Docking

Design, Synthesis and Evaluation of Some Novel
Pyrazoloquinolines as Anticancer Agents
Thesis submitted to
J.S.S. UNIVERSITY, MYSORE

in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY.
by
Ms. Roshini Chandrasekhar, M.Sc.,
July, 2013
Under the Supervision of
Prof. M.J. Nanjan
J.S.S. College of Pharmacy

(Off Campus, JSS University, Mysore)
Udhagamandalam 643 001, The Nilgiris
Tamilnadu, India
ACKNOWLEDGEMENTS
I wish to express my deep sense of gratitude to Prof. M.J. Nanjan, Director, PG Studies
and Research/TIFAC CORE, J.S.S. College of Pharmacy. Ootacamund, under whose
active guidance, innovative ideas, constant inspiration, suggestions and encouragement
this research work was carried out.
I would like to thank Dr. B.Suresh, Vice Chancellor, JSS University, Mysore and Dr. K.
Elango, Principal, J.S.S. College of Pharmacy, Ootacamund, for providing all the
necessary facilities for the successful completion of my work.
I sincerely thank, Prof. S.P. Dhanabal, HOD, Dept. of Pharmacognosy and
Phytopharmacy for all his support. I am especially grateful to Dr. T.K. Praveen, for
helping me at every stage of my work with support and advice for the betterment of my
research.
I would like to thank Prof. S.Chandrasekaran, Dept. of Organic Chemistry, for giving me
an oppurtunity to carry out a part of my work at the Indian Institute of Sciences,
Bangalore and providing me with all the facilities for my research work. It is my pleasure
to thank Mr. Ganesh Venkataraman, Ms. Bandita Datta, Mr. Kishore Gadde, Ms. Divya
for their support and advice throughout my work in IISc., Bangalore.
My sincere thanks to Mrs. Mahalakshmi, Mr. Prashath Francis, Ms. Bincy, Mr. Ashish
Wadhwani, Mr. Viral Patel, Mr. Ankur Gupta, Ms. Bagyashree Kamble, Mr. Pankaj
Masih, Ms. Charitha, Ms. Santilna, my batch mates, Mr. Manikandan and Ms. Rubha,
my lab mates, for their encouragement.
I also would like to thank laboratory technicians and assistants, Mr. Gopal, Mr.
Subramanium and Mr. Madappa for their support and help given throughout my project.
I thank my dad, Mr. C.Chandraseharan, my mom, Mrs. Kumari Chandraseharan and my
sister, Reshna Chandrasekhar for being with me through thick and thin. Their
unconditional love and constant inspiration and blessing given throughout my work was,
no doubt, a great support to me.I am thankful to GOD ALMIGHTY with whose mercy I
have reached so far .
(Roshini Chandrasekhar)
LIST OF TABLES
Table 1: Typical properties of fragments, lead-like and drug-like molecules.
Table 2: Some of the patents related to pyrazoloquinolines.
Table 3: Data on 4,5-dihydro[1,2,3]oxadiaz-olo[3.4-a]quinolin-10-ium-3-olate

and its derivatives.
Table 4: Data on 4,5-dihydropyrazolo[1,5-a]quinolines.
Table 5: Docking parameters of selected ligands.
Table 6:Anticancer activity of 4,5-dihydro[1,2,3]oxadiazolo[3.4-a]quinolin-10-

ium-3-olate, its derivatives and 5FU against selected human cancer and
normal cell lines IC50 (µg/mL). (21-24)
Table 7:Anticancer activity of phenyl-4,5-dihydropyrazolo[1,5-a]quinoline-2-

carboxylate and its derivatives against selected human cancer cell lines
IC50 (µg/mL). (26-29)
Table 8:Anticancer activity of dimethyl 4,5-dihydropyrazolo[1,5-a]quinoline-2,3-

dicarboxylate and its derivatives against selected human cancer cell lines
IC50 (µg/mL). (31-34)
Table 9:Anticancer activity of (4,5-dihydropyrazolo[1,5-a]quinolin-3-yl)methyl

benzoate and its derivatives against selected human cancer cell lines
IC50 (µg/mL). (36-43)
LIST OF FIGURES
Figure 1: The progress of development of new molecular target therapy, gene to
drug.
Figure 2: Human topoisomerase IIα (1ZXM) from the Protetin Data Bank.
Figure 3: Mechanism of action of topoisomerase II.
Figure 4: The catalytic cycle of topoisomerase II. The individual steps of the
catalytic
cycle of topoisomerase II are shown. These are: (1) DNA binding; (2)
pre-strand passage cleavage/religation; (3) DNA strand passage; (4)
post-strand passage cleavage/religation; (5) ATP hydrolysis; and (6)
enzyme recycling.
Figure 5: Synthetic topoisomerase II inhibitors.
Figure 6: Some potent inhibitors of topoisomerase II.
Figure 7: Some biologically active sydnones.
Figure 8: Some important tetrahydroquinolines.
Figure 9: Some important pyrazoloquinolines.
Figure 10: Ramachandran Plot- 1ZXM.
Figure 11: Hydrogen bonding interactions of ANP, the co-crystallised ligand.
Figure 12: Hydrophobic interactions of salvicine-S in the binding site of topo IIα.
Figure 13: Hydrophobic interactions of the ligands in the binding site of topo IIα.
Figure 16: IR spectra of Compound 21.
Figure 17: MASS spectra of Compound 21.
Figure 18: 1H NMR spectra for Compound 21.
Figure 19: 13C NMR spectra for Compound 21.
Figure 20: IR spectra for Compound 22 .
Figure 21: MASS spectra for Compound 22.
Figure 28: IR spectra for Compound 24.
Figure 35: NOESY spectra for Compound 26.
Figure 39: 1H NMR for Compound 27.
Figure 40: 13C NMR for Compound 27.
Figure 53: IR for Compound 32.
Figure 54: MASS for Compound 32.
Figure 70: NOESY for Compound 36.
Figure 93: Graph representing the IC50 values of compounds 21-24.
Figure 96: Graph representing the IC50 values of compounds 36 to 43.
Figure 97 :(A) HeLa cells: Control cells and cells treated with 23 (B) MCF-7
cells: Control and cells treated with 27 (C) HeLa cells: Control cells
and cells treated with 28 (D) MCF-7 cells: Control and cells treated
with 41.
CONTENTS
Page no.
1. INTRODUCTION
1.1 Cancer and Cancer Therapy 1
1.2 Bioinformatics, Chemoinformatics and Drug Discovery 6
1.3 Some Novel Anticancer Targets 6
1.4 Topoisomerase Inhibitors 10
1.5 Type II Topoisomerase as a Drug Target 11
1.6 Sydnones and Sydnoquinolines 13
1.7 Quinolines and Tetrahydroquinolines 15
1.8 Cycloaddition Reactions 16
1.9 Pyrazoles and Pyrazoloquinolines 18
1.10 Review of Literature 21
2. SCOPE AND OBJECTIVES 63
3. EXPERIMENTAL
3.1 Chemicals and Reagents 69
3.2 Docking and Molecular Modeling Studies 70
3.3 Synthesis and Purification 73
3.4 In vitro Cell Proliferation MTT Assay 81
4. RESULTS AND DISCUSSIONS

4.1 Docking Studies 85
4.2 ADME Studies 91
4.2 Synthesis and Characterization 92
4.3 In vitro Anticancer Studies 148
5. SUMMARY AND CONCLUSIONS 156
6. BIBLIOGRAPHY 160
7. ANNEXURES 170
Introduction
1. INTRODUCTION
1.1 Cancer and Cancer Therapy
The first description of cancer dates back to 1600 BC and was found in an Egyptian
papyrus. It was regarded as an incurable disease until the 19th Century when surgical
removal was made possible. Radiation therapy started being used after 1960. Over time it
was realized that neither surgery nor radiation, or both in combination, was effective in
treating cancer as this could not control metastastic cancer.
Cancer is medically known as malignant neoplasm and is a group of various diseases,
all related to unregulated cell growth. Cancer is not caused by one agent, a virus or a
bacterium, that can be flushed or crushed. It is an intricate and potentially lethal
collaboration of genes gone awry, absence of some growth inhibitors or changing
hormones and epigenomes (Alice Park, 2013). In cancer, cells divide and grow
uncontrollably, forming malignant tumors, and invade nearby parts of the body. Cancer
may also spread to more distant parts of the body through the lymphatic system or
bloodstream. There are over 200 different known cancers that afflict humans
(cancerresearchuk.org). Cancer thus remains one of the most life threatening diseases. A
tremendous amount of research is, therefore, being carried out today for the prevention,
diagnosis and treatment of cancer.
The rationale for the use of conventional cytotoxic drugs is based on the theory that
rapidly proliferating and dividing cells are more sensitive to these compounds than the
normal cells. The hall mark of treatment for cancer has, therefore, been intravenous
cytotoxic chemotherapy. But then, cancer chemotherapy is a very difficult task. The
problems associated with chemotherapy are, non-specific toxicity and multi drug
1
Introduction
resistance (MDR) (who.int/mediacentre/factsheets/fs297/en). Many patients experience
the classic toxicities of alopecia, gastrointestinal symptoms and myelosuppression.
Advances that are taking place today in understanding the molecular mechanisms
underlying cancer development and progression are expected to lead to more efficacious
and less toxic drugs. Investigation are being conducted adapting a rational approach to
drug discovery in which a new generation of small molecules are designed and generated
to inhibit specific proteins or pathways abnormally expressed in cancerous cells. This
approach, termed Targeted therapy, is widely being applied today to discover new targets
and develop new anticancer drugs (Collins and Workman, 2006). Although traditional
cytotoxic chemotherapy remains the treatment of choice for many malignancies, targeted
therapies are now a component of treatment for many types of cancers, including breast,
colorectal, lung, and pancreatic cancers, as well as lymphoma, leukemia and multiple
myeloma.
Of the several new anticancer drugs approved by the U.S. Food and Drug Administration
(FDA) since 2000, 15 are targeted therapeutic agents compared to only five traditional
chemotherapeutic agents (centerwatch.com/patient/drugs/Druglist.html)
A large number of novel targets and approaches to anticancer therapy have emerged in
recent years. These approaches include targeting to the growth signal transduction
machinery within cancer cells, processes involved in cell invasion and metastatic spread,
apoptosis and tumor-related angiogenesis. Other approaches focus on tumor-specific
antigens, targeted poisons, stimulation of the immune system and gene therapy.
2
Introduction
Targeted therapies work by influencing the processes that control growth, division and
spread of cancer cells, as well as the signals that cause cancer cells to die naturally, the
way normal cells do when they are damaged or old. The development of new molecules
that bind to targets of biological interest is a breakthrough in anticancer research. The
knowledge of cell cycle mechanism and pathways of cancer pathogenesis has lead to
novel strategies and targets like Topoisomerases, Check Point Kinases (CPK’s), Cyclin
Dependent Kinases (CDK’s), Oncogenic Human Papiloma virus (HPV), ABC
transporters, etc.(Antony et al.,2009). One way of achieving this is through in silico
methods and Structure Based Drug Design (SBDD) methods.
Newer, safer and effective drugs, however, for cancer are yet to be explored due to the
fact that, drug discovery and development is cost and time intensive. It involves much
considerations right from Structure Based Drug Design (SBDD) or Ligand Based Drug
Design (LBDD) to synthesis, preclinical testing to clinical testing from cellular/molecular
to global effects on population. Only 20% of drug discovery projects are reported to lead
to clinical candidates of which only 10% compounds achieve registration. Analysis of the
reasons for this apparently low success is due to poor pharmacokinetics, toxicity and lack
of efficacy. Issues like target specificity and affinity must also be dealt for improving the
success rates (Shaikh et al.,2007). In recent years, continuous efforts have been made
both via computer aided drug design and reverse pharmacology using natural resources to
discover and develop drugs capable of specifically binding to the proteins/DNA
responsible for cancer pathogenesis (Spychala et al.,2009; Bonjean et al.,1998;
Yamamoto et al.,1990).
3
Introduction
SBDD has been widely used in ligand design and discovery (Kuntz 1992, Klebe 2000). In
particular, ligands can be identified among a large library of small molecules by virtual
screening. Each library molecule is docked into a binding site, then scored and ranked by
its ability to compliment the protein. High-ranking docked molecules are then tested
experimentally (Figure 1). The two critical parts of this process are docking to search
through plausible binding modes for candidate compounds and scoring to distinguish
ligands from nonbinding “decoys” (Collins and Workman, 2006).
Figure 1: The progress of development of new molecular target therapy, gene to drug
(Collins and Workman, 2006)
Not all small molecule leads are suitable as drugs. Profiling of potential leads against
multiple chemical, physicochemical and biological criteria has been adopted to select the
4
Introduction
best chemical starting point to maximize the probability of clinical success (Table 1)
(Davis et al., 2005).
In addition to target potency and selectivity, cellular efficacy and preliminary ADMET
properties are evaluated for promising compounds. This is called lead profiling. Lead
optimization involves addition of functional groups to increase the efficacy and
biological activity (Opera et al., 2001).
Table 1: Typical properties of fragments, lead-like and drug-like molecules. (Collins

and Workman, 2006)
Fragment Lead Drug
Physicochemical
Molecular weight (Da) <300 <400-450 <500
Lipophilicity (LogP) <3 <4 <5
H-bond donors (OH,NH) <3 <4-5 <5
H-bond acceptors (N,O) <3 <8-9 <10
Polar Surface areas NA NA <140-150Å2
Rotatable bonds NA <8 <10
Chemically reactive NA None present None present
groups
Biological
Target activity (IC50 or >>10-5M 10-6-10-7M 10-8-10-9M
K1) >0.3 >0.3 >0.3
Ligand efficiency (Kcal NMR or X-ray data SAR established SAR understood
atom-1)
SAR
ADMET NA Membrane permeable Bioavailable

Absorption NA Distributes to tissues Sufficient tumor
Distribution NA Low intrinsic levels
Metabolism/Excretion No common clearance Sufficient half life
Toxicology toxicophore Off-target Therapeutic window
pharmacology
5
Introduction
1.2 Bioinformatics, Chemoinformatics and Drug Discovery
Bioinformatics in drug discovery has been used for finding new targets. It predicts the
biological functions at the macromolecular level and considers gene as a fundamental
unit of genetic material. Chemoinformatics studies the relation between chemical
structures and properties of molecules, the interaction between small molecules and
macromolecules and the construction of chemical libraries. Today bioinformatics and
chemoinformatics overlap more and more (XU Jun et al., 2012).
Docking is a computational tool of structure–based drug design to predict the protein-
ligand interaction geometries and binding affinities. Docking program can be utilized to
discover and refine leads. Small molecule structures are bound with the target protein, the
energies of the resulting complexes are evaluated and those that show promising values
are tested as lead molecules. Here, the scoring method plays a very crucial role in
evaluating the molecules. The primary forces of attraction are the electrostatic
interactions. Among these, hydrogen bonds are of special interest (Jeffery et al., 1997,
Morimoto et al., 1991, Michael et al., 1997).
1.3 Some Novel Anticancer Targets
Some novel anticancer targets are, Tyrosine Kinase (TK) (Vlahovic and Crawford, 2008),
Farnesyl transferase (FTase) (Rowinsky et al., 1999), DNA topoisomerase.
Tyrosine Kinase (TK): TK are a family of mono-transmembrane -helix proteins
including membrane receptor tyrosine kinase and cytoplasm non-receptor tyrosine kinase.
The membrane receptor TK related to tumorigenesis includes epidermal cell growth
6
Introduction
factor receptor (EGFR) tyrosine kinase and vascular endothelial growth factor receptor
(VEGFR) tyrosine kinase, while the non-receptor tyrosine kinases include Src kinase and
Bcr-abl kinase, etc.
Farnesyltransferase (FTase): Ras protein, a low-molecular-weight GDP/GTP-binding
guanine triphosphatase encoded by Ras gene, plays a critical role in signal transduction of
cell growth and differentiation. In malignant transformation, invasion and spread of
cancer, Ras mutations or constitutive activation have been described. The continuous cell
growth signals are out of control causing cell differentiation and proliferation excessively
resulting in tumorigenesis.
DNA Topoisomerase: DNA is one of the most important molecules in cell biology.
Topoisomerases are the enzymes responsible for the changes in the DNA structure. These
enzymes accomplish this feat by either passing one strand of the DNA through a break in
the opposing strand (type I) or by passing a region of duplex from the same or a different
molecule through a double-stranded gap generated in a DNA (type II). Topoisomerases
are also known to relax only negative supercoils that relax supercoils of both signs or that
introduce either negative (bacterial DNA gyrase) or positive supercoils into the DNA
(reverse gyrase). They play an important role in regulating cellular processes such as
replication, transcription and chromosomal segregation by altering DNA topology. Most
type I DNA topoisomerases modify DNA topology in an ATP independent fashion by
creating single strand breaks in DNA whereas type II DNA topoisomerases do so in an
ATP-dependent fashion by creating double strand breaks in DNA (Schoeffler et al.,
2008). Among topoisomerases, topo II is a well-known anticancer target. Agents that
7
Introduction
target topo II are among the most effective anticancer drugs currently available for the
treatment of human cancers. Nevertheless, topo II based chemotherapy remains
associated with incidences of life-threatening toxic side effects like drug-induced
secondary malignancies. Intriguingly, previous studies suggest that suppressing topo II ,
a subtype of topo II, is responsible for the development of secondary malignancy
associated with etoposide treatment (Azarova et al., 2007). Drugs like etoposide and
doxorubicin may, however, have potent anticancer activity because they target another
subtype of topo II, namely topo II . Hence, agents specifically targeting topo II may
potentially be efficacious and safe chemotherapeutic drugs with a reduced risk of
treatment related to secondary malignancies (Toyoda et al., 2008).
Human Topoisomerase II (topo II ): Topo II is a homodimeric enzyme that requires
ATP for function (Figure 2). Topo II enzymes pass one duplex DNA through a second
duplex. To accomplish this reaction, the enzyme forms a reversible bond with 5’-
Phosphate of the DNA strand thereby creating a transient break in each phoshodiester
backbone. These breaks create a protein linked gate through which the second DNA
duplex can be pass (Bekhit et al., 2005). The whole process and functioning of topo II is
controlled by the hydrolysis of ATP to ADP and inorganic phosphate which leads to an
asymmetric retraction of Lys-378. This opens the enzyme-bridged middle gate of the
protein-DNA complex and movement of the T-segment from the upper cavity formed in
the ATPase domain into the lower cavity formed by the cleavage-reunion core (Figure 3
and 4). The inhibition of topo II leads to cell death and apotosis.
8
Introduction
Figure 2: Human topoisomerase II (1ZXM) from the Protetin Data Bank.
Figure 3: Mechanism of action of topoisomerase II
9
Introduction
Figure 4: The catalytic cycle of topoisomerase II. The individual steps of the catalytic
cycle of topoisomerase II are shown. These are: (1) DNA binding; (2) pre-strand passage
cleavage/religation; (3) DNA strand passage; (4) post-strand passage cleavage/religation;
(5) ATP hydrolysis; and (6) enzyme recycling.
1.4 Topoisomerase inhibitors
The topoisomerases were discovered in 1971 but it was not until 1980 that the
significance of these enzymes and their therapeutic potential were identified (Wang,
1985). Topoisomerases are the enzymes that regulate the overwinding and unwinding of
DNA. They control and modify the extracellular functions involved in replication,
recombination and transcription by catalyzing the passage of individual DNA strands
(topo I) or double helices (topo II) through one another. Topo I causes a break in a single
DNA strand, while topo II breaks both strands of DNA and requires ATP for activity
(Pinar et al., 2004). A variety of anticancer agents used in chemotherapy or those
10
Introduction
evaluated for clinical trials are known to inhibit topo I or topo II. Some synthetic Topo II
inhibitors are given in Figure 5
Figure 5: Synthetic topoisomerase II inhibitors.
1.5 Type II topoisomerase as a drug target
Eukaryotic Topo I and II and bacterial DNA gyrase (topo II) are the targets for a number
of clinically important drugs. Bacterial DNA gyrase is the target for quinolone synthetic
antibiotics. Quinolones potentially interact with the host, topoisomerase, and their affinity
to eukaryotic topoisomerase is thought to be relatively low. The precise mechanism of
quinolone-topoisomerase interaction is unknown. It is believed that the DNA and
11
Introduction
topoisomerase complex causes a conformational change in the "quinolone pocket", and
drugs bound at this site stabilize the complex. The stabilization of this complex inhibits
DNA rejoining and brings about the subsequent DNA damage. It is likely that quinolone
resistance is not transmitted by plasmids or transposons. Rare point mutations at the
quinolone pocket might decrease quinolone affinity and show drug resistance. Topo II is
essential for cell proliferation. In rapidly dividing mammalian cells, the total amount of
Topo II is dramatically increased. Hence topo II is a good target for cancer
chemotherapy. Drugs such as etoposide, doxorubicin, daunomycin and mitoxantrone are
widely used for systemic treatment of malignant neoplasms. Furthermore, the
chemotherapeutic regimens either include or are based on agents targeted to topo II. Topo
II poisons can be categorized into three groups; intercalators (such as doxorubicin and m-
AMSA), non-DNA-intercalators (etoposide,teniposide) and catalytic inhibitors of the
topoisomerase (aclarubicin and bisdioxopiperidines like ICRF-19363) (Figure 6). The
first two groups trap topo II on the DNA in a covalently bound state, often referred to as
the "cleavable complex". Catalytic inhibitors act on topo II without forming the cleavable
complex. It is not entirely clear what causes cell death following topoisomerase
stabilization. DNA damage provoked by topo II poisons has been shown in some cases to
result in apoptosis.
12
Introduction
Figure 6: Some potent inhibitors of topoisomerase II
1.6 Sydnones and Sydnoquinolines
Sydnones were first synthesized by Earl and Mackney in 1935. The structure 2 was
assigned to them (Scheme 1).
Later Baker, Ollis and Poole suggested the resonance stabilized 5-membered heterocyclic
system, 3, as the structure of sydnones (Baker et al.,1949).
13
Introduction
Sydnones are unique, dipolar heteroaromatic members of the general class of mesoionic
compounds. Chemically, they are 1, 2, 3-oxadiazolium-5-olates (Ollis et al.,1976). A
large number of sydnone derivatives have been synthesized (Kier et al., 1967;
Ackermann 1967; Pandey et al., 2006) and reported to possess a wide spectrum of
biological activities such as antiviral, antimicrobial, antiinflammatory, analgesic,
anthelmintic (Kalluraya et al., 2002), antitumor (Dunkley et al., 2003), free radical
scavenging (Mallur et al.,2007) and nitric oxide donor (Satyanarayana et al., 2002)
activity. The potential value of sydnones as biologically active substances is found in
their planar aromatic character, their relatively small size and the variation in electron
density around the ring. It is believed that the ionic resonance structures of sydnones
promote significant interactions with biological molecules. The stimulant drugs,
feprosidine and mesocarb, are substructures of sydnones imine in which the keto group of
sydnones (=O) is replaced by imino group (=NH). Some sydnones of biological
importance are shown in Figure 7.
14
Introduction
Figure 7: Some biologically active sydnones
1.7 Quinolines and Tetrahydroquinolines
Quinoline and tetrahydroquinoline derivatives have attracted a lot of interest due to their
applications as synthetic intermediates for a wide range of biologically active molecules.
Quinolines are an important scaffold in medicinal chemistry due to their occurrence in
natural products (Campbell et al.,1998) and drugs (Jenekhe et. al., 2001, Agrawal et al.,
1991, Jegou et al., 2001), polymer chemistry, electronics and optoelectronics for their
excellent mechanistic properties (Niwa et al.,1987). A large number of
tetrahydroquinolines have gained interest due to their biological activities. They are
found widely in nature. 2-Methyl-1,2,3,4-tetrahydroquinoline is present in the human
brain (Hadden et al., 2001). Dynemycin, a natural antitumor antibiotic based on
tetrahydroquinoline nucleus, the 2,4,6-trisubstituted tetrahydroquinoline, isolated from

15
Introduction
Martinella iquitosensis (Stewert, 1964), exhibits activity as a bradykinin antagonist and
with -adrenergic, histaminergic and muscarinic receptors. Some of the important
tetrahydroquinolines are shown in Figure 8.
Figure 8: Some important tetrahydroquinolines
Though sydnones are molecules which have been widely studied, sydnones fused with
tetrahydroquinolines have been studied only sparingly. There are only a few novel
synthetic routes for the synthesis of fused ring sydnoquinolines.
1.8 Cycloaddition Reactions
Cycloaddition reactions have always played an important role in the field of medicinal
chemistry. Woodward and Hoffmann first laid the fundamental basis for these concerted
16
Introduction
reactions (Woodward et al., 1965). Cycloaddition reactions belong to the class of
pericyclic reactions.
Pericyclic reactions are defined as the reactions that occur by a concerted cyclic shift of
electrons. This definition states two key points that characterize a pericyclic reaction.
Firstly, the reaction is concerted. In a concerted reaction, reactant bonds are broken and
product bonds are formed at the same time without intermediates. Secondly, these
reactions involve a cyclic shift of electrons and thus are characterized by a cyclic
transition state involving the bonds. The energy of activation of pericyclic reactions is
supplied by heat (Thermal Induction), or by UV light (Photo Induction). A cycloaddition
is a reaction between two compounds with bonds to form a cyclic product with two new
bonds as shown in Scheme 2.
Scheme 2
Sydnones undergo cycloaddition reaction with alkenes and alkynes to give pyrazolines
and pyrazoles, respectively. This is one of the most important synthetic applications of
sydnones. Cycloaddition with alkynes delivers pyrazoles via a
cycloaddition/retrocycloaddition with the expulsion of carbon dioxide (Scheme 3).
Recent years have seen a growing interest in pyrazole chemistry from both industrial and
academic standpoint.
17
Introduction
1.9 Pyrazoles and Pyrazoloquinolines
Pyrazoles and their derivatives have gained considerable importance over the years due to
their wide range of biological activities like antibacterial (Tandon et al., 2005), anticancer
(El-Deeb et al., 1962), antiinflammatory (Godaginamath et al., 2003), antitumor (Salgin-
Goksen et al., 2007), anticonvulsant(Onkol et al., 2004), etc. Pyrazoloquinolines,
quinolines fused with pyrazoles, are an important class of compounds which have been
studied extensively. Pyrazoloquinolines are known to possess anticancer (Mark et al.,
1995), antipsychotic (Shu-Wei Yang et al., 2012) and antiviral among other activities
Some biologically active pyrazoloquinolines are shown if Figure 9.
18
Introduction
19
Introduction
Though pyrazoloquinoline drugs are not in the market, KW-2170 and PD-115934 are two
such topoisomerase inhibitors which have reached phase II clinical trials. KW-2170 is a
pyrazoloacridine which is a topo II inhibitor of synthetic origin whereas PD-115934 is a
dual topo I and topo II inhibitor (Avendano and Menendez, 2008).
Although pyrazoloquinolines have been widely studied, reports on 4,5-
dihyropyrazolo[1,5-a]quinolines are very limited.
In the present investigation it was proposed, therefore, to design some fused ring
sydnoquinolines and pyrazolo[1,5-a]quinolines for the target, Topoisomerase II (pdb id:
1ZXM). The designed molecules will be synthesized and evaluated for their
antiproliferative activity on cervical cancer (HeLa), larynx epidermoid carcinoma (HEp-
2) and breast cancer (MCF-7) cell lines. The results of the in vitro studies and docking
studies will then be correlated to establish a structure-activity relationship for the
molecules.
20
Review of literature
1.10 Review of literature
The investigations carried out by earlier workers on the synthesis, pharmacological
properties and structure-activity relationships of sydnoquinolines and pyrazoloquinolines
including some of the related patents will now be reviewed for the period 1977 to 2013.
Chemistry and Pharmacology of Sydnoquinoline Derivatives.
Sydnones are a class of compounds that have been widely studied due to their diverse
biological properties and their betain like character. The synthesis of these compounds
was first carried out by the dehydration of N-nitroso-N-aryl (or –alkyl) glycines (Baker et
al.,1950). Nassoy et al., (2013) have carried out the synthesis and cycloaddition of
sydnones to obtain oxetanyl-substituted sydnones 2 (Scheme 4). They focused on
oxetanes due to their favourable pharmacological properties. The synthesized oxetane
substituted sydnones were further subjected to cycloaddition reaction with commercially
available alkynes (Scheme 5). Microwave assisted cycloaddition was found to result in
higher yields in lesser times with good regiocontrol. A higher yield of the isomer 4b was
observed.
Scheme 4
21
Scheme 5
They also studied the intramolecular cycloaddition using microwave method (Scheme
6)which revealed that all the pyrazoles are formed in good yields in short times.
Scheme 6
The authors thus effectively proved that oxetane-substituted sydnones are effective
precursors for the synthesis of pyrazoles via cycloaddition. They also successfully
synthesized spiro-fused intermediates (6) via intramolecular cycloaddition of sydnones
and alkynes.
22
Nitinchandra et al.,(2012) have synthesized a group of sydnones containing new mannich
21 and schiff’s bases 20 and evaluated them for their analgesic and antiinflammatory
activity (Scheme 8). The compounds containing schiff’s bases showed surprisingly good
antiinflammatory and analgesic activity on the introduction of electron donating groups.
In the case of compounds containing mannich bases significant antiinflammatory and
analgesic activity were observed and the presence of a piperidine or morpholine ring was
found to enhance the activity.
Scheme 7
23
Scheme 8
Chandrasekhar et al.,(2011) have synthesized 4, 5-dihydro[1,2,3] oxadiazolo [3,4-
a]quinolin-10-ium and its fluoro derivative 28 (Scheme 9). This preliminary
24
communication reports the synthesis of fused ring quinolines. There are only a very few
reports on the synthesis of these type of molecules. The authors have also published a
review on the synthesis and biological activites of sydnones and their derivatives
(Chandrasekhar et al.,2012).
Scheme 9
Tegginamath et al.,(2011) have reported a one-pot synthesis of sydnones fused with
coumarin ring via thiazoles (Scheme 10). The reaction was carried out in presence of
reusable silica sulphuric acid. The synthesized compounds 32a-r were tested for their α-
amylase inhibition and DNA cleavage properties. The compounds with electron
withdrawing groups exhibited stronger α-amylase inhibitory activity when compared to
the control enzyme inhibitor. The in vitro studies revealed that among the synthesized
molecules chlorine substituted derivatives exhibit good α-amylase inhibitory and also
DNA cleavage activity.
25
Scheme 10
Taj et al.,(2011) have utilized 3-aryl sydnones and exploited their ability to undergo
electrophilic substitution at the 4th position in order to couple benzotriazole,
benzothiazolin-2-thione, morpholine and diphenylamine to 4-bromo-3-arylsydnones to
obtain the desired molecules 35, 37, 39 and 41 (Scheme 11). The synthesized molecules
were studied for their potential antitubercular activity. The compounds containing
electron withdrawing groups exhibited excellent inhibition at a concentration of less than
or equal to 5μg/mL.
26
Scheme 11
Zhang et al.,(2002) have efficiently synthesized 3-(3-hydroxypropyl)sydnones 50 and
studied their intramolecular electrophilic aromatic substitution (Scheme 12). They
exploited the intramolecular Friedel-Crafts acylation reaction in order to obtain seven
membered fused ring sydnones 51. The Friedel-Crafts reaction was carried out in the
presence of H2SO4 or BF3.Et2O.
27
Scheme 12
Butkovioc et al.,(2011) have synthesized a series of stilbene-sydnones from methyl
anthranilate (Scheme 13) and evaluated them for their cytotoxic properties in five cell
lines, namely HeLa (cervical carcinoma), MCF-7 (breast carcinoma), SW 620 (colon
carcinoma), MiaPaCa-2 (pancreatic carcinoma) and H 460 (lung carcinoma). All the
compounds, however, showed low antiproliferative effect. An interesting observation
made was that though trans stilbenes generally exhibit high biological activity, the trans
derivatives synthesized by them showed low activity when compared to the cis
derivatives. This method was found to be convenient for the synthesis of both
diastereomers.
28
Scheme 13
Deshpande et al.,(2010) have synthesized sydnones containing a chalcone moiety
(Scheme 14) and evaluated them for their antiinflammatory, analgesic and antibacterial
properties. The synthesized molecules exhibited low antibacterial and antiinflammatory
activities.
29
Dunkley et al.,(2003) have synthesized N-(4’-substituted-3’-nitrophenyl)sydnones 69a-d
(Scheme 15) and evaluated them for their anticancer activity. These molecules were
synthesized based on the work of Grynberb et al., who synthesized a series of 4’-
substituted-3’-nitrophenylsydnones, evaluated them for their anticancer activity and
found that the 4’- chloro and 4’-pyrrolidino compounds had good activity when
compared to others.
Scheme 15
The authors also synthesized various substituents of Grynberg’s compounds and studied
the role of inductive effect or the ring size but were not successful in suggesting the
actual mechanism for the activity of these compounds. However, based on the findings of
Fliedner, Dunkley and coworkers suggested a mechanism for the anticancer activity of
sydnones. Fliedner has found that the pKa of 3-sydnonyl acetic acid and nitroacetic acid
are similar which suggests that the 3-sydnonyl moiety has electron withdrawing character
as the nitro group. The nitro group and the sydnonyl moiety thus act as o,p-directing
30
group in a normal nucleophilic aromatic substitution. They suggested that the
nucleophilic groups on DNA may get attached to the sydnones and get deactivated.
Satyanarayana et al.,(1995) have synthesized 3-phenylsydnones (Scheme 16) and
evaluated them for their antiinflammatory, antiarthritic and analgesic activity. The
antiinflammatory activity was evaluated using carrageenen induced paw edema method.
All the synthesized molecules exhibited low activity when compared to the standard,
phenylbutazone. These compounds, when evaluated for their antiarthritic activity in
adjuvant induced arthritis model in rats, exhibited significant activity. The compounds
were also more active compared to the standards, phenylbutazone and indomethacin,
though the bromo derivative showed activity similar to phenylbutazone. The compounds
were also evaluated for analgesic activity using acetic acid induced assay in mice. The
fluoro derivative exhibited very high activity when compared to the standard, aspirin. The
authors also evaluated the active compounds for their ulcerogenic effect as gastric effects
are the major side effects of antiinflammatory drugs. All the compounds were found to be
ulcerogenic though not significantly.
31
Senff-Ribeiro et al.,(2004) have studied the cytotoxic and antiproliferative activity of MI-
D (72) on human melanoma cell lines and found that MI-D could be potent against
human melanoma cells.
Sydnones as synthons for other biologically active molecules
Latthe et al.,(2011) have reported the synthesis of 1,2-diaza-five membered heterocyclic
systems. They synthesized 4-(hydrazinocarbonyl)phenylhydrazine 74 and used it as a
synthon for the synthesis of bismesoionic compounds (Scheme 17). The synthesized
molecules were then evaluated for their antibacterial activity. They reported the synthetic
utility of the bisfunctional compound, for the synthesis of novel bis azide–4-
(azidocarbonyl)phenyl azides 75, that were a synthetic challenge till then. The azides
were then used for the synthesis of compounds 81a-c and 82a-c. These compounds were
found to show moderate antibacterial activity. All the phenylcarbamates 77a-c showed
considerably good antifungal activity when compared to the standard, griseofulvin.
32
Scheme 17
Foster et al.,(2011) have synthesized azine-substituted pyrazoles via the cycloaddition of
sydnones (Scheme 18). This synthesis is novel as not many N-azine-substituted sydnones
have been reported in literature. The authors studied the cycloaddition of sydnones with
alkylboronates for the synthesis of pyrazole boronic acid derivatives 88-91. These studies
are of importance due the the boronate motif. The authors have thus efficiently
33
demonstrated that alkyne/sydnone cycloaddition reactions are a direct and convenient
method for the preparation of azine-substituted pyrazoles.
34
Scheme 18
35
Wu et al.,(2010) have reported the synthesis of 2H-indazoles via the [3+2] cycloaddition
of arynes to sydnones (Scheme 19). They carried out the cycloaddition reaction with
different precursors of arynes and observed that the reaction proceeded to completion
with good yields with single products 107. The reaction conditions were mild. A detailed
study on the mechanism required was also in progress by the authors.
Sateesha Rai et.al.,(2008) have synthesized a series of novel 1-aryl-3-(5-nitro-2-thienyl)-
4-aroyl-pyrazoles 121 (Scheme 22)via 1,3-dipolar cycloaddition of 3-arylsydnones 113
(Scheme 20) with 1-aryl-3-(5-nitro-2-thienyl)-2-propyn-1-ones 118 (Scheme 21). The
synthesized molecules were evaluated for their antibacterial (Escherichia coli ATCC-
25922, Staphylococcus aureus ATCC-25923, Pseudomonas aeruginosa ATCC-27853,
recultured Bacillus subtilis) and antifungal activity [(Candida albicans (NCIM No.
3100)] by serial dilution method. In an attempt to increase the activity of the molecules
they introduced 5-nitrofuran and 5-nitrothiophene moiety. The results revealed that some
of the tested compounds had good activity. The compounds with methyl and chloro
derivatives were found to show excellent antibacerial activity. The compounds with
methyl, chloro and methoxy substituents were found to show exceptionally good
antifungal activity when compared to the standard, fluconazole.
36
Scheme 20
Scheme 21
37
Scheme 22
Ganesha Rai et al.,(2006) have synthesized a series of 1-aryl-3-(5-nitro-2-furyl)-4-
aroylpyrazoles 124 via 1,3-dipolar cycloaddition reaction of 3-arylsydnones 123 and α,β-
acetylenic ketones 122 containing nitrofuran moiety (Scheme 23). Although 1,3-dipolar
cycloaddition of sydnones is well known and well studied, the authors felt that less
attention has been given to the regiochemistry of 1,3-dipolar cycloaddition reaction with
more complex dipolarophiles. Hence, they made an attempt to study the cycloaddition
reaction with more complex dipolarophiles like 1-aryl-3-(5-nitro-2-furyl)propynones 122.
They found that the reaction of these dipolarophiles with 3-arylsydnones resulted in
regiospecific 1-aryl-3-(5-nitro-2-furyl)-4-aroylpyrazoles in good yields. In order to study
the regiospecificity in more detail the authors used different para substituted aryl
compounds and in all the cases the reactions were found to be highly regioselective. They
38
found the formation of single products and this was confirmed using X-ray
crystallographic studies.
Scheme 23
Chemistry and Pharmacology of Pyrazoloquinoline derivatives.
Faidallah et al.,(2013) have studied the DNA binding property of tetrahydroquinolines
125, tetrahydropyrimidino[4,5-b]quinolines 126 and tetrahydropentaazacyclopenta[a]
anthracenes 127. All the synthesized compounds displayed good antitumor activity and
good DNA binding activity. Tricyclic tetrahydropyrimidino [4,5-b]quinolines, however,
showed better activity.
Jun Ya Kato et al.,(2013) have reported a novel method for the synthesis of pyrazolo[1,5-
a]quinolines 130 under transition metal free conditions (Scheme 24). This method
involved the synthesis of pyrazoloquinolines via a combination of aromatic nucleophilic
substitution and knovenagel condensation.
39
Scheme 24
Mishra (2012) has studied the 2D- structure-activity relationship (QSAR) of 2,5-
dihydropyrazolo[4,3-c]quinolines on the inhibition of phosphodiesterase-4 (PDE-4). The
models were checked for the observed biological activity and the predicted activity. The
biological activities of the selected compounds were shown to be due to the
hydrophobicity, electrostatic and topological properties of the molecules. The authors
concluded that the increase PDE-4 inhibitory activity of 2, 5-dihydropyrazolo [4, 3-c]
quinoline-3-one derivatives was due to the presence of groups contributing to flexibility
in chain length and lipophilicity of molecule.
Chang et al.,(2012)have efficiently synthesized two regioisomers of 2-arylpyrazolo[3,4-
c]quinolin-4(5H)-ones 134 and 2-arylpyrazolo[4,3-c]quinolin-4(5H)-ones 135 from 3-
arylsydnones 133, ethyl 3-bromopropynoate, and 2-aminophenylboronic acid pinacol
40
ester using Pd(PPh3)4 as catalyst (Scheme 25). This efficient one-pot synthesis
methodology involved 1,3-dipolar cycloaddition, Suzuki coupling reaction and
intramolecular cyclization.
Abu-Hashem et al.,(2012) have synthesized a series of 2-
hydrazinyltetrahydropyrimido[4,5-b]quinolin-4(3H)-ones 138 by desulphurization of S-
and N-dimethyl derivatives 137 with hydrazine hydrate (Scheme 26).
These molecules on reacting further with malonitrile, carbondisulphide, potassium
thiocyanate, pthalic achydride and aromatic aldehydes gave 3,5-di
aminopyrazolopyrimido[4,5-b]quinolines 139, triazolotetrahydropyrimido[4,5-
b]quinolines 140, aminotriazolopyrimido[4,5-b]quinolines 141,
aminopthalimidopyrimido[4,5-b]quinolines 142 and N-arylidene hydrazinepyrimido[4,5-
b]quinoline derivatives, respectively 146 (Scheme 27). The synthesized molecules were
41
evaluated for their antitumor potential and a few of them proved to be potent antitumor
agents.
Scheme 27
42
The in vitro cytotoxicity studies of the synthesized molecules revealed that the
pyrimidoquinoline when introduced into the basic scaffold helped improve the antitumor
activity. Compounds 139, 140, 141 and 146d showed cytotoxicity against KB, MGC-803
and MCF-7 cell lines and compound 139 showed potent cytotoxicity against CNE2
cancer cell lines. The structure-activity relationship revealed that the presence of 3,5-
diaminopyrazolo, 2-amino-1,3,4-triazolo, 1,3,4-triazolo and triazolothiazolidine moieties
linked to the pyrimido[4,5-b]quinolines enhanced the cytotoxicity of the molecules.
Reis et al.,(2011) have synthesized and evaluated some novel 2-(benzo[d]thiazol-2-yl)-8-
substituted-2H-pyrazolo[4,3-c]quinolin-3(5H)-ones 151 (Scheme 28) for their anticancer
activity against MDA-MB-435, HL-60, HCT-8 and SF-295 cell lines. The results
revealed that compounds 151b and 151c exhibited good anticancer activity on all three
cell lines with IC50 values less than 5μg/mL. Molecular modeling studies were also
carried out by these authors using Osiris programs to evaluate the electronic properties,
hydrogen bonding, molecular weight and theoretical toxicity properties.
Scheme 28
43
Hemolytic assay studies revealed that none of the molecules were capable of causing
hemolysis in mouse erythrocytes even at high concentrations.
Lenzi et al.,(2011) have designed and synthesized 2-phenyl and 2-methylpyrazolo[3,4-
c]quinolines-4-one 155, 156 (Scheme 29) and 4-amine derivatives 163, 167 (Scheme 30)
as adenosine receptor antagonists. The synthesized molecules were evaluated for their
ability to displace specific [3H]DPCPX, [3H]ZM241385 and [125I]AB-MECA binding
from cloned hA1, hA2A and hA3 receptors, respectively. The results revealed that the
synthesized molecules showed A1 receptor affinity and selectivity. Molecular docking
studies were also carried out in order to define the structural features of the binding (pdb
code: 3EML). The docking studies revealed that the introduction of the functional group
at the 6th position leads to enhanced affinity towards the A1 receptor and also selectivity.
44
45
Imbriglio et al.,(2011) have worked on a series of amino-anthranilic acid derivatives as a
new class of low serum-shifted high affinity full agonists of the human orphan G-protein-
coupled receptor, GPR109a, with improved ADME profile. They designed a series of
GPR109a receptor antagonists. A few pyrazoloquinolines based on these series of
compounds were found to show a 10-fold reduction of the serum shift.
The anthranilic acid derivatives were found to have a 10,000 fold shift of serum-potency,
excellent in vitro profile and modest ADME properties. Considering the importance of
the anthranilic acid moiety and the terminal phenol group they designed molecules
keeping these two moieties intact and modifying the rest of the molecule in order to
increase the efficacy. They successfully synthesized a new class of aminoanthranilic acid
agonists of GPR109a with potent agonists, reduced serum shift and excellent ADME
properties.
Hang et al.,(2011) have carried out a facile copper catalyzed tandem reaction for the
synthesis of 4,5-dihydropyrazolo[1,5-a]quinoline 176 (Scheme 32) and pyrazolo[1,5-
46
a]indoles 173 (Scheme 31). They found that the yields pyrazolo[1,5-a]quinolines were
found to be better than the indoles. This was explained on the basis of the steric
hinderance. They also came up with an efficient method for the synthesis of some fused
ring indoles and pyrazoles.
47
Colotta et al.,(2009) have reported a series of pyrazoloquinolines (Scheme 33) and found
them to exhibit a high affinity for adenosine receptors and were active in nanomolar
quantities.
Scheme 33
Scheme 34
48
Scheme 35
The target compounds were synthesized from 2-arylpyrazolo-[3,4-c]quinolines-4-amines,
200-203 (Scheme 34, 35), which were prepared from 3-ethoxalylindole. The final
molecules 178-197, 198 and 199 were synthesized from 4-amino derivatives by reacting
with suitable carboxylic acid in DMF in the presence of 1- hydroxybenzotriazole,
triethylamine, and 4-(dimethylamino)pyridine. 4-Diaroylamino derivatives, 198 and 199,
were obtained by refluxing the 4-amino derivative, 200, with an excess of 3-
49
trifluoromethylbenzoyl chloride and 4-trifluoromethylbenzoyl chloride, respectively, in
anhydrous methylene chloride and pyridine.
The structure-activity relationship of the synthesized compounds revealed that
introducing aroyl ring in place of the benzoyl moiety increases the binding affinity of the
synthesized molecules. They also evaluated the effect of various heterocyclic rings in the
basic scaffold and found that the 2-furyl and 2- or 3- or 4-pyridyl rings were the most
beneficial. The introduction of a methyl group to the furyl moiety increased the affinity
further. The presence of a Me or OMe, either in the para or meta position, while
maintaining a high hA3 affinity, reduced the hA3 versus hA1 selectivity. The authors
also carried out the docking studies in order to obtain a structure based pharmacophore
model (PDB id: 1L9H). The docking scores were compared with the binding assay
results. Based on these results a pharmacophore model was developed which may be of
help in designing molecules for this receptor.
The authors have also reported a novel group of compounds as adenosine receptor
antagonists, efficiently correlated the in silico and in vitro studies and explained the
structure-activity relationships of the synthesized molecules.
Duggineni et al.,(2006) have reported a novel application of a pictet Spengler reaction for
the synthesis of some pyrazoloquinolines and thiazoloquinolines (Scheme 36). Thiazole
and pyrazole based arylamine substrates were used for the reaction unlike the
conventional method. The studies carried out by this group proved that arylamines linked
to an activated heterocyclic ring can lead to a variety of second-generation substrates for
the Pictet–Spengler cyclisation (Scheme 37, 38).
50
Scheme 36
The authors worked on this concept with the hope to synthesize benzannulated
heterosystems and avoid the stereochemical issues associated with the traditionally used
pictet Spengler reactions. Their work helped to prove that aryl amine derived substrates
are likely to undergo pictet Spengler reaction faster than the substrate derived from
aliphatic amines. They also worked out an efficient synthetic strategy for the synthesis of
dihydropyrazoles (pyrazolines) and also successfully modified the problem faced during
cyclisation when an electron withdrawing group is attached to the aldehyde.
51
Scheme 38
Selvi et al.,(2006) have synthesized a series of pyrimido[4,5-b] 239, 240 (Scheme 39)
and pyrazolo[3,4-b]quinolines (Scheme 40,41) and evaluated them for their antimicrobial
activity. They carried out the synthesis using environmentally benign solvent-free
conditions using p-tolylsulphonic acid as catalyst and found that compounds 239a-g and
240a-g had significant effect on the inhibition of Bacillus subtilis, Escherichia coli,
Pseudomonas aeruginosa, compounds 239a–g exhibited good antifungal activity against
Candida albicans whereas the compounds 240a–g were active against Aspergillus flavus.
Compounds 242a–g were found to exhibit good antibacterial activity against E. coli and
52
P. aeruginosa in addition to a pronounced effect on the growth of fungi like Rhodotorula
rubra, C. albicans and Lipomyces lopofera whereas compounds 244a–g were active
against Staphylococcus aureus, Staphylococcus albus, E. coli and P. aeruginosa.
53
Kalayanov et.al.,(1998) have synthesized a series of 1-aryl-1H-pyrazolo[4,3-c]quinolines
and 2-aryl-2H-pyrazolo[4,3-c]quinolines (Scheme 42) and evaluated them for their
H+/K+- ATPase activity. They synthesized these molecules based on the reversible proton
pump inhibitor, SK and F 96067.
The carbonyl group present in the SK and F 96067 was found to be responsible for the
restriction of the NH group conformation by forming a hydrogen bond with the carbonyl
and also by increasing the conjugation between the nitrogen and quinolines ring. 1H-
Pyrazolo[4,3-c]quinolines were, therefore, synthesized in order to reduce the flexibility of
the molecule.
All the synthesized molecules were evaluated for their antiulcer activity using SK and F
97067 was used as the standard. The activity of the synthesized molecules was found to
be lower than that of the standard.
54
Wojciechowska et al.(1998) have reported some pyrazoloquinolines having affinity for
benzodiazepine receptors. They synthesized pyrazoloquinoline based on the scaffold
pyrazolo[4,3-c]quinolin-3-ones, known for their affinity for benzodiazepine receptors.

55
Studies were carried out to explain the effect of different substituents in pyrazolo[4,3-
c]quinolin-3-ones. A series of 6- and 7-substituted-2-arylpyrazolo[4,3-c]quinolin-3-ones
were synthesized (Scheme 43) and evaluated for their benzodiazepine receptor binding in
competition with flunitrazepam. The target molecules were synthesized from diethyl
ethoxymethylenemalonate via condensation with a suitable aniline. The product was
further treated with POCl3 and phenylhydrazine to obtain the final compounds. The
partition coefficient and electronic parameters used in correlation regression were
compared with the experimental data obtained. The results of the QSAR studies revealed
that the hydrophobicity and the position of the bicyclic core are both important for the
binding affinity for the benzodiazepine receptor. The authors also successfully evaluated
the structure-activity relationship of pyrazoloquinolines and explained the effect of the
substituents on the pyrazoloquinoline scaffold.
56
Scheme 43
David Barrett et al.(1996) have reported a novel synthesis of pyrazolo[1,5-a] quinolines
in excellent yields via a tandem Michael reaction of N-methylaminoquinolones with
various acrylate derivatives in the presence of NaH (Scheme 44). The synthesized
compounds were converted to DNA gyrase inhibitors by reaction with secondary amines
(Scheme 45). The in vitro antibacterial studies carried out on the synthesized molecules,
however, revealed that these derivatives were weak when compared to the standard,
levofloxacin.
Scheme 44
57
Scheme 45
Wentland et al.,(1995) have studied the topoisomerase II inhibitory activity of a
quinolone derivative and related compounds (Scheme 46). They demonstrated that
significant enhancement in topoisomerase II inhibition on introducing a keto group and
carboxylate group in the pyrazole ring.
Scheme 46
One of the earliest reports on the synthesis of 4,5-dihydropyrazolo[1,5-a]quinolines was
by Deshayes et al., (1983). They explored the photo reactivity of a series of 5-alkenyl or
dialkenyl-1-phenylpyrazoles and carried out the reaction under N2 atmosphere using
benzene as the solvent (Scheme 47). They did not, however, carry out the biological
evaluation of these molecules
58
Scheme 47
Table 2: Some of the patents related to pyrazoloquinolines are summarized below,
Patent number Biological activity Compound
EP 2 520 577 A1 Central cannabinoid receptor
Nov 7, 2012 (CB1) antagonizing activity.
US 7 863 266 B2 GABA receptor modulator Pyrazolo[4,3-c]quinolin-3-one
Jan 4 2011
US 7 858 614 B2 GABA receptor modulator Pyrazolo[4,3-c]quinolin-3-one
Dec 28, 2010
59
US 7 081 456 B2 Immunomodulation,

Rheumatoid arthritis,
July 25, 2006 multiple sclerosis, diabetes,
asthma, psoriasis.
US 6 642 249 B2 Immunomodulation,

Rheumatoid arthritis,
Nov 4, 2003
multiple sclerosis, diabetes,
asthma, psoriasis.
US 5 442 065 Antiinflammatory agents 1H-pyrazolo[4,3-c]quinol-4(5H)-one-

carboxylicacid
Aug 15, 1995
US 4 560 689 Benzodiazepine Pyrazolo[4,3-c]quinolin-3-one
Dec 24, 1985 receptor modulators
US 4 312 870 Psychoactive drug for 2-arylpyrazolo[4,3-c]quinolin-3-one

treatment of anxiety and
Jan 26 1982 depression.
60
US 4 268 516 Immuno regulators. Benzothiopyrano[4,3-c]pyrazoles
May 19, 1981
US 4 076 818 Bronchodialators, Pyrazolo[1,5-c]quinazoline
Feb 28, 1978 antihistamine,Antiinflammat
ory agent and for
rheumatoid arthritis.
US 4 024 149 Antifertility drug Triazolo[1,5-a]quinolines
May 17, 1977
Conclusions and Future Perspectives
In conclusion, sydnones are versatile and privileged structures which belong to the
mesoionic class of compounds. They possess a wide variety of biological activities and
undergo a large number of reactions like cycloaddition, alkylation, arylation, lithiation,
etc. Cycloaddition reactions have been one of the most exploited reactions of sydnones.
This has been a stepping stone for the discovery of lead molecules. Among these, the
reaction of sydnones to form pyrazoles are of considerable importance. The synthesis of
pyrazoloquinolines have lead to the discovery of several biologically active molecules.
Several pyrazoloquinolines have been evaluated for their anticancer, anticonvulsant,
antibacterial and antifungal activities among others. Although sydnones and
pyrazoloquinolines have been studied, fused ring derivatives of these molecules have
61
been reported only sparingly. In Particular, sydnoquinolines and 4,5-
dihydropyrazoloquinolines are yet to be explored, synthetically as well as biologically.
Pyrazoloquinolines are known for their ability to bind with Adenosine receptor,
benzodiazepine receptor, Chk1kinase, phosphodiesterase, ras and topoisomerase II.
Although sydnones and pyrazoloquinolines have been widely studied, reports on
sydnoquinolines and 4,5-dihydropyrazolo[1,5-a]quinolines are sparingly found in
literature. There is good scope to study these molecules adopting new synthetic strategies.
It was proposed, therefore, to explore the synthetic routes for 4,5-dihydrosydnoquinolines
and 4,5-dihydropyrazoloquinolines. The structural features of these molecules and their
capability to bind with receptors have increased the writer’s interest in them.
62
Scope and Objectives
2. SCOPE AND OBJECTIVES
The problems associated with the current cancer chemotherapy are non-specific toxicity
and multi drug resistance (MDR). Rapid advances are being made today in understanding
the molecular mechanisms underlying cancer development and progression.
Simultaneously, investigations are being conducted adapting a rational approach to drug
discovery in which a new generation of small molecules are designed and synthesized to
inhibit specific proteins or pathways abnormally expressed in cancerous cells. This
approach termed Targeted therapy is widely being studied today to discover new targets
and develop new anticancer drugs.
Targeted therapy offers a number of potential advantages over conventional
chemotherapy including an increased therapeutic index (i.e., effective cancer treatment
with less side effects), better tolerability due to a better toxicity profile, better target
selectivity, availability for chronic treatment and in some cases oral administration.
The principal criterion applied in modern anticancer drug design is the principle of
selective toxicity which requires activity restricted exclusively to the cancer cells. From
the point of view of selective activity directed on tumour cells and mechanisms of
carcinogenesis, different classes of modern drugs can be distinguished like inhibitors of
Cyclin Dependent Kinases (CDK’s), Check Point Kinases (CPK’s), Oncogenic Human
pappiloma virus (HPV), Topoisomerases, Cdc25 Phosphatase and ABC transporters.
DNA topoisomerases are enzymes that control and modify the topological states of DNA.
They can catalyze several interconversions between topological isomers of DNA by
transiently breaking single or double strands, and resealing them after reorganization of
63
the topology. Topoisomerases are important drug targets for anticancer therapeutics due
to the vital role played by them in the survival of the cells.
Topoisomerase I (topo I) breaks a single DNA stand, while topoisomerase II (topo II)
breaks both strands but requires ATP for full activity. Since the activity of
topoisomerases is essential for several cellular processes such as replication, transcription
and chromosome condensation, investigation of the inhibitory activities of eukaryotic
topoisomerases is widely studied in anticancer drug development. Topo II is the target for
some of the most active anticancer drugs such as etoposide, teniposide, and doxorubicin
used in the treatment of human malignancies.
Compounds of the mesoionic class have interesting structural features due to their betain-
like character. They consist of a five membered ring associated with a sextet of p and Π
electrons, supported by a partial positive charge in the heterocyclic ring and
counterbalanced by a formal negative charge. The association of these characteristics
suggests a high probability of strong interactions with biomolecules such as DNA and/or
proteins. Sydnones belong to this class of compounds.
Sydnones, therefore, have been widely studied due to their unique heteroaromatic
character and their biological applications. Apart from their biological activity, another
attractive feature of these molecules is their application as synthetic precursors for other
molecules like pyrazolines and pyrazoles.
64
Various 4-substituted sydnones have been studied over the years for anticancer,
antiinflammatory and analgesic activities among others. Some of these have shown
reasonably good biological activity. Sydnones have also been fused with quinolines or
tetrahydroquinolines. The tetrahydroquinoline ring is present in numerous biologically
active compounds. Due to the importance of these privileged molecules, numerous
synthetic routes have been reported for these molecules. Derivatives containing fused
ring sydnones, however, have been sparingly reported.
Further, a broad spectrum of biological activity has been reported for pyrazoloquinolines.
No pyrazoloquinolines have, however, been identified so far that show clinical promise
although the p-chloro derivative, CGS 9896, has been reported to be a potent
anticonvulsant.
65
Would pyrazoloquinolines be effective against multidrug resistance? Numerous tri- and
tetracyclic planar nitrogen containing heterocycles are well known as topoisomerase
inhibitors. On the basis of some known topoisomerase II catalytic inhibitors like
benzophenanthridine, psorospermin, intoplicine, etc., it was hypothesized that the
presence of a planar ring system and 3 to 4 fused aromatic rings would result in a better
binding to the DNA.
Pyrazoloquinolines are known to be made up of 3 planar ring systems.
Pyrazoloquinolines were, therefore, chosen as the target molecules for topo II because the
fused polycyclic part can bind to the ATP site via van der waals forces and hydrogen
bonding interactions. It is also likely that the pyrazole ring when attached to the quinoline
ring may help in increasing the rigidity of the side chains attached to the quinoline
nucleus thus increasing its the efficacy for the ATP binding site of topoisomerase II.
66
The basic scaffold of pyrazoloquinolines are,
Prazolo[3,4-c]quinolines, pyrazolo[4,3-c]quinolines and pyrazolo[1,5-c]quinolines have
been studied for their antitumor and adenosine receptor antagonist properties. Although a
few attempts have been made to synthesize 4,5-dihydropyrazolo[1,5-a]quinolines their
biological evaluation have not been reported so far.
Based on a detailed literature review, it was hypothesized that the desired
pyrazoloquinolines could be synthesized by the reterosynthetic Scheme 48.
Sydnoquinolines can be synthesized from anilines and crotonaldehyde via Doebner-
miller reaction. Further, the obtained molecules may be reduced and nitrosated. The N-
67
nitroso derivatives so obtained can then be converted to sydnones using acetic anhydride
cyclisation. The sydnoquinolines so obtained can then be converted to some novel
pyrazoloquinolines via cycloaddition reaction. Sydnoquinolines are known to undergo
this reaction.
In the present work it was proposed, therefore, to synthesize some sydnoquinolines,
namely sydnones fused with tetrahydroquinoline ring. Attempts would also be made
to synthesize some fused ring pyrazoloquinolines from the sydnoquinolines via [3+4]
dipolar cycloaddition reaction. The sydnoquinolines and pyrazoloquinolines
obtained would then be evaluated for their anticancer activity in vitro using various
cell lines.
The objectives of the present study are to,
· design molecules for the topoisomerase II target.
· carry out docking studies of the designed molecules on the topoisomerase II (pdb
id:1ZXM)
· synthesize fused ring sydnoquinolines using feasible synthetic strategies.
· synthesize pyrazoloquinolines from sydnoquinolines via the cycloaddition
reaction.
· characterize all the synthesized molecules by MASS, 1H NMR, 13

C NMR,
NOESY spectroscopy.
· evaluate the synthesized molecules for their in vitro anticancer potential using
various cell lines.
68
Experimental
3. EXPERIMENTAL
3.1 Chemicals and Reagents
All the solvents and reagents were purified and dried according to the procedures given
in Vogel’s text book of Practical Organic Chemistry. All the reactions were monitored by
thin layer chromatography (TLC) on aluminum plates precoated with silica gel GF.
Reduction of C=C double bonds were carried out under H2 atmosphere. All the
synthesized compounds were characterized based on TLC, physical nature, melting point
(mp), Infrared Spectroscopy (IR), Nuclear Magnetic Spectroscopy (NMR) and Mass
Spectroscopy (MS).
The melting points were determined in open capillaries using Veego VMP-1 melting
point apparatus and are uncorrected. The temperatures are expressed in °C.
The IR spectra of the compounds were recorded on Shimadzu and Perking-Elmer FT-IR
spectrophotometer using neat technique and were expressed in cm-1. 1H NMR, 13C NMR,
1
H-1H HOMO COSY spectra were recorded on Bruker 400MHz spectrometer, using
CD3OD as solvent and TMS as internal standard. The chemical shifts were expressed in δ
ppm and the following abbreviations were used, s=singlet, bs= broad singlet, d = doublet,
t = triplet, q = quartet, m = multiplet. Attempts were made to quantify and identify the
peaks in 13C NMR spectra.
Mass spectra were recorded using high resolution mass spectrometer (HRMS) and liquid
chromatography mass spectroscopy (LCMS) under electron spray ionization technique,
using time of flight (TOF) and triple quardrupole mass analyzers, respectively.
69
Experimental
3.2 Docking and Molecular Modeling Studies
The molecular modeling studies were carried out on Linux Fedora 7.0 workstation using
GLIDE, version 5.7, Schrodinger suit 2011, LLC, New York, on a Maestro graphical user
interface.
3.2.1 Ligand Preparation
The structures of the ligands were generated using Chem Draw Ultra Version 8.0. These
ligands were converted to mol2 format and subjected to ligprep module of Maestro in
Schrodinger suite of tools. They were converted from 2D to 3D structures by including
stereochemical, ionization, tautomeric variations as well as energy minimization. They
were then optimized for their geometry, desalted and corrected for their chiralities and
missing hydrogen atoms. The bond orders of these ligands were fixed and the charges
neutralized. The ionization and tautomeric states were generated between pH of 6.8 to 7.2
using Epik module. In the final stage of Ligprep, compounds were minimized using
Optimized Potential for Liquid Simulations-2005 (OPLS-2005) force field in Impact
package of Schrodinger until a root mean square deviation of 1.8 Å was achieved.
Steepest descent algorithm was used for minimization, followed by conjugate gradient
method. A single low energy ring conformation per ligand was generated and the
optimized ligands were used for docking.
3.2.2 Protein Preparation
The crystal structure of the topoisomerase II was downloaded from RCSB Protein Data
Bank (Collaboratory for Structural Bioinformatics, PDB) and was used for the modeling
studies. Refinement of bond orders, formal charges and missing hydrogen atoms,
70
Experimental
incomplete and missing residues and terminal amide groups were carried out. Water
molecules beyond 5 Å of the hetero atom were removed. The possible ionization states
were generated for the heteroatoms in the protein and the most stable state was chosen.
The hydrogen bonds were assigned and orientations of the retained water molecules were
corrected. A restrained minimization of the protein molecule was carried out using OPLS
2005 force field to reorient side chain hydroxyl groups and alleviate potential steric
clashes. The minimization was restrained to the input protein coordinated by a predefined
Root Mean Square Deviation (RMSD) tolerance of 0.3 Å.
3.2.3 Receptor Grid Generation
The ligand was retained in the crystal structure of the prepared protein which was used
for the receptor grid construction. The grid dimensions (within which the docked pose is
confined) of the protein was set to 14 Å X 14 Å X 14 Å.
3.2.4 Validation of the docking programme
The accuracy of the docking procedure was determined by comparing the lowest energy
pose of the co-crystallized ligand predicted by the object scoring function, Glide score,
and an experimental binding mode as determined by X-ray crystallography. Extra
precision Glide procedure was validated by removing the co-crystallized ligand from the
binding site and redocking the ligand. The hydrogen bonding interactions and the root
mean square deviation (RMSD) between the predicted conformation and the observed X-
ray crystallographic conformation was used to analyze the results.
71
Experimental
3.2.5 Glide ligand docking
The docking of the molecules were carried out using the prepared receptor grid and the
ligand molecules. The favorable interactions between the ligand and receptor were scored
using Glide. All the docking calculations were performed using extra precision (XP) and
OPLS 2005 force field. The program was run in the flexible mode which automatically
generates conformations for each input ligand. The poses generated were run through a
series of filters to evaluate the receptor-ligand interactions. The initial filter tests the
spatial fit of the ligand to the defined active site and examines the ligand –receptor
interactions using a grid based method patterned after the empirical ChemScore function.
The algorithm recognizes hydrophobic interactions, hydrogen bonding, metal-ligand
interactions. Poses that pass this initial screening enter the final stage which involves
evaluation and minimization of a grid approximation OPLS nonbonded ligand-receptor
interaction energy. Finally the minimized poses were rescored using Glide Score
function. Glide Score is based on chemscore, but includes a steric-clash term, adds buried
polar terms to penalize electrostatic mismatches and has modifications to other terms:
GScore = 0.065 * vdw + 0.1130 * Coul + Lipo +Hbond + Metal + BuryP + RotB +
Site
Where, Vdw = Vander waals energy, Coul = Coloumb energy, Lipo = Lipophilic term,
Hbond = Hydrogen Bonding term, Metal = metal binding term, BuryP = penalty for
buried polar groups, RotB = Penalty for freezing rotatable bonds, Site = Polar
interactions in the active site.
72
Experimental
3.2.6 General procedure for molecular modeling studies for the identification of
potent ligands
Docking studies on ligands were carried out in the active site using Glide. The ligands
prepared were docked using High-Thorughput Virtual Screening (HTVS) which is the
least computationally intense process intended for rapid screening of ligands. From this,
ligands were selected for further steps based on their Glide energy and ranking. The top
100 ligands were selected and docked in the standard precision (SP) mode from which
30 ligands were selected and docked using extra precision (XP). From this result
obtained the ligands with the best score were selected.
3.3 Synthesis and Purification
3.3.1 General procedure for preparation of quinolines
Aniline (1.12g, 4.46mmol) was heated with 6M HCl (22.4mL) at 100°C (bath
temperature). Toluene (5.8mL) and then crotonaldehyde (0.74mL, 8.92mmol) were added
dropwise at 100°C. Stirring was continued for 2h at 100°C and the mixture was allowed
to cool to room temperature. The lower aqueous layer was separated and neutralized with
aqueous NaOH. The neutralized layer was extracted with ethyl acetate (3X5mL). The
ethyl acetate layer was dried and concentrated to afford the crude 2-methylquinoline. The
crude product was purified using flash column to give 70% yield of the pure compound.
73
Experimental
3.3.2 General procedure for preparation of quinoline-2-carboxylic acids
Selenium dioxide (0.36g, 3.3mmol) was added to a solution of 2-methylquinoline (0.63g,
3.1mmol) in dry 1,4-dioxan (30mL). The reaction mixture was refluxed for 2h and then
cooled to room temperature and filtered. The filtrate was evaporated to give a residue
which was dissolved in formic acid (1mL) and cooled to 0°C. Hydrogen peroxide (1.7mL
of 30% solution in water, 15.5mmol) was slowly added and the mixture was allowed to
stand overnight between 0°C and 10°C. The precipitate formed was collected by
filtration, washed with cold water and dried to obtain 3-hydroxyquinaldic acid as an
yellow solid. This was then purified using flash column by elution with CHCl3 to get 75%
yield of the pure compound.
3.3.3 General procedure for preparation of 1,2,3,4-tetrahydroquinoline-2-
carboxylic acids
Quinaldinic acid (1g, 5.77mmol) in methanol (6mL) was hydrogenated over platinum
oxide (0.3g) under atmospheric pressure at room temperature until the theoretical amount
of hydrogen was consumed. The mixture was filtered through celite and the filtrate was
concentrated and used as such for the next step.
74
Experimental
3.3.4 General procedure for preparation of N-nitroso quinoline-2-carboxylic acids
In a 250mL round bottom flask tetrahydroquinoline-2-carboxylic acid (4.60g, 19.1mmol),
THF (10mL), NaNO2 (1.51g, 22.0 mmol) and HCl (3M, 8.8mL) were placed. The
reaction mixture was stirred for 18h at room temperature. The completion of the reaction
was confirmed using TLC. The reaction mixture was concentrated and used as such for
the next step.
3.3.5 General procedure for preparation of sydnoquinolines
The residue obtained from scheme 3.3.4 (3.6g) was treated with acetic anhydride (30mL)
and pyridine (2mL). The completion of reaction was monitored using TLC. Water was
added to the reaction mixture to neutralize the acetic anhydride and extracted with ethyl
acetate (3X5mL). The ethyl acetate layer was dried and concentrated and purified using
flash column to get 70% yield of the pure compound. The structures and data of the
sydnoquinolines obtained are given in Table 3.
75
Experimental
Table 3: Data on 4,5-dihydro[1,2,3]oxadiazolo[3,4-a]quinolin-10-ium-3-olate and its

derivatives
Comp Molecular Rf
Structure IUPAC Name formulae/
no. value
Weight
4,5-dihydro[1,2,3]oxadiaz-
C10H8N2O2
21 -olo[3,4-a]quinolin-10- 0.48
188.1827
ium-3-olate
7-methoxy-4,5-dihydro-
C11H10N2O3
22 [1,2,3]oxadiazolo[3,4- 0.50
218.2087
a]quinolin-10-ium-3-olate
7-fluoro-4,5-dihydro- C10H7FN2O
23
[1,2,3]oxadiazolo[3,4- 2 0.50
a]quinolin-10-ium-3-olate 206.1732
7-methyl-4,5-dihydro-
24 C11H10N2O2
[1,2,3]oxadiazolo[3,4- 0.60
202.2093
a]quinolin-10-ium-3-olate
76
Experimental
3.3.6 General procedure for preparation of pyrazoloquinolines
A solution of the sydnone (0.05g) and ethylphenylacetylene (0.05g) in Xylene (2mL) was
stirred under N2 atmosphere at reflux for 4-5h. The solvent was removed in vacuo to give
the crude product. Further purification by flash chromatography on silica gel gave the
product 66% yield of the pure compound.
The structures and data on pyrazoloquinolines are given in Table 4.
Table 4: Data on 4,5-dihydropyrazolo[1,5-a]quinolines
Comp Molecular Rf
Structure IUPAC name
no. formulae/weight value
ethyl 3-
phenyl-4,5-
dihydropyrazo
C20H18N2O2 0.40
26 lo[1,5-
318.3691
a]quinoline-2-
carboxylate
ethyl 7-
methoxy-3- C21H20N203
27 phenyl-4,5- 348.3951
0.50
dihydropyrazo
lo[1,5-
a]quinoline-2-
carboxylate
77
Experimental
28
ethyl 7-fluoro- C20H17FN2O2
3-phenyl-4,5- 0.50
dihydropyrazo 336.3596
lo[1,5-
a]quinoline-2-
carboxylate
ethyl 7-
methyl-3-
phenyl-4,5- C21H20N2O2
29 dihydropyrazo 0.54
lo[1,5- 332.3957
a]quinoline-2-
carboxylate
dimethyl 4,5-
dihydropyrazo
lo[1,5- C15H14N2O4
31 0.52
a]quinoline- 286.2827
2,3-
dicarboxylate
dimethyl 7-
methoxy-4,5-
dihydropyrazo C16H16N2O5
32 lo[1,5- 0.48
2,3-
dicarboxylate
78
Experimental
dimethyl 7-
fluoro-4,5-
dihydropyrazo C15H13FN2O4
33 lo[1,5- 0.50
2,3-
dicarboxylate
dimethyl 7-
methyl-4,5-
34 lo[1,5- 0.46
2,3-
dicarboxylate
Benzyl 4,5-
36 lo[1,5- 0.62
a]quinoline-3- 304.3425
carboxylate
Benzyl 4,5-
40 lo[1,5- 0.58
a]quinoline-2- 304.3425
carboxylate
79
Experimental
benzyl 7-
methoxy-4,5-
37 0.64
lo[1,5- 334.3685
a]quinoline-3-
carboxylate
benzyl 7-
methoxy-4,5-
41 0.59
lo[1,5- 334.3685
a]quinoline-2-
carboxylate
Benzyl 7-
fluoro-4,5-
dihydropyrazo C19H15 FN2O2
38 0.60
lo[1,5- 322.3330
a]quinoline-3-
carboxylate
Benzyl 7-
fluoro-4,5-
dihydropyrazo C19H15 FN2O2
42 0.55
lo[1,5- 322.3330
a]quinoline-2-
carboxylate
80
Experimental
Benzyl 7-
methyl-4,5-
39 0.60
lo[1,5- 318.3692
a]quinoline-3-
carboxylate
Benzyl 7-
methyl-4,5-
43 0.57
lo[1,5- 318.3692
a]quinoline-2-
carboxylate
3.4 In vitro cell proliferation MTT assay
3.4.1 Principle
Measurement of cell viability and proliferation forms the basis of numerous in vitro
anticancer assays. The yellow coloured MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) is reduced to the purple coloured formazan in living cells.
A solubilization solution (usually either dimethyl sulfoxide, an acidified ethanol solution,
or a solution of the detergent sodium dodecyl sulfate in diluted hydrochloric acid) is
added to dissolve the insoluble purple formazan product into a colored solution. From the
absorbance of this colored solution the product obtained can be quantified by measuring
at a certain wavelength (usually between 500 and 600 nm) using a spectrophotometer.
81
Experimental
The absorption maximum is dependent on the solvent employed. The intensity of the
colour of the product is directly proportional to the number of living cells in the culture.
Reduction of MTT into formazan crystals by mitochondrial dehydrogenase.
3.4.2 Maintenance of cell lines
The cell lines were maintained using Eagle’s MEM supplemented with 10% FBS and 50
μg/mL gentamycin sulphate at 37°C in CO2 incubator in an atmosphere of humidified 5%
CO2. The cells were maintained by routine sub culturing in 25 cm2 tissue culture flasks.
3.4.3. Sub culturing process of cell lines
· The culture media from the flasks containing the monolayer culture was aspirated
and washed with sterile phosphate buffered saline (PBS).
· To the flasks, 2 mL of 0.1% trypsin-EDTA solution was added. After few seconds
it was aspirated and the flask was kept in incubator for 2-3 minutes for cell
detachment.
· The flasks were then removed from the incubator and the cell detachment was
confirmed by observing under an inverted microscope (Olympus).
82
Experimental
· Once the cells were completely detached from the flasks 2-3 mL of MEM media
containing 10% FBS was added and mixed well.
· Cell viability was checked with a small sample of the suspension by tryptan blue
dye exclusion test.
· From the stock cell suspension, 1x104 viable cells/mL suspended in media were
seeded in 25 cm2 tissue culture flask containing about 4 mL of fresh media and
incubated until the flasks attained 60-70% confluence.
3.4.4. Preservation of the tumor cells
Tumor cells from the first and second passage of transplantation were stored in liquid
nitrogen in cryovials at a concentration of 1x106 cells/mL containing respective media
supplemented with 20% serum and 10% DMSO as a preservative. This constitutes the
tumor bank. After every ten passages, the tumor cells were discarded and new passage
was started using the original tumor cells from the tumor bank.
3.4.5. Trypsinisation
To obtain a single cell suspension from monolayer culture, cells were dislodged from the
culture flasks by trypsinisation.
· From a 60-70% confluent flask, the culture media was aspirated out using
micropipette. Cells were washed with 3 mL of PBS to remove the trace amount of
media.
83
Experimental
· To each culture flask 2 mL of trypsin-EDTA was added and after few seconds it
was aspirated and the flask was kept in the incubator for 3-4 minutes for cell
detachment.
· The culture flasks were observed under an inverted microscope to ensure that the
cells were completely dislodged.
· Trypsin activity was stopped by adding 2-3 mL media containing 10% FBS.
3.4.6 In vitro cell proliferation MTT assay method
The selected compound was dissolved in 1mL of distilled dimethylsulphoxide (DMSO)
and the volume was made up to 10mL with maintenance medium to obtain a stock
solution of 1mg/mL concentration, sterilized by filteration and further dilutions were
made from the stock solution. The cytotoxicity assay was carried out using 0.01mL of
cell suspension, containing 10,000 cells/well of a 96 well microtitre plate. Fresh medium
containing different concentrations of the test compounds were added after 24h of partial
monolayer. Control cells were incubated with the test compounds and MEM. The
microtitre plates were incubated at 37°C in a humidified incubator with 5% CO2 for a
period of 72h. MTT solution (20µL, 2mg/mL in PBS) was added to the plates and
incubated for 4h at 37°C. MTT- formazon crystals formed were dissolved in 100 µL of
DMSO and the optical density was read with a microtitre plate reader at 570nm. The
percentage cytotoxicity of each compound at various concentrations was calculated using
the following equation;
84
Results and Discussion
4. RESULTS AND DISCUSSION
4.1 Docking Studies
The Ramachandran diagram was studied to visualize the backbone dihedral angle of the
amino acid residues in the protein (Figure 10). The plot showed about 87.6% residues in
the most favorable regions.
Figure 10: Ramachandran Plot- 1ZXM
The validation results of Glide docking procedure for topo IIα revealed a very good
agreement between the localization of the ligand upon docking as compared to the crystal
structure. The re-docked ligand, phosphoaminophosphonic acid-adenylate ester (ANP),
showed similar hydrogen bonding interactions with Asn91, Asn120, Ser148, Ser149,
Asn150, Arg162, Asn163, Gly164, Tyr165, Gly166, Ala167, Lys168, Gln376, Lys378
and Mg2+ residues of the protein, suggesting the reliability of the Glide docking in
85
reproducing the experimentally observed binding mode for ANP (Figure 11). The
parameters set for Glide docking are, therefore, reasonable to reproduce the X-ray
structure.
Figure 11: Hydrogen bonding interactions of ANP, the co-crystallised ligand.
A series of 180 molecules were designed for both sydnoquinolines and 4,5-
dihydropyrazoloquinolines. All these molecules were screened in silico for their ability to
inhibit the topo IIα at the ATP site. Among them a few of them showed a good docking
score (Table 5). Salvicine, a well known potent topo IIα inhibitor, was used as the
standard. Salvicine is currently in Phase II clinical trials. The docking scores of the
designed molecules were compared with the score of salvicine.
86
Table 5: Docking parameters of selected ligands.
Title Docking score Glide energy

C49 -7.384656 -45.113769
C36 -7.088328 -52.495523
C45 -7.064577 -42.567197
Salvicine -6.909206 -41.849454
C48 -6.801888 -48.801266
C37 -6.757759 -50.040877
C44 -6.608523 -55.093816
C41 -6.320824 -47.07366
C39 -6.195273 -52.644247
C40 -5.948458 -49.987325
C43 -5.462206 -47.60748
C47 -5.460709 -46.249083
C35 -5.322588 -48.209609
C34 -5.007437 -40.726292
C46 -4.3637 -31.648829
C42 -4.040974 -30.734029
C38 -1.193795 -32.451712
87
Both the R and S isomers of salvicine were docked to determine which isomer binds
better with the ATP site. It was observed that the S-isomer (Figure 12) had a better score
(-6.90921) than that of the R-isomer (-4.5461).
Figure 12: Hydrophobic interactions of salvicine-S in the binding site of topo IIα
The molecules used for docking were designed based on the basic scaffold given below.
The results of the docking studies revealed that on the sydnoquinoline ring (3) the change
in the functional group R does not bring any major change to the binding affinity. The
presence of the sydnone ring attached to the tetrahydroquinoline enhances the rigidity of
the molecule. The docking score of the sydnoquinoline was found to be -4.046974.
Although the change in the substituents bring about a slight variation in the glide score, it
was not significant. The scores of the pyrazoloquinoline (1) were found to be higher with
88
the presence of a phenyl ring attached to the nucleus at the R1 position (Figure 13). This
could probably be due to the enhanced resonance in the aromatic ring system. The
presence of an electron donating group attached to the –COOH was found to enhance the
binding affinity. On increasing the length of the side chain increased binding affinity than
the standard selected with a glide score of -7.3846. In the case of the pyrazolines (2) the
scores were comparatively low, probably because of the absence of aromaticity in the
pyrazole ring. The aromaticity of the ring is thus a crucial factor for binding.
The results of the docking studies showed very good glide score indicating a good
interactions with the ATP binding site (Figure 13-15).The docking scores revealed that
the designed molecules have good hydrogen bonding interactions with the important
amino acids in the ATP binding site. The molecules were found to interact with Asn 91,
Ser 148, Ser 149, Asn 150 Ala 167 and Lys 168 residues. The scores of a few designed
molecules were better than salvicine, whereas, others were comparable with that of
salvicine. A total of 20 molecules were selected for synthesis based on the glide scores.
The glide scores of the ligand C49 (-7.3846) were higher to that of salvicine and that of
C48 (-6.80188) is similar to the standard, salvicine. In comparison with salvicine, the test
molecules, C49, C36 and C35 showed better glide score and glide energy.
89
Figure 13 : Hydrophobic interactions of the ligands in the binding site of topo IIα
Figure 14: Hydrophobic interactions of the ligands in the binding site of topo IIα
90
Figure 15: Hydrophobic interactions of the ligands in the binding site of topo IIα
4.2 ADME studies
QikProp, the prediction program designed by Prof. William L. Jorgensen (Jorgenson et
al.,2004), was used to calculate ADME (Absorption, Distribution, Metabolism, and
Excretion) properties. Qikprop is quick, accurate and predicts physically significant
descriptors and pharmaceutically relevant properties of organic molecules. Ligprep
minimized ligands were given as a source in Qikprop 3.2. Qikprop modules provide the
ranges of molecular predicting properties for comparing the properties of a particular
molecule with those of 95% of known drugs. This helps in screening the proposed
molecules for various properties “drug-like” molecules should follow. Using the
compounds designed by SBDD, the physiochemical properties were, therefore, calculated
in Qikprop simulation. All the compounds obeyed the Lipinski's rules: molecular weight
below 500 Da, hydrogen bond donor (less than five) and acceptor (less than ten). Also
91
QPlogP o/w (octanol/water partition coefficient) for all the compounds was less than five.
Total solvent accessible surface area (SASA), hydrophobic component of the SASA
(FOSA) and hydrophilic component of the SASA (FISA) were analyzed for the
compounds that were abiding the ranges in Qikprop physiochemical properties.
Qualitative human oral absorption was predicted. Polar nitrogen and oxygen van der
waals surface area (PSA) of SBDD compounds fullfilled the limit in physicochemical
calculations. All the compounds satisfied the values of partition coefficient of octanol/gas
(QPlogPoct), water/gas (QPlogPw) and brain/blood (QPlogBB). Aqueous solubility
(QPlogS) and skin permeability (QPlogKp) predicted for ligands also lie in the allowed
solubility and permeability range.
4.3 Synthesis and Characterization
Scheme 49 gives the synthesis of 4,5-dihydro-[1,2,3]oxadiazolo[3,4-a]quinolin-10-
ium-3-olates:
92
2-Methylquinoline was synthesized by Doebner Miller reaction. In this method the
reaction takes place in a two phase solvent system which reduces polymerization of the
aldehyde (Matsugi et al.,2000). Aniline and crontonaldehyde were heated to reflux along
with 6M HCl and toluene for 3h. The product, 2-methyl quinoline, was isolated in good
yields after purification by flash column. The solvent system used was PE:EtOAc (7:3).
The structure of the product was confirmed using 1H NMR and mass. The proton NMR
reveals, three methyl protons (-CH3) at δ 2.75 ppm and 6 aromatic protons appear in the
region δ 7.26 - 8 ppm. In the mass spectra M+1 (144 amu) is observed.
Mechanism of for step 1 of Scheme 49.
2-Methyl quinoline obtained was oxidized to quinoline-2-carboxylic acid using selenium
dioxide and dioxan followed by hydrogen peroxide and formic acid (Riego et al.,2005).
This reaction takes place in two stages. First, conversion of the methyl group of 2-methyl
quinoline to an aldehyde on treatment with SeO2 and dioxan, followed by conversion to
the acid when treated with H2O2 and HCOOH. Generally to carry out the oxidation of the
methyl group, a strong oxidizing agent like KMnO4 would be used. But oxidation with
93
KMnO4 leads to cleavage of the double bonds and hence KMnO4 was not used. The
quinoline-2-carboxylic acid obtained was purified by eluting with CHCl3 to obtain the
pure product. The product was confirmed using 1H NMR which shows 6 aromatic protons
between δ 7.72-8.43 ppm.
Mechanism of step 2 of Scheme 49
The double bond of the quinoline-2-carboxylic acid was reduced to afford 1,2,3,4-
tetrahydroquinoline-2-carboxylic acid. This was carried out using platinum oxide as the
catalyst (Kim et al.,2007). The reaction was carried out under hydrogen atmosphere. The
product obtained was used as such for the next step. Efforts were made to isolate the pure
product but the product was found to degrade on eluting with the solvent during column
chromatography. Hence, the catalyst was filtered out and the solvent was removed and
the residue was dried thoroughly and used for the next step. The formation of the product
was confirmed by using ninhydrin solution as the staining agent.
94
Mechanism of step 3 of Scheme 49.
The tetrahydroquinoline was converted to its N-nitroso derivative using NaNO2, 3M HCl
and THF (Banerjee et al.,2010). The N-nitroso product was confirmed using ninhydrin
solution as the staining agent. The N-nitroso derivative was isolated by extracting with
EtOAc which was removed in vacuo and dried thoroughly to get a residue which was
used as such for the next reaction.
Mechanism of step 4 of Scheme 49.
The synthesis of the sydnoquinoline was carried out by treating the N-nitroso derivative
with Ac2O and a drop of pyridine. In this reaction, acetic anhydride and pyridine react to
form 1-acetylpyridinium, which in turn reacts with the N-nitroso quinoline-2-carboxylic
acid to form an acetyl derivative of the N-nitroso quinoline-2-carboxylic acid. With the
elimination of –OAc, cyclisation takes place to give the sydnoquinoline. The reaction
mixture was stirred overnight and purified using flash column to give the sydnone in
95
good yield. The reaction was carried out under N2 atmosphere. The mechanism for the
above reaction may be given as follows;
Mechanism of step5 of Scheme 49.
The following is the physical and analytical data of the compounds 4,5-dihydro[1,2,3]
oxadiazolo[3,4-a]quinolin-10-ium-3-olates;
4,5-dihydro[1,2,3]oxadiazolo[3,4-a]quinolin-10-ium-3-olate(21): brown sticky solid;
Rf : 0.48 (hexane/EtOAc, 5:5); Purified using flash column; Yield: 70%; IR (neat) :
2936, 2863, 1739, 1507, 1243, 767, 722cm-1; 1H NMR (400 MHz, CDCl3) δ ppm: 7.94-
7.96(d, 1H, J=8Hz), 7.43-7.55(m, 3H), 3.06-3.10(t, 2H), 2.93-2.97(t, 2H);13C NMR (100
96
MHz,CDCl3):167.57, 131.69, 130.11, 129.52, 128.29, 117.57, 102.48, 24.7, 16.77; HR-
MS m/z:calcd for C10H8N2O2Na [M+Na]: 211.0483; found: 211.0491.
7-methoxy-4,5-dihydro-[1,2,3]oxadiazolo[3,4-a]quinolin-10-ium-3-olate (22): Light
brown sticky solid; Rf : 0.5 (hexane/EtOAc, 5:5); Purified using flash column; Yield: 72%;
IR (neat) : 2936, 2863, 1741, 1717, 1265, 767, 722cm-1; 1H NMR (400 MHz, CDCl3) δ
ppm: 7.87-7.90(d, 1H, J=12Hz), 6.93-6.95(m,1H, J= 8Hz), 6.89(m, 1H), 3.88(s, 3H),
3.01-3.04 (t, 2H), 2.91-2.95 (t, 2H);13C NMR (100 MHz, CDCl3): 167.50, 161.84, 132.03,
119.22, 114.35, 113.35, 101.43,55.73, 25.22, 16.88; HR-MS m/z:calcd for C11H10N2O3Na
[M+Na]: 241.0589; found: 241.0596.
7-Fluoro-4,5-dihydro-[1,2,3]oxadiazolo[3,4-a]quinolin-10-ium-3-olate (23): Brown
sticky solid; Rf : 0.5 (hexane/EtOAc, 5:5); Purified using flash column; Yield: 75%; IR
(neat) : 3085, 1739, 1495, 1249,767, 722cm-1; 1H NMR (400 MHz, CDCl3) δ ppm: 7.96-
13
7.99(s, 1H), 7.15-7.19(m, 2H), 3.07-3.11 (t, 2H), 2.95-2.98 (t, 2H); C NMR (100 MHz,
CDCl3):167.36, 165.11, 162.58, 133.05, 132.96, 127.64, 119.97, 119.88, 116.69, 116.46,
115.77, 115.53, 102.10, 24.96, 16.65;HR-MS m/z:calcd for C11H10N2O3Na [M+Na]:
229.0389; found: 229.0388.
7-Methyl-4,5-dihydro-[1,2,3]oxadiazolo[3,4-a]quinolin-10-ium-3-olate(24): Brown
sticky solid; Rf :0.6 (hexane/EtOAc, 5:5); Purified using flash column; Yield: 67%; IR
(neat) : 1743, 1739, 1493,1238, 767, 722cm-1; 1H NMR (400 MHz, CDCl3) δ ppm: 7.81-
7.83(d, 1H, J=8Hz), 7.23-7.27(m, 2H), 3.00-3.04 (t, 2H), 2.90-2.94 (t, 2H), 2.43 (s,
3H);13C NMR (100 MHz, CDCl3):167.57, 142.41, 129.97, 128.97, 128.92, 117.41,02.07,
24.77, 21.43, 16.89;HR-MS m/z:calcd for C11H10N2O2Na [M+Na]: 225.0640; found:
225.0641.
97
Figure 17: MASS spectra of Compound 21.
98
99
100
101
102
103
104
105
Scheme 50 gives the synthesis of Ethyl 3-phenyl-4,5-dihydropyrazolo[1,5-

a]quinoline-2-carboxylates:
The cycloaddition reaction of the sydnones was carried out with three different alkynes.
The reaction takes place via [3+2] cycloaddition. The elimination of CO 2 results in the
final pyrazole ring. The reaction of sydnone with ethylphenyl acetylene results in only
one product, ethyl 3-phenyl-4,5-dihydropyrazolo[1,5-a]quinoline-2-carboxylate.
Mechanism of Scheme 50.
This was confirmed using Mass, IR, 1H NMR, 13

C NMR, 2D-NMR. The mass spectra
shows the [M+H] peak of 319.1449 amu. The proton NMR shows, 3 methyl protons of
the terminal –CH3 at δ 1.22-1.26 ppm as a triplet due to the adjuscent –CH2. The –CH2
appears as a quartet at δ 4.22-4.27 ppm due to the –CH3. The –CH2 of the
106
tetrahydroquinoline ring appear as triplets at δ 3.42-3.38 ppm, δ 3.04-3.01 ppm,

13
respectively. The C NMR shows aromatic carbon at 163.71,
154.06,143.98,135.68,132.97,129.52,128.41,128.29,127.83,127.68,126.72,126.15,116.63,
109.18 and aliphatic carbons at 60.05,24.69, 21.55,14.11.
The 13C NMR shows all the aromatic protons appear between δ 7.70-7.18 ppm. The 2D-
NMR spectrum revealed the interaction between the proton of the tetrahydroquinoline
ring and the proton of the phenyl ring as shown below.
The following are the physical and analytical data for Ethyl 3-phenyl-4,5-dihydropyra-
-zolo[1,5-a]quinoline-2-carboxylates;
Ethyl 3-phenyl-4,5-dihydropyrazolo[1,5-a]quinoline-2-carboxylate (26): White solid;
Rf : 0.4 (hexane/EtOAc, 9:1); m.p.: 87-90°C; Yield:66 %; IR (neat) : 2931, 1719, 1708,
1418, 1288, 1155, 1130, 1065, 765, 696 cm-1;1H NMR (400 MHz,CDCl3) δ ppm: 7.70-
7.72(m, 1 H), 7.40-7.45(d, 2 H), 7.37-7.39(m, 3 H), 7.28-7.36(m, 1H), 7.25-7.26(d, 1H),
107
7.18-7.22(d, 1H, J=8Hz), 4.22-4.27 (q, 2H),3.42-3.38 (t, 2H),3.04-3.01(t, 2H),1.22-
1.26(t,3H);13C NMR(100 MHz, CDCl3):163.71, 154.06, 143.98, 135.68, 132.97, 129.52,
128.41,128.29,127.83,127.68, 126.72, 126.15, 116.63, 109.18, 60.05, 24.69, 21.55,14.11;
HR-MS m/z: calcd for C20H18N2O2H [M+H]: 319.1447; found: 319.1449.
7-Methoxy-3-phenyl-4,5-dihydropyrazolo[1,5-a]quinoline-2-carboxylate (27): White
solid; m.p.: 128-130°C; Rf : 0.5(hexane/EtOAc, 9:1); Yield:68 %; IR (neat) : 2951 ,1741,
1720, 1736, 1477, 1294, 1226, 1077, 760 cm-1;1H NMR (400 MHz,CDCl3) δ ppm: 7.89(d,
1H), 7.70-7.72(d,2H, J=8Hz), 7.40-7.42 (d, 2H, J=8Hz), 7.26(s, 2H), 6.82-6.87(t, 1H),
4.23-4.25(q, 2H), 3.83(s, 3H), 2.98-3.02(t, 2H),1.22-1.26(t, 3H);13C NMR(100
MHz,CDCl3):163.76,157.83,153.69,143.00,129.51, 128.29,127.64,117.79,113.94,112.54,
108.79,59.95,55.52,25.08,21.55,14.10;HR-MS m/z: calcd for C21H20N2O3Na[M+Na]:
317.1372; found: 371.1372.
Ethyl 7-fluoro-3-phenyl-4,5-dihydropyrazolo[1,5-a]qunoline-2-carboxylate (28):
White solid; m.p.: 20-121°C; Rf :0.5(hexane/EtOAc, 9:1); Yield:70 %; IR (neat) : 3372,
2018, 1701, 1158, 1018, 764 cm-1;1H NMR (400MHz,CDCl3) δ ppm: 7.91-8.00(m, 1H),
7.70-7.71(m, 2H), 7.26(m, 3H), 6.99-7.05(m, 2H), 4.22-4.27(q, 2H),3.38-3.42(t, 2H),3.00-
3.04(t, 2H),1.22-1.25(t, 3H);13C NMR(100 MHz,CDCl3):163.59,161.91,159.46,154.06,
143.38, 132.84, 132.01, 129.48, 128.86, 128.45, 127.69, 118.23, 115.31, 115.08, 114.59,
114.36, 109.22, 60.07, 24.84, 21.35, 14.09; HR-MS m/z: calcd for C20H17 FN2O2H[M+H]:
337.1352; found: 337.1354.
108
Ethyl 7-methyl-3-phenyl-4,5-dihydropyrazolo[1,5-a]quinoline-2-carboxylate (29):
White solid; m.p.:79-80°C, Rf : 0.54(hexane/EtOAc, 9:1); Yield:63 %; IR (neat) : 2980,
1715,1503,1147cm-1;1H NMR (400 MHz,CDCl3) δ ppm:7.86(m, 1H), 7.84(m,2H), 7.71-
7.72(m,3H), 7.12-7.14(d,1H,J=8Hz),7.07(s,1H),4.23-4.24(q,2H),3.35-3.39(t,2H),2.95-
2.99(t,2H),2.35(s,3H),1.24(q,3H);13C NMR(100 MHz,CDCl3):163.76, 153.84, 143.63,
135.93, 133.46, 133.06,129.53, 128.89, 128.32, 127.67, 116.47, 108.95, 24.70, 21.62,
21.01, 14.21;HR-MS m/z: calcd for C21H20N2O2H[M+H]: 333.1603; found: 333.1601.
109
110
Figure 35: NOESY spectra for Compound 26.
111
112
113
114
115
116
Scheme 51 gives the synthesis of Ethyl dimethyl 4,5-dihydropyrazolo[1,5-

a]quinoline-2,3-dicarboxylates:
On reaction of the sydnoquinoline with dimethyl acetylene dicarboxylate only one single
product was formed and this was confirmed using 1H NMR, 13

C NMR and Mass. The
mass spectra shows an [M+Na] peak of 309.0854.The proton NMR shows methyl
protons of the –COOCH3 at 3.98 and 3.88 as singlets. The –CH2 of the
tetrahydroquinoline ring were split into triplets and appeared at δ 3.33-3.29 ppm and δ
117
13
3.02-2.98 ppm. The aromatic protons appear between δ 7.97-7.21 ppm. The CNMR
shows 9 aromatic carbon atoms at 144.53, 143.27, 135.17, 128.44, 127.98,127.03,
117.04, 111.25, 4 aliphatic carbon atoms at 52.66,51.80,24.46,20.94 and carboxylic
carbons at 162.89 and 162.80.
The following are the physical and analytical data for dimethyl 4,5-dihydropyrazolo[1,5-
a]quinoline-2,3-dicarboxylates;
Dimethyl 4,5-dihydropyrazolo[1,5-a]quinoline-2,3-dicarboxylate (31): White solid;
m.p.: 127-128°C, Rf : 0.5 (hexane/EtOAc, 8:2); Yield:80%; IR (neat) : 2951, 1720, 1736,
1477, 1294, 1226, 1077, 760 cm-1;1HNMR (400MHz, CDCl3) δ ppm: 7.95-7.97(d, 1 H,J
=8Hz),7.33-7.37(m, 1 H),7.21-7.28 (m, 2 H), 3.98(s, 1 H), 3.88(s, 1 H), 3.20-3.33(t, 2 H),
2.98-3.02 (t, 2 H);13C NMR(100 MHz,CDCl3):162.89,162.80,144.53,143.27,135.17,
128.44,127.98,127.03,117.04,111.25,52.66,51.80,24.46,20.94; HR-MS m/z: calcd for
C15H14N2O2Na [M+Na]:309.0851;found: 309.0854.
Dimethyl 7-methoxy-4,5-dihydropyrazolo[1,5-a]quinoline-2,3-dicarboxylate (32):
White solid;m.p.: 116-117°C, Rf: 0.48(hexane/EtOAc, 8:2);Yield: 80%;IR (neat) : 2916,
2847,2615,1717,1522,1223,1021, 772cm-1;1H NMR(400 MHz,CDCl3) δ ppm:7.87-7.89
118
(d,1H),6.85-6.89(d,1H,J=8Hz),6.80(s,1H),3.98(s,3H),3.87(s,3H),3.83(s,3H),3.27-3.31(t,
2H),2.95-2.99(t,2H);13CNMR(100MHz,CDCl3):162.91,158.44,143.99,142.36,128.96,
128.62,118.31,113.97,112.71,111.06,55.52,52.62,51.76,24.87,20.96;HR-MS m/z:calcd
for C16H16N2O5Na [M+Na]:339.0957; found: 339.0956.
Dimethyl 7-fluoro-4,5-dihydropyrazolo[1,5-a]quinoline-2,3-dicarboxylate(33): pale
brown solid; m.p.:110-111°C; Rf :0.5(hexane/EtOAc, 8:2); Yield: 80%; IR(neat) : 2918,
2954,1739,1731,1471,1225,1077cm-1;1H NMR(400 MHz,CDCl3)δppm:7.90-7.94(m,1H),
6.99-7.06(m,2H),3.98(s,3H),3.87(s,3H),3.29-3.33(t,2H),2.98-3.01(t,2H);13C NMR(100
MHz,CDCl3):162.76,162.65,162.32,159.86,144.49,142.69,131.43,129.29,129.21,118.79,
118.70,115.48,114.80,114.58,111.27, 52.64, 51.80,24.56,20.70;HR-MS m/z calcd for
C15H13FN2O4Na [M+Na]:327.0757; found: 327.0752.
Dimethyl 7-methyl-4,5-dihydropyrazolo[1,5-a]quinoline-2,3-dicarboxylate (34): pale
brown solid; m.p.: 82-83°;Rf : 0.46 (hexane/EtOAc, 8:2); Yield:80%; IR (neat) : 2954,
1742,1719,1476,1224,1079cm-1;1HNMR(400MHz,CDCl3)δppm:7.83-7.85(d,1H,J=8Hz),
7.14-7.16(d, 1H, J=8Hz),7.08(s,1H),3.98(s, 3H),3.87(s, 3H),3.27-3.31(t, 2H),2.94-2.97(t,
2H),2.36(s, 3H);13C NMR(100 MHz,CDCl3):162.88, 144.22, 142.96, 136.97, 132.95,
129.00, 128.49, 126.87, 116.90, 111.14, 52.63, 51.77, 24.48, 21.02.; HR-MS m/z:calcd
for C16H16N2O4Na [M+Na]:323.1008; found: 323.1012.
119
120
121
122
123
124
125
126
127
Scheme 52 gives the synthesis of benzyl 4,5-dihydropyrazolo[1,5-a]quinoline-3-

carboxylate:
The scheme shows the cycloaddition of sydnoquinoline with benzyl propiolate. The
reaction leads to two isomers A and B shown below. This was due to the less steric
hinderance on one side of the triple bond. Hence, the cycloaddition could result in both
isomers.
Both the isomers were isolated by column chromatograpy using PE:EtOAc as solvent
system. The products were confirmed using Mass, 1H NMR and 13

C NMR. The mass
spectra shows [M+Na] of 327.1107 for the isomer (A) and [M+H] of 305.1292 for the
isomer (B). The proton NMR shows the protons of the –CH2 in the tetrahydroquinoline
ring as triplets at δ 2.91 ppm and δ 3.01 ppm for isomer A and δ 2.96 ppm and δ 3.30
ppm for the isomer B. A single proton was present at δ 8.89 ppm for isomer A and δ 6.69
128
ppm for isomer B. This was used to differentiate between the isomers. The aromatic
13
protons are found between δ 7-8 ppm. The C NMR reveal aromatic carbons at
163.23,142.70,142.18,136.18,135.79,128.58,128.40,128.18,128.11,127.89,126.63,126.28,
116.50,111.27 and aliphatic carbons at 65.83,24.63,20.87 for isomer A and aromatic
carbons at 162.35,143.71, 139.78,135.97,128.51,128.33,128.18,127.90,127.12,126.37,
117.78,106.78 and aliphatic carbons at 66.52,25.39,21.26 for isomer B.
1
H NMR
13
C NMR
The orientation of the isomers were confirmed by 2D-NMR. Interactions between the
proton of the tetrahydroquinoline and H of the benzyl propiolate is observed for one
isomer (B) whereas no interaction is observed for the other isomer (A).
129
The following is the physical and analytical data of the compounds benzyl 4,5-dihydro
pyrazolo[1,5-a]quinolinecarboxylates;
Benzyl 4,5-dihydropyrazolo[1,5-a]quinoline-3-carboxylate (36): Light brown solid;
m.p.: 88-89°C; Rf : 0.62(hexane/EtOAc, 8:2); Yield:35%; IR (neat) :2928, 1719, 1577,
1253, 756cm-1;1H NMR (400 MHz, CDCl3) δ ppm:8.89(s,1H),8.61-8.64(d,1H,J=12Hz),
7.25-7.44(m,6H),7.18-7.21(m, 2H),5.32(s, 2H),3.33-3.36(t, 2H),2.99-2.96(t, 2H);13C
NMR(100 MHz,CDCl3):163.23, 142.70, 142.18, 136.18, 135.79, 128.58, 128.40, 128.18,
128.11,127.89,126.63,126.28,116.50,111.27,65.83,24.63,20.87;HR-MS m/z: calcd for
C19H16N2O2H [M+H]: 305.1290; found: 305.1292.
benzyl 4,5-dihydropyrazolo[1,5-a]quinoline-2-carboxylate (40): Light brown solid;
m.p.: 110-112°C;Rf : 0.58(hexane/EtOAc, 8:2); Yield:25%;1H NMR (400 MHz, CDCl3) δ
ppm: 8.05-8.03 (d,1H,J=8Hz), 7.46-7.48 (d, 2H, J=8Hz), 7.32-7.46 (m, 4 H), 7.17-7.25
(m, 2H), 6.69 (s,1H), 5.4(s, 2H), 3.01-3.04(t, 2H),2.95-2.98 (t, 2H);13C NMR(100
MHz,CDCl3): 162.35, 143.71,139.78, 135.97, 128.51, 128.33, 128.18, 127.90, 127.12,
126.37, 117.78, 106.78, 66.52,25.39,21.26; HR-MS m/z: calcd for C19H16N2O2Na
[M+Na]: 327.1109; found: 327.1107.
130
Benzyl 7-methoxy-4,5-dihydropyrazolo[1,5-a]quinoline-3-carboxylate (37): White
solid; m.p.: 89-90°C;Rf : 0.64 (hexane/EtOAc, 8:2); Yield:40%; IR (neat) : 2928, 1719,
1577,1253,756cm-1;1H NMR (400MHz,CDCl3)δ ppm: 8.02(s,1H),7.80-7.82(d,1H,J=8Hz),
7.33-7.44(m,5H),7.25(s,1H),6.84-6.87(d,1H,J=12Hz),5.32(s, 2H),3.82(s, 3H), 3.30-3.34
(t,2H),3.82(s,3H),3.30-3.34(t, 2H),2.92-2.96(t, 2H);13C NMR(100 MHz,CDCl3):163.31,
157.89, 141.78,136.24,129.65,128.57,128.15,128.08,117.64,114.03,112.60,110.93,65.77,
55.52,25.01,20.87; HR-MS m/z: calcd for C20H18N2O3Na[M+Na]:357.1215; found:
357.1216.
Benzyl 7-methoxy-4,5-dihydropyrazolo[1,5-a]quinoline-2-carboxylate (41): White
solid; m.p.: 106-107°C; Rf : 0.59(hexane/EtOAc, 8:2); Yield:30%;1H NMR (400 MHz,
CDCl3) δ ppm:7.95-7.97(m,1H),7.46-7.48(m,2H),7.32-7.39(m,3H),6.84-6.87(d,1H,
J=12),6.77-6.78(s,1H), 6.68(s,1H),5.40(s,2H),3.82(s,3H),2.00-3.02(t,2H),2.91-2.94
(t, 2H);13C NMR(100 MHz,CDCl3):162.42, 157.97,143.09,138.98,136.03,129.89,128.71,
128.49,128.31,128.15,118.25,113.95,112.60,106.64,66.44,55.50,25.73,21.29.; HR-MS
m/z: calcd for C20H18N2O3Na [M+Na]: 357.1215; found: 357.1216.
Benzyl 7-fluoro-4,5-dihydropyrazolo[1,5-a]quinoline-3-carboxylate (38): Pale brown
solid; m.p.:93-94°C; Rf : 0.60(hexane/EtOAc, 8:2); Yield:38%;1H NMR (400 MHz,
CDCl3) δ ppm: 8.03(s,1H),7.37-7.4(m,6H),7.25(m,2H),5.31(s,2H),3.32-3.34(t,2H),2.96-
2.98(t,2H);13C NMR(100 MHz,CDCl3):142.14, 136.12, 128.59, 128.21, 128.11, 118.20,
115.43,115.20,114.67,114.44,111.33, 65.88, 24.78, 20.67; HR-MS m/z: calcd for
C19H15FN2O2H [M+H]: 323.1196; found: 323.1197.
Benzyl 7-fluoro-4,5-dihydropyrazolo[1,5-a]quinoline-2-carboxylate (42): Pale brown
solid; m.p.: 112-113°C; Rf :0.55(hexane/EtOAc, 8:2); Yield:25%,IR (neat) :2918,1713,

131
1567,1503,1254,1239,1095,1055cm-1;1HNMR(400MHz,CDCl3)δppm:7.46-7.48(m,2H),
7.33-7.39(m,4H), 7.26(s, 1H),6.96-7.05(m, 2H),5.4(s, 2H),3.02-3.04(t, 2H),2.96-2.98(t,
2H);13C NMR(100Hz,CDCl3):162.25,162.01,143.69,139.25,135.92,128.51,128.33,118.80,
115.35,115.12,114.72,114.50,106.86,66.56,60.37; HR-MS m/z: calcd for C19H15FN2O2Na
[M+Na]: 345.0757; found: 345.1018.
Benzyl 7-methyl-4,5-dihydropyrazolo[1,5-a]quinoline-3-carboxylate (39): Pale brown
solid; m.p.:87-88 °C; Rf : 0.60(hexane/EtOAc, 8:2); Yield:40% IR (neat) : ,2952, 2921,
2119,1714,1561, 1507, 1256, 1237, 1096cm-1;1H NMR (400 MHz, CDCl3) δ ppm: 8.03
(s,1H), 7.76-7.78(d,1H, J=8), 7.34-7.44(m,5H), 7.25(s,1H), 7.13-7.15(d, 1H, J=8), 7.07
(s,1H),5.31(s,2H),3.30-3.32(t,2H),2.93-2.95(t,2H),2.35(s,3H);13C NMR(100MHz,CDCl3):
162.41,143.37,139.5,136.19,136.02,133.80,128.92,128.49,128.41,128.32,128.15,126.98,
116.89,106.67,66.96,25.39,21.34,20.99; HR-MS m/z: calcd for C20H18N2O2Na [M+Na]:
341.1266; found: 341.1265.
Benzyl 7-methyl-4,5-dihydropyrazolo[1,5-a]quinoline-2-carboxylate (43): Pale brown
solid; m.p.:126-127°C; Rf : 0.57(hexane/EtOAc, 8:2); Yield:30%;1H NMR (400 MHz,
CDCl3) δ ppm:7.91-7.93(d,1H, J=8Hz),7.46-7.48(m, 2H), 7.34-7.37(m,3H), 7.13-7.15
(d,1H,J=8Hz),7.05(s,1H),6.68(s,1H),5.41(s,2H), 2.99-3.01(t,2H),2.92-2.94(t,2H),2.34
(s,3H);13CNMR(100MHz,CDCl3):162.41,143.37,139.5,136.19,136.02,133.80,128.2,
128.49, 128.32,128.41,126.98,106.67,66.46,25.39,21.34,20.99; HR-MS m/z: calcd for
C20H18N2O2Na [M+Na]: 341.1266; found: 341.1262.
132
133
134
135
136

137
138
139
140
141
142
143
144
145
146
147
4.4 In vitro anticancer studies
The synthesized molecules were subjected to in vitro cytotoxicity studies against HEp-2,
MCF-7, HeLa and HBL-100 cell lines. 5-FU was used as the standard drug for
comparison. The cytotoxicity was performed by the MTT assay (Carmichael et al.,1987).
The results are given in Tables 6-9 and Figures 94-97.
Table 6 and Figure 93 gives the IC50 values of 4,5-dihydro[1,2,3]oxadiazolo[3.4-
a]quinolin-10-ium-3-olates. The IC50 value of 5-FU is 7.8 µg/mL, 11.7 µg/mL and 9.1
µg/mL for HEp-2, MCF-7 and HeLa cell lines, respectively. The IC50 values of the
sydnoquinolines range from 30 µg/mL to 100 µg/mL. The methoxy substituted
sydnoquinoline (22) is active against MCF-7 (30 µg/mL) and HeLa (30 µg/mL). The
fluoro substituted sydnoquinoline (23) is active against HeLa (30 µg/mL). Compounds 21
and 24 were found to be moderately active against all the cell lines.
Compound no. HEp-2 MCF-7 HeLa HBL-100 cells

µg/mL µg/mL µg/mL µg/mL
21 80 60 60 70
22 70 30 30 50
23 50 60 30 40
24 60 100 50 90
Table 6: IC50 (µg/mL) values for 4,5-dihydro[1,2,3]oxadiazolo[3,4-a]quinolin-10-ium-3-

olates against the selected human cancer and normal cell lines.
148
Figure 93: IC50 values of compounds 21-24
Table 7 and Figure 94 gives the IC50 values of phenyl-4,5-dihydropyrazolo[1,5-
a]quinoline-2-carboxylates. The IC50 values of the pyrazoloquinolines 26 to 29 range
from 20 µg/mL to 90 µg/mL. The fluoro derivative (28) is found to be active against
HEp-2 and HeLa with IC50 values of 30 µg/mL and 20 µg/mL, respectively. When the
cytotoxicity was checked against breast cancer cells (MCF-7), it is found to be less active
when compared with HEp-2 and HeLa. The other molecules are found to be moderately
active.
149
Compound no. HEp-2 MCF-7 HeLa HBL-100
26 50 40 70 60
27 40 30 40 50
28 30 90 20 70
29 70 50 70 80
Table 7: IC50 (µg/mL) values for phenyl-4,5-dihydropyrazolo[1,5-a]quinoline-2-

carboxylates against the selected human cancer cell lines.
Figure 94: IC50 values of compounds 26 to 29
150
Table 8 and Figure 95 gives the IC50 values of 4,5-dihydropyrazolo[1,5-a]quinoline-2,3-
dicarboxylates. The IC50 values for compounds 31 to 34 range from 40 µg/mL to 80
µg/mL. All the compounds are found to be active against HEp-2 cells with an IC50 value
of 40 µg/mL except 32 (70 µg/mL).
Compound no. HEp-2 MCF-7 HeLa HBL-100

31 40 50 70 80
32 70 40 70 80
33 40 70 70 80
34 40 50 40 60
Table 8: IC50 (µg/mL) values for dimethyl 4,5-dihydropyrazolo[1,5-a]quinoline-2,3-
dicarboxylates against the selected human cancer cell lines.
151
Table 9 and Figure 96 gives the IC50 values of benzyl 4,5-dihydropyrazolo[1,5-
a]quinolinecarboxylates. The synthesis of these molecules resulted in two isomers. This
was due to the less steric hinderance on one side of the alkyne used for the cycloaddition.
Both the isomers were evaluated for their anticancer activity. The IC50 values of the
evaluated molecules range from 20 µg/mL to 90 µg/mL. The methoxy substituted
pyrazoloquinoline, 41 shows good activity with an IC50 value of 20 µg/mL against MCF-
7 cells and 30 µg/mL against HEp-2 cells when compared to the standard, 5-FU with an
IC50 value of 11.7 µg/mL and 7.8 µg/mL, against MCF-7 and HEp-2 cells, respectively.
Compound MCF-7
HEp-2 cells HeLa HBL-100 cells
no. cells
ug/mL ug/mL ug/mL ug/mL
36 50 70 60 80
40 60 50 40 60
37 50 60 60 70
41 30 20 50 60
38 80 90 60 80
42 60 60 80 90
39 90 40 80 70
43 60 60 50 80
Table 9: IC50 (µg/mL) values for benzyl 4,5-dihydropyrazolo[1,5-
a]quinolinecarboxylates against the selected human cancer cell lines.
152
The in vitro results reveal that methoxy and fluoro derivatives show good activity when
compared to the compounds with other substituents. The pyrazoloquinolines show better
activity when compared to the sydnoquinolines. This could be due to the increased
functionalization of the pyrazole ring. Benzyl 7-methoxy-4,5-dihydropyrazolo[1,5-
a]quinoline-3-carboxylate is active against HEp-2 and MCF-7 cell lines with IC50 values
of 30 µg/mL and 20 µg/mL, respectively. In the methoxy substituted pyrazoloquinolines
with benzyl acetate group attached to the pyrazole nucleus seems to be essential for the
activity because an increase in the IC50 value is observed in the presence of these groups
in the basic scaffold.
A comparison between the in silico and in vitro studies reveal that the synthesized
compounds, with the phenyl and ethylcarboxylate groups exhibit good activity as
predicted by the docking scores.
The images of the cytotoxicity studies of compounds 23, 27, 28 and 41 are given in
Figure 97. These compounds show considerably good activity when compared to others.
153
The images show the morphological difference in the treated cells after 48h of exposure
to the synthesized molecules at their IC50 values. The images show a loss in morphology
and the complete detachment of the cells after treatment.
D
Figure 97 :(A)Images of HeLa cells: Control cells and cells treated with 23 (B) Images
of MCF-7 cells: Control and cells treated with 27 (C) Images of HeLa cells: Control cells
and cells treated with 28 (D) Images of MCF-7 cells: Control and cells treated with 41.
154
FUTURE PERSPECTIVES
In the present study all the synthesized compounds show moderate to good in
vitro cytotoxic activity. Compounds 22, 23, 28 and 41 possess potent in vitro
cytotoxic activity. It is worthwhile, therefore, to study the compounds synthesized
in the present investigation for their activity in vivo.
The synthesized molecules can be taken further to design and synthesize more
potent topo II inhibitors and subject them to detailed in vivo and toxicity studies.
A detailed topo II inhibiton studies may also be carried out. QSAR and CoMFA
studies on these molecules are under progress in order to identify the active
pharmacophore. This will be effective in designing more potent molecules.
155
Summary and Conclusions
5. SUMMARY AND CONCLUSIONS
Cancer is one of the most dreaded diseases. Multidisciplinary scientific investigations are
being made today to combat this disease, but perfect cure is yet to be brought into the
world of cancer medicine. The problems associated with the current cancer chemotherapy
are non-specific toxicity and multi drug resistance (MDR).
DNA topo II inhibitors are a major target for antineoplastic agents used in the treatment
of breast, lung, and prostate cancer, sarcomas, and hematological malignancies because
of the important role played by them in the survival of cells.
Sydnones and pyrazoles are known for their broad range of biological activities. 4-
Substituted sydnones and pyrazoloquinolines have been evaluated for their anticancer,
antiinflammatory, antihypertensive, antimicrobial, antidiabetic, DNA cleavage activity
and antitubercular activities.
Although pyrazoloquinolines have been widely studied, drugs based on this nucleus are
rare. Various synthetic routes for the synthesis of sydnones and pyrazoloquinolines have
been reported. A thorough review of literature, however, revealed that there are only a
few reports on the synthesis and biological evaluation of fused ring sydnones. Sydnones
can undergo cycloaddition reactions to give pyrazoles. Fused ring sydnoquinolines and
dihydropyrazolo[1,5-a]quinolines have not so far been explored thoroughly.
The objective of the present study was, therefore, to design, synthesize and evaluate some
novel sydnoquinolines and pyrazoloquinolines for their anticancer activity in vitro using
various cell lines.
The following are some of the important finding made from the present study;
156
· A total of 180 compounds including 4,5-dihydro[1,2,3]oxadiazolo[3,4-a]quinolin-
10-ium-3-olates and 4,5-dihydropyrazolo[1,5-a]quinolines were designed for the
target, topo II and docking studies were carried out for the designed molecules.
The scores were compared with that of salvicine, the known topo II inhibitor.
Molecules C49, C36 and C45 showed a glide score slightly higher than that of the
salvicine.
· Post docking analysis showed various interactions of the ligands with the protein.
The molecules C48, C37, C44, C41, C39, C40, C43, C47, C35, C34, C46 and
C42 showed good glide score. The scores of these molecules are in the range of
-7.3846 to -4.0409 as compared to salvicine which is -6.9092.
· The ADME properties of the designed molecules were analyzed in silico using the
Qikprop programme in Glide, Schrodinger. All the molecules showed good
ADME predictions for all the parameters although slight deviations were
observed for a few.
· Based on the glide scores and synthetic considerations, a total of 16 novel 4,5-
dihydro[1,2,3]oxadiazolo[3,4-a]quinolin-10-ium-3-olates and 4,5-
dihydropyrazolo[1,5-a]quinolines were selected and successfully synthesized
using various synthetic schemes. They were purified and characterized using IR,
MASS, 1H NMR and 13C NMR.
· A total of 4 novel sydnoquinolines were synthesized and characterized using IR,
MASS, 1H NMR and 13C NMR.
· The synthesized sydnoquinolines were subjected to cycloaddition reaction and
successfully converted to 4,5-dihydropyrazolo[1,5-a]quinolines. The reaction was

157
found to be a concerted reaction as observed by the formation of single products.
No intermediates were formed or observed. The synthesized 4,5-
dihydropyrazolo[1,5-a]quinolines were characterized using IR,MASS, 1H NMR

13
and C NMR. A total of 16 novel pyrazoloquinolines were synthesized and
characterized and being reported for the first time.
· In the case of cycloaddition of pyrazoloquinolines with benzyl propiolate two
isomers were formed. These isomers were confirmed using NOESY. This helped
in confirming the interactions between the protons and thus confirming the
structures of the isomers.
· All the synthesized molecules were subjected to in vitro cytotoxicity studies
against HEp-2 (Human larynx cancer) cells, MCF-7 (breast cancer) cells, HeLa
(Cervical cancer) cells and HBL-100 (Human breast normal epithelial) cells.
· Among all the compounds screened for cytotoxicity activity, the methoxy
substituted pyrazoloquinoline 41 shows good activity with an IC50 value of 20
µg/mL against MCF-7 cells and 30 µg/mL against HEp-2 cells when compared to
the standard, 5-FU which has an IC50 value of 11.7 µg/mL and 7.8 µg/mL, against
MCF-7 and HEp-2 cells, respectively.
· The in vitro results reveal that methoxy and fluoro derivatives have good activity
when compared to other compounds.
· The pyrazoloquinolines were found to have better activity when compared to the
sydnoquinolines. This could be due to the increased functionalization of the
pyrazole ring. Benzyl 7-methoxy-4,5-dihydropyrazolo[1,5-a]quinoline-3-
158
carboxylate was found to be active against HEp-2 and MCF-7 cell lines with IC50
value of 30 µg/mL and 20 µg/mL, respectively.
· The in vitro results and the in silico results correlate reasonably well. Compounds
22, 23, 26, 27, 28, 31, 33, 34, 40, 41 and 39 show good in vitro cytotoxicity. Most
of these compounds show good glide score as well.
· The in silico-in vitro correlation reveal that the substitution at the R1 and R2
position of the pyrazoloquinoline is crucial for the activity. These substituents
were also important for the increase in the binding affinity in silico.
In conclusion, in the present study, 20 compounds were synthesized, characterized
and are being reported for the first time. The compounds show moderate to good
anticancer activity both in silico and in vitro. Among these compounds, compound 41
shows good activity with an IC50 value of 20µg/mL against MCF-7 cells when
compared to 5-FU which shows an activity of 11.7µg/mL. Compounds 22, 23, 28 and
41 were also found to possess fairly good in vitro anticancer activity. With the
correlated outcome of in silico and in vitro results, it may be concluded that these
compounds possess anticancer activity via topo IIα inhibition at the ATP site. These
compounds can be further taken up for the in depth in vivo studies.
159
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Design, Synthesis and Evaluation of Some Novel

Pyrazoloquinolines as Anticancer Agents

J.S.S. UNIVERSITY, MYSORE

Ms. Roshini Chandrasekhar, M.Sc.,

Under the Supervision of

Prof. M.J. Nanjan

J.S.S. College of Pharmacy

Table 2: Some of the patents related to pyrazoloquinolines.

Table 3: Data on 4,5-dihydro[1,2,3]oxadiaz-olo[3.4-a]quinolin-10-ium-3-olate

Table 4: Data on 4,5-dihydropyrazolo[1,5-a]quinolines.

Table 5: Docking parameters of selected ligands.

Table 6:Anticancer activity of 4,5-dihydro[1,2,3]oxadiazolo[3.4-a]quinolin-10-

Table 7:Anticancer activity of phenyl-4,5-dihydropyrazolo[1,5-a]quinoline-2-

Table 8:Anticancer activity of dimethyl 4,5-dihydropyrazolo[1,5-a]quinoline-2,3-

Table 9:Anticancer activity of (4,5-dihydropyrazolo[1,5-a]quinolin-3-yl)methyl

4. RESULTS AND DISCUSSIONS

1.1 Cancer and Cancer Therapy

treating cancer as this could not control metastastic cancer.

Cancer is medically known as malignant neoplasm and is a group of various diseases,

bacterium, that can be flushed or crushed. It is an intricate and potentially lethal

collaboration of genes gone awry, absence of some growth inhibitors or changing

diagnosis and treatment of cancer.

resistance (MDR) (who.int/mediacentre/factsheets/fs297/en). Many patients experience

the classic toxicities of alopecia, gastrointestinal symptoms and myelosuppression.

to inhibit specific proteins or pathways abnormally expressed in cancerous cells. This

chemotherapeutic agents (centerwatch.com/patient/drugs/Druglist.html)

apoptosis and tumor-related angiogenesis. Other approaches focus on tumor-specific

that bind to targets of biological interest is a breakthrough in anticancer research. The

Dependent Kinases (CDK’s), Oncogenic Human Papiloma virus (HPV), ABC

transporters, etc.(Antony et al.,2009). One way of achieving this is through in silico

methods and Structure Based Drug Design (SBDD) methods.

Design (LBDD) to synthesis, preclinical testing to clinical testing from cellular/molecular

discover and develop drugs capable of specifically binding to the proteins/DNA

responsible for cancer pathogenesis (Spychala et al.,2009; Bonjean et al.,1998;

ligands from nonbinding “decoys” (Collins and Workman, 2006).

(Davis et al., 2005).

Table 1: Typical properties of fragments, lead-like and drug-like molecules. (Collins

ADMET NA Membrane permeable Bioavailable

1.2 Bioinformatics, Chemoinformatics and Drug Discovery

biological functions at the macromolecular level and considers gene as a fundamental

unit of genetic material. Chemoinformatics studies the relation between chemical

macromolecules and the construction of chemical libraries. Today bioinformatics and

chemoinformatics overlap more and more (XU Jun et al., 2012).

Docking is a computational tool of structure–based drug design to predict the protein-

Morimoto et al., 1991, Michael et al., 1997).

1.3 Some Novel Anticancer Targets

Farnesyl transferase (FTase) (Rowinsky et al., 1999), DNA topoisomerase.

Tyrosine Kinase (TK): TK are a family of mono-transmembrane -helix proteins

The membrane receptor TK related to tumorigenesis includes epidermal cell growth

Bcr-abl kinase, etc.

Farnesyltransferase (FTase): Ras protein, a low-molecular-weight GDP/GTP-binding

cell growth and differentiation. In malignant transformation, invasion and spread of

molecule through a double-stranded gap generated in a DNA (type II). Topoisomerases

replication, transcription and chromosomal segregation by altering DNA topology. Most

type I DNA topoisomerases modify DNA topology in an ATP independent fashion by

creating single strand breaks in DNA whereas type II DNA topoisomerases do so in an

ATP-dependent fashion by creating double strand breaks in DNA (Schoeffler et al.,

2008). Among topoisomerases, topo II is a well-known anticancer target. Agents that

treatment of human cancers. Nevertheless, topo II based chemotherapy remains

associated with incidences of life-threatening toxic side effects like drug-induced

secondary malignancies. Intriguingly, previous studies suggest that suppressing topo II ,

a subtype of topo II, is responsible for the development of secondary malignancy

potentially be efficacious and safe chemotherapeutic drugs with a reduced risk of

treatment related to secondary malignancies (Toyoda et al., 2008).