
Carbamate linker
p-Nitrophenylcarbamate derivative
Scheme 18. Benzyl type linkers
B) PAM linker (Scheme 19) is usually used in SPPS to increase the stability of the
growing peptide chain and less than 0.2%54 cleavage of the peptide occurs during the
procedure. Esters of the PAM linker are obviously more stable toward acids.
R O O
N
H
PS
PAM
Scheme 19
Several novel types of linkers were developed for a specific type of synthesis of
small organic compounds and they worth mentioning. Such linkers are the photocleavable
linkers which should release a target compound only by absorbing the used light for
cleavage without to affect other groups. These types of linkers are attractive for the
combinatorial synthesis due to the mild cleavage conditions - products can be released
directly into neutral aqueous solution suitable for direct screening27. Safety catch linkers
based on the functional group that is uncreative during synthesis but is activated by
chemical transformations prior to cleavage. “Traceless” linkers forming C-H bond upon
cleavage are mostly silicon based linkers and are based on a acidic removal of a silicon
group and cleavage of C-Si bond. Cyclative cleavage has been used for preparing small
heterocyclic rings by cyclization and cleavage from the resin at the same time. Literature24
precisely states how the method can be used for preparing of cyclic olefins upon
cyclization/cleavage approach.
26
4. Solid Phase Synthesis and its application
Traditional solution chemistry is sure a chemistry of a “clean” high yielding single
compound but often accompanied with complicating and time consuming purification
process. As the saying goes “time is money” traditional chemistry can be replaced with a
novel method not with only purpose to be fast and simple but also to be efficient and
library trained. We can agree that research and discovery of biological active compounds is
a “hit” and foundation for research and development of novel drugs. That and many other
already mentioned reasons in previous chapters, led to a overcoming of solution chemistry
and a use of solid phase combinatorial chemistry which has mainly one goal, synthesizing
hundreds or even thousands biological active compounds in a short period.
The overview of important methods used in SP synthesis we can start with
Geysen55 who first developed peptide parallel synthesis for precisely peptide epitope
mapping. Method was applicable in that matter that he managed to synthesize 208
hexapeptides with sufficient purity in order to help and improve understanding of
antibody-antigen interactions, since the epitope is an antigen which triggers the production
of antibody of the immune system. The importance of that discovery lies in immunogenic
epitope of the important protein of foot and mouth disease virus. Bunin56,57 et al. prepared a
chemical library of 192 diverse 1,4 - benzodiazepines using Geysen55 pin method.
The application did not stop there, it goes further even to analyzing and comparing
of nucleic acids sequences with a macroscopic DNA arrays while preparing an array
displaying 256 octapurines using mask plates40.
By reading and evaluating accomplished results we can claim that the nineties were
years of discovery of novel methods in SP combinatorial chemistry. Fodor58 et al. draw
remarkable attention with developing light-directed spatially addressable parallel chemical
synthesis in combination with use of photolabile protecting groups and photolithography.
An array of 1024 peptides was prepared only in several steps. Method today known
VLSIPS – very large-scale immobilized polymer synthesis based on the peptides-on-chip
concept first described by Pirrung and Leighton40.
Method of light – directed synthesis is based on the surface which is glass
microscope slide derivatized with silane linker with an amino protected acid. As protecting
groups was used photolabile group Nvoc which can be removed with use of light source
and only at the exposed areas.
27
The method is based on repeating several steps: exposure of light on the pattern of
the mask and deprotection at the selected area. Coupling process occurs only at the
deprotected amino acid. Former named steps of masking, light exposure and coupling are
repeated until desired number of peptides is formed (Picture 2).
Picture 2
http://biocourse.org/images/e/e0/Photolithography.gif
Novel approach to SP combinatorial chemistry led to developing of a method

known as SPOT. It is based on use of cellulose paper sheets as membranes. Peptides are
building upon a spotting of solutions containing the activated amino acid derivative on to
the sheets (Picture 3.)
28
Picture 3
http://bioinformatics.charite.de/superficial/peptide_lib.htm
Method allows relatively facile and rapid preparation of hundreds of peptides.
SP combinatorial chemistry is inevitable when it comes to the desire for

effectiveness, simplicity and speed. Currently inevitable also in the synthesis of novel
derivatives of diverse small organic molecules and even of complex targets. It does not
require prior knowledge, it is applicable in every organic laboratory with or without
expensive equipment. The possibility of automatisation has an enormous impact on
accelerating the research process. All characteristics justified its purpose and existence, and
make her biggest concurrent to traditional chemistry which until recently held a
"monopole". Explosion of interest, even by those who looked at it sceptically and avoided
it, promises many more variations of existing methods for synthesis of small to large
organic molecule libraries, surely synthesis of novel drug-like molecules and overall
efficacy in the research and development.
29
5. Biological part
Target molecules 3-hydroxyquinolinones are known to express various biological
properties as antiprotozoal, antiviral, immunosuppressive and anticancer. This is not
surprising due to their relation to the nature occurring biological active flavones 4 known
as class of flavonoids. That is why the 3-hydroxy-2-phenyl-4-quinolinones are also known
as aza-flavones or aza-analogues 5 regarding to the similarity of the structures (Scheme
20.)
O O
OH OH
R1 R1
O R2 N R2
R3
4 5
Scheme 20. Structure of flavones 4 and aza-flavones 5
Flavones have been studied frequently for a long period while 3-hydroxy-2-phenyl-
4-quinolinones became a target of interest recently, when the simple synthetic route for
their preparation was developed. Anticancer activity of quinolinones 5 is recently
extensively studied combining the use of SP combinatorial chemistry and correlation to the
previous results of SAR studies to obtain novel derivatives with better results from
screening tests. Before we get to the biological properties it is surely interesting to get
insight into the synthetic preparation of hydroxyquniolinones.
5.1. Some routes to the 3-Hydroxyquinolin-4(1H)-ones

The previously mentioned largely applicable synthetic method for the preparation
of 3-hydroxy-2-phenyl-4(1H)-quinolinones 9 was developed in the year 1995, by
cyclization of phenacyl esters 8 in polyphosphoric acid59. Corresponding phenacyl esters
were obtained after alkylation of anthranilic acid 6 with bromoacetophenones 7 (Scheme
21).
30
Br
R R
O
COOH O 7
O
O
NH2 NH2
8
6
PPA
O
OH
N R
H
9
R=H, Cl, Br, NO2 , NH2, NHiPr
Scheme 21
This method affords 3-hydroxy-2-phenyl-4(1H)-quinolinones 9 with high yields,

using available starting materials and simple procedure. The method was further modified
to prepare variously substituted derivatives 60,61,62.
3-hydroxy-2-phenyl-4-quinolinones are also available through the method
described in literature63 by cyclization of suitable ketones 10 with sodium ethoxide to
methoxy derivatives 11 which by demethylation with HBr provide hydroxyderivatives 12
(Scheme 22).
31
R1 O R1
OMe O
NaOEt OMe
R2 NH
R2 N
H
O 11
R3
R3
HBr
10
R1
O
OH
R2 N
H
R3
12
Scheme 22. Preparation method of 3-hydroxy-2-phenyl-4-quinolinones63
Slight modification of the synthetic route led even to the novel amino derivatives 13
described in reference64 and carboxylic acid derivatives 14 described in literature65
(Scheme 23.)
O O
NH2 OH
HO
N N
H R H R
O
13 14
Scheme 23. Prepared derivatives referring to literature64,65
Solid-phase synthesis of 3-hydroxyquinolinone-carboxamides present a new area of

exploring as in chemical field referring to the synthesis and as well in the biological field
regarding their biological properties. We already briefly described the route of SP synthesis
at the beginning of the present work in the Scheme 1 and that is why the detailed
description of the chemistry will be omitted and discussed in the experimental section of
the work. In the following chapter we will briefly describe how the biological properties of
the 3-hydroxyquinolinones are influenced by the various substitution of the scaffold.
32
5.2. Biological properties of 3-Hydroxyquinolin-4(1H)-ones
We can say that everything started in year 1986 with the first used term of
biological activity with the isolated 3-hydroxygraveoline 15 from the arial parts of Ruta
66
chalepensis (Scheme 24), plant already known and used in traditional medicinal for
treatment of many disorders67.
O
O
N
CH3
O
15 OH
Scheme 24. Isolated 3-hydroxygraveoline66
The first research has started a new era in the detection of important biological
properties of 3-hydroxyquinolinones as immunosuppressive properties68, the inhibition of
topoisomerase63 and inosine monophosphate dehydrogenase69. Among the mentioned
biological properties the anticancer activity is the most frequently studied and especially in
our interest.
The cytotoxic activity of hydroxyquinolinones was first described for the dichloro
and monochloroderivatives61.
Compounds were screened against 3-cancer cell lines: breast cancer MCF7, non –
small cell lung cancer NCI-H460 and brain cancer SF-268. The most in-vitro active
compound 5,8-dichloro-2-phenyl-3-hydroxy-4(1H)-quinolinone 16 was further tested against 60
cancer lines exhibiting log GI50 between –5.48 to –5.93 for human acute lymphocytic leukemia
(ALL) cell line- CCRF-CEM and myeloid leukemia cell line K562.
Cl O
OH
N
H
Cl
16
Introduction of the carboxylic group in the position 7 should help by increasing the
possibilities of the modification of the quinolinone scaffold and a possible insertion of
various substituents. As far the results there was no significant cytotoxic activity against
33
cancer cell lines although one derivative with the dichloroaminophenyl moiety 17
exhibited IC50 between 9.83-11.98 µM for T-lymphoblastic cell line- CEM and K562.
Prepared compounds were also investigated on their solubility. Results showed better
solubility in common organic solvents than in those derivatives without the carboxylic
group65.
O
OH
HO Cl
N
H
O
NH2
17 Cl
Surprisingly the cytotoxic activity of the compounds increased rapidly by

introducing the phenacyl ester group instead of the unsubstituted carboxylic group65.
Among others the most active derivative 18 exhibited IC50 = 0.76-3.6 µM against CEM and
K562 cell lines.
NH2
Cl Cl
O
OH
O
O Cl
N
H
O
NH2
18 Cl
As is it obvious from the gained results the substitution at the position 3 and 4 in the
2-phenyl moiety has a large effect on the activity on the cell except of those nitro
derivatives.
Somewhat very different and interesting occurred when the hydroxyquinolinones
bearing nitro group at the 2-phenyl moiety showed significant cytotoxic activity68 (Scheme
25.)
34
O
O
OH
OH
NO2
N NO2
H N
OH H
N
N
CEM : IC50 = 0.25
CEM : IC50 = 0.72
K562 = Inactive OH
K562 : IC50 = 0.57
O
OH
NO2
N
H
N
H
CEM : IC50 = 0.77
K562 : IC50 = 0.66
Scheme 25. Examples of derivatives with high cytotoxic activity68
In combination with previous biological results and respecting the developed

synthesis route, efficient SP synthesis of 3-hydroxyquinolinone derivatives3, with
carboxamide at position 7 ensured new approach for discovery of target compounds with
potential high biological activity especially the cytotoxic one.
The prepared novel carboxamide derivatives were analyzed in detail with the SAR
approach. Results of such analysis are the synthesis of three generation libraries1 under the
systematical studying of compounds, evaluation of their biological properties and
thoroughly optimization of the structure toward the activity and vice versa. The cytotoxic
activity was analyzed against three cell lines: CEM, K562 and colorectal carcinoma
HCT116. The most active derivatives were also tested on the normal human cells – BJ
(normal cycling fibroblasts) and PBMC (human peripheral blood mononuclear).
Not to go into details or too comprehensively we will only briefly discuss the main
outcome of the previous mentioned SAR studies of the 3-hydroxyquinolinones.
35
The first generation library was based on the structure 19.
O
R1 OH
HN
N R2
H
O
19
Two approaches were used in the preparation of the library:

A) approach combines variously substituted phenyls (R2) with invariable R1, which
was the propyl ligand in this case,
B) approach combines various substituents such as H, propyl, hydroxypropyl,
hydroxyethyl, imidazol-N-propyl, piperidine-N-ethyl and 4-methylbenzyl (represented by
the position R1), with invariable R2 (the 4-methylphenyl moiety).
The main outcome from the first generation was that the results of the cytotoxic
screening test did not really depend on the used substitutions at the 2-phenylmoiety. Best
results were found for the structure 20.
CH3 O
OH
CEM: IC50 = 0.98
HN Cl
N K562: IC50 = 9.6
H
O HCT116: IC50 = 7.9
NH2
20 Cl
In the following procedure (the second generation library) the most active 2-
phenylmoiety was fixed as R2 ligand. For the carboxamide substitution (R1) variously
ligands were used such as: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
methyl, isobutyl, pentyl, Bn, 2-methyl-Bn, 3-methyl-Bn and 4-methyl-Bn 21. Results of
few derivatives from the biological test are shown in Table 1. Data are expressed as IC50
(µmol/l).
36
O
R1 OH
HN Cl
N
H
O
NH2
21 Cl
Table 1. Results of few derivatives from the biological test1
21 R1 CEM K562 HCT116

A Cyclopentyl 1.1 15.3 12.9
B Cycloheptyl 1.0 9.5 10.9
C Methyl 3.9 25.8 32.1
C Pentyl 1.0 7.5 5.6
D Benzyl 0.94 6.5 6.6
E 3-methyl-Bn 0.75 3.1 4.1
Result of the second generation library led to the discovery that the carbocyle
slightly increases the activity toward CEM lines and the elongation of the aliphatic chain
from methyl to pentyl increases the activity toward all lines. From that point of view the
ligands for the third generation were practically elected. Longer aliphatic chains and larger
cyclic hydrocarbons were chosen to form the R1 position. The outcome of the third library
indicated that the longer aliphatic chains as well as the larger sizes of the carbocyle did not
significantly increase the cytotoxicity of the prepared derivatives.
In our present work we tried to synthesize novel derivatives of 3-
hydroxyquinolinones using ligands which provided best results in the above mentioned
studies (Scheme 25 and Structure 21).
37
6. Results and Discussion
The aim of the present work was preparation of novel derivatives.
Regarding the applied SP synthesis, it is already well known since 2007 when it
was first introduced for the synthesis of 3-hydroxyquinolinones3. To provide an easier
overview this work could be divided into several stages or parts:
- SP synthesis of novel derivatives of 3-hydroxyquinolin-4(1H)-on-

carboxamides
- Optimization of conditions regarding the SP synthesis chemistry
and the occurred problems during synthesis
a) Attempt for synthesis of the diethanolamine derivative
b) Attempt of reduction of the nitrogroup
c) Attempt for synthesis of the benzimidazole derivative
- Evaluation of biological properties of the synthesized compounds
6.1. SP Synthesis of Novel Derivatives of 3-Hydroxyquinolin-4(1H)-on-

carboxamides
Applied SP synthesis is based on several steps already well known to us3 and will
be described briefly in the following text. The individual results as yield or purity and
specific details will be listed later in the experimental part of the work.
First step constitutes from forming a connection of a PS, in our case
aminomethylated polystyrene resin, to a linker. For our synthesis we used BAL – acid
cleavable linker which features are already described in the chapter 3.3.
Starting aminomethylated resin 25 is long-term stored and stabilized as
hydrochloride salt. To release an amine linkage it was first shaken with a 10% solution
of Et3N in DMF. Acylation of the resin linker (4 - (4-formyl-3-methoxy-
phenoxy) butanoic acid 26 was performed using in situ prepared HOBt active ester
(Scheme 26). The outcome of the reaction was evaluated by mixing a small sample of the
resulting resin with a solution of bromophenol blue in a mixture of NMP and DCM. The
proof of absence of primary amino group was only slightly green colour of the resin.
38
H3C
O O
NH2 + O
PS HO =L
25 26 O
Et3N
DMF
DCM DIC
HOBt
rt, overnight
L O
PS
Scheme 26. Preparation of PS-BAL resin
Subsequent reductive amination, described in Scheme 27, provided immobilized

secondary amines 27.
R1 NH2
10 % AcOH in DMF L N
L O PS R1
PS rt, overnight
H
NaBH(OAc)3
propyl 5 % AcOH in DMF
pentyl 5 hours
decyl rt
R1 = dodecyl
cyclopentyl
benzyl
H +NH -
N 10 % PIP in DMF L 2CH3COO
L
PS R1 PS
R1
10min., rt H
27
Scheme 27. Reductive amination step
Reaction proceeded with all five amines without any problems and the course of
reaction was evaluated quantitatively by LC-MS using reaction with Fmoc-OSu with
defined amount of the resin and Fmoc-Ala as an external standard compound. The next
step involved an acylation of polymer bounded secondary amines 27 with 1-methyl-2-
39
aminoterephtalate 28, using DIC and HOBt as activation reagents for in situ formed HOBt
ester. The reaction afforded desired carboxamides 29 (Scheme 28).
O
O
CH3
O CH3
O
HO
NH2 O
H NH2
L N O 28
PS R1 L N
DIC, HOBt PS R1 29
27 in DMF, DCM
rt, overnight
propyl
pentyl
R1 = decyl
dodecyl
cyclopentyl
benzyl
Scheme 28. Acylation step
After the saponification of the methyl ester 29 with KOTMS, the obtained
carboxylic acids 30 were esterified with a bromoketone, in our case 4-chloro-3-nitro-
acetophenone 31, to form appropriate phenacyl esters 32 (Scheme 29).
40
O
O
CH3
O
OH
KOTMS in THF
O
NH2 O
NH2
L N rt, overnight
PS R1 L N
PS R1
30
29
O
propyl
pentyl Br NO2
decyl DIEA
R1 =
dodecyl DMF
cyclopentyl 31 Cl
benzyl
NO2
Cl
O
O O
NH2
L N
PS R1
32
Scheme 29. Saponification and esterification step
The described procedure did not exhibit any problems or side reactions. The
reaction conditions differed only by the reaction times ranging from 3h - 24h.
Nucleophilic substitution of the chlorine atom at the phenacyl moiety 32 afforded a
second diversity position for several amines 33. For sure we can say that at this point this
step was the only one which has cause us some serious problems mainly concerning the
conversion of the initial material to product and impact on the yield and purity of the
obtained precursors.
In those cases, in which it was necessary, we modified the procedure to gain a
better purity of the products. The main problem that appeared was sudden cleavage of the
ester bond and formation of the intermediate 33a, even up to 50 % (Scheme 30).
41
NO2
Cl
O
R1
O
H2N O
NH2
32
O
DMSO
R2NH2
NO2
R2 O
R1
O
R1 OH
+
O H2N
NH2
H2N O
NH2 O
O
33 33a
Scheme 30
Use of dry DMSO, repetition of reactions with prolonged reaction time, or use of
catalyst, have provided us enough results to be able to continue with synthesis.
Unfortunately, cyclopentylamine and isopropylamine gave very low percentages of the
corresponding product so the synthesis was not continued in these cases. Final optimized
conditions which were successful and led to the desired derivatives 33 can be seen in Table
2, 3 and 4. From the presented results we can claim that prolonged reaction time
(overnight) and the use of dry DMSO gave the desired derivatives in sufficient purity. In
one derivative the use of DIEA was required.
Table 2. Optimized conditions for derivatives 33
R1 R2 Dipropylamine Piperidine Pyrrolidine Diethanolamine Isopropylamine

DMSO, rt, DMSO, rt, DMSO, rt, Dry DMSO, rt,
Pentyl Unsuccessful
overnight 3hours 3hours overnight
R1 R2 Diethanolmine Pentylamine
Propyl Dry DMSO, rt, overnight Dry DMSO, rt, 2hours
42
R1 R2 Dipropylamine Piperidine Pyrrolidine

Dry DMSO, rt,
Benzyl Dry DMSO, rt, Dry DMSO, rt,
repeated,
overnight overnight
catalyst DIEA
Cyclopentyl Dry DMSO, rt, Dry DMSO, rt,
Unsuccessful
overnight overnight
Dry DMSO, rt,
Decyl Dry DMSO, rt, Dry DMSO, rt,
overnight,
overnight overnight
repeated
Dry DMSO, rt, Dry DMSO, rt, Dry DMSO, rt,
Dodecyl overnight, overnight, overnight,
repeated repeated repeated
Target compounds 34 were obtained after the cleavage of the ester precursors 33
and their cyclization in TFA or AcOH (Scheme 31).
NO2
NO2 R2
R2 O
O R1
O
R1
PS O HN O
NH2
L N O
NH2 TFA/DCM O
rt, 30 min.
O 33
AcOH, reflux
TFA, reflux
O
R1 OH
HN NO2
N
H
O
R2
34
Scheme 31
We chose mainly AcOH for the cyclization due the unsatisfying results when using
TFA. The cyclization step was performed under reflux in an appropriate acid. Although the
reaction took place in both acids, the purity of the final product after TFA cyclization was
significantly lower compared to the use of acetic acid. The latter method afforded products
in very good crude purity containing only carboxylic acid as a side product (a by-product
formed after the previous reaction step – substitution with amines). After the addition of
43
diethyl ether to the cleaved material, the final products precipitated (while the side products
33a were dissolved) and were isolated by filtration in sufficient purity over 90%.
Regarding the results themselves we can state that the cyclization step was mostly
successful except for derivatives 35 (Scheme 32) and 36 (Scheme 33) which did not
cyclize under various conditions or gave side products that we did not manage to separate.
NO2
NO2 R2
R2 O
O
O
PS O HN O
NH2
L N O
NH2 TFA/DCM O
rt, 30 min.
O
R2 = piperidine AcOH, reflux

pyrrolidine
O
OH
HN NO2
N
H
O
R2
35
Scheme 32
OH
NO2
N
O OH
R1
PS O
L N O
NH2
O
AcOH, reflux
R1 = pentyl TFA, reflux
propyl H2SO4
O
R1 OH
HN NO2
N
H
O OH
N
36
OH
Scheme 33
44
In the following chapter 6.1.1. we will give description of the occurred problems at
the cyclization step including attempt to synthesize benzimidazole derivative 37,
diethanolamine derivative 36 and also reduction of the nitro group to amine derivative 38.
O O
R1 R1 OH
OH
HN HN NH2
N N
N H
H R3 O
O
N R2
37 38
R2
6.2. Optimalization of conditions regarding the SP Synthesis Chemistry

and the occurred problems during Synthesis
a) Synthesis of the Diethanolamine Derivative 39

Problems, which occurred during the last step, cannot be really considered as the
cyclization problems. We have been successful in obtaining the target derivative but not in
a desired quality. Instead of one derivative we obtained a mixture of three compounds.
What really happened during the synthesis can be represented by the Scheme 34
45
OH
NO2
N
O OH
R1
O
L N O
PS NH2
O
R1 = pentyl R-COOH
propyl reflux
R = CH3, CF3
O
R1 OH
HN NO2
N
H
O OH
N
39
OH
O
+
R1 OH O
HN NO2 R1 OH
N
H HN NO2
O O R N
N H
O O R
40 O N
41
O O O
R OH
Scheme 34
The LC-MS analysis of our first attempt using cyclization in AcOH confirmed the
presence of assumed product and two more derivatives: monoacetyl derivative 41- and
diacetylderivative 40. We tried to remove the unwanted acetyl groups with saponification
using KOH in MeOH at very low concentration bearing in mind that the
hydroxyquinolinones are in general not very stable in strong bases. Mixture was stirred for
1h, at rt but unfortunately LC-MS analysis did not show any promising results. We
repeated the reaction for one more time but the result were the same, the mono – and
diacetylderivatives were not hydrolysed and according the LC-MS analysis the quinolinone
scaffold was partially decomposed after further exposure. Our second attemp was based on
the use of KOTMS in THF but the LC-MS analysis showed that the quinolinone scaffold
rather decomposed again and appearances of some undefined products was observed.
Prevoius mentioned attemps are shown at the Scheme 35.
46
O
1. 1 % KOH in MeOH R1 = pentyl
2. 1 % KOTMS in THF R1 OH propyl
40, 41
R = CH3 HN NO2
N
H
rt, 1hour O OH
42 N
OH
Scheme 35
Due to above described problems we changed the acid used for the cyclization. For
the second attempt we have chosen the TFA. LC-MS analysis also confirmed the presence
of three derivatives, similar as in the AcOH cyclization case. However, their content in a
mixture was reduced due to a partial decomposition of the product during TFA cyclization
and appearance of other unknown compounds was observed. Despite this fact we tested
possible hydrolysis of trifluoroacetyl esters. For this we used MeOH for 1h, overnight, and
overweekend (Scheme 36). Best results were obtain after overweekend exposure, when we
did not observe any mono- nor bis-trifluoracetylderivative, but the overall purity of the
final compound was less than 50% and we did not manage to purify the compound to a
sufficient quality for biological experiments. From the above described results it is obvious
that the reaction sequence is not suitable for the preparation of derivatives that contain
hydroxygroup in a side chain of the 2-phenyl moiety.
CH3 O
MeOH OH MeOH 40, 41
40, 41
R = CF3 R = CF3
HN NO2 overweekend
N
H
overnight O OH
43 N
OH
Scheme 36
b) Reduction of the Nitro group

We also tried to prepare several derivatives with the nitro group reduced to amino
group using SnCl2*2H2O. Specific type of problem occurred during the cleavage
47
procedure. With use of a standard cleavage cocktail, TFA / DCM (1:1), at rt for 30 min. we
observed the formation of amides 45 (Scheme 37).
NH2
R2
O
NO2
SnCl2*2H2O
R2 O
O DIEA, DMF
O
O
NH2
O rt, 2-24hours N 44
O R1
O
NH2 L F
L N PS
PS R1 O
F
F
NH
R2
O
TFA/DCM O
30min, rt O
44 O
NH2
L N
R1 = benzyl, decyl PS R1
45
R2 = piperidine
pyrrolidine
dipropylamine
Scheme 37
44
a) MeOH / HCl, 30min., rt

b) MeOH / HCl, 2h, 400 C
c) MeOH / HCl, overnight, rt
d) MeOH / HCl, 30min., 400 C
e) MeOH / HCl, 15min., 400 C
R1 = benzyl, decyl
R2 = piperidine
pyrrolidine
NH2 dipropylamine
R2
O
O
O
O
NH2
HN
R1
46
Scheme 38
We tried to cleave the target compounds with alternative procedures but none of
these was successful (Scheme 38) due to the cleavage of the ester bond which led to very
low yields of target compounds 46. In our last attempt we tried to prepare the
corresponding 3-hydroxyquinolinones directly by reflux of the resin in AcOH without
previous TFA cleavage but the LC-MS analysis was unfortunately unreadable not
confirming the presence of the target compound nor intermediate 44.
48
c) Attempt for the Synthesis of the Benzimidazole Derivative 48
Our last goal of the thesis was to test the possible preparation of the benzimidazole
derivative.
SnCl2*2H2O
NO2
H DIEA, DMF
N 2hours, rt
O CH3
O
O
O
NH2
L N DMF
PS NH2 rt, overweekend
CH3
N
CH3 O H
CHO
O
O
O
NH2
L N 47
PS
CH3
N
N
O
O
O
O
NH2
L N CH3
PS 47a
CH3
Scheme 39
As we already knew that the reduction of the nitro group was successful we
focused on the cyclization of the o-phenylenediamine derivative to the benzimidazole
scaffold. For this purpose, after the reduction step, we used benzaldehyde for the
condensation leading to derivative 47 (Scheme 39). The LC-MS analysis confirmed the
structure of the intermediate 47 a without presence of the starting material but the crude
purity of the compound was < 40 %.
Further step involved a cyclization with AcOH (Scheme 40). At that point we
experienced a problem with the LC-MS analysis. It was highly probable that the
cyclization afforded the corresponding derivative but it was not possible to determine its
quality with the LC-MS analysis because there was no reasonable peak in UV spectrum.
However, in a mass spectrum we detected the corresponding ion spreaded over large area
(over several minutes). Such behaving has already been observed before for several
49
hydroxyquinolinones of various structure and refers to their different and difficult
chromatographic properties. To validate assumptions we decided to repeat the reaction
with higher quantity of the resin and subject the isolated material for the 1H NMR analysis.
Unfortunately we were not able to specify the outcome of the reaction due to the
unreadable NMR spectrum.
1. TFA/DCM
30min., rt CH3 O
2. AcOH, reflux
OH
47a
HN N
N
H
O
N
48
CH3
Scheme 40
6.3. Biological properties of the synthesized Compounds

Prepared derivatives were tested for their anticancer activity in vitro with use of
MTT screening test on the following cell lines: CEM, CEM-DNR-bulk: (CEM cells
daunorubicin resistant), K562, K562-Tax (K562 cells paclitaxel resistant), A549 (Human
lung adenocarcinoma epithelial cell line), HCT116p53 wt (colon cancer cell lines p53 wild
type) and HCT116p53 mut (colon cancer cell lines p53 mutated). Activity is expressed as
IC50 value in µM.
From the results of the MTT test quite surprising information were obtained.
Compound 49a do not show any activity, which means the combination of the
corresponding substituents R1 and R2 totally changes a biological feature of the molecule.
On the other hand, other screened compounds exhibited in specific cases promising activity
less than 10 µmol, however the best results (about 2 µmol) do not reach the IC50 obtained
for the model compounds (see Scheme 26 and Table 1 in the Theoretical part). Compounds
49b and 49c gave very similar results for leukemic cells and lung adenocarcinoma which
50
indicates that the difference in a size of an aliphatic cycle (piperidine vs. pyrrolidine) does
not have much influence on a cytotoxic properties of the molecule.
On the other hand, an exception can be observed for the colon cancer cell lines:
compound 49c exhibits a reasonable selectivity between p53 wild type and p53 mutated
subclon. The same selectivity can be observed for similar derivatives 50a, 50b, 51a which
indicates that this biological feature is probably not much influenced by the substitution of
the carboxamide group. Interestingly, when the aliphatic cycle is replaced by the aliphatic
chain (see 49a), the selectivity as well as the total activity is completely lost.
Unfortunately, the results obtained from the prepared set of derivatives cannot be
applied more generally due to a limited number of the studied compounds, however, some
conclusions can be done: (i) the combination of the most active ligands selected from the
model compounds does not significantly increase the activity of the molecules, (i)
activity/selectivity cannot be reasonably predicted as it is highly dependent on the
structural features of the specific parts of the molecule.
Results of the in-vitro cytotoxicity screening for derivatives of structures 49, 50 and
51, are summarized in the appropriate Tables 5, 6 and 7.
O
R1 OH
HN NO2
N
H
O
R2
49: R1= pentyl 50: R1= benzyl 51: R1= decylamine

R2 = dipropylamine (49a) R2 = pyrrolidine (50a) R2 = pyrrolidine (51a)
pyrrolidine (49b) piperidine (50b)
piperidine (49c)
51
Table 5. Results of the in-vitro cytotoxicity screening for derivatives of structures 49
No. 49 a 49 b 49 c
CEM 182,93 ± 8,45 3,02 ± 0,46 3,02 ± 0,2
CEM–DNR-bulk 150,25 ± 10,95 9,97 ± 0,87 9,28 ± 0,8
K 562 152,67 ± 7,98 4,14 ± 4,05 2,68 ± 0,93
K 562-Tax 150,69 ± 32,83 4,66 ± 0,97 11,1 ± 11,07
HCT 116p53 wt 177,07 ± 10,14 3,13 ± 0,18 30,04 ± 66,04
HCT 116p53 mut 179,8 ± 4,82 2,43 ± 1,19 2,42 ± 1,15
A 549 168,83 ± 21,78 2,36 ± 1,32 5,23 ± 4,22

No. 50 a 50 b
CEM 6,73 ± 5,35 9,05 ± 4,51
CEM–DNR-bulk 41, 71 ± 2,36 40,97 ± 4,53
K 562 11,39 ± 1,72 39,45 ± 5,86
K 562-Tax 67,62 ± 88,32 62,62 ± 67,83
HCT 116p53 wt 26,98 ± 12,75 40 ± 22,45
HCT 116p53 mut 3,3 ± 0,34 4,55 ± 2,49
A 549 8,73 ± 1,28 10,89 ± 1,04

No. 51a
CEM 2,97 ± 0,3
CEM–DNR-bulk 9,85 ± 1,96
K 562 7,24 ± 4,05
K 562-Tax 9,41 ± 11,86
HCT 116p53 wt 18,63 ± 21,1
HCT 116p53 mut 2,11 ± 0,94
A 549 2,95 ± 0,15
52
7. Experimental part
The SP synthesis was performed with the use of polypropylene fritted syringes as
reaction vessels. All reactions were carried out at room temperature if not stated otherwise.
After each step resins were washed three times (shaken for at least 1 min.) with the solvent
used for the reaction and subsequently with DCM. Solvents for the SP synthesis were used
in an equivalent of 1:10 which means that for 1g of resin we used 10 ml of solvent.
Washing proceeded on a specialized tumbler (Domino block synthesizer). Resin bound
derivatives were dried in a stream of nitrogen for quantitative analysis.
The LC-MS analysis required the treatment of the resin (approximately 5 – 10 mg)
with the cleavage cocktail (50 % TFA in DCM) and shaking for 30 min. at rt. Cleavage
cocktail was subsequently evaporated in a stream of nitrogen, 1 ml of MeOH was added to
a released intermediates and the solution sample was analyzed. Evaluation of the analysis
proceeded further with the help of the XcaliburIM software.
o Analysis were performed with HPLC (High Performance Liquid

Chromatogram) connected to a Triple Quadrupole Mass Spectrometer
Quantum Access (both Thermo Scientific)
o Detector TSQ Quantum Access (Thermo Scientific, USA, SN:
TQU01482)
• Mobile phases: acetonitrile (B) and 10 mM ammonium

acetate in water (A)
• Gradient: – 10 A : 90 B (0 min); 80 A : 20 B (2,5 min);
80 A : 20 B (4 min); 10 A : 90 B (4,5 min); 10 A : 90 B (5
min)
o NMR 1H/13C spectra were obtained with a Bruker AMX 300 Instrument
and in d6-DMSO as a solvent. Chemical shifts δ are reported in ppm and
coupling constants J in Hz. Spectra were evaluated with the help of
ACD LABS (NMR Assistant) software.
53
A) Preparation of PS-BAL resin (Scheme 26)
Aminomethylated resin 25 (loading 0.78 mmol/g) was mixed with the solution of
Et3N in DMF (10 %, 10 ml) and the mixture was shaken for 10 min. at rt. Subsequently the
resin was washed three times with DMF. Then we added an acylation mixture which was
prepared by dissolving 4(4-formyl-3-methoxy-phenoxy) butanoic acid 26 (1.56 mmol) and
HOBt (1.56 mmol) in DMF (5 ml) with the activating agent DIC (1.56 mmol) and DCM (5
ml). Resin was shaken for 24 hours, then washed three times with DMF, DCM and MeOH
and dried in stream of nitrogen. Acylation step was evaluated by using a sample of the
resulting resin with a solution of bromophenol blue in a mixture of NMP and DCM. The
proof of absence of primary amino group was only slightly green colour of the resin.
B) Reductive amination step 27

Resin prepared in a previous step was washed 2 times with dry DMF and shaken for
24 hours at rt with solution of amine (5 equivalents) in a mixture of 10 % AcOH in
anhydrous DMF. Then NaBH(OAc)3 (five equivalents) in a mixture of 5 % AcOH in
anhydrous DMF was added in three portions (1 hour intervals) . After the last addition the
resin was left on a tumbler at rt for three hours (total reduction time 5 hours). The resin
was washed with DMF and shaken in a mixture of 10 % piperidine in DMF for 10 minutes.
Finally the resin was washed three times in DMF, DCM and MeOH and dried in stream of
nitrogen.
C) Quantitative evaluation of reductive amination step

From each prepared resin a sample of 30 mg was taken and reacted with Fmoc-OSu
(0.26 mmol) and DIEA (0.26 mmol) in DCM (0.5 ml). After 30 minutes of shaking at rt the
resin was washed three times with DCM, three times with MeOH and dried in the stream
of nitrogen. Two samples (each 10 mg) were taken and cleaved in a cocktail of TFA in
DCM. To a cleaved intermediates 1 ml MeOH was added for the LC-MS analysis. External
standard was prepared by dissolving Fmoc-Ala (1 mg) in MeOH (1 ml). The loading of
each resin was calculated from UV spectra after integration of the corresponding peaks at
300 nm. The results are summarized in the Table 8.
54
Table 8. Results of loading for PS-BAL secondary amines 27
R1 Loading
Pentylamine 0.40 mmol/g
Decylamine 0.25 mmol/g
Dodecylamine 0.30 mmol/g
Benzylamine 0.30 mmol/g
Cyclopentylamine 0.30 mmol/g
Propylamine 0.41 mmol/g
D) Acylation step – resins 29

The above prepared resins 27 were reacted with the solution of two equvivalents of
1-methyl-2-aminotherphtalate 28, HOBt and DIC in DMF/DCM (Scheme 28). Resins were
kept on a tumbler overnight at rt and after it washed three times with DMF, DCM, MeOH
and dried. Purity of cleaved intermediates is summarized in Table 9.
Table 9. Purity of intermediates after the acylation step 29
R1 Purity % ms [ M+ H]+
Pentylamine 95 % 265
Decylamine 84 % 335
Dodecylamine 60 % 363
Benzylamine 95 % 285
Cyclopentylamine 90 % 263
Propylamine 93 % 237
E) Saponification of methylesters - resins 30
Resins 29 were reacted with solution of 0.2 M TMSOK in THF and kept on a
shaker overnight at rt (Scheme 29). After it resins were washed three times with DMF and
three times with DCM. Purity results can be seen in Table 10.
55
Table 10. Purity of compounds 30 after the saponification step
+
R1 Purity % ms [ M+ H]
Decylamine 90 % 321
F) Esterification step – preparation of phenacyl esters 32
Resins 30 were reacted with solution of three equivalents of 3-chloro-4-nitro-

phenacylbromide in DMF. Resins were shaken for 3h (in the case of resin R1 =
propylamine) or 24h, after it washed three times with DMF and three times with DCM.
LC-MS purities of intermediates are shown in Table 11.
Table 11. Purity of compounds 32 after the esterification step
R1 Purity % ms [ M+ H]+
Decylamine 98 % 518
G) Substitution of the chlorine atom at the phenacyl moiety - resins 33

Resins 32 were split to five parts (200 mg; Table 12), three parts (400 mg; Table
13) and two parts (250 mg; Table 14). Polypropylene fritted syringes were charged with
resins and appropriate solution of 0.2M of amines for the second diversity position (R2
substituents) in DMSO and kept on the shaker for 2h - overnight (Scheme 30). Conditions
for the reaction were already shown in Table 2, 3 and 4 (pages 42 and 43). For the resin 33
56
(R1/R2 = benzyl / dipropylamine) an equivalent of DIEA was added. After the reaction
resins were washed three times with DMF and three times with DCM. Purity of
intermediates is shown in Tables 12, 13 and 14.
Table 12. LC-MS purity
33 /R1 R2
Pentyl Dipropylamine Piperidine Pyrrolidine Diethanolamine Isopropylamine
Purity % 85 % 98 % 96 % 80 % 50 %
ms [ M+ H]+ 513 497 483 517 471
R2
33 / R1 Dipropylamine Piperidine Pyrrolidine
Purity %
Benzyl 67 % 90 % 85 %
ms [ M+ H]+ 533 517 503
Cyclopentyl < 10 % 68 % 60 %
ms [ M+ H]+ 511 495 477
Decyl 65 % 91 % 91 %
ms [ M+ H]+ 583 567 553
Dodecyl 70 % 77 % 78 %
ms [ M+ H]+ 611 595 585
R2
33 / R1
Propyl Pentylamine Diethanolamine
Purity % 70 % 79 %
ms [ M+ H]+ 471 489
57
H) Reduction of the nitro group – resins 46 and 47
Resins 33 were reacted with 1M solution of SnCl2*2H2O and equivalent of DIEA in

degassed DMF. Resins were kept on a tumbler at rt for 2 – 24 hours (24h for derivative 44
a; R1 = benzyl). After reaction the resins were washed three times with DMF and three
times with DCM. Purity of derivative 47 is lower due to the cleavage of the ester bond
during the cleavage procedure. Results are shown in Table 15, 16 and 17.
Table 15. Results for derivative 47
47 Purity % ms [ M+ H]+
Attempt I 37 % 441
II 31 % 441
Table 16. Results for derivatives 44
a)Dipropylamine Piperidine Pyrrolidine

44 / R1 R2
Purity %
Benzyl 50 % 21 % 20 %
ms [ M+ H]+ 503 487 473
Decyl 44 % 14 % 34 %
+
ms [ M+ H] 553 537 523
Table 17. Results for derivatives 45
Dipropylamine Piperidine Pyrrolidine

45 / R1 R2
Purity %
Benzyl 50 % 43 % 50 %
+
ms [ M+ H] 599 583 569
Decyl 45 % 85 % 40 %
ms [ M+ H]+ 649 634 619
I) Cyclization to hydroxyquinolinones
Cleavage cocktail (50 % TFA in DCM) was added to the resin and it was shaken at
rt for 30 min. Cleavage cocktail was collected and the resin washed two more times with
58
additional 50 % TFA in DCM. Washes were collected and evaporated in a stream of
nitrogen. To the resulting material AcOH or TFA was added and the solution was heated to
reflux. Successful reactions are shown as + sign.
i) Derivatives 49, 50 and 51 were cyclized by dissolving a residual material, after

the cleavage, in AcOH and refluxed for 1 – 2h. AcOH was evaporated under the stream of
nitrogen and to the residual materials diethyl ether was added and sonified for ~ 5 min.
Precipitated solids were collected by suction, washed with fresh diethyl ether and dried.
LC-MS purity of the derivatives is summarized in Tables 18, 19 and 20 (a, b and c).
Table 18. Cyclization results for the derivatives 34
34 / R1 R2
Pentyl Dipropylamine Piperidine Pyrrolidine Isopropylamine
In AcOH + + + -
Purity % 95 % 98 % 96 % 40 %
ms [ M+ H]+ 495 465 479 453
Table 19. Cyclization results for the derivatives 34
R2
34 / R1 Dipropylamine Piperidine Pyrrolidine
In AcOH
Benzyl + + +
ms [ M+ H]+ 515 499 485
Purity % 38 % 93 % 76 %
Cyclopentyl - + +
ms [ M+ H]+ 493 477 459
Purity % - 32 % 45 %
Decyl + + +
ms [ M+ H]+ 565 549 535
Purity 85 % 89 % 87 %
Dodecyl + + +
ms [ M+ H]+ 593 577 567
Purity % 64 % 60 % 70 %
59
Table 20 a, b and c. Cyclization results for the derivative 39
a
39 AcOH
Purity %
R1= Pentyl +
ms [ M+ H]+ / 39 499 10 %
ms [ M+ H]+ / 40 583 27 %
ms [ M+ H]+ / 41 541 34 %
b
39 AcOH
Purity %
R1= Propyl +
ms [ M+ H]+ / 39 471 4%
+
ms [ M+ H] / 40 555 35 %
ms [ M+ H]+ / 41 513 30 %
c
39 AcOH
Purity %
R1= Propyl +
ms [ M+ H]+ / 39 471 4%
ms [ M+ H]+ / 40 663 31 %
ms [ M+ H]+ / 41 567 28 %
J) Condensation and cyclization step to the Benzimidazole Derivative –resin

47a and 48
To the resin 47 solution of benzaldehyde (0.2 mmol) in DMF was added. Resin was
kept on a shaker at rt for 6h (for the first attempt) and overweekend for the last attempt.
Then the resin was washed three times with DMF and three times with DCM. LC-MS
purity of the compound 47a for the first attempt was 48 % and second 30 %. After the
cleavage the residual material was dissolved in AcOH and the reaction mixture was
refluxed for 1h. LC-MS analysis of the compound 48 was in both cases unreadable.
60
7.1. Analytical data of 3-Hydroxyquinolinones
2-[4-(dipropylamino)-3-nitrophenyl]-3-hydroxy-4-oxo-N-pentyl-1H-quinoline-
7-carboxamide 49 a
Yield of the product 20 %, purity of the product 95 %, MS= 495, [M + H]+. 1H
NMR (300 MHz, DMSO-d6) δ ppm 0.77 - 0.95 (m, 9 H) 1.31 (m., 4 H) 1.44 - 1.62 (m, 6
H) 3.12 - 3.41 (m, 6 H) 7.46 (d, J=8.96 Hz, 1 H) 7.65 (d, J=8.42 Hz, 1 H) 7.97 (d, J=8.23
Hz, 1 H) 8.11 - 8.26 (m, 3 H) 8.65 (br. s., 1 H) 11.68 (s, 1 H)
3-hydroxy-2-[3-nitro-4-(piperidin-1-yl)phenyl]-4-oxo-N-pentyl-1H-quinoline-7-
carboxamide 49 b
Yield of the product 39 %, purity of the product 98%, MS=465, [M + H]+. 1H NMR
(300 MHz, DMSO-d6) δ ppm 0.89 (m., 3 H) 1.31 (m., 3 H) 1.55 (m., 2 H) 1.88 - 2.03 (m, 3
H) 3.17 - 3.44 (m, 8 H) 7.22 (d, J=8.78 Hz, 1 H) 7.65 (d, J=8.60 Hz, 1 H) 7.99 (d, J=8.05
Hz, 1 H) 8.13 - 8.24 (m, 2 H) 8.29 (s, 1 H) 8.65 (br. s., 1 H) 11.64 (br. s., 1 H)
3-hydroxy-2-[3-nitro-4-(pyrrolidin-1-yl)phenyl]-4-oxo-N-pentyl-1H-quinoline-
7-carboxamide 49 c
Yield of the product 38 %, purity of the product 98%, MS=479, [M + H]+. 1H NMR
(300 MHz, DMSO-d6) δ ppm 0.89 (br. s., 3 H) 1.31 (m., 4 H) 1.47 - 1.72 (m, 7 H) 3.10
(m., 3 H) 3.20 - 3.42 (m, 4 H) 7.43 (d, J=8.60 Hz, 1 H) 7.66 (d, J=8.60 Hz, 1 H) 8.02 (d,
J=8.23 Hz, 1 H) 8.17 (m, 2 H) 8.29 (s, 1 H) 8.66 (br. s., 1 H) 11.70 (br. s., 1 H)
2-[4-(isoproylamino)-3-nitrophenyl]-3-hydroxy-4-oxo-N-pentyl-1H-quinoline-
7-carboxamide 34
LC-MS purity of the product 40 %, MS=453
2-{4-[bis(2-hydroxyethyl)amino]-3-nitrophenyl}-3-hydroxy-4-oxo-N-pentyl-
1H-quinoline-7-carboxamide 39
LC-MS purity of the product 10 %, MS=499
N-benzyl-2-[4-(dipropylamino)-3-nitrophenyl]-3-hydroxy-4-oxo-1H-quinoline-
7-carboxamide 34
Yield of the product 20 %, purity of the product 55 %, MS=515, [M + H]+.
N-benzyl-3-hydroxy-2-[3-nitro-4-(piperidin-1-yl)phenyl]-4-oxo-1H-quinoline-
7-carboxamide 50 b
N-benzyl-3-hydroxy-2-[3-nitro-4-(pyrrolidin-1-yl)phenyl]-4-oxo-1H-quinoline-
7-carboxamide 50 a
61
N-decyl-2-[4-(dipropylamino)-3-nitrophenyl]-3-hydroxy-4-oxo-1H-quinoline-7-
carboxamide 34
LC-MS purity of the product 85 %, MS=565, [M + H]+.
N-decyl-3-hydroxy-2-[3-nitro-4-(piperidin-1-yl)phenyl]-4-oxo-1H-quinoline-7-
carboxamide 51 b
N-decyl-3-hydroxy-2-[3-nitro-4-(pyrrolidin-1-yl)phenyl]-4-oxo-1H-quinoline-7-
carboxamide 51 a
N-cyclopentyl-3-hydroxy-2-[3-nitro-4-(piperidin-1-yl)phenyl]-4-oxo-1H-
quinoline-7-carboxamide 35
LC-MS purity 32 %, MS=495, [M + H]+.
N-cyclopentyl-3-hydroxy-2-[3-nitro-4-(pyrrolidin-1-yl)phenyl]-4-oxo-1H-
LC-MS purity 40 %, MS=481, [M + H]+.
2-{4-[bis(2-hydroxyethyl)amino]-3-nitrophenyl}-3-hydroxy-4-oxo-N-propyl-
1H-quinoline-7-carboxamide 39
LC-MS purity 4 %, MS=471, [M + H]+.
3-hydroxy-4-oxo-2-(1-pentyl-2-phenyl-1,3-benzimidazol-5-yl)-N-propyl-1H-
LC-MS analysis was unreadable, MS=509, [M + H]+.
62
8. Conclusion
Thesis in general is focused on the preparation of novel derivatives of 3-
hydroxyquinolinones. Target compounds 34 represent combination of derivatives 1 and 2
which were with use of biological screening identified as compounds with high anticancer
activity.
Present thesis consisted of several concrete targets:

a) Preparation of 3-hydroxyquinolinones with use of SP synthesis
b) Isolation and biological screening of the prepared derivatives and
c) Focus on optimalization of conditions regarding specific derivatives.
Another task was also preparation of benzimidazole derivative 48 with help of SP

synthesis, including the reduction of nitro group which led us to the o-phenylendiamine 47
intermediates which are ideal precursors for the synthesis of nitrogen containing
heterocyclic moiety.
In conclusion we have prepared small set of 3-hydroxyquinolin-4(1H)-one

carboxamides with use of efficient and simple SP synthesis procedure. Successfully
isolated compounds were sent for evaluation of their biological properties. Results of the
screening test can be seen in Tables 5, 6 and 7 and are commented in chapter 6.3.
The unsuccessful cyclization of several derivatives stays a challenging task for
further research as well as more detailed evaluation of the structure-reactivity relationship
that would require synthesis of larger set of diverse derivatives with use of developed
method.
63
9. Literature
1. Soural M., Hlaváč, J., Funk, P., Džubák, P., Hajdúch, M. ACS Comb. Sci. 2011, 13,
39-44
2. Krejčí P., Disertační práce, UP Olomouc, 2006
3. Soural, M., Krchňák, V. J. Combinatorial Chem. 2007, 9, 793-796.
4. Olbe L., Enar C., Lindberg P. Nature Reviews Drug Discovery, 2003, 2, 132-139
5. Merrifield R.B. Solid phase peptide synthesis. I. The synthesis of a Tetrapeptide.
J.Am.Chem.Soc., 1963, 85, 2149-2154
6. Hlaváč J., Soural M., Krchňák V., Practical Aspects of Combinatorial Solid-Phase
Synthesis, Department of Organic Chemistry, Palacky University, 2011
7. Bergmann M., Zervas L. Berichte der Deutschen Chemischen Gesellschaft, 1932, 65,
1192-1201
8. Hermkens, P.H.H., Ottenheijm, H.C.J., Rees.D. Tetrahedron Report 394, Tetrahedron,
1996, 52, 4527-4554
9. Hermkens P.H.H., Ottenheijm, H.C.J., Rees.D. Tetrahedron Report 418, Tetrahedron,
1997, 53, 5643-5678
10. Leznoff C.C. Acc. Chem. Res. 1978, 11, 327-333
11. Crowley J.I., Rapoport H. Acc. Chem. Res. 1976, 9, 135-144
12. Fridkin M., Patchornik A., Katachalski E. J. Am. Chem. Soc., 1966, 88, 3164-3165
13. Merrifield R. B., Stewart J. M., Jernberg N. Anal. Chem., 1966, 38, 1905-1914
14. Letsinger L. R., Mahadevan V. J. Am. Chem. Soc., 1965, 87, 3526-3527
15. Letsinger L. R., Mahadevan V. J. Am. Chem. Soc., 1966, 88, 5319-5324
16. Frechet M. J., Schmerch C. J. Am. Chem. Soc., 1971, 492-496
17. Zehavi U., Patchornik A. J.Am.Chem.Soc., 1973, 95, 5673-5677
18. Nicolaou K. C., Winsinger N., Pastor J., DeRoose F. J. Am. Chem. Soc., 1997, 119,
449-450
19. Soural M., Bouillon I., Krchňák V. J. Comb. Chem., 2008, 10, 923-933
20. Vaňková B., Hlaváč J., Soural M. J. Comb. Chem., 2010, 12, 890-894
21. Phillips G. B., Wei G. P. Tetrahedron Letters, 1996, 37, 4887-4890
22. Sim M. M., Cheng L., Ganesan A. Tetrahedron Letters, 1998, 39, 6399-6402
23. Arya P., Bonnaire S. C., Durieux P., Laforce D., Kumar R., Leek M. D. J. Comb.
Chem., 2004, 6, 735-745
64
24. Van Maarseveen J. H., den Hartog J. A. J., Engelen V., Finner E., Visser G., Kruse C.
G. Tetrahedron Letters, 1996, 37, 8249-8252
25. Buckman O. B., Mohan R. Tetrahedron Letters, 1996, 37, 4439-4442
26. Yun W., Mohan R. Tetrahedron Letters, 1996, 37, 7189-7192
27. Brown R. A., Hermkens H. H. P., Ottenheijm J. C. H., Rees C. D. Synlett, 1998, 8,
817-827
28. Ashis K. S., Liu L., Simoneaux L. R. J. Comb. Chem., 2001, 3, 181-188
29. Wang S. S. J. Am. Chem. Soc., 1973, 95, 1328-1333
30. Kates A. S., Cammish E. L., Albericio F. J. Peptide Res., 1999, 53, 682-683
31. Bouillon I., Soural M., Miller J. M., Krchňák V. J. Comb. Chem., 2009, 11, 213-215
32. McAlpine R. S., Schreiber L. S. Chem. Eng. J., 1999, 5, 3528-3532
33. Dorwald Z. F. “Organic Synthesis on Solid Phase, Supports, Linkers, Reactions”,
2ndEdition, Wiley-VCH Verlag Gmbh, Weinheim, 2002, p22.
34. Lam S. K., Lebl M., Krchňák V. Chem. Rev., 1997, 97, 411-448
35. Roice M., Johannsen I., Meldal M. QSAR&Comb.Science, 2004, 23, 662-673
37. Seneci P.”Solid Phase Synthesis and Combinatorial Technologies”, John Wiley&Sons
Inc., 2000, p4
39. Albericio F., Pons M., Pedroso E., Siralt E. J. Org. Chem., 1989, 54, 360-366
40. Pirrung C. M. Chem. Rev., 1997, 97, 473-488
41. Guilier F., Orain D., Bradley M. Chem. Rev., 2000, 100, 2091-2157
42. Kerschen A., Kanizsai A., Batros I., Krchnak V. J. Comb. Chem., 1999, 1, 480-484
43. Nouvet A., Lamaty F., Lazaro R. Tetrahedron Letters, 1998, 39, 3469-3472
44. Gordeev F. M., Dinesh V. P., Wu J., Gordon W. E. Tetrahedron Letters, 1996, 37,
4643-4646
45. Seneci P.”Solid Phase Synthesis and Combinatorial Technologies”, John Wiley&Sons
Inc., 2000, p11
46. Blackburn C. Biopolymers (Peptide Science), 1998, 47, 311-351
47. Jensen J. K., Alsina J., Songster F. M., Vagner J., Albericio F., Barany G. J. Am.
Chem. Soc., 1998, 120, 5441-5452
65
48. Boas U., Brask J., Jensen J. K. Chem. Rev., 2009, 109, 2092-2118
49. Albericio F., Cordonier-Kneib. N., Biancalana S., Gera L., Masada I. R., Hudson D.,
Barany G. J. Org. Chem., 1990, 55, 3730-3743
50. Boas U., Brask J., Christensen B. J., Jensen J. K. J. Comb. Chem., 2002, 4, 223-228
51. Boojamra G. C., Burow M. K., Thompson A. L., Ellman A. J. J. Org. Chem., 1995,
60, 5742-5743
52. Boojamra G. C., Burow M. K., Thompson A. L., Ellman A. J. J. Org. Chem., 1997,
62, 1240-1256
53. Merrifield B. R., Biochemistry, 1964, 3, 1385-1390
55. Geysen M. H., Meloen H. R., Barteling J. S. Proc. Natl. Acad. Sci., 1984, 81, 3998-
4002
56. Bunin A. B., Plunkett J. M., Ellman A. J. Proc. Natl. Acad. Sci., 1994, 91, 4708-4712
57. Stevens Y. S., Bunin A. B., Plunkett J. M. Swanson C. P., Ellman A. J., Glick D. G. J.
Am. Chem. Soc., 1996, 418, 10650-10651
58. Fodor A. P. S., Read L. J., Pirrung C. M., Stryer L., Lu T. A., Solas D., Science, 1991,
251, 767-773
59. Hradil J., Jirman J. Collect. Czech. Chem. Commun., 1995, 60, 1357-1366
60. Hradil P., Hlaváč J., Lemr K. J. Heterocyclic Chem., 1999, 36, 141-144
61. Hradil P., Krejčí P., Hlaváč J., Wiedermannová., Lyčka A., Bertolasi V. J. Heterocyclic
Chem., 2004, 41, 375-378
62. Spáčilová L., Hlaváč J., Hradil P., Fryšová I., Maloň M., Soural M., Krejčí P. J.
Heterocyclic Chem., 2006, 43, 1065-1070
63. Sui Z. H., Nguyen V. N., Altom J., Fernandez J., Hilliard J. J., Bernstein J. I., Barett J.
F., Ohemeng K. A. Eur. J. Med. Chem, 1999, 34, 381-387
64. Hradil P., Grepl M., Hlaváč J., Soural M., Maloň M., Bertolasi V. J. Org. Chem., 2006,
71, 819-822
65. Soural M., Hlaváč J., Hradil P., Fryšová I., Hajdúch M., Bertolasi V., Maloň M. Eur. J.
Med. Chem., 2006, 41, 467-474
66. Ulubelen A., Terem B., Tuzlaci E., Cheng K. F., Kong Y. C. Phytochemistry, 1986, 25,
2692-2693
66
67. lauk L., Flores M., Ragusa S., Rapisarda A., Greco A. M., Minardi R., Oliveri S.
Etudes chimiques et pharmacologique - Antimycotic activity of Ruta chalepensis L.,
From the sources of knowledge to the medicines of the future, 436-439
68. Krejčí P., Hradil P., Hlaváč J., Hajdúch M., Intl. Patent WO 2008028427, 2008
69. Iwanowicz E. J., Watterson S. H., Dhar T. G. M., Pitts W. J., Gu H. H., Intl. Patent WO
2001081340, 2001
67

Kadric-Master Thesis PDF

Hochgeladen von

Dokumentinformationen

Originaltitel

Copyright

Verfügbare Formate

Dieses Dokument teilen

Dokument teilen oder einbetten

Freigabeoptionen

Stufen Sie dieses Dokument als nützlich ein?

Sind diese Inhalte unangemessen?

Copyright:

Verfügbare Formate

Kadric-Master Thesis PDF

Hochgeladen von

Copyright:

Verfügbare Formate

PALACKÝ UNIVERSITY OLOMOUC

Synthesis of Novel Derivatives of 3-Hydroxyquinolin-4(1H)-on-

Student: Jasna Kadric

In Olomouc Jasna Kadric

AcOH Acetic acid

As our previous experience showed a different variations of structure can have

Overview of the used amines:

PS-polymer support L-linker

Scheme 1. Synthesis of 3- hydroxyquinolinones with SP synthesis

3.2.1. Solid Supports – type of Resins

Scheme 4. Polystyrene cross-linked with divinylbenzene

Merrifield PS Aminomethylated Bromomethylated PS

Scheme 6. TentaGel Resins

Scheme 7. ArgoGel Resins

The PEGA35 resin, as hydrophilic resin, is a poly (ethylene glycol)

3.2.1.4. Other Polymer Supports

Picture 1. Type of PepSets

Scheme 8. Wang resin linker, Fmoc protecting strategy and cleavage

B) SASRIN – super sensitive acid linker is a modification of the previous Wang

Scheme 9. Preparation of highly substituted pyridines44 with Sasrin or Wang linker

Scheme 10. HAL – hypersensitive acid unloaded linker

Scheme 12. PAL – peptide amide-unloaded linker

F) BAL – backbone amide linker allows immobilization of amines by reductive

Scheme 14. Preparation of PALdehayde 49

Scheme 15. Proposed mechanism for the cleavage step48

SCAL - for SPPS

Scheme 18. Benzyl type linkers

Novel approach to SP combinatorial chemistry led to developing of a method

Method allows relatively facile and rapid preparation of hundreds of peptides.

SP combinatorial chemistry is inevitable when it comes to the desire for

Scheme 20. Structure of flavones 4 and aza-flavones 5

5.1. Some routes to the 3-Hydroxyquinolin-4(1H)-ones

This method affords 3-hydroxy-2-phenyl-4(1H)-quinolinones 9 with high yields,

Scheme 22. Preparation method of 3-hydroxy-2-phenyl-4-quinolinones63

Scheme 23. Prepared derivatives referring to literature64,65

Solid-phase synthesis of 3-hydroxyquinolinone-carboxamides present a new area of

Scheme 24. Isolated 3-hydroxygraveoline66

Surprisingly the cytotoxic activity of the compounds increased rapidly by

K562 : IC50 = 0.66

Scheme 25. Examples of derivatives with high cytotoxic activity68

In combination with previous biological results and respecting the developed

Two approaches were used in the preparation of the library:

Table 1. Results of few derivatives from the biological test1

21 R1 CEM K562 HCT116

- SP synthesis of novel derivatives of 3-hydroxyquinolin-4(1H)-on-

6.1. SP Synthesis of Novel Derivatives of 3-Hydroxyquinolin-4(1H)-on-

Scheme 26. Preparation of PS-BAL resin

Subsequent reductive amination, described in Scheme 27, provided immobilized

Scheme 27. Reductive amination step

Scheme 28. Acylation step

Scheme 29. Saponification and esterification step

Table 2. Optimized conditions for derivatives 33

R1 R2 Dipropylamine Piperidine Pyrrolidine Diethanolamine Isopropylamine

Table 3. Optimized conditions for derivatives 33

R1 R2 Dipropylamine Piperidine Pyrrolidine

R2 = piperidine AcOH, reflux

6.2. Optimalization of conditions regarding the SP Synthesis Chemistry

a) Synthesis of the Diethanolamine Derivative 39

b) Reduction of the Nitro group

a) MeOH / HCl, 30min., rt

6.3. Biological properties of the synthesized Compounds

49: R1= pentyl 50: R1= benzyl 51: R1= decylamine

Table 6. Results of the in-vitro cytotoxicity screening for derivatives of structures 50