Download Synthesis and Pharmacological Evaluation of

Synthesis and Biological Evaluation of
Pteridine Derivatives
M. Pharm Dissertation Protocol Submitted to
Rajiv Gandhi University of Health Sciences, Karnataka
Bangalore – 560 041
By
Mr. BHAMARE RAKESH ASHOK
Under the Guidance of
Mr. D. GILES
Asst. Professor
Department of Pharmaceutical Chemistry,
Acharya & B.M. Reddy College of Pharmacy,
Soldevanahalli, Chikkabanavara Post,
Bangalore -560090.
Page | 1
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE.
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1
Name of the candidate and
Mr. Bhamare Rakesh Ashok,
address
Shakti Hostel,
Acharya & B.M. Reddy college of Pharmacy,
Soladevanahalli, Hesaraghatta main road,
Bangalore-560090.
2
Name of the institution
ACHARYA & B.M. REDDY COLLEGE
OF PHARMACY.
Soldevanahalli, Chikkabanavara post,
Hesaraghatta main road,
Bangalore – 560090.
Office: 080-65650815. Fax :080-28393541.
3
Course of study and subject
MASTER OF PHARMACY
(PHARMACEUTICAL CHEMISTRY)
4
Date of the admission
5
Title of the topic:
15th July 2010
“Synthesis and Biological Evaluation of
Pteridine Derivatives”.
Page | 2
6.0 Brief resume of the intended work
6.1 Need for study:
Pteridine is a bicyclic planer compound composed of pyrimidine & pyrazine ring fused
together. Modifications of the pteridine ring have resulted in a large number of compounds
having diverse pharmacological activity. The pteridine nucleus is found to be essential
component of various compounds possessing biological activities like anti-inflammatory4-5,
analgesic6, anti-microbial7-10, anti-hepatitis11, neurodegenerative12, immunosuppressive13-14,
anti-tumor15-82 activities.
7
N
8
10
9
N
1
6
N
5
4
N
2
3
Pteridine
Cancer, a disease of worldwide importance, is a group of diseases characterized by
uncontrolled growth and spread of abnormal cells. It is now generally accepted that a
neoplastic transformation is related to genes alteration or oncogene activation, allowing
progress in the development of new treatments for malignant diseases, both by revealing the
pathobiology of the disease and the discovery of new drugs. Moreover, the role of many
proteins has been identified as novel targets in cancer therapy allowing the design of more
selective agents. The classical anticancer agent methotrexate (MTX) and aminopterine which
are folic acid anti-metabolites having pteridine nucleus owe their cytotoxicity by inhibiting
the enzyme dihydrofolate reductase (DHFR) 1. DHFR catalyzes NADPH dependent reduction
of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate. It also catalyzes reduction of folate to 7,8dihydofolate by NADPH. Without the function of DHFR, cells are deprived of key metabolic
intermediates needed to form nucleotides and ultimately nucleic acids required for
proliferation of cells. Many heterocycles have showed interesting cytotoxic activity and
different mode of action in cancer therapy. Methotrexate is also known to act partly through
inhibition of thymidylate synthase (TS), which catalyses the methylation of deoxyuridylate
monophosphate (dUMP) to thymidylate, which is then incorporated into DNA. More specific
inhibitors of TS have been developed, which target the folate-binding site of the enzyme.2
Page | 3
O
O
NH2
HO
NH
N
N
N
H2N
N
N
HO
O
H3C
Methotrexate
Numerous experimental, epidemiologic, and clinical studies suggest that nonsteroidal
anti-inflammatory drugs (NSAIDs), particularly the highly selective cyclooxygenase (COX)2 inhibitors, have promise as anticancer agents. Many epidemiologic studies have found that
long-term use of NSAIDs is associated with a lower risk of colorectal cancer, adenomatous
polyps, and, to some extent, other cancers. Two NSAIDs, sulindac and celecoxib, have been
found to inhibit the growth of adenomatous polyps and cause regression of existing polyps in
randomized trials of patients with familial adenomatous polyposis3.
6.2 Compounds having Pteridine nucleus:
O
OH
O
OH
NH2
NH
N
O
N
H2N
N
CH
N
Pralatrexate
Page | 4
CH3
NH2
N
N
N
H2N
N
O
N
HN
COOH
COOH
Edatrexate
O
COOH
NH
NH2
COOH
N
N
H2N
NH
N
N
Aminopterin
Bearing in mind the importance of folate metabolism in the proliferation of cells, we
planned to synthesize derivatives of pteridines analogues as anticancer, anti-inflammatory and
analgesic agents.
6.3 Review of literature:

Hoffmann G. et al studied the potential role of immune system activation-associated
production of neopterin derivatives in human. They focuses on the clinical utility of
measuring neopterin levels in inflammatory diseases and the potential functions of
neopterin as a mediators and or modulator in the course of inflammatory and infectious
processes.4
O
OH
N
HN
OH
OH
H2N
N
N
Neopterin

The neopterin and 7,8 dihydroneopterin in Th1- type immune response was reported
by Wirleitner B et al. Main triggers for neopterin synthesis of interferon-γ, tumor
Page | 5
necrosis α (TNF-α), lypopolysacharides ( LPS), IFN- α and IFN-β. They carried out in
vivo anti inflammatory activity.5
OH
O
CH3
N
HN
OH
H2N
N
N
H
7,8 Dihydroneopterin

Gomtsyan A et al reported the investigation of three new approaches of modifying
existing pyridopyrimidine and alkynylprimidine adenosine kinase inhibitors. The 4amino- substituted pteridines were less active than corresponding 5- and 6- substituted
pyridopyrimidines . They carried out in vitro analgesic and anti inflammatory activity.6

Antimycobacterial
activity
of
1-7,8-dihydropteridine
derivatives
against
Mycobacterium tuberculosis and mycobacterium avium complex in vitro was carried
out by Suling JW et al.7
NH2
N
N
1
R HN
N
R
3
2
R
6-phenyl-2,4-diamino-1deaza-7,8-dihydropteridine
Compound No.2 R 1 = -COOCH 2 CH 3 , R 2 =CH 3, R3 = -H 2
Compound No.6 =-H, R 2 =H 2 , R 3 = -CH 3

Didier SE et al reported the screening of purine and pteridine compounds for
antimicrosporidial activity in vitro against Encephalitozoon intestinalis and vittaform
corneae. 12 out of 44 purines and pteridine with putative tubulin binding activity
inhibited microsporidial replication by more than 50% at concentrations that were not
toxic to the host cells.8

The synthesis of S-glycopyranosyl-6,7-diarylthiolumazines and test their nematocide
activity was reported by Alho MAM et al. They performed in vitro tests against
Caenorhabditis elegans and it was found that monosubstituted derivatives showed
higher activity.9
Page | 6

Suling JW et al reported the evaluation of 2,4-diamino-5-methyl-5-deazapteridines
derivatives for their in vitro antimycobacterial activity against Mycobacterium avium
complex. Some of the derivatives were >100-fold more active against MAC rDHFR
than against human rDHFR.10

The synthesis of pteridine derivatives as potent inhibitors for hepatitis C virus. They
carried out SAR studies at the 4-position of pteridine compound was reported by Ding
Y et al. It bears certain resemblance to a benzimidazole 5-carboxylate NS5B RdRp
inhibitor. They conclude that small group at para position is needed for optimal
activity.11
R
N
N
N

N
Prins HAL et al synthesized pteridine analogues and evaluated for monoamine
oxidase B and nitric oxide synthase inhibition activity. They found that
mitochondrial
fractions of baboon liver as pteridine 2,4-dione analogues were
active.12

Modification of pteridine ring of γ-fluoromethotrexate and in vitro study for
immunosuppressive activity was reported by Korkuryo Y et al. They found
Pyrrolopyrimidine derivatives 1e and 1t were exhibit potent suppressive effects on
the responses of both T and B cells to mitogens.13
NH2
N
H2N
O
N
N
H
1e- X= F
1t- X=  F
HN
CO 2H
CO 2H X
.
Page | 7

Shen C et al carried out immunosuppressive activity of a new pteridine derivative.
Natural occurring pteridines such as tetrahydrobiopterin (BH4) and Neopterin have
been reported to have immune modulating activities. In vitro activity on T-cells and
in vivo activity of trinitrobenzenesulfonate (TNBS) in mice were carried out. They
identified a pteridine analogue 4AZA1378 with immunosuppressive activity. They
also carried out ELISA test by using IL-2 and INF-γ in the supernatants of human T
cell cultures (after 24 h co-stimulation).14

The design and synthesis of several diamino pteridine-benzene sulfonamide and
benzenesulfonate conjugates was reported by Marques MS et al, which differ in the
nature and size of the spacer group between the two key moieties. Some inhibitors
showed activities only in millimolar range and revealed their potential as anticancer
agents.
NH2
N
N
H2N
N
N
O
spacer
SpacerH3C
S
X
O
The inhibition of isoenzyme carbonic anhydrase II, the activities presented a more
pronounced variation, the highest activities being observed for compound 1a in which
at spacer NH and at X=NH2 group is present and compound 4a in which at spacer
NMepABA-NH-CH2-CH2 and at X=NH2 group is present with KI of 4.5 and 260 nM,
respectively.15

The synthesis of several pteridine analogues were screened in vitro against three cancer
cell lines, MCF7 (Breast), NCI (lung) and SF-268 (CNS) was reported by Chauhan
MSP et al. All these were tested as novel templates for anticancer chemotherapy.
Page | 8
N
O
S
N
N
N
N
N
R
N
R
N
R
S
N
R
OH
OH
4.R=H
5.R=CH 3
11. R=CH
3
They have described pteridine compounds as potential anticancer agents and identified
analogues in particular 4,5,11 that can be novel templates for lead optimization
purpose in cancer chemotherapy.16

Ge P et al designed and synthesize 3,5-Dialkylamino substituted 8H, 10H3(R),15(S)-2,3,6,7-Tetrahydro-1,5,3-Dioxazepino [3,2-c] Indolo[3,2-g] pteridine-7Ones derivatives. Preliminary result showed that they were active a inhibitors of the
growth of murine leukemia L1210 cells in vitro.17
 1,2-substituted 2-deoxoflavin-5-oxides, 2-deoxoalloxazine-5-oxides, and their 5-deaza
analogs were synthesized by Ali IH et al . They carried out antitumor activities against
CCRF-HSB-2 and KB tumor cells, in vitro and many compounds showed promising
antitumor activities. SAR studies showed that the higher binding affinities were
obtained with the structure features on the flavins or 5-deazaflavin skeleton; NH2 or Ph
group at the C2-position, H (oralloxazine conformation) or Ph group at the N10Position.18

Srivastava V et al described the docking of 2,4-diamino-5-methyl-5-deazapteridine
(DMDP) derivatives as inhibitors to human dihydrofolate reductase (DHFR). The
chloro substituted naphthyl ring compound makes significant hydrophobic contact with
Leu 22, Phe 31 and Pro 61 of the DHFR active site leading to enhanced inhibition of
the enzymes.19
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NH2
CH3
N
H2N
NH
N
N
Cl

The synthesis of highly functional 6-substituted pteridine was carried out by Guiney D
et al. Alkene, esters, ketone, amide, cyano, oxime, methoxy and dihydroxy functional
groups were introduced principally through improved methodologies for Witting
reactions using 2-thioalkyl-6-formyl pteridines as substrates.20

Mcandless JM et al carried out deuterium exchange of C-methyl protons in lumazine
derivatives. The catalysis of 6,7,8-trimethyllumazine by the phosphate buffer species
as well by hydronium ion (H3O+) was studied.21

A new series of quinazoline analogs to resemble methotrexate structure and fitted with
functional groups to enhance inhibition of mammalian DHFR was synthesized and
designed by Sarah T et al. They synthesized compounds were evaluated for their
ability to inhibit mammalian DHFR in vitro tissue culture assay panel. Some of the
compounds were found to be most active DHFR inhibitors.22

McKie HJ et al reported design and synthesis of inhibitors which are able to overcome
malarial pyrimethamine resistance by using homology model of the 3-D structure of
DHFR-TS, rational drug design techniques. The results showed that the m-chloro
analogue of pyrimethamine was a stronger inhibitor of both wild-type DHFR and the
doubly mutant purified enzyme.23
 The docking and database screening reveal new classes of Plasmodium falciparum
dihydrofolate reductase Inhibitors was reported by Rastelli G et al. They synthesize
twelve new compounds whose structures are completely unrelated to known antifolates
were identified and found to inhibit, at the micromolar level, the wild-type and resistant
mutant PfDHFRs harboring A16V, S108T, A16V + S108T,C59R + S108N + I164L, and
N51I + C59R + S108N + I164L mutations.24

Deng Y et al reported the synthesis and biological activity of a novel series of seven 2amino-4-oxo-6-substituted thieno[2,3-d]pyrimidines with bridge length variations (from
Page | 10
2 to 8 carbon atoms) as selective folate receptor (FR) α and β substrates and as
antitumor agents.25
 Synthesis and biological evaluation of two analogues of dihydrofolic acid possessing a
7,8-dihydro-8-oxapterin ring system was carried out by Nair MG et al. These
compounds,N-[(2-amino-4-hydroxy-7,8-dihydro-8-oxa-6-pteridinyl)benzoyl]-glutamic
acid & N-[ (2-amino-4-hydroxy-7,gdihydro-8-ox8-oxa-6-pte~dinyl)methyl]benzoyl]glutamic acid, were synthesized by reacting the appropriately substituted α-halo ketones
with 2,5-diamino-4,6-dihydroxypyrimidine. Compound 4 showed activity against
Streptococcus faecium (ATCC 8043).26
COOH
O
H
N
H
H
NH
HN
COOH
O
N
H
N
H2N
Compound No.4

Gangjee A et al carried out synthesis of N-[4-[1-ethyl-2-(2,4-diaminofuro[2,3d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid as an antifolate. They carried out
evaluation of the cytotoxicity of 3 in methotrexate-resistant CCRF-CEM cell sublines
and metabolite protection studies implicated DHFR as the primary intracelluar target.
Thus, alkylation of the C9 position in the C8-C9 bridge of the classical 5-substituted
2,4-diaminofuro-[2,3-d]pyrimidine is highly conducive to DHFR and tumor inhibitory
activity as well as FPGS substrate efficiency.27
NH2
NH
N
H2N
N
O
H3C
O
COOH
COOH
Compound No.3

The synthesis of the reduced derivatives of 11-deazahomofolic acid, 10-Methyl- 11deazahomofolic
acid,
and
their
evaluation
as
inhibitors
of
Glycinamide
Ribonucleotide Formyltransferase was carried out by Nair MG et al. Compound
Page | 11
(6R,S)-ll-deazatetrahydrohomofolate
(4)
show
the
potent
inhibition
of
on
Lactobacillus casei glycinamide ribonucleotide (GAR) formyltransferase.28
COOH
O
H
N
H
H
NH
HN
COOH
O
H2N

N
H
N
Compound No.4
Piper JR et al carried out synthesis and antifolate evaluation of the 10-Propargyl
derivatives
of
5-Deazafolic
Acid,
5-Deazaaminopterin,
and
5-Methyl-5-
deazaaminopterin. The 2,l-diamino compounds 5 and 6 were as potent inhibitors of
DHFR from L1210 cells as MTX and 7-and 35fold, respectively, more inhibitory
than MTX toward L1210 cell growth.29
NH2
R
O
N
NH
H2N
N
HO
N
O
O
HO
5 R=H
6 R = CH 3

Synthesis of 7-acyl-2,4-disubstituted pteridine by nucleophilic substitution was carried
out by Thomas D et al. They carried out various acetylation reactions by acetic
anhydride (Ac2O) and gave 89-93 and reduction of pyrazine moiety to the 5,6,7,8tetrahydropteridine derivatives 94-96.30
Page | 12
NHAc
N
N
R
NH2
N
N
N
O
Compound No.89 R=OAc
H3 CS
N
Compound No.90 R=NHAc
H
H
H
N
H
OH
Compound No.94
Compound No.91 R=N(CH 3 )

H
N
Thomas RJ et al reported that small molecule binders to thymidylade synthsase (TS)
mRNA. In order to identify the ligand with higher affinity. They tested all DNA
binding compounds and concluded all were able to compete for paromycin – binding
site of TS mRNA and suggested that this compounds bind 1X1 internal loop region.
Hoechst 33258 (compound 13) was the most effective at displacing paromomycin form
the internal loop.31
N
HO
N
N
H
NH
N
N
Hoechst3325

Synthesis and biological activity of 5-phenylselselenenyl-substituted pyrimidine
nucleosides was carried out by Sehinazi FR et al in which 5-phenylselenenyl
derivatives of pyrimidine nucleosides were synthesized by electrophilic substitution of
phenylselenyl chloride under basic condition. These compounds may serve as
inhibitors of thymidylate synthase, as potential antiviral and anticancer agents.32

Tolleson HW et al reported the determination of enzymes in cellular extracts by active
site titrations or by catalytic activity measurements. They gave the example of how an
Page | 13
enzyme- linked immunosorbent assay (ELISA) was used to determine the true enzyme
concentration which was compared to the effective enzyme concentration obtained by
ligand binding and catalytic assay methods in a crude bacterial cell extract.33

The antibody directed enzyme prodrug therapy (ADEPT) technique for to increase the
antitumor selectivity in cancer chemotherapy was reported by Wolfe LA et al. They
designed a mutant of human carboxypeptidase A (hCPA1-T268G) which is capable of
hydrolyzing in vivo stable prodrugs of methotrexate and targeting this enzyme to
tumors on an Ep-CAM1- specific antibody. In current report, prodrugs of the
thymidylate synthase inhibitors GW1031 and GW1843 and the dihydrofolate reductase
inhibitor methotrexate were studied for their wild-type and mutant hCPA enzyme
hydrolysis, there in vivo stability and their use in therapy.34
O
O
HO
HO
O
O
O
O
N
NH
R
R
F
NH
NH
O
O
HN
HN
N
GW1843

N
GW1031-R
Mohsen AAW et al reported ligand-conformational changes of thymidylate synthase
from lactobacillus casei and detected by limited tryptic proteolysis. Most of the
identified sites of proteolysis were between R72 and R178, a region that includes a
large loop containg residues 90-139 that is absent in thymidylate synthase from most
other sources. R-17 Argenine residues that bind to the phosphate of dUMP. The
peptide that have been indentified reveal that R72, R78, K89, R107, K124, R128,
K138,R151, K157, K172, R178, K269 and R274 are sites of tryptic hydrolysis. R78 is
Page | 14
involved in electrostatic binding of one glutamyl residues of cofactor analogues,
polygluatamyl 10-prapargyl 5,8 dideazafloate so this should be exposed in absence of
folate.35

Stereoelectronic activation of methylenetetrahydrofolate by thymidylate synthase by
using resonance Raman Spectroscopy was reported by Austin JC et al. The spectra are
reported for the ternary complex of Escherichia coli thymidylate synthase with the
cofactor 5,10-methylenetetrahydrofolate (CH2-H4-folate) and the inhibitor 5-fluoro-2’deoxyuridylate, excited at 337 or 356 nm, in resonance with perturbed absorption
bands of the p-aminobenzoylglutamate (PABA-Glu) portion of the cofactor. They also
explained drugs which are act by forming covalent inhibitor complexes with
thymidylate synthase36.

Austin JC et al reported the characterization of the Binary and ternary complexes of
thymidylate synthase with 5-Nitrodeoxyuridylate. The TS/NDU binary complex RR
spectrum shows many similarities to the RR spectra of thiol adducts of NDU or of 5nitro-1-methyluracil formed in solution, providing strong evidence in support of the
formation of a covalent link between Cys 146 of TS and C6 of NDU.37

Structure of thymidylate synthase with a C-terminal deletion and the role of the Cterminus
in
alignment
of
2̍-deoxyuridine
5̍-monophosphate
and
5,10-
methylenetetrahydrofolate was reported by Perry MK et al. Thymidylate synthase
undergoes a major conformational change upon ligand binding, where the carboxyl
terminus displays the largest movement. The critical aspect of reaction is large
conformational change where the segment of Protein move from “open” conformation
to form “closed” active site cavity.38

Liu L et al reported the exclusion of 2̍-deoxycytidine 5̍-monophosphate by asparagines
229 of thymidylate synthase. They concluded that TS discriminates binding of dUMP
versus dCMP by a 3-4 kcal mol-1 difference in binding energy by exclusion of dCMP
from active site. They proposed that this exclusion id a consequence of untoward
interactions between dCMP and the side chain carboxamide group of the Asn or Gln at
position 229 of TS.39

The replacement of Val3 in human thymidylate synthase affects its kinetic properties
and Intracellular stability was reported by Huang X et al. Human and other mammalian
Page | 15
thymidylate synthase (TS) enzymes have an N-terminal extension of 27 amino acids
that is not present in bacterial TSs. In mammalian cells the variant V3A has a half –life
similar to that of wild-type human TS(wt hTS) while V3T is much more stable, V3L,
V3F and V3Y have half - lives approximately half of that for wt hTS. They observed
that N-terminal extension affects the conformational state of the hTS catalytic region.40

Trujellio M et al reported the heterologous expression and characterization of the
bifunctional dihydrofolate reductase- thymidylate synthase enzymes of toxoplasma
gondii. They expressed catalytically active toxoplasma gondii dihydrofolate
thymidylate synthase (DHFR-TS) and the individuals TS and DHFR domains in
Escherichia coli using the T7 promoter of pET-15b. they concluded that properties
such as yield, stability, and activities of the recombinant T. gondii DHFR-TS provide
clear advantages over other bifunctional DHFR-TSs as a model for future studies.41

Complete restoration of activity to inactive mutants of Escherichia coli thymidylate
synthase was carried out by Maley F et al . Escherichia coli thymidylate synthase (TS)
is a dimeric protein containing identical subunits. They carried out R126E, an inactive
mutant of this enzyme, was incubated at room temperature with other inactive mutants
of E. coli, TS enzyme activity gradually reappeared. They concluded that other mutant
dimmers that contain both active site cysteines such as (R126E)-(Y94A)-(I264Am) are
also fully active, even though one of the subunits is functionally inactive. Whether TSs
in generally catalyze their reactions via a half-the-sites activity mechanism remains to
be resolved , they suggested that E.coli TS employs this mechanism.42

Gokhale SR et al carried out covalent reinforcement of a fragile region in the dimeric
enzyme stabilizes the protein against chaotrope-induced unfolding. Urea and
guanidinium chloride induced unfolding of thymidylate synthase, a dimeric enzyme.
And engineered interface mutants have been monitored by circular dichroism,
fluorescence, and size exclusion chromatography. The chaortope-induced denaturation
of TS appears to proceed through a partially unfolded intermediate that is stabilized by
aggregation. They concluded that dissociation and loss of structure occur
concomitantly at high denaturant concentrations.43

The thermodynamic stabilization of Nucleotide binding to thymidylate synthase by a
potent Benzoquinazoline folate analogue inhibitor was reported by Chen CH et al. The
Page | 16
stabilization of dUMP, FdUMP, and dGMP binding to Escherichia coli thymidylate
synthase (TS) in the presence and absence of a folate analogue inhibitor of TS, 1843U,
was determined by differential scanning calorimetry. They concluded that tightness of
the complex is due to the stacking energy that results from Vander Waals contacts
between the nucleotide purine or pyrimidine ring and the benzoquinazoline ring of
1843U.44

Sotelo MR et al reported the crystal structures of rat TS inhibited by tomudex, a potent
anticancer drug. Two crystal structure of rat thymidylate synthase are complexed with
dUMP and anticancer tomudex have been determined to resolutions of 3.3 and 2.6 A o.
Tomudex is a new antifolate targeted to TS and the first approved for clinical use. The
structures represent the first views of any mammalian TS bound to ligands and suggest
that the rat protein undergoes a ligand-induced conformational change similar to that of
the Escherichia coli protein.45
 The X-ray crystal-structure based design, synthesis, and biological activity of a novel
family of benz[c,d]indole-containing inhibitors of thymidylate synthase (TS) was
described by Varney DM et al. The structure-activity of the lead compound was studied
by conceptually dividing the molecule into four regions and independently optimizing
the substituent’s for each region. They synthesized inhibitors from substituted 6aminobenz [cd]indol-2(lH)-ones by alkylation with both a simple alkyl group and a
substituted benzylic portion. The 2,6-diaminobenz indoles were prepared from the
corresponding ladams by conversion to the thiolactam, alkylation to the methylated
thiolactam, and then displacement with a substituted or unsubstituted amine.46
 Steadman JD et al carried out substitution at residue 214 of human thymidylate synthase
which alters nucleotide binding. A glutamine corresponding to residue 214 in human TS
(hTS) is located in a region that is postulated to be critical for conformational changes
that occur upon ligand binding. Crystallographic studies of E. coli TS revealed that
glutamine at the position corresponding to 214 in hTS is located at a β-bulge that is
postulated to be important for ligand-induced conformational changes.47

An hTS variant, R163K, in which the inactive conformation is destabilized was
designed and expressed by Gibson ML et al. Crystal structures of R163K in two
different crystal forms, with six and two subunits per asymmetric part of the unit cells,
Page | 17
have been determined. All subunits of this mutant are in the active conformation while
wt hTS crystallizes as the inactive conformer in similar mother liquors.48
 Webber ES et al reported the design, synthesis and biological evaluation of a new class
of inhibitors of thymidylate synthase (TS). The molecular design was carried out by a
repetitive crystallographic analysis of protein-ligand structures. The synthetic strategy
was based on the displacement of a halogen at the 5-position of a quinazolinone by
various aryl thioanions. The compounds were tested for inhibition of purified E. coli
and/or human TS, and were assayed for cytotoxicity against three tumor cell lines in
vitro. Significant thymidine protection effects were observed with several of the
inhibitors, indicating that TS was the intracellular locus of activity.49
 Quantum mechanics or molecular mechanics study of thymidylate synthase was carried
out by Kannan N et al. A theoretical study of the reduction of an exocyclic methylene
intermediate by hydride transfer from the 6S position of 5,6,7,8-tetrahydrofolate
(H4folate), to form 2′-deoxyuridine 5′-monophosphate (dTMP) and 7,8-dihydrofolate
(H2folate), has been carried out by using hybrid quantum mechanics/molecular
mechanics methods.50
 Ostrowski T et al reported the synthesis, antiviral activity, conformational analysis,
and interaction with Viral Thymidine Kinase of 5-substituted Pyrimidines with a 1,5anhydro-2,3-di-deoxy-D-arabino-hexitol Moiety at N-1. The results shown that the
vinyl, propynyl and in particular, the 5-trifluoromethyl analogue showed potent activity
against herpes simplex virus (HSV).51

Cyclopenta[g]quinazoline-based antifolates as inhibitors of Thymidylate Synthase and
potential antitumor agents were designed and synthesized by Basselios V et al. The
synthesis of non polyglutamatable inhibitors of TS that do not use the reduced folate
carrier (RFC) for cellular entry should provide compounds which overcome
mechanisms of resistance to folate-based inhibitors of TS that are associated with
decreased/altered folylpolyglutamate synthetase (FPGS) expression and/or an impaired
RFC.52

Yoo BC et al reported the application of the phosphoramidate ProTide
technology to improve the metabolism of the DNA methytransferase inhibitor,
zebularine.53
Page | 18

The Ab initio molecular orbital calculations are used to study the Michael additions of
sulfur and oxygen anionic nucleophiles to acrolein was carried out by Thomas EB.54

A novel and simple method of preparation of 2-alkyl amino quinazolin-4-ones with
fused hetero aromatic rings from easily accessible (hetero) aromatic amines by
intramolecular Friedel Craft’s type substitution was reported by Zeghida W et al.55

Kannan N et al reported a theoretical study of the reduction of an exocyclic methylene
intermediate by hydride transfer from the 6S position of 5,6,7,8-tetrahydro folate, to
form 2′-deoxyuridine 5′-monophosphate (dTMP) and 7,8-dihydrofolate, carried out
using hybrid quantum mechanics/ molecular mechanics methods.56

Kannan N et al reported a theoretical study of dynamic effects on the rate-limiting step
of the thymidylate synthase catalyzed reaction has been carried out by means of Grote
Hynes theory.57

The design and synthesis of a series of sulfonyl- containing 5-fluoro-2′-deoxyuridine
(FdU) phosphotriester and phosphor- amidate analogues as anticancer prodrug of
FdUMP was reported by Sun YW et al.58
 Steadman JD et al studied the crystal structures of thymidylate synthase (TS) and
revealed that a kink is present in β-sheets that form the core of the enzyme. The data are
consistent with the hypothesis that residue 214 is involved in maintaining the enzyme
in a conformation that facilitates nucleotide binding and catalysis. They concluded that
substitutions at position 214 of TS may be altering nucleotide binding through effects
on enzyme conformation.59

Synthesis and characterization of two “flexible” bisubstrate analogues of the
intermediate in the thymidylate synthase reaction reported by Yang IY et al. They
minimized steric constraints and avoided diasteromeric mixtures by using a
pyrimidine-based analogue as the folate portion of the inhibitor while retaining all
known important binding sites. They concluded that compounds are shown to be
potent competitive inhibitors with respect to dUMP or 5,l0-CHz- H4PteGlu but gave
mixed kinetics with respect to 5,l0-CH2-H4PteGlu5 for human thymidylate
synthase.60

Pendergast W et al carried out several folate-like thymidylate synthase inhibitors are
described in which the pteridine nucleus of the folic acid molecule is replaced by a
Page | 19
benzoquinazoline moiety, which in turn is attached to the benzoylglutamate side chain
by a sulfonamide link. The most potent compounds had Ki values as low as 2.5 nM
against the human enzyme, were good substates for the cellular reduced folate
transport system and for folylpolyglutamate synthetase, and had IC50 values for
growth inhibition of tumor cell lines as low as 70 nM. Compound 5a-f are show
antitumor activity.61
R
1
SO 2 NR
O
2
CONH
H
3
CO 2R
HN
4
CO 2 R
Y
R
CPD
5a
5b
5c
5d
5e
5f

N
X
R1
NH2
NH2
NH2
NH2
CH3
CH3
R2
H
H
8 Br
H
H
H
R3
H
Propargyl
H
H
H
H
R4
H
H
H
H
H
H
X-Y
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
Design, synthesis, and biological activities of classical N-4-[2-(2-amino-4ethylpyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl-L-glutamic acid and its 6-methyl
derivative as potential dual inhibitors of Thymidylate Synthase and Dihydrofolate
Reductase as potential antitumor agents was carried out by Gangjee A et al.62
O
O
HN
N
COOH
H2N
NH
N
HC
COOH
CB3717

The mode of action of site-directed irreversible folate analogue inhibitors of
thymidylate synthase.5,8-dideazafolate analogues are tight binding but not irreversible
Page | 20
inhibitors of thymidylate synthase (TS) reported by Lobo PA et al. They synthesize 2desamino-2-methyl-5,8-dideazafolate (DMDDF). They concluded that a sulfhydryl
reactive compound that is directed to the folate binding site of TS may diffuse to the
active site cysteine, and form a covalent bond with this residue.63

Anderson CA et al reported Thymidylate synthase (TS), a half-the-sites reactive
enzyme, catalyzes the final step in the de novo biosynthesis of deoxythymidine
monophosphate, dTMP, required for DNA replication and co crystal structure of TS
from Pneumocystis carinii (PcTS), a new drug target for an important pathogen, with
its substrate, deoxyuridine monophosphate (dUMP), and a cofactor mimic, and they
determine CB3717.64

Morse JR et al studied that in thymidylate synthase, four conserved arginines provide
two hydrogen bonds each to the oxygens of the phosphate group of the substrate, 2̍deoxyuridine-5̎-monophosphate. Of these, R23, R178, and R179 are far removed from
the site of methyl transfer and contribute to catalysis solely through binding and
orientation of ligands. They concluded that three of the four phosphate-binding
arginines in TS, R23, R178, and R179, are not essential for activity and contribute to
catalysis through ligand binding.65

Trapping of the C5 methylene intermediate in thymidylate synthase using the cofactor
N5,10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate) carried out by Barrett JE et
al . They concluded that Trp82Tyr, which has a kcat of 0.034 s-1 for the production of
dTMP (100-fold slower than wild-type enzyme), was determined to be the most
efficient producer of the new product.66

Barrett JE et al represent a model for hydride transfer in thymidylate synthase based
on unnatural amino acid mutagenesis. They observe in wild type Escherichia coli TS,
the primary isotope effect observed for (6R)-5,10-CH2-[6-2H]H4-folate is large,
indicating that hydride transfer may also contribute to the rate-limiting step in the
wild-type enzyme. To minimize the effects of steric perturbation, a variety of
tryptophan analogues containing fluorine, methyl, or ring heteroatom substituents
were substituted for Trp82.67

De Novo design of enzyme inhibitors by Monte Carlo ligand generation is carried out
by Gehlaar KD et al. They describe MCDNLG (Monte Carlo De Novo Ligand
Page | 21
Generator), a computer program for the de novo generation of ligands within the
vacant binding site of a protein. The method has been evaluated using two wellstudied systems, dihydrofolate reductase and thymidylate synthase. The method has
also been used to guide improvements to inhibitors of HIV-1 protease. One such
improvement resulted in a compound selected for preclinical studies as an antiviral
agent against AIDS.68

Marsham RP et al carried out the synthesis of 16 new N10-propargyl
quinazoline
antifolates
with
methylamino,ethylamino,(2-aminoethyl)amino,[2-
(dimethylamino)ethyl]amino,(2-hydroxyethyl)amino,(carboxymethyl)amino, dimethyl
amino, imidazol-1-yl, methoxy,ethoxy, phenoxy, 2-methoxyethoxy, 2-hydroxyethoxy,
mercapto, methylthio, and chloro substituents at C2 is described. In general, the
synthetic route involved the coupling of diethyl N-[4-(prop-2-ynylamino)benzoyl]Lglutamate with 6
(bromomethyl)-2-chloro-3,4-dihydro-4-oxoquinazoliinne N ,N-
dimethyl formamide with calcium carbonate as the base, displacement of the C2chloro substituent with nitrogen and sulfur nucleophiles and deprotecton using mild
alkali. The C2-methoxy analogue lla was tight bind with TS inhibitor N10-propargyl5,8-dideazafolic acid (CB3717, IC1 155387,) against the TS enzyme and exhibited
enhanced potency in culture.69
O
NH
N
HN
H3C
CO 2 H
R
O
O
CH 2 CH 2 CO 2 H
N
11a=CH 2 CCH

In thymidylate synthase (TS), the invariant residue Asp-221 provides the only side
chain that hydrogen bonds to the pterin ring of the cofactor, 5,10-methylene-5,6,7,8tetrahydrofolate reported by Sage RC et al. All mutants of D221 except cysteine
abolish activity. They have determined the crystal structures of two ternary complexes
of the Escherichia coli mutant D221N. In a complex with dUMP and the antifolate
10-propargyl-5,8-dideazafolate (CB3717), dUMP is covalently bound to the active
site cysteine, as usual. CB3717, which has no imidazolidine ring, is also bound in the
usual productive orientation, but is less ordered than in wild-type complexes.70
Page | 22
O
CH
glu
N
O
HN
H2N
N
CB3717

Wang Z et al reported the nature of different bond activations along the same catalytic
path is of general interest in chemistry and biology. They compare the physical nature
of two sequential H-transfers in the same enzymatic reaction. Thymidylate synthase
(TSase) catalyzes a complex reaction that involves many chemical transformations
including two different C-H bond cleavages, a rate-limiting C-H-C hydride transfer
and a non-rate-limiting C-H-O proton transfer. They concluded that TSase optimizes
the donor-acceptor geometries for the slower and overall rate-limiting hydride transfer
but not for the faster proton transfer.71

The structural determinants for the intracellular degradation of human thymidylate
synthase was carried out by Forsthoefel MA et al. Thymidylate synthase (EC 2.1.1.45)
(TS) catalyzes the conversion of dUMP to dTMP and is therefore indispensable for
DNA replication in actively dividing cells. Molecular dynamics (MD) simulations
showed that the nonadditive behavior was due to the interference between the way
different substituents interacting with key protein side chains. For the compounds, the
interference between 4-nitro substitution and 5,6-difluoro substitution is the smallest.
In this substituents at 5- and 6-position are able to provide extra binding enhancement
which could be a new direction for TS inhibitors.72

L-y-L-Linked dipeptide and L-y-amide analogues of 2-desamino-2-methyl-N10propargyl-5,8-dideazafo allied were synthesized and evaluated for thymidylate
synthase inhibitory activity by Bisset FMG et al. All N10-propargyl dipeptide
analogues were potent inhibitors of TS (IC50 2-24 nM) and inhibited growth of L1210
cells in the range 0.1-10 pM. In the block the possibility of poly-y-glutamation, the
propionate side chain of the terminal amino acid was substituted by hydrogen, alkyl
groups, or branched alkyl groups, giving a series of dicarboxylates with activity
Page | 23
against TS in the range 10-24 nM. Replacing the propionate chain with aromatic or
polar groups again produced no substantial increase in binding affinity for TS.73

Bretner M et al synthesized 2-thio derivatives of dUrd and 5-fluoro-dUrd and their 5'monophosphates and interaction with Tumor Thymidylate Synthase, and evaluated for
in vitro antitumor activity. 2-Thio dUMP is a good substrate and its affinity for the
enzyme being only 2-fold lower than that of dUMP and 2-thio-FdUMP is a potent,
competitive slow-binding inhibitor, only 1 order of magnitude weaker than FdUMP.74

Jones RT et al carried out synthesis of quinazoline antifolates inhibiting Thymidylate
Synthase: 2-Desamino derivatives with enhanced solubility and potency. The poor
solubility of the thymidylate synthase (TS) inhibiting antifolate 10-propargy1-5,8dideazafolic acid has posed problems for its clinical use and is probably responsible
for its renal toxicity.75
O
O
COOH
HN
N
NH
R
N
COOH
Compound No.8e=propargyl

Design and synthesis of novel N-{2-amino-4-methyl[(pyrrolo[2,3-d]pyrimidin-5yl)ethyl]benzoyl}-L-glutamic acid (3a) as a potent dual inhibitor of thymidylate
synthase (TS) and dihydrofolate reductase (DHFR) and as an antitumor agent was
carried out by Gangje A et al. Compounds 3a and 3b were more inhibitory against TS
from Lactobacillus casei and Escherichia coli. Analogue 3a was also more inhibitory
against DHFR from human, Toxoplasma gondii, and Pneumocystis carinii.76
CH3
O
COOH
N
H2N
NH
N
N
R
COOH
Compound No.3a R=H
Compound No.3b R=CH 2 Ph
Page | 24

Jones RT et al carried out synthesis of quinazoline antifolates inhibiting Thymidylate
Synthase by variation at the N10 substituent. The synthesis of 12 new 5,8 didewafolates
with isopropyl, cyclopropylmethyl, 2-fluoroethyl, carbamoylmethyl, phenacyl,3fluorobenzyl, 5-uracilylmethyl, carboxymethyl, 2-carboxyethyl, 3-cyanopropyl, 3hydroxypropyl, and cyanomethyl substituents at N10. The compounds were tested as
inhibitors of purified L1210 thymidylate synthase. They concluded that
N10
substitution in general reduced DHFR inhibitory activity.77
O
O
COOH
HN
N
NH
R
H2N
N
N
COOH

Complete replacement set of amino acids at the C-Terminus of Thymidylate Synthase
was reported by Climie CS et al. The C-terminal residue of thymidylate synthase (TS)
is highly conserved and has been implicated in cofactor binding, catalysis, and a
conformational change.78

Agrawalla S et al reported the X-ray crystal structures of binary complexes of dUMP
or dCMP with the Lactobacillus casei TS mutant N229D, a dCMP methylase,
revealed that there is a steric clash between the 4-NH2 of dCMP and His 199, a
residue which normally H-bonds to the 4-O of dUMP but is not essential for activity.
As a result, the cytosine moiety of dCMP is displaced from the active site and the
catalytic thiol is moved from the C6 of the substrate about 0.5 Å further than in the
wild-type TS-dUMP complex. They concluded that structures of TS H199A/N229D in
complex with dCMP and dUMP confirmed that the position and orientation of bound
dCMP closely approaches that of dUMP in wild-type TS, whereas dUMP was
displaced from the optimal catalytic.79

Discovery of antibacterial agent using Thymidylate Synthase biolibrary screening was
carried out by Costi PM et al. Thymidylate synthase (TS, ThyA) catalyzes the
Page | 25
reductive methylation of 2̍-deoxyuridine 5̍-monophosphate to 2̍-deoxythymidine 5̍monophosphate, an essential precursor for DNA synthesis. A specific inhibition of
this enzyme induces bacterial cell death.80
 Jackman LA et al carried out in vitro activity of 2-desamino-5,8-dideazafolate and 2desamino-N10-propargyl-5,8-dideazafolate (desamino-CB3717), the more water
soluble
2-desamino
analogues
of
5,8-dideazafolate
and
N10-propargyl-5,8-
dideazafolic acid (CB3717). They reported that, removal of 2-amino function from
CB3717 resulted in an increase in water solubility (because of decreased potential
hydrogen bonding) with only an 8-fold loss in TS-inhibitory activity and concomitant
10-fold increase in L1210 cytotoxicity.81
 Shyam KS et al prepared and evaluated a novel folic acid analogue, Nα- (5-deaza5,6,7,8- tetrahydropteroyl) – L – ornithine (3) by multistep synthetic sequence. Results
shown that compound 3 was an effective inhibitor of hog liver folylpolyglutamate
synthetase and showed retardation of polyglutamates of a structurally related folic
acid analogues in HCT – 8 cells in vitro.82
Page | 26
6.4 Objectives of the study
1) To synthesize some newer derivatives of pteridine.
2) To characterize the synthesized compounds by different analytical techniques such
as IR, NMR and Mass spectral data.
3) To screen the synthesized compounds for their in vivo anti-inflammatory, in vivo
analgesic, in vivo anticancer and in vitro antimicrobial activities.
4) To publish the research works in peer reviewed journals.
ACTIVITIES
Literature survey
Synthesis and collection of analytical
data
Pharmacological activities
Typing of thesis book and sending for
publication
Total
DURATION
Till the completion of project
6 Months
2 Months
1 Month
9 Months
Page | 27
7.0
Materials and methods:
7.1 Sources of data
Databases like Chemical abstracts, Biological abstracts, Medline, and Journal of
Chemistry section B, Indian Journal of Heterocyclic Chemistry, European Journal of
Medicinal Chemistry, Bioorganic and Medicinal Chemistry Letters, Acta crystallographica,
through Helinet of RUGHS etc.
7.2 Method of collection of Data
A) Synthesis of the compounds:
Chemicals and other reagents required for the synthesis will be procured from
standard company sources. Compounds will be synthesized by using standard
procedures. The reactions will be monitored by TLC and purification of the compounds
will be carried out by recrystallization method using suitable solvent.
Scheme:R
R
O
R
NH2
substituted 4,5-diaminopyrimidine
-2H 2 O
3
N
R
N
R
N
+
N
R
3
NH2
N
1
2
2
O
4
R
1
R
substituted
1,2-dicarbonyl
N
4
substituted pteridine
R1=NH2 , R2=NH2 or OH , R3= PABA derivatives ,R4=alkyl group
B) Characterization of the compounds:
The synthesized compounds will be characterized by preliminary laboratory
techniques such as melting point, boiling point etc and by FTIR, Mass Spectroscopy
and NMR spectral data.
C) 1)) Screening of anti-inflammatory activity:1,4,5
In vivo anti-inflammatory activity study:
Method Used: Carrageenan-induced paw edema model.
Page | 28
Animals Used: C57 BL/6 male mice
Number of animals used: 72 numbers
Carrageenan-induced paw edema model:
A 1% w/v suspension of carrageenan will be prepared freshly in normal saline
and injected into subplantar region of left hind paw (usually 0.1 mL in rats and 0.0250.05 mL in mice). In control group animals, only vehicle will be injected. Test drug is
usually administered orally or intraperitoneally, according to body weight immediately
or half an hour or one hour before (depending on the expected peak effect)
carrageenan challenge. A mark will be made on the ankle joint of each rodent. Paw
volume up to the ankle joint will be measured in drug treated and untreated groups
before and after 3 h of carrageenan challenge using a plethysmograph filled with
mercury.
2) Screening of anticancer activity:22,24.
Anticancer activity study: Against Ehrlich Ascites Carcinoma (EAC cells)
Method Used: In-vivo anticancer activity
Animals Used: Swiss Albino mice
Number of animals used: 72 numbers
Anticancer activity against Ehrlich Ascites Carcinoma:
The animals will be dividing into twelve groups of 6 animals
each. The EAC cell containing phosphate buffer saline (106 cells/0.1 mL) will be
injected into the peritoneal cavity of test group animals and treatment will be started
24 h after inoculation of tumor cells, (once daily as single dose) for 10 days. Group I
will serve as control and will receive 0.3% CMC suspension. Group II will serve as
standard and will receive vincristine (ip, 520 µg/kg body weight). Groups III-XII will
serve as test groups and receive test compound, administered intraperitoneally.
Antitumor activity will be screened by determining different parameters like body
weight analysis, mean survival time and percent increase in life span.
3) Screening of analgesic activity:3
In vivo analgesic activity study:
Method Used: Acetic acid Writhing method.
Page | 29
Animals Used: Swiss Albino mice.
No. of animals used: 72 nos.
Acetic-acid induced writhing in mice:
The animals will be weighed, numbered and divided into two groups. Each group
contains five animals. Appropriate volume of acetic acid solution will be administer to
the first group (which serves as control) and placed individually under glass jar for
observation. The onset on Writhes will be noted. The number of abdominal
contractions, trunk twist response and extension of hind limbs as well as the number of
animals showing such response during a period of 10 min is recorded. The test
compound will be injected to the second group of animals. Fifteen minutes later, acetic
acid solution will be administered to these animals. The onset and severity of writhing
response will be noted. The mean writhing scores in control and compound treated
groups are calculated & inhibition of pain response by compound will be noted.
7.3 Does the study require any investigation or interventions to be
conducted on patients or other humans or animals?
YES
7.4 Has ethical clearance been obtained from your institution in case of
7.3?
OBTAINED & ENCLOSED
Page | 30
8.0
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9.0
Signature of the Candidate
10.0
Remarks of the Guide
11.0
Name and Designation of
11.1Guide
Mr. D. Giles
Asst. Professor,
Department of Pharmaceutical Chemistry,
Acharya & B.M. Reddy College of Pharmacy,
Soldevanahalli,
Bangalore-90
11.2 Signature
11.3 Co-Guide
NIL
11.4 Signature
11.5 Head of the
Department
Dr. Amit Kumar Das
Professor,
Department of Pharmaceutical Chemistry,
Acharya & B.M. Reddy College of Pharmacy,
Soldevanahalli,
Bangalore-90
11.6 Signature
12.0
12.1Remarks of Principal
12.2 Name of the Principal
Dr. Divakar Goli
Principal,
Acharya and B.M. Reddy College of Pharmacy,
Soldevanahalli,
Bangalore-90
12.3 Signature
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