Novel 2-amino-4-oxo-5-arylthio-substituted-pyrrolo[2,3-d]pyrimidines as nonclassical antifolate inhibitors of thymidylate synthase

Article abstract of DOI:10.1016/j.bmcl.2005.03.029

A series of 17 novel 2-amino-4-oxo-5-[(substituted phenyl)thio]pyrrolo[2,3- d]pyrimidines were synthesized as potential inhibitors of thymidylate synthase (TS) and as antitumor agents. The analogues contain a variety of electron withdrawing substituents on the phenyl ring of the side chain and were evaluated as inhibitors of human TS (hTS) and Escherichia coli TS and of human and E. coli dihydrofolate reductase (DHFR). The analogues 14, 17, and 18 were potent inhibitors of hTS with IC₅₀ values of 0.28, 0.21, and 0.22 μM, respectively, and were more potent than the clinically used ZD1694, 2 and LY231514, 3 against human TS.

Full text of DOI:10.1016/j.bmcl.2005.03.029

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2225–2230
Novel 2-amino-4-oxo-5-arylthio-substituted-
pyrrolo[2,3-d]pyrimidines as nonclassical antifolate
inhibitors of thymidylate synthase
Aleem Gangjee,a,* Hiteshkumar D. Jaina and Roy L. Kisliukb
aDivision of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA 15282, USA
bDepartment of Biochemistry, Tufts University School of Medicine, Boston, MA 02111, USA
Received 14 February 2005; revised 4 March 2005; accepted 7 March 2005
Abstract—A series of 17 novel 2-amino-4-oxo-5-[(substituted phenyl)thio]pyrrolo[2,3-d]pyrimidines were synthesized as potential
inhibitors of thymidylate synthase (TS) and as antitumor agents. The analogues contain a variety of electron withdrawing substit-
uents on the phenyl ring of the side chain and were evaluated as inhibitors of human TS (hTS) and Escherichia coli TS and of human
and E. coli dihydrofolate reductase (DHFR). The analogues 14, 17, and 18 were potent inhibitors of hTS with IC50 values of 0.28,
0.21, and 0.22 lM, respectively, and were more potent than the clinically used ZD1694, 2 and LY231514, 3 against human TS.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Thymidylate synthase (TS) catalyzes the reductive meth-
ylation of 20-deoxyuridine-50-monophosphate (dUMP)
to 20-deoxythymidine-50-monophosphate (dTMP) utiliz-
ing 5,10-methylenetetrahydrofolate as the source of the
methyl group as well as the reductant.1 This represents
the sole de novo source of dTMP and hence inhibition
of TS, in the absence of salvage, leads to Ôthymineless
deathÕ. Thus inhibition of TS is an attractive goal for
the development of antitumor agents.2,3
PDDF,7 ZD1694, and LY231514 are classical antifo-
lates. They contain a benzoyl L-glutamic acid side chain
similar to natural folates. The presence of the benzoyl-L-
glutamic acid side chain usually provides excellent sub-
strate properties for the enzyme folylpoly-c-glutamate
synthetase (FPGS).6,7 FPGS catalyzes the formation of
poly-c-glutamates, which lead to high intracellular con-
centrations of these antitumor agents and at the same
time, increases TS inhibitory activity for certain antifo-
lates (ZD1694 and LY231514). Although polyglutamyl-
ation of some classical antifolates is necessary for
cytotoxicity, it has also been implicated as a possible
cause of toxicity to host cells and in the development
of resistance in tumors with low levels of FPGS. An addi-
tional problem associated with classical antifolates is
their dependence on a reduced folate carrier (RFC) sys-
tem to gain access into the cell. The impairment of this
carrier system can also lead to drug resistance.8
5-Fluorouracil (5-FU), a mechanism based inhibitor of
TS, is a prominent antitumor agent. The emergence of
resistance as well as the insensitivity of certain tumor
types to 5-FU has prompted the design of novel folate
analogues as potential TS inhibitors and antitumor
agents.4 Several TS inhibitors, related to folic acid nota-
bly ZD16945 (Tomudex,TM Raltitrexed) and LY2315146
(Alimta,TM Pemetrexed) (Fig. 1) have been approved in
Europe and the US, respectively, as antitumor agents.
The recent approval of LY231514 for the treatment of
malignant pleural or peritoneal mesothelioma in combi-
nation with cisplatin and as a single-agent in the treat-
ment of locally advanced or metastatic nonsmall cell
lung cancer (NSCLC) has provided additional stimulus
for the design of novel TS inhibitors as antitumor agents.
The problems associated with classical antifolates, such
as resistance due to decreased FPGS activity and/or inef-
ficient uptake could be overcome by nonclassical lipo-
philic analogues, which would not be dependent on
FPGS for their potency and could passively diffuse into
cells.8 AG337 (Thymitaq) (Fig. 1) is the first nonclassical
TS inhibitor to reach clinical trials.9 Gangjee et al.10
designed the 2-amino-4-oxo-5-thiopyridyl-6-methylpyr-
rolo[2,3-d]-pyrimidine, 5 as a potent TS inhibitor (human
TS IC50 = 0.34 lM), based on the X-ray crystal structure
*
Corresponding author. Tel.: +1421 396 6070; fax: +1421 396
5593; e-mail: gangjee@duq.edu
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.029

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A. Gangjee et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2225–2230
COOH
O
O
O
O
N
N
H
COOH
S
N
HN
H3C
N
COOH
HN
H2N
HN
CH3
N
2 ZD1694
COOH
OMe
1 PDDF
N
OMe
OMe
NH2 CH3
O
N
O
N
S
CH3
N
N
HN
H2N
HN
H2N
H
O
H2N
N
N
H
HN
COOH
3 LY231514
4 AG337
Trimetrexate (TMQ)
HOOC
R
N
O
N
O
S
S
HN
H2N
HN
CH3
CH3
N
N
H2N
N
H
H
6-24
5
6
R = 4'-NO2
R = 3',4'-diCl
R = 3'-Cl
13 R = 4'-CF3
14 R = 4'-CN
15 R = 4'-OCF3
16 R = 3'-NO2, 4'-Cl
17 R = 3'-NO2, 4'-Br
18 R = 3'-Cl, 4'Br
19 R = 3'-Cl, 4'-F
20 R=3',4'-diF
21 R=3',5'-diF
22 R=3',5'-diCF3
23 R=3',5'-diNO2
24 R=3',5'-diCl
7
8
9
R = 3'-Br
10 R = 3'-F
11 R = 4'-F
12 R = 4'-Br
Figure 1.
of TS and AG337.8 Molecular modeling of compound 5
in human TS indicated that the 6-methyl group makes
an important hydrophobic contact with a Tryptophan
(Trp109) of human TS and it also sterically restricts the
rotation of the 5-position side chain so that it adopts
the most favorable conformation for binding to human
TS. SAR studies on 5 afforded 6 and 7 (Fig. 1), which were
somewhat more potent inhibitors of human TS with IC50
values of 0.15 and 0.13 lM, respectively.11 Compounds 6
and 7, with electron withdrawing nitro- or chloro- substit-
uents in the 30- and/or 40-positions of the side chain phenyl
ring, were found to be much more potent than the unsub-
stituted phenyl (IC50 = 30 lM) or an analogue containing
classical antifolates.8,11 In contrast, para substituted elec-
tron donating groups on the phenyl ring have been
reported to be detrimental to TS inhibitory activity.8,12,13
In previous studies Gangjee et al.10,11 have shown that
TS inhibitors demonstrate differences in inhibitory
potency between TS from different sources. We reasoned
that lipophilic nonclassical inhibitors of TS may also
provide for selective inhibition of bacterial TS. Thus
the target nonclassical TS inhibitors, 8–24 (Fig. 1), in
addition to their antitumor effects, were also of interest
as potential selective antibacterial agents.
30,40-dimethoxy, electron donating, substituents (IC50
=
Thus both the position and nature of the electron with-
drawing groups present on the phenyl ring of the side
chain influences human TS inhibitory activity.10,11 In
an attempt to determine the optimum electron with-
drawing substituent(s) on the phenyl ring of the side
chain for human TS inhibitory activity compounds 8–
24 were synthesized with a variety of electron withdraw-
ing groups including chlorine, fluorine, nitrile, bromine,
nitro, trifluoromethyl, and trifluoromethoxy at the 30
and/or 40-positions of the phenyl side chain. The pur-
pose of this study was to determine the optimum loca-
tion and nature of the electron withdrawing group(s)
on the phenyl side chain for human TS and bacterial
TS inhibitory activity.
2.4 lM) against human TS. The 30,40-dichloro analogue,
7, was 8-fold more potent than the 40-chloro analogue
(IC50 = 1 .0lM) against human TS. Thus the introduc-
tion of an additional chlorine atom at the 30-position re-
sulted in increased activity against human TS. The
increased activity could be attributed to the presence of
additional bulk at the 30-position and/or reinforcement
of the direction and magnitude of the dipole in the mole-
cule.8 The nature of the electron withdrawing group pres-
ent at the 40-position also influences the inhibitory activity
against human TS. Thus the 40-nitro analogue, 6, was
about 7-fold more potent than the 40-chloro analogue
(IC50 = 1 .0lM) against human TS. Compounds 6 and 7
were also more potent than the classical analogues
PDDF, ZD1694, and LY231514 against human TS. In
addition, potent nonclassical TS inhibitors reported
in the literature possess electron withdrawing groups in
the phenyl ring in place of the benzoyl-L-glutamate of
Recently, Gangjee et al.14 suggested dual binding modes
for 2-amino-4-oxo-pyrrolo[2,3-d]pyrimidines. These
consist of the 2-amino-4-oxo mode (Fig. 2), which is
the usual binding mode of antifolates for TS inhibition19

A. Gangjee et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2225–2230
2227
CH3
R
2 equiv of I2 and reflux maintained for 3–4 h. Purifica-
tion via addition of NaS2O7, to remove the excess I2,
was an additional modification, which significantly re-
duces the volume of eluent (CHCl3) necessary in the sub-
sequent chromatographic purification. Evaporation of
the solvent under reduced pressure gave a residue, which
was purified by column chromatography to afford the
target compounds 8–24 in 9–55% yields. The absence
of the 5-aromatic proton and the presence of the appro-
priate protons of the phenyl side chain for 8–24 con-
firmed that substitution had occurred. This method
has the advantage of decreasing the reaction steps to
one and 3–4 h for completion with an average yield of
39% compared to the previous method with an overall
average yield of only 25% over two steps. The aryl thiols
28–30 were not commercially available and were synthe-
sized15 from the precursor amines 31–33. The method
involved diazotization of the amine with NaNO2 in aq
HCl acid, followed by nucleophilic displacement of the
diazonium moiety with ethyl xanthic acid potassium
salt, and hydrolysis of the resulting xanthate ester with
KOH (Scheme 2). Acidification of the xanthic acid ob-
tained resulted in the evolution of carbon oxysulfide
and the formation of an oil, which was extracted with
ether. The crude oil obtained on evaporation of the
ether contained the desired aryl thiols, which were used
without further purification.
O
N
HN
R
HN
H2N
N
CH3
N
H2N
N
H
O
H
2-Amino-4-Oxo Mode
2,4-Diamino Mode
Figure 2.
and the flipped 2,4-diamino mode, which is the usual
binding mode of antifolates for DHFR inhibition. This
flipped mode is obtained by a 180ꢁ rotation about the
2-NH2–C2 bond of the 2-amino-4-oxo mode. In this bind-
ing mode the pyrrole NH mimics the 4-amino moiety of
the 2,4-diamino pyrimidine such as trimetrexate. Since
compounds 8–24 are pyrrolo[2,3-d]pyrimidines and
could adopt both binding modes discussed above, these
compounds were also of interest as potential DHFR
inhibitors.
Compounds 8–24 were synthesized from the key inter-
mediate
2-amino-4-oxo-6-methylpyrrolo[2,3-d]pyrim-
idine, 2711 (Scheme 1), to which various aryl thiols
were conveniently attached at the 5-position via a mod-
ification of an oxidative thiolation procedure reported
by Gangjee et al.10,11 The original procedure of 5-thio-
arylation involved a two step sequence in which the 2-
amino group of compound 27 was protected with a piva-
loyl group, in order to increase its solubility, followed by
oxidative addition of the aryl thiols to the 5-position of
the pivaloyl protected compound. This oxidative addi-
tion step also provide for concurrent deprotection of
the 2-amino group.10,11 The disadvantage of the re-
ported method is that it involves a tedious protection
and deprotection of the 2-amino group. The protection
requires 48 h while the deprotection requires an addi-
tional 12–16 h. To provide a more efficient procedure
the oxidative addition of the aryl thiols was attempted
on 27 without 2-amino protection.10,11 Compound 27
was obtained by the addition of chloroacetone, 26, to
a solution of 2,6-diamino-4-hydroxypyrimidine, 25, in
NaOAc and water as reported previously.11 Following
several variations of reaction time, temperature and se-
quence of addition of the reactants, it was determined
that the addition of I2 after, rather than before, the addi-
tion of the thiophenol affords better yields of the target
compounds without the necessity of 2-amino protection
compared with the previously reported method.10,11
Thus 8–24 were synthesized in a one-step oxidative addi-
tion, of the appropriately substituted aryl thiols, to the
5-position of 27. The optimized procedure involves heat-
ing a mixture of 27 with the appropriate aryl thiol in eth-
anol/water (2:1) at reflux followed by the addition of
Compounds 8–24, along with LY231514 and ZD1694
were evaluated16,17 as inhibitors of human TS and Esch-
erichia coli TS (Table 1). All of the compounds were
moderate to potent inhibitors of human TS except 22,
which was inactive at the concentration tested. Com-
pounds 14, 17, and 18 were the most potent inhibitors
of human TS and were more potent than the classical,
clinically used analogues ZD1694 and LY231514. They
were also comparable in potency to the previously syn-
thesized compounds, 6 and 7.11 In general, all com-
pounds (except 22 and 23) were more potent than
LY231514 against human TS. Compounds 14, 17, and
18 were approximately 34-, 43-, and 45-fold more potent
R
R
a-d
NH2
SH
28 R=3'Cl-4'-Br
29 R=3',5'-diF
30 R=3',4'-diF
31 R=3'Cl-4'-Br
32 R=3',5'-diF
33 R=3',4'-diF
Scheme 2. Reagents and conditions: (a) NaNO2, concd HCl, 0 ꢁC; (b)
ethyl xanthic acid potassium salt, 60 ꢁC; (c) KOH, reflux; (d) concd
HCl.
R
O
O
O
S
R
Cl
O
SH
HN
H2N
HN
H2N
HN
H2N
CH3
CH3
+
a
b
N
N
N
N
N
NH2
CH3
H
H
25
26
27
8-24
Scheme 1. Reagents and conditions: (a) NaOAc/H2O, 100 ꢁC; (b) I2, EtOH/H2O (2:1), 100–110 ꢁC.

2228
A. Gangjee et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2225–2230
Table 1. Inhibitory concentration (IC50s in M) against Isolated TS and DHFRa
Compd
TS
DHFR
Humanb ·10ꢀ6
E. colib ·10ꢀ5
E. coli c ·10ꢀ5
Humand ·10ꢀ5
5
0.34e
0.15f
0.13f
1.4
6
7
8
>3.3(0)g
>2.8(22)
>3.3(0)
>3.3(0)
>2.8(0)
>2.9(23)
>3.4(0)
>2.8(18)
>2.8(6)
>2.5(18)
>2.6(41)
>3.1(12)
>3.2(10)
>3.2(0)
>3.2(0)
9
1.2
2.8
>2.4(17)
>2.8(9)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
ZD1694h
1.4
>3.2(0)
>2.8(20)
>2.9(0)
>3.4(33)
0.46
0.8
0.28
2.3
>2.3(18)
2.3
1.1
0.46
0.21
0.22
0.83
0.9
>2.6(0)
>3.1(12)
>3.2(14)
>3.2(10)
2.7
3)
>21>2.5(39)
23
2.4
>2.5(1
>2.3(10)
>2.4(26)
5.7
>2.7(0)
>2.9(15)
0.38
9.5
LY231514i
MTX
76
230
6.6 · 10ꢀ4
6.6
2.2 · 10ꢀ3
a The percent inhibition was determined at a minimum of four inhibitor concentration within 20% of the 50% point. The standard deviations for
determination of IC50 values were within 10% of the given value.
b Enzymes kindly supplied by Dr. F. Maley, New York State Dept. Health, Albany, NY.
c Kindly provided by Dr. R. L. Blakley, St. Jude ChildrenÕs Hospital, Memphis, TN.
d Kindly provided by Dr. J. H. Freisheim, Medical college of Ohio, Toledo, OH.
e The data has been taken from Ref. 11.
f The data has been taken from Ref. 12.
g Numbers in parentheses indicate % inhibition at the given concentration.
h Kindly provided by Dr. Ann Jackman, Institute of Cancer Research, Sutton, Surrey, UK.
i Kindly provided by Dr. Chuan Shih, Eli Lilly & Co.
than LY231514, respectively. Compounds 12 and 16
were equipotent with ZD1694, while 19 and 20 were
approximately one-half as potent as ZD1694 against
human TS. In general compounds bearing an electron
withdrawing group at both the 30- and 40-positions
(16–20) were the most potent inhibitors of human TS.
Monosubstitution of an electron withdrawing group at
either the 30- or 40-position (8–15) was found to be more
favorable for human TS inhibition than having substitu-
tion at both the 30- and 50-positions (21–24). The nature
of the halogen substituent at the 30-position has little
influence on human TS inhibition, (compare compounds
8, 9, and 10) However in the 40-position the nitrile (14) is
the most favorable substituent and is equipotent with
40-nitro analogue 6.11 Thus large electron withdrawing
groups at the 40-position afford potent human TS inhibi-
tion. The trend at the 40-position substitution for human
TS inhibitory activity is as follows: nitro > nitrile >
bromine > trifluoromethyl > fluorine > trifluoromethoxy.
This indicates that size along with strong electron with-
drawing properties at the 40-position is conducive to po-
tent human TS inhibition. In addition, a strong electron
withdrawing group at the 40-position is better than at
the 30-position (compare 10 and 11; and 12 and 9). In
the 30,50-disubstituted series electron withdrawing chlo-
rine and fluorine are more favorable than nitro or triflu-
oromethyl substituents for human TS inhibition. The
replacement of a chlorine (16) with a bromine (17) re-
sults in a 2-fold increase in activity against human TS,
again suggesting that size along with an electron with-
drawing effect at the 40-position is conducive for potent
TS inhibition. The same trend was observed in 18 and 19
where the replacement of an electron withdrawing fluo-
rine (19) with bromine (18) results in a 4-fold increase in
human TS inhibitory activity. In the 30,40-disubstituted
compounds the nature of the electron withdrawing
group present at the 40-position dictates the activity
against human TS. Thus 17 and 18 with bromine at
the 40-position but different electron withdrawing
groups at the 30-position have similar potency, however
compounds 16 and 17 have the same electron withdraw-
ing nitro group at the 30-position but differ in the 40-hal-
ogen substitution and have different activities.
Compound, 17 (40-Br) is approximately 2-fold more po-
tent than 16 (40-Cl). A similar trend was observed for
compounds 18 and 19 where a 40-bromine compared
to fluorine imparts a 4-fold increase in TS inhibitory
activity. Compounds 19 and 20 which have fluorine at
the 40-position but different electron withdrawing
groups at the 30-position are similar in activity to 17
and 18. The position of the electron withdrawing sub-
stituents on the phenyl ring also plays an important role
in human TS inhibition. The 30,50-difluoro substituted
21 was approximately 3-fold less active than the 30,40-

A. Gangjee et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2225–2230
2229
Figure 3. Stereoview showing the interaction of the 6-methyl moiety (van der walls dot surface in violet) of compound 14 with Trp 109 in human TS
(PDB2KCE)20 and the hydrogen bonding between 7NH and d1-O of Asn 112. The Trp83 of E. coli TS in yellow.
difluoro 20 against human TS. Similarly the 30,50-di-
chloro 24 was approximately 18-fold less active than
the previously synthesized 30,40-dichloro 7 suggesting
that the 30,40-disubstitution is more conducive to human
TS inhibitory activity. Selected analogues were tested
against human DHFR and were found to be poorly ac-
tive (Table 1) suggesting that perhaps these compounds
do not flip to adopt the Ô2,4-diaminoÕ binding mode
where the pyrrole NH mimics the 4-amino moiety of
DHFR inhibitors. All of the compounds tested were
also inactive against E. coli DHFR (Table 1).
In summary, the SAR for 5-phenylsubstituted-2-amino-
4-oxopyrrolo[2,3-d]pyrimidines against human TS indi-
cates that the nature and position of the substituent(s)
on the phenyl ring is important. In general, electron
withdrawing 30,40-disubstituted compounds (7, 17, and
18) have the highest potency. Disubstitution, at both
the 30- and 50-positions (22 and 23) is detrimental to po-
tent TS inhibition. In addition, the nature of the electron
withdrawing group at the 40-position is important while
the nature of the electron withdrawing group at the 30-
position is less important. The analogues synthesized
in this series demonstrates that strong electron with-
drawing groups in the 30- and 40-positions of the phenyl
side chain can provide nonclassical antifolates that are
better human TS inhibitors than the clinically used clas-
sical compounds ZD1694 and LY231514.
Compounds 16 and 17 were moderately potent inhibi-
tors of E. coli TS. All of the analogues tested were more
potent against human TS than E. coli TS indicating a
difference in the architecture of human TS and E. coli
TS. Molecular modeling using SYBYL 6.9118 and superim-
position of the 6-5 pyrrolo[2,3-d]pyrimidine of com-
pounds 8–24 on the 6-5 ring system of ZD1694 in the
crystal structure of ZD1694 in human TS (PDB
2KCE)19 suggested that the 7NH of compounds 8–24
can hydrogen bond with the d1-O of Asn 112 in human
TS.19 This interaction is not possible in E. coli TS in
which Asn 112 is replaced by Trp 83. Stroud and co-
workers20 have shown these interactions in the crystal
structure of LY231514 in human TS and E. coli TS
and this could account for the increased activity of 8–
24 against human TS compared to E. coli TS. The inter-
action of the 6-methyl moiety of compounds 8–24 with
Trp 109, which enhances TS inhibitory activity, is illus-
trated by van der Waals dot surface shown in Figure 3.
The lipophilic analogue, 17, was selected for evaluation
by the National Cancer Institute in its preclinical in vitro
tumor screening program and was found to be moder-
ately cytotoxic with a growth inhibitory concentration
GI50 = 4.23 lM against Molt-4 leukemia cancer cells,
6.18 lM against HT29 colon cancer cells, and 6.55 and
3.28 lM against MDA-MB-231and MDA-MB-435
breast cancer cell lines, respectively.
Acknowledgements
This work was supported, in part, by NIH Grants from
the National Cancer Institute CA98850 (A.G.) and
CA10914 (R.L.K.).
Supplementary data
Details of the synthesis and characterization of all new
compounds is provided. Supplementary data associated
with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2005.03.029.
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