Potent dual thymidylate synthase and dihydrofolate reductase inhibitors: Classical and nonclassical 2-amino-4-oxo-5-arylthio-substituted-6- methylthieno[2,3-d]pyrimidine antifolates

Article abstract of DOI:10.1021/jm8006933

N-{4-[(2-Amino-6-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-5-yl) sulfanyl]benzoyl}-L-glutamic acid (4) and nine nonclassical analogues 5-13 were synthesized as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors. Th

Full text of DOI:10.1021/jm8006933

J. Med. Chem. 2008, 51, 5789–5797
5789
Potent Dual Thymidylate Synthase and Dihydrofolate Reductase Inhibitors: Classical and
Nonclassical 2-Amino-4-oxo-5-arylthio-substituted-6-methylthieno[2,3-d]pyrimidine Antifolates
Aleem Gangjee,*,† Yibin Qiu,† Wei Li,† and Roy L. Kisliuk‡
DiVision of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne UniVersity, 600 Forbes AVenue, Pittsburgh,
PennsylVania 15282, and Department of Biochemistry, Tufts UniVersity School of Medicine, Boston, Massachusetts 02111
ReceiVed June 6, 2008
N-{4-[(2-Amino-6-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-5-yl)sulfanyl]benzoyl}-L-glutamic acid
(4) and nine nonclassical analogues 5-13 were synthesized as potential dual thymidylate synthase (TS) and
dihydrofolate reductase (DHFR) inhibitors. The key intermediate in the synthesis was 2-amino-6-
methylthieno[2,3-d]pyrimidin-4(3H)-one (16), which was converted to the 5-bromo-substituted compound
17 followed by an Ullmann reaction to afford 5-13. The classical analogue 4 was synthesized by coupling
the benzoic acid derivative 19 with diethyl L-glutamate and saponification. Compound 4 is the most potent
dual inhibitor of human TS (IC50 ) 40 nM) and human DHFR (IC50 ) 20 nM) known to date. The nonclassical
analogues 5-13 were moderately potent against human TS with IC50 values ranging from 0.11 to 4.6 µM.
The 4-nitrophenyl analogue 7 was the most potent compound in the nonclassical series, demonstrating potent
dual inhibitory activities against human TS and DHFR. This study indicated that the 5-substituted 2-amino-
4-oxo-6-methylthieno[2,3-d]pyrimidine scaffold is highly conducive to dual human TS-DHFR inhibitory
activity.
Introduction
transported into cells via the reduced folate carrier (RFC) and
undergo rapid polyglutamylation by the enzyme folylpoly-
glutamate synthetase (FPGS).13–17
Most eukaryotic organisms synthesize the essential metabolite
thymidylate via the thymidylate cycle which consists of three
enzymes, serine hydroxymethyltransferase (SHMTa), thymidy-
late synthase (TS), and dihydrofolate reductase (DHFR). SHMT
catalyzes the interaction of serine and tetrahydrofolate to form
5,10-methylenetetrahydrofolate (5,10-MTHF). TS then catalyzes
the reductive methylation of 2′-deoxyuridine-5′-monophosphate
(dUMP) to 2′-deoxythymidine-5′ monophosphate (dTMP), with
5,10-MTHF as the cofactor, and the formation of dihydrofolate.
DHFR then completes the cycle by catalyzing the conversion
of dihydrofolate to tetrahydrofolate using NADPH as the
reductant.1–5 Inhibition of TS or of DHFR leads to “thymineless
death” in the absence of salvage,6,7 and inhibition of these
enzymes has found clinical utility as antitumor, antimicrobial,
and antiprotozoal agents.8,9
As shown in Figure 1, folate analogues that inhibit TS
generally contain a 2-amino-4-oxo or 2-methyl-4-oxo substitu-
tion in the pyrimidine ring, for example, the clinically used
pemetrexed (PMX)10 and raltiterxed (RTX).11 In contrast, folate
analogues that inhibit DHFR generally contain 2,4-diamino
substitution in the pyrimidine ring, typified by methotrexate
(MTX),12 a DHFR inhibitor that has been a mainstay in cancer
chemotherapy. Raltiterxed (Figure 1) is a quinazoline based
analogue that has been approved as a first-line agent for
advanced colorectal cancer in several European countries,
Australia, Canada, and Japan. PMX, RTX, and MTX are
Pemetrexed, a 6-5 pyrrolo[2,3-d]pyrimidine represents an
important example of a clinically used classical antifolate that
has reported dual TS and DHFR inhibitory activity. Pemetrexed
and its polyglutamylated metabolites are reported to be inhibitors
of several important folate-dependent enzymes including TS,
DHFR, glycinamide ribonucleotide formyltransferase (GARFT),
and aminoimidazole carboxamideribonucleotide formyltrans-
ferase (AICARFT). Pemetrexed is designated as a multitargeted
antifolate (MTA).18,19 In combination with cisplatin, pemetrexed
has been approved for malignant pleural mesothelioma and for
non-small-cell lung cancer. The clinical success of pemetrexed
has generated renewed interest in the design of single agents
that function as dual inhibitors against TS and DHFR.20,21 Such
inhibitors could circumvent pharmacokinetic, drug-drug inter-
actions, and/or toxicity disadvantages of administering two
separate agents in combination chemotherapy protocols. In
addition, the cost of a single agent might be less than two
separate agents and might also improve patient compliance.
It has been our long-standing goal to design and synthesize
single agents that are potent dual inhibitors against TS and
DHFR. As part of a continuing effort to develop dual TS and
DHFR inhibitors, Gangjee et al.20 reported the synthesis of a
classical antifolate N-{4-[(2-amino-6-methyl-4-oxo-4,7-dihydro-
3H-pyrrolo[2,3-d]pyrimidin-5-yl)thio]benzoyl}-L-glutamic acid,
1 (Figure 2). Compound 1 is a potent inhibitor of human TS
(IC50 ) 54 nM) and a moderate inhibitor of human DHFR (IC50
) 2.2 µM), in its monoglutamate form, thus providing dual
inhibition of human TS and human DHFR. More importantly,
compound 1 was not a substrate for human FPGS at concentra-
tions up to 1045 µM, indicating that 1, unlike pemetrexed or
raltitrexed does not require polyglutamylation for its potent
inhibition of human TS. Molecular modeling using (SYBYL
6.91)22 indicated that the 6-methyl group in 1 makes important
hydrophobic contacts with Trp109 in human TS and also
* To whom correspondence should be addressed. Phone: 412-396-6070.
Fax: 412-396-5593. E-mail: gangjee@duq.edu.
†
Duquesne University.
Tufts University School of Medicine.
‡
a Abbreviations: SHMT, serine hydroxymethyltransferase; TS, thymidy-
late synthase; DHFR, dihydrofolate reductase; dUMP, deoxyuridylate;
dTMP, deoxythymidylate; 7,8-DHF, 7,8-dihydrofolate; MTX, methotrexate;
RFC, reduced folate carrier; FPGS, folylpolyglutamate synthetase; GARFT,
glycinamide ribonucleotide formyltransferase; AICARFT, aminoimidazole
carboxamideribonucleotide formyltransferase; AIDS, acquired immuno-
deficiency syndrome; P. carinii, Pneumocystis carinii; T. gondii, Toxoplasma
gondii; E. coli, Escherichia coli; PTX, piritrexim.
10.1021/jm8006933 CCC: $40.75
2008 American Chemical Society
Published on Web 08/23/2008

5790 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Gangjee et al.
Figure 1
sterically restricts the rotation of the 5-position side chain so
that it could adopt a favorable conformation for binding to
human TS. In addition, molecular modeling of 1 suggested that
the 6-methyl group makes hydrophobic contact with Val115 in
human DHFR, which is absent in pemetrexed modeled in human
DHFR. Thus, 1 is a promising lead compound that can be further
structurally modified to optimize dual human TS and human
DHFR inhibitory activities.
phobic contacts with Trp109 in human TS and also serves to
lock the 5-position side chain into favorable, low-energy
conformations for TS binding. Both these aspects were antici-
pated to contribute to potent inhibitory activity of 4 against
human TS in its monoglutamate form.
Molecular modeling revealed that compound 4 could also bind
to human DHFR in either the “normal” folate binding mode to
DHFR (Figure 5) or the “flipped” folate binding mode to DHFR
(Figure 6). In the “normal” binding mode (Figure 5), compound
4 binds just like folic acid (PDB 1DRF). Superimposition onto
folate (not shown) shows the 2-NH2 and N3 moieties form
hydrogen bonds with Glu30 (2.53 and 2.70 Å, respectively).
The R-carboxyl group of 4 interacts with Arg70 in an ionic
bond, and the thieno[2,3-d]pyrimidine and the phenyl ring make
hydrophobic contacts with Phe31, Phe34, and Ile60 (Figure 5)
in the binding pocket.
In the course of our structure-based drug design program,
we investigated the thieno[2,3-d]pyrimidine scaffold, which
could be considered an isostere of the pyrrolo[2,3-d]pyrimidine
system, to develop potential dual TS and DHFR inhibitors. Thus,
compound 4 (Figure 2) was designed as an isostere of 1 to
determine the importance of the pyrrole 7-NH in 1 for binding
to human TS and DHFR. The replacement of the NH of a
pyrrolo[2,3-d]pyrimidine with a S to afford the thieno[2,3-d]-
pyrimidine was also anticipated to evaluate the importance of
a hydrogen bond donor (NH) versus a hydrogen bond acceptor
(S). In addition, the larger size of the sulfur atom compared to
the nitrogen allows the thieno[2,3-d]pyrimidines to more closely
mimic the size of the 6-6 pteridine system of folates. Molecular
modeling using SYBYL 7.1 indicated that compound 4 should
bind to human TS in the “normal” mode (Figure 3) and bind to
human DHFR in the “flipped” mode similar to that proposed
for pemetrexed,20 thereby also allowing for human DHFR
inhibition. We23,24 and others25,26 have shown, via X-ray crystal
structures, that both the “normal” and “flipped” modes are viable
binding modes for DHFR of similar analogues. Figure 4 shows
the superimposition of 4 on pemetrexed in the X-ray crystal
structure with human TS (PDB code 1JU6). Like compound 1,
the 6-methyl moiety of compound 4 makes important hydro-
Additionally, molecular modeling indicated that compound
4 could also bind to the human DHFR in the “flipped” mode
(Figure 6) compared to folic acid, in which the sulfur atom of
the thieno ring is superimposed onto the 4-oxo moiety of folate.
This is the “flipped” mode with respect to folate and is the
binding mode for MTX to human DHFR, in its X-ray crystal
structure (PDB 1U72). Superimposition of 4 onto MTX (not
shown) in human DHFR (PDB 1U72) with the sulfur atom
superimposed onto the 4-NH2 group of MTX is shown in Figure
6. In this binding mode, the 4-oxo moiety of compound 4 makes
hydrogen bonding with the backbone of Val115, Ile7, and
Tyr121. The Glu30 residue also interacts with 2-NH2 and N1
moieties of 4. The p-aminobenzoyl ring along with thieno[2,3-
d]pyrimidine ring makes hydrophobic interactions with Phe31,
Figure 3. Proposed binding modes of 2-amino-4-oxo-5-substituted-
6-methylthieno[2,3-d]pyrimidines. The “normal” mode is defined
as the binding mode of pemetrexed and raltitrexed to human TS
and folic acid (a 2-amino-4-oxopyrimidine system) to human
DHFR. The “flipped” mode is defined as the binding mode when
pemetrexed, raltitrexed, or folic acid is rotated about the C2-NH2
bond by 180°.
Figure 2

6-Methylthieno[2,3-d]pyrimidine Antifolates
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5791
Figure 4. Stereoview of compound 4 superimposed on pemetrexed (in green) in human TS (PDB code 1JU6).
Figure 5. Stereoview: compound 4 bound to human DHFR in “normal” mode (PDB code 1DRF), superimposed on folic acid (not shown).
Phe34, and Ile60, and the R-COOH forms an ionic bond with
Arg70 just as MTX does with human DHFR.
is a desirable goal to overcome tumor resistance because of low
or deficient FPGS. A further disadvantage of some classical
antifolates as antitumor agents is that they require an active
transport mechanism to enter tumor cells, which, when impaired,
causes tumor resistance.29–32 In addition, cells that lack these
transport mechanisms, including several bacterial and protozoan
cells, are not susceptible to the action of classical antifolates.
In an attempt to overcome these potential drawbacks associated
with classical antifolates, lipophilic nonclassical antifolates have
also been designed and synthesized.3,33–35 These lipophilic
nonclassical antifolates such as nolatrexed (Figure 1) lack the
polar glutamate moiety and hence do not depend on FPGS for
their inhibitory activity.36,37 In addition, nonclassical antifolates
do not require the RFC system for active uptake into the cell,
On the basis of molecular modeling, the thieno[2,3-d]pyri-
midine classical antifolate 4 can bind to human DHFR in either
mode.Thus,N-{4-[(2-amino-6-methyl-4-oxo-3,4-dihydrothieno[2,3-
d]pyrimidin-5-yl)sulfanyl]benzoyl}-L-glutamic acid, 4 (Figure
2), was designed and synthesized as a potential dual TS-DHFR
inhibitor and an antitumor agent.
Tumor cells develop resistance to classical antifolates such
as pemetrexed and raltitrexed, which depend on polyglutamy-
lation for their antitumor effects by producing low or defective
folylpoly-γ-glutamate synthetase (FPGS).27,28 Thus, the syn-
thesis of classical antifolates that are potent inhibitors of TS
and DHFR that do not require FPGS for their antitumor activity

5792 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Gangjee et al.
Figure 6. Stereoview of compound 4 bound to human DHFR in the “flipped” mode (PDB code 1U72).
since they are lipophilic and are passively transported into cells.
Gangjee et al.38 described the design and synthesis of nonclas-
alkyl-substituted-thiophene-3-carboxylates on cyclization with
chlorformamidine hydrochloride (generated from aminonitrile
and HCl in ethyl ether as previously described46) afforded the
corresponding 2-amino-6-alkyl-substituted-thieno[2,3-d]pyri-
midin-4(3H)-ones in reasonably good yield. Thus, we envisioned
that compound 16 could be obtained in a single step from
chlorformamidine hydrochloride and ethyl 2-amino-5-methyl-
thiophene-3-carboxylate 15 using similar reaction conditions as
reported previously. Treatment of commercially available pro-
pionaldehyde, 14 (Scheme 1), with ethylcyanoacetate, sulfur,
and triethylamine in DMF under nitrogen for 2 h afforded 15
in 56% yield via a Gewald47 reaction. Heating a mixture of 15,
chlorformamidine hydrochloride and DMSO2 at 125 °C for 30
min gave 16 in 87% yield.
With the key intermediate 16 in hand, it was initially
anticipated that different arylthiols could be easily attached to
the 5-position of 16 via an oxidative addition reaction using
iodine in ethanol/water at reflux as reported by Gangjee et
al.20,21,38 Unfortunately, all attempts at this oxidative addition
using a variety of reaction conditions of time and temperature
variations were without success. Failure of the above method
led us to explore a new alternative strategy that involved the
bromination of the 5-position of intermediate 16 followed by
displacement with arylthiols using the Ullmann coupling reac-
tion. The first approach for the synthesis of 2-amino-5-bromo-
6-methylthieno[2,3-d]pyrimidin-4(3H)-one, 17, was to utilize
NBS in a variety of solvents under different temperature
conditions. When DMF or chloroform was used as solvent at
room temperature or under reflux for 24 h, no new spot was
detected on TLC, indicating that no reaction had occurred.
However, when acetic acid was used as the solvent at reflux
for 24 h, the brominated product 17 was obtained in a yield of
14%. The 1H NMR of 17 in deuterated dimethyl sulfoxide
indicated the absence of the 5-proton at 6.77 ppm. This along
with elemental analysis indicated that bromination had occurred.
In an attempt to circumvent the harsh reaction conditions (reflux
24 h) and to improve the overall yield, bromine instead of NBS
was utilized as the brominating reagent. When DMF was used
as the solvent at 50 °C for 24 h, no product was found (TLC).
sical antifolate analogues 2 (IC50 ) 0.15 µM) and 3 (IC50
)
0.13 µM) as potent inhibitors of human TS, which were more
potent against human TS than the clinically used pemetrexed.
Thus, in addition to the classical analogue 4, it was also of
interest to synthesize nonclassical analogues 5-13 (Figure 2)
with the thieno[2,3-d]pyrimidine scaffold as potential antitumor
agents.
An additional aspect of our interest in nonclassical dual
TS-DHFR inhibitors lies in the treatment of opportunistic
infections in immunocompromised patients such as those with
acquired immunodeficiency syndrome (AIDS).39 The principal
cause of death in patients with AIDS is opportunistic infections
caused by Pneumocystis carinii (P. carinii)40 and Toxoplasma
gondii (T. gondii).41 It should be noted that the thieno[2,3-
d]pyrimidine scaffold has been examined previously for anti-
bacterial and antimalarial activity.42–46 Several noncalssical
thieno[2,3-d]pyrimidine analogues have also been evaluated as
inhibitors of P. carinii DHFR and T. gondii DHFR.43–46 Some
of these analogues have been found to be selective for DHFR
from these pathogens.44,46 However, none of these studies
addressed the dual inhibitory effects of thieno[2,3-d]pyrimidines
on TS and DHFR from T. gondii. Thus, we were also interested
in evaluating the nonclassical analogues 5-13 as dual inhibitors
of T. gondii DHFR and T. gondii TS.
Chemistry
The synthetic method for the classical compound 4 and the
nonclassical compounds 5-13 are outlined in Schemes 1 and
2. The key intermediate in the synthesis is 2-amino-6-methyl-
thieno[2,3-d]pyrimidin-4(3H)-one, 16 (Scheme 1), which could
be converted to the 5-bromo substituted compound 17 by
bromination. Subsequent Ullmann coupling reaction with ap-
propriate thiophenols would afford the target compounds 5-13
and the intermediate 18 for the classical target compound 4. A
literature search revealed that 16 has not been previously
reported. Gangjee et al.46 had reported that ethyl 2-amino-5-

6-Methylthieno[2,3-d]pyrimidine Antifolates
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5793
Scheme 1a
a Reagents and conditions: (a) ethylcyanoacetate, TEA, sulfur, DMF, 50 cC to room temp, 2 h; (b) chlorformamidine hydrochloride, DMSO2, 125 °C, 30
min; (c) Br2, AcOH, microwave 150 °C, 30 min; (d) thiols, Cu2O, K2CO3, DMF, microwave 180 °C, 30 min.
Scheme 2a
a Reagents and conditions: (a) ethyl 4-mercaptobenzoate, Cu2O, K2CO3, DMF, microwave 180 °C, 30 min; (b) 1 N NaOH, EtOH, room temp, 18 h; (c)
L-glutamic acid diethyl ester hydrochloride, 2-chloro-4,6-dimethoxy-1,3,5-triazine, N-methylmorpholine, DMF, room temp, 5 h; (d) 1 N NaOH, EtOH, room
temp, 24 h.
However, with acetic acid as solvent at reflux for 12 h a new
spot was obtained on TLC with the disappearance of 16,
indicating that the reactant had been consumed. The isolated
yield for 17 was 35%. Inspired by these initial results, the
bromination of 16 was also attempted under microwave irradia-
tion with bromine in acetic acid at 150 °C for 30 min, which
afforded 17 in a yield of 80%. At this stage, with the
intermediate 17 in hand, attention was turned to the synthesis
of target compounds 5-13 (Scheme 1) and intermediate 18
(Scheme 2). The Ullmann coupling with 17 and appropriate
arylthiols in the presence of Cu2O and K2CO3 in DMF under
microwave irradiation at 180 °C for 30 min afforded 5-13 in
yields of 20-68%.
For the synthesis of the classical compound 4, the required
intermediate 4-(2-amino-6-methyl-4-oxo-3,4-dihydro-thieno[2,3-
d]pyrimidine-5-ylsulfanyl)benzoic acid ethyl ester, 18 (Scheme
2), was prepared, using the same synthetic strategy as shown
for 5-13 (Scheme 1) using ethyl 4-sulfanylbenzoate to afford
18 in a yield of 34%. Ester hydrolysis of 18 with 1 N NaOH at
room temperature for 18 h afforded the corresponding acid 19
in 99% yield. Coupling of the acid 19 (Scheme 2) using
conventional peptide coupling methods with L-glutamic acid
diethyl ester hydrochloride and 2-chloro-4,6-dimethoxy-l,3,5-
triazine as the activating agent followed by chromatographic
purification afforded 20 in 65% yield.20,48 The 1H NMR of 20
revealed the newly formed peptide NH proton at 8.61 ppm as
a doublet, which exchanged on addition of D2O. Hydrolysis of
20 with aqueous NaOH at room temperature, followed by
acidification with 3 N HCl under ice cold conditions, afforded
compound 4 in 60% yield.
trimethoprim. The classical analogue 4 was an excellent dual
inhibitor of human TS (IC50 ) 40 nM) and human DHFR (IC50
) 20 nM). Thus, compound 4 is a novel dual TS-DHFR
inhibitor. To our knowledge this is the first example of a
classical 2-amino-4-oxo-thieno[2,3-d]pyrimidine antifolate that
possesses dual TS-DHFR inhibitory activity.
Against human TS, compound 4 was similar in potency to
the previously reported 1 and about 2-fold more potent than
PDDF and 238-fold more potent than pemetrexed. Against
human DHFR, compound 4 was similar in potency to clinically
used MTX (Table 1) and was 330-fold more potent than
pemetrexed. These results indicate that isosteric structural
modification of the pyrrolo[2,3-d]pyrimidine scaffold to a
thieno[2,3-d]pyrimidine maintains high potency against human
TS and remarkably increases human DHFR inhibition by 105-
fold for 4 compared with compound 1. Thus, the principal goal
to increase the human DHFR inhibitory activity of 1 without
compromising the potent human TS inhibitory activity of 1 was
accomplished. Thus, on the basis of our results, the replacement
of the smaller hydrogen bond donor pyrrolo NH with larger
hydrogen bond acceptor S has little or no effect on human TS
inhibition but increases human DHFR inhibitory potency by
105-fold for classical antifolates. The thieno[2,3-d]pyrimidine
scaffold is the most conducive classical scaffold known to date
for potent dual human TS/DHFR inhibitory activity.
On the basis of our molecular modeling, we have suggested
the possibility of different binding modes of 4 to human DHFR.
The actual mode of binding awaits the X-ray crystal structure
that is currently underway. In addition to these molecular
modeling studies there are three possible reasons for the better
activities of thieno[2,3-d]pyrimidines than pyrrolo[2,3-d]pyri-
midines against human DHFR. The first reason is that the larger
6-5 thieno[2,3-d]pyrimidine ring system more closely ap-
proximates the size of 6-6 bicyclic pteridine ring and 5-deaza
folate systems of DHFR inhibitors MTX and 5-deaza folic acid
than the pyrrolo[2,3-d]pyrimidine of 1. Second, thieno[2,3-
Biological Evaluation and Discussion
The classical and nonclassical analogues 4-13 were evaluated
as inhibitors of human, Escherichia coli (E. coli), and T. gondii
DHFR49 and TS.50 The inhibitory potencies (IC50) are listed in
Table 1 and compared with pemetrexed, PDDF, MTX, and

5794 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Gangjee et al.
Table 1. Inhibitory Concentrations (IC50 in µM) against TS and DHFRa
TS (µM)
DHFR (µM)
E. colie
compd
humanb
E. colib
T. gondiic
humand
T. gondiic
rh/tgf
1g
2h
3h
4
5
6
7
8
9
10
11
12
13
0.042
0.15
0.13
0.04
1.2
0.26
0.11
4.6
0.11
1.0
0.12
0.28
1.1
ndl
13
45
0.04
29
ndl
2.2
ndl
ndl
ndl
ndl
ndl
2.5
29
48
10
393
100
88
66
74
54
15.4
8.6
0.6
>50
ndl
ndl
ndl
ndl
0.2
ndl
ndl
0.036
8.4
ndl
0.02
3.5
1.5
0.56
22
2.8
5.6
2.9
2.5
ndl
0.008
0.12
0.031
0.056
0.056
0.028
0.064
0.044
0.034
0.048
0.43
0.22
0.033
6.8
>35 (18)i
28
5.2
1.2
2.6
1.2
1.2
>23 (36)i
2.3
>23 (0)i
1.2
>28 (0)i
>2.8 (0)i
>2.8 (0)i
58
23
2.4
1.1
10
2.3
1.2
1.4
4.0
35
32
230
23
0.0088
0.01
2.6
6.6
1.9
pemetrexedj
PDDFk
MTX
trimethoprim
9.5
76
2.8
0.085
0.019
0.43
ndl
ndl
ndl
0.02
ndl
ndl
ndl
>340 (22)i
a The percent inhibition was determined at a minimum of four inhibitor concentrations within 20% of the 50% point. The standard deviations for determination
of 50% points were within (10% of the value given. b Kindly provided by Dr. Frank Maley, New York State Department of Health. c Kindly provided by
Dr. Karen Anderson, Yale Univerisy, New Haven, CT. d Kindly provided by Dr. J. H. Freisheim, Medical College of Ohio, Toledo, OH. e Kindly provided
by Dr. R. L. Blakley, St. Jude Children’s hospital, Memphis, TN. f rh/tg is selectivity ratio for T. gondii DHFR and is (IC50 against rhDHFR)/(IC50 against
T. gondii DHFR). g Data derived from ref 20. h Data derived from ref 38. i Numbers in parentheses indicate the % inhibition at the stated concentration.
j Kindly provided by Dr. Chuan Shih, Eli Lilly and Co. k Kindly provided by Dr. M. G. Nair, University of South Alabama. l nd ) not determined.
d]pyrimidines are more aromatic like MTX and 5-deaza folic
acid than pyrrolo[2,3-d]pyrimidines. Lastly, like the pteridine
ring of MTX and 5-deaza folic acid, the thieno[2,3-d]pyrimidine
has a hydrogen bond acceptor in the five-member heterocyclic
ring rather than a hydrogen bond donor as in the pyrrolo[2,3-
d]pyrimidine ring. Thus, the increased similarity of the thieno[2,3-
d]pyrimidine ring system to the pteridine ring may explain the
higher potency of 4 compared to 1 against human DHFR.
The nonclassical analogues 5-13 were also evaluated as
inhibitors of TS and DHFR (Table 1). All of the nonclassical
analogues were reasonably potent inhibitors of human TS with
IC50 values ranging from 0.11 to 4.6 µM. The electronic nature
of the substitutent on the side chain phenyl was an important
factor in determining inhibitory potency. Analogues with
electron withdrawing substitutions on the phenyl ring were more
potent than analogues with electron donating substitutions or
the unsubstituted phenyl. Electron withdrawing 4-chloro, 4-nitro,
3,4-dichloro, and 4-fluoro substituents in analogues 6, 7, 9, and
13, respectively, showed the most potent inhibition against
isolated human TS. Compounds 7 and 9 were comparable in
potency to the pyrrolo[2,3-d]pyrimidine analogues 2 and 3,
respectively. These data are consistent with SAR studies
previously reported for the pyrrolo[2,3-d]pyrimidine series.38
Like the classical analogue 4, all of the nonclassical analogues
were also more potent than pemetrexed as inhibitors of human
TS. This result indicates that isosteric structural modification
of the pyrrolo[2,3-d]pyrimidine to a thieno[2,3-d]pyrimidine
maintains potent human TS inhibitory activity. Compounds
5-13 are also moderately potent inhibitors of human DHFR
with IC50 values ranging from 0.56 to 5.6 µM. Analogue 7 (IC50
) 0.56 µM) was the most potent compound in this series against
human DHFR. Compound 7 was 28-fold less potent against
human DHFR than MTX but was more than 12-fold more potent
than pemetrexed. In addition, all the nonclassical analogues were
more potent human DHFR inhibitors than pemetrexed. These
results demonstrate that the nonclassical analogues follow the
same trend of dual inhibition against human TS and DHFR as
the classical analogue 4 albeit with lower potency. Interestingly,
all the nonclassical compounds were potent inhibitors of T.
gondii DHFR with IC50 values ranging from 0.028 to 0.12 µM.
The IC50 values of compounds 6-13 against T. gondii DHFR
were similar in potency to MTX and were about 243-fold more
potent than the clinically used trimethoprim (Table 1). In
addition, all the nonclassical compounds showed good to
excellent selectivity against T. gondii DHFR compared to human
DHFR. Compound 8 with a 2,5-dimethoxy substitution on the
phenyl ring was marginally active against human DHFR (IC50
) 22 µM) but very potent against T. gondii DHFR (IC50 ) 56
nM) exhibiting 393-fold selectivity, which indicated a distinct
species difference in DHFR from different sources. This 2,5-
dimethoxyphenyl substitution occurs in several other potent
DHFR inhibitors that usually lack selectivity such as piritrexim
(PTX). In this series of compounds, potency and selectivity were
also found with the unsubstituted phenyl analogue and analogues
with electron withdrawing substitutions. These result parallel
the structure-activity relationship (SAR) we recently reported
for the nonclassical N5-substituted 2-amino-4-oxo-6-methylpyr-
rolo[3,2-d]pyrimidines against DHFR.48 The excellent selectivity
of compound 8 against T. gondii DHFR attests to the fact that
differences in mammalian and pathogen DHFR can be exploited
with nonclassical DHFR inhibitors. We are in the process of
developing other nonclassical TS inhibitors with potential
selectivity toward nonmammalian DHFR and TS and other
analogues as antitumor agents.
In summary, the 5-substituted 2-amino-4-oxo-6-methyl-
thieno[2,3-d]pyrimidine classical antifolate 4 and nine nonclas-
sical analogues 5-13 were designed and synthesized as potential
dual TS-DHFR inhibitors. Compound 4 maintained the potent
human TS inhibitory activity of 1 and significantly increased
the human DHFR inhibition of 1 by 105-fold. Compound 4, to
our knowledge, is the most potent dual TS-DHFR inhibitor
known to date. Compound 7 was the most potent compound in
the nonclassical series, also demonstrating potent dual inhibitory
activities against human TS (IC50 ) 0.11 µM) and human DHFR
(IC50 ) 0.56 µM). In addition, excellent potency and high
selectivity for T. gondii DHFR compared to human DHFR were
observed for all the analogues (except 4 and 7). This study
indicated that the 5-substituted 2-amino-4-oxo-6-methylthieno-

6-Methylthieno[2,3-d]pyrimidine Antifolates
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5795
pressure. The crude product was purified by flash chromatography
on silica gel (1% MeOH/CHCl3) to afford 0.57 g (80%) of 17 as
a yellow solid: Rf ) 0.60 (MeOH/CHCl3, 1:7); mp 254-256 °C;
1H NMR (DMSO-d6) δ 2.33 (s, 3 H), 6.60 (s, 2 H), 10.97 (s, 1 H).
Anal. (C7H6BrN3SO) C, H, N, Br, S.
[2,3-d]pyrimidine scaffold is most conducive to dual human
TS-DHFR inhibitory activity.
Experimental Section
2-Amino-4-oxo-6-methyl-5-phenylsulfanylthieno[2,3-d]pyrimi-
dine (5). To a microwave reaction vial was added 17 (0.15 g, 0.6
mmol), K2CO3 (0.25 g, 1.8 mmol), and dry DMF (15 mL). The
mixture was evacuated and back-filled with nitrogen (three cycles).
Cu2O (89 mg, 0.6 mmol) and benzenethiol (0.27 g, 2.4 mmol) were
added, and the reaction mixture was degassed twice. The reaction
mixture was irradiated in a microwave apparatus at 180 °C for 30
min. After the reaction mixture was cooled to ambient temperature,
the mixture was filtered; the filtrate was concentrated under reduced
pressure. The crude product was purified by flash chromatography
on silica gel using 2% MeOH in CHCl3 as the eluent. Fractions
containing the product (TLC) were combined and evaporated to
afford 0.12 g (68%) of 5 as a white solid: Rf ) 0.60 (MeOH/CHCl3,
1:7); mp 291-294 °C; 1H NMR (DMSO-d6) δ 2.38 (s, 3 H), 6.56
(s, 2 H), 7.00-7.22 (m, 5 H), 10.77 (s, 1 H); HRMS (EI) calcd for
C13H11N3OS2 m/z ) 289.0343, found m/z ) 289.0351.
2-Amino-5-[(4-chlorophenyl)sulfanyl]-6-methylthieno[2,3-d]py-
rimidin-4(3H)-one (6). Compound 6 was synthesized as described
for 5 with 4-chlorobenzenethiol and was obtained as a white solid
(yield 49%): Rf ) 0.70 (MeOH/CHCl3, 1:7); mp >330 °C; 1H NMR
(DMSO-d6) δ 2.40 (s, 3 H), 6.58 (s, 2 H), 7.02 (d, 2 H, J ) 7.2
Hz), 7.27 (d, 2 H, J ) 7.2 Hz), 10.77 (s, 1 H); HRMS (EI) calcd
for C13H10N3OS2Cl m/z ) 322.9953, found m/z ) 322.9944.
2-Amino-6-methyl-5-[(4-nitrophenyl)sulfanyl]thieno[2,3-d]pyri-
midin-4(3H)-one (7). Compound 7 was synthesized as described
for 5 with 4-nitrobenzenethiol and was obtained as a yellow solid
(yield 20%): Rf ) 0.70 (MeOH/CHCl3, 1:7); mp >300 °C; 1H NMR
(DMSO-d6) δ 2.41 (s, 3 H), 6.63 (s, 2 H), 7.16-7.19 (d, 2 H, J )
7.8 Hz), 8.06-8.09 (d, 2 H, J ) 7.8 Hz), 10.83 (s, 1 H). Anal.
(C13H10N4O3S2 ·H2O) C, H, N, S.
2-Amino-5-[(2,5-dimethoxyphenyl)sulfanyl]-6-methylthieno[2,3-
d]pyrimidin-4(3H)-one (8). Compound 8 was synthesized as
described for 5 with 2,5-dimethoxybenzenethiol and was obtained
as a white solid (yield 26%): Rf ) 0.70 (MeOH/CHCl3, 1:7); mp
>300 °C; 1H NMR (DMSO-d6) δ 2.34 (s, 3 H), 3.52 (s, 3 H), 3.79
(s, 3 H), 5.93 (s, 1 H), 6.55 (s, 2 H), 6.58 (d, 1 H, J ) 8.7 Hz),
6.86 (d, 1 H, J ) 8.7 Hz), 10.77 (s, 1 H). Anal. (C13H10N4-
O3S2 ·0.7H2O) C, H, N, S.
2-Amino-5-[(3,4-dichlorophenyl)sulfanyl]-6-methylthieno[2,3-
d]pyrimidin-4(3H)-one (9). Compound 9 was synthesized as
described for 5 with 3,4-dichlorobenzenethiol and was obtained as
a white solid (yield 48%): Rf ) 0.64 (MeOH/CHCl3, 1:7); mp
297-300 °C; 1H NMR (DMSO-d6) δ 2.41 (s, 3 H), 6.59 (s, 2 H),
6.95 (dd, 1 H, J ) 1.5 Hz, J ) 6.3 Hz), 7.23 (d, 1 H, J ) 1.5 Hz),
7.45 (d, 1 H, J ) 6.3 Hz), 10.79 (s, 1 H); HRMS (EI) calcd for
C13H9N3OS2Cl2 m/z ) 356.9564, found m/z ) 356.9567.
2-Amino-5-[(3,5-dichlorophenyl)sulfanyl]-6-methylthieno[2,3-
d]pyrimidin-4(3H)-one (10). Compound 10 was synthesized as
described for 5 with 3,5-dichlorobenzenethiol and was obtained as
a white solid (yield 54%): Rf ) 0.70 (MeOH/CHCl3, 1:7); mp >300
°C; 1H NMR (DMSO-d6) δ 2.42 (s, 3 H), 6.64 (s, 2 H), 6.98 (s, 2
H), 7.33 (s, 1 H), 10.84 (s, 1 H). Anal. (C13H9N3OS2Cl2) C, H, N,
S, Cl.
Analytical samples were dried in vacuo (0.2 mmHg) in a CHEM-
DRY drying apparatus over P2O5 at 80 °C. Melting points were
determined on a MEL-TEMP II melting point apparatus with
FLUKE 51 K/J electronic thermometer and are uncorrected. Nuclear
magnetic resonance spectra for proton (1H NMR) were recorded
on a Bruker WH-300 (300 MHz) spectrometer. The chemical shift
values are expressed in ppm (parts per million) relative to
tetramethylsilane as an internal standard: s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet; br, broad singlet. Mass spectra were
recorded on a VG-7070 double-focusing mass spectrometer or in
a LKB-9000 instrument in the electron ionization (EI) mode.
Chemical names follow IUPAC nomenclature. Thin-layer chroma-
tography (TLC) was performed on Whatman Sil G/UV254 silica
gel plates with a fluorescent indicator, and the spots were visualized
under 254 and 366 nm illumination. Proportions of solvents used
for TLC are by volume. Column chromatography was performed
on a 230-400 mesh silica gel (Fisher, Somerville, NJ) column.
Elemental analyses were performed by Atlantic Microlab, Inc.,
Norcross, GA. Element compositions are within 0.4% of the
calculated values. Fractional moles of water or organic solvents
frequently found in some analytical samples of antifolates could
not be prevented in spite of 24-48 h of drying in vacuo and were
confirmed where possible by their presence in the 1H NMR spectra.
Microwave-assisted synthesis was performed utilizing an Emrys
Liberator microwave synthesizer (Biotage) utilizing capped reaction
vials. All microwave reactions were performed with temperature
control. All solvents and chemicals were purchased from Aldrich
Chemical Co. or Fisher Scientific and were used as received.
Ethyl 2-Amino-5-methylthiophene-3-carboxylate (15). In a 250
mL round-bottom flask, under nitrogen, were placed triethylamine
(4.7 g, 51 mmol), ethylcyanoacetate (9.7 g, 90 mmol), sulfur (2.8
g, 90 mmol), and dry DMF (30 mL). The reaction mixture was
stirred for 30 min at room temperature, and then propionaldehyde
(5.0 g, 90 mmol) was added dropwise to this suspension while
maintaining the temperature at 50 °C. When the addition was
complete, the reaction mixture was allowed to cool to room
temperature and then stirred for another 2 h. The reaction mixture
was diluted with EtOAc (120 mL) and washed sequentially with
H2O (30 mL) and brine (30 mL). The organic layer was separated
and dried over MgSO4, filtered, and concentrated under reduced
pressure to afford yellow oil. The crude product was purified by
flash chromatography on silica gel (gradient, 5% EtOAc/hexane to
10% EtOAc/hexane) to afford 8.9 g (56%) of 15 as a yellow solid:
Rf ) 0.6 (hexane/EtOAc, 3:1); mp 45-47 °C, (lit.47 mp 46 °C);
1H NMR (DMSO-d6) δ 1.22 (t, 3 H, J ) 7.2 Hz), 2.17 (s, 3 H),
4.14 (q, 2 H, J ) 7.2 Hz), 6.47 (s, 1 H), 7.06 (s, 2 H).
2-Amino-6-methylthieno[2,3-d]pyrimidin-4(3H)-one (16). In a
100 mL round-bottom flask, under nitrogen, were added 15 (0.67
g, 3 mmol), chlorformamidine hydrochloride (0.35 g, 4.5 mmol),
and DMSO (10 g, 102 mmol). The resulting mixture was heated
to 120-125 °C for 30 min. The reaction mixture was quenched
with water (15 mL). The resulting solution was cooled in an ice
bath, and the pH was adjusted to 8-9 with dropwise addition of
NH4.OH. This suspension was left at 5 °C for 1 h. The precipitate
obtained was collected by filtration, washed with brine, dried in
vacuo, and purified by flash chromatography on silica gel (gradient,
2% MeOH/CHCl3 to 5% MeOH/CHCl3) to afford 0.56 g (87%) of
16 as a light-yellow solid: Rf ) 0.54 (MeOH/CHCl3, 1:7); mp
370-372 °C; 1H NMR (DMSO-d6) δ 2.35 (s, 3 H), 6.43 (s, 2 H),
6.77 (s, 1 H), 10.82 (s, 1 H). Anal. (C7H7N3SO) C, H, N, S.
2-Amino-5-bromo-6-methylthieno[2,3-d]pyrimidin-4(3H)-one (17).
To a microwave reaction vial was added 16 (0.5 g, 3 mmol),
bromine (1 g, 333 µL), and AcOH (10 mL). The reaction mixture
was irradiated in a microwave apparatus at 150 °C for 30 min.
After the reaction mixture was cooled to ambient temperature, the
mixture was filtered and the filtrate was concentrated under reduced
2-Amino-6-methyl-5-(2-naphthylsulfanyl)thieno[2,3-d]pyrimidin-
4(3H)-one (11). Compound 11 was synthesized as described for 5
with naphthalene-2-thiol and was obtained as a white solid (yield
31%): Rf ) 0.70 (MeOH/CHCl3, 1:7); mp 300-303 °C; 1H NMR
(DMSO-d6) δ 2.43 (s, 3 H), 6.57 (s, 2 H), 7.16 (d, 1 H, J ) 7.2
Hz), 7.38-7.48 (m, 3 H), 7.72-7.83 (m, 3 H), 10.75 (s, 1 H);
HRMS (EI) calcd for C17H13N3OS2 m/z ) 339.0466, found m/z )
339.0504.
2-Amino-6-methyl-5-(pyridin-4-ylsulfanyl)thieno[2,3-d]pyrimi-
din-4(3H)-one (12). Compound 12 was synthesized as described
for 5 with pyridine-4-thiol and was obtained as a yellow solid (yield
56%): Rf ) 0.69 (MeOH/CHCl3, 1:7); mp >300 °C; 1H NMR
(DMSO-d6) δ 2.32 (s, 3 H), 6.62 (s, 2 H), 6.92 (d, 2 H, J ) 6.9

5796 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Gangjee et al.
Hz), 8.29 (d, 2 H, J ) 6.9 Hz), 10.83 (s, 1 H); HRMS (EI) calcd
for C12H10N4OS2 m/z ) 290.0296, found m/z ) 290.0302.
Supporting Information Available: Results from elemental
analysis and high resolution mass spectrometry. This material is
available free of charge via the Internet at http://pubs.acs.org.
2-Amino-5-[(4-fluorophenyl)sulfanyl]-6-methylthieno[2,3-d]py-
rimidin-4(3H)-one (13). Compound 13 was synthesized as described
for 5 with 4-fluorobenzenethiol and was obtained as a white solid
(yield 59%): Rf ) 0.70 (MeOH/CHCl3, 1:7); mp >300 °C; 1H NMR
(DMSO-d6) δ 2.41 (s, 3 H), 6.56 (s, 2 H), 7.07-7.09 (m, 4 H),
10.76 (s, 1 H). Anal. (C13H10N3OS2F·0.3H2O) C, H, N, S, F.
References
(1) Chan, D. C. M.; Anderson, A. C. Towards Species-Specific Antifolates.
Curr. Med. Chem. 2006, 13, 377–398.
(2) Hawser, S.; Lociuro, S.; Islam, K. Dihydrofolate Reductase Inhibitors
as Antibacterial Agents. Biochem. Pharmacol. 2006, 71, 941–948.
(3) Gangjee, A.; Kurup, S.; Namjoshi, O. Dihydrofolate Reductase as a
Target for Chemotherapy in Parasites. Curr. Pharm. Des. 2007, 13,
609–639.
(4) Berman, E. M.; Werbel, L. M. The Renewed Potential for Folate
Antagonists in Contemporary Cancer Chemotherapy. J. Med. Chem.
1991, 34, 479–485.
(5) Blakley, R. L. Dihydrofolate Reductase. In Folate and Pterins; Blakley,
R. L., Benkovic, S. J. Eds.; Wiley-Interscience: New York, 1984; Vol.
I, pp 191-253.
(6) MacKenzie, R. E. Biogenesis and Interconversion of Substituted
Tetrahydrofolates. In Folates and Pterins Chemistry and Biochemistry;
Blakley, R. L., Benkovic, S. J. Eds.; Wiley: New York, 1984; Vol. I,
pp 255-306.
(7) Petero, G. J.; Kohne, C. H. Fluoropyrimidines as Antifolate Drugs.
Antifolate Drugs Cancer Ther. 1999, 101–145.
(8) Rosowsky, A. Chemistry and Biological Activity of Antifolates. In
Progress in Medicinal Chemistry; Ellis, G. P., West, G. B. Eds.;
Elsevier Science Publishers: Amsterdam, 1989; pp 1-252.
(9) Gangjee, A.; Elzein, E.; Kothare, M.; Vasudevan, A. Classical and
Nonclassical Antifolates as Potential Antitumor, Antipneumocystis and
Antitoxoplasma Agents. Curr. Pharm. Des. 1996, 2, 263–280.
(10) Taylor, E. C.; Kuhnt, D.; Shih, C.; Rinzel, S. M.; Grindey, G. B.;
Barredo, J.; Jannatipour, M.; Moran, R. A Dideazatetrahydrofolate
Analogue Lacking a Chiral Center at C-6, N-[4-[2-(2-Amino-3,4-
dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-y1)ethylbenzoyl]-L-glutam-
ic Acid, Is an Inhibitor of Thymidylate Synthase. J. Med. Chem. 1992,
35, 4450–4454.
4-(2-Amino-6-methyl-4-oxo-3,4-dihydro-thieno[2,3-d]pyrimidine-
5-ylsulfanyl)benzoic Acid Ethyl Ester (18). Compound 18 was
synthesized as described for 5 with ethyl 4-sulfanylbenzoate and
was obtained as a yellow solid (yield 34%): Rf ) 0.60 (MeOH/
1
CHCl3, 1:7); mp 282-284 °C; H NMR (DMSO-d6) δ 1.26 (t, 3
H, J ) 6.8 Hz), 2.39 (s, 3 H), 4.23 (q, 2 H, J ) 6.8 Hz), 6.59 (s,
2 H), 7.05 (d, 2 H, J ) 8.1 Hz), 7.77 (d, 2 H, J ) 8.1 Hz), 10.79
(s, 1 H); HRMS (EI) calcd for C16H15N3O3S2 m/z ) 361.0554, found
m/z ) 361.0558.
4-[(2-Amino-6-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-
5-yl)sulfanyl]benzoic Acid (19). To a solution of 18 (0.25 g, 0.7
mmol) in ethanol (10 mL) was added aqueous 1 N NaOH (10 mL),
and the reaction mixture was stirred at room temperature for 18 h.
The reaction mixture was evaporated to dryness under reduced
pressure. The residue obtained was dissolved in water (5 mL), the
resulting solution was cooled in an ice bath, and the pH was adjusted
to 3-4 with dropwise addition of 3 N HCl. This suspension was
left at 5 °C for 24 h. The precipitated solid was collected by
filtration, washed with brine, and dried in vacuo to afford 0.23 g
(99%) of 19 as a light, white solid: mp >300 °C; 1H NMR (DMSO-
d6) δ 2.51 (s, 3 H), 6.61 (s, 2 H), 7.47-7.79 (m, 4 H), 10.82 (s, 1
H); HRMS (EI) calcd for C14H11N3O3S2 m/z ) 333.0241, found
m/z ) 333.0227.
Diethyl-N-{4-[(2-amino-6-methyl-4-oxo-3,4-dihydrothieno[2,3-
d]pyrimidin-5-yl)thio]benzoyl}-L-glutamate (20). To a solution of
19 (142 mg, 0.43 mmol) in DMF (8 mL) were added 6-chloro-
2,4-dimethoxy-1,3,5-triazine (80 mg, 0.45 mmol) and N-methyl-
morpholine (52 mg, 0.52 mmol) at 0 °C. After the mixture was
stirred at 0 °C for 2 h, N-methylmorpholine (52 mg, 0.52 mmol)
and dimethyl-L-glutamate hydrochloride (95 mg, 0.45 mmol) were
added together. The mixture was stirred at 0 °C for 2 h and at
room temperature for 12 h. The solvent was removed under reduced
pressure and the crude product was purified by flash chromatog-
raphy on silica gel with 5% MeOH/CHCl3 as the eluent to afford
140 mg (65%) of 20 as a light-yellow solid: Rf ) 0.50 (MeOH/
CHCl3, 1:7); mp 211-212 °C; 1H NMR (DMSO-d6) δ 1.01-1.20
(m, 6 H), 1.98-2.12 (m, 2 H), 2.27-2.32 (m, 2 H), 2.42 (s, 3 H),
4.00-4.13 (m, 4 H), 4.35-4.40 (m, 1 H), 6.60 (s, 2 H), 7.03 (d, 2
H, J ) 7.8 Hz), 7.70 (d, 2 H, J ) 8.1 Hz), 8.61 (d, 1 H, J ) 7.5
Hz), 10.79 (s, 1 H). HRMS (EI) calcd for C23H26N4O6S2 m/z )
518.1293, found m/z ) 518.1316.
(11) Jackman, A. L.; Taylor, G. A.; Gibson, W.; Kimbell, R.; Brown, M.;
Calvert, A. H.; Judson, I. R.; Hughes, L. R. ICI D1694, a Quinazoline
Antifolate Thymidylate Synthase Inhibitor That Is a Potent Inhibitor
of L1210 Tumour Cell Growth in Vitro and in Vivo: A New Agent
for Clinical Study. Cancer Res. 1991, 51, 5579–5586.
(12) Bertino, J. R.; Kamen, B.; Romanini, A. Folate Antagonists. In Cancer
Medicine; Holland, J. F., Frei, E., Bast, R. C., Kufe, D. W., Morton,
D. L., Weichselbaum, R. R. Eds.; Williams and Wilkins: Baltimore,
MD, 1997; Vol. 1, pp 907-921.
(13) Nair, M. G.; Galivan, J.; Maley, F.; Kisliuk, R. L.; Ferone, R.
Transport, Inhibition of Tumor Cell Growth and Unambiguous
Synthesis of 2-Desamino-2-methyl-N10-propargyl-5,8-dideazafolate
(DMPDDF) and Related Compounds. Proc. Am. Assoc. Cancer Res.
1989, 30, 476.
(14) Gibson, W.; Bisset, G. M. F.; Marsham, P. R.; Kelland, L. R.; Judson,
I. R.; Jackman, A. L. The Measurement of Polyglutamate Metabolites
of the Thymidylate Synthase Inhibitor, ICI D1694, in Mouse and
Human Cultured Cells. Biochem. Pharmacol. 1993, 45, 863–869.
(15) Sikora, E.; Jackman, A. L.; Newell, D. F.; Calvert, A. H. Formation
and Retention and Biological Activity of N10-Propargyl-5,8-didea-
zafolic Acid (CB 3717) Polyglutamates in L1210 Cells in Vitro.
Biochem. Pharmacol. 1988, 37, 4047–4054.
N-{4-[(2-Amino-6-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimi-
din-5-yl)sulfanyl]benzoyl}-L-glutamic Acid (4). To a solution of 20
(0.1 g, 0.19 mmol) in ethanol (15 mL) was added 1 N NaOH (12
mL), and the solution was stirred at room temperature for 24 h.
The solution was evaporated under reduced pressure, and the residue
was dissolved in water (10 mL) and stirred for a further 24 h. The
solution was then cooled in an ice bath and acidified carefully to
pH 4.0 with dropwise addition of 3 N HCl. This suspension was
left at 0-5 °C for 24 h and filtered. The residue was washed well
with water and dried over P2O5/vacuum to afford 71 mg (93%) of
4 as a yellow solid: mp 188-190 °C; 1H NMR (DMSO-d6) δ
1.88-2.10 (m, 2 H), 2.27-2.33 (t, 2 H, J ) 7.2 Hz), 2.39 (s, 3 H),
4.36 (m, 1 H), 6.59 (s, 2 H), 7.02 (d, 2 H, J ) 8.4 Hz), 7.70 (d, 2
H, J ) 8.4 Hz), 8.49 (d, 1 H, J ) 7.2 Hz), 10.79 (s, 1 H,), 12.38
(s, 2 H). Anal. (C19H18N4O6S2 ·1.5H2O) C, H, N, S.
(16) Jackman, A. L.; Newell, D. R.; Gibson, W.; Jodrell, D. I.; Taylor,
G. A.; Bishop, J. A.; Hughes, L. R.; Calvert, A. H. The Biochemical
Pharmacology of the Thymidylate Synthase Inhibitor 2-Desamino-2-
methyl-N10-propargyl-5,8-dideazafolic Acid (IC1 198583). Biochem.
Pharmacol. 1991, 41, 1885–1895.
(17) Nair, M. G.; Abraham, A.; McGuire, J. J.; Kisliuk, R. L.; Galivan, J.
Polyglutamylation as a Determinant of Cytotoxicity of Classical Folate
Analogue Inhibitors of Thymidylate Synthase and Glycinamide
Ribonucleotide Formyltransferase. Cell. Pharmacol. 1994, 1, 245–
249.
(18) Mendelsohn, L. G.; Shih, C.; Chen, V. J.; Habeck, L. L.; Gates, S. B.;
Shackelford, K. A. Enzyme Inhibition, Polyglutamation, and the Effect
of LY231514 (MTA) on Purine Biosynthesis. Semin. Oncol. 1999,
26, 42–47.
(19) Shih, C.; Chen, V. J.; Gossett, L. S.; Gates, S. B.; Mackellar, W. C.;
Habeck, L. L.; Shackelford, K. A.; Mendelsohn, L. G.; Soose, D. J.
LY231514, a Pyrrolo[2,3-d]pyrimidine-Based Antifolate That Inhibits
Multiple Folate-Requiring Enzymes N-[4-[2-(2-Amino-3,4-dihydro-
4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic Acid
(LY231514), Is a Novel Pyrrolo[2,3-d]pyrimidine-Based Antifolate
Currently Undergoing Extensive Phase II Clinical Trials. Cancer Res.
1997, 57, 1116–1123.
Acknowledgment. This work was supported, in part, by a
grant from the National Institutes of Health, National Institute
of Allergy and Infectious Diseases (Grant AI069966 (A.G.)).

6-Methylthieno[2,3-d]pyrimidine Antifolates
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5797
(20) Gangjee, A.; Devraj, R.; McGuire, J. J.; Kisliuk, R. L. 5-Arylthio
Substituted 2-Amino-4-oxo-6-methylpyrrolo[2,3-d]pyrimidine Anti-
folates as Thymidylate Synthase Inhibitors and Antitumor Agents.
J. Med. Chem. 1995, 38, 4495–4502.
(21) Gangjee, A.; Jain, H. D.; McGuire, J. J.; Kisliuk, R. L. Benzoyl Ring
Halogenated Classical 2-Amino-6-methyl-3,4-dihydro-4-oxo-5-sub-
stitutedthiobenzoyl-7H-pyrrolo[2,3-d]pyrimidine Antifolates as Inhibi-
tors of Thymidylate Synthase and as Antitumor Agents. J. Med. Chem.
2004, 47, 6730–6739.
(22) Tripos Inc., 1699 South Hanley Road, St. Louis, MO 63144.
(23) Gangjee, A.; Yu, J.; McGuire, J. J.; Cody, V.; Galitsky, N.; Kisliuk,
R. L.; Queener, S. F. Design, Synthesis, and X-ray Crystal Structure
of a Potent Dual Inhibitor of Thymidylate Synthase and Dihydrofolate
Reductase as an Antitumor Agent. J. Med. Chem. 2000, 43, 3837–
3851.
(24) Cody, V.; Galitsky, N.; Luft, J. R.; Pangborn, W.; Gangjee, A.; Devraj,
R.; Queener, S. F.; Blakley, R. L. Comparison of Ternary Complexes
of Pneumocystis carinii and Wild-Type Human Dihydrofolate Re-
ductase with Coenzyme NADPH and a Novel Classical Antitumor
Furo[2,3-d]pyrimidine Antifolate. Acta Crystallogr.,Sect. D 1997, 53,
638–649.
(25) Sawaya, M. R.; Kraut, J. Loop and Subdomain Movements in the
Mechanism of Escherichia coli Dihydrofolate Reductase: Crystal-
lographic Evidence. Biochemistry 1997, 36, 586–603.
(26) Davies, J. F.; Delcamp, T. J.; Prendergast, N. J.; Ashford, V. A.;
Freisheim, J. H.; Kraut, J. Crystal Structures of Recombinant Human
Dihydrofolate Reductase Complexed with Folate and 5-Deazafolate.
Biochemistry 1990, 29, 9467–9479.
(27) McGuire, J. J.; Hsieh, P.; Coward, J. K.; Bertino, J. R. Enzymatic
Synthesis of Folypolyglutamates. Characterization of the Reaction and
Its Products. J. Biol. Chem. 1980, 255, 5776–5788.
(28) McCloskey, D. E.; McGuire, J. J.; Russell, C. A.; Rowan, B. G.;
Bertino, J. R.; Pizzorno, G.; Mini, E. Decreased Folypolyglutamate
Synthetase Activity as a Mechanism of Methotrexate Resistance in
CCRF-CEM Human Leukemia Sublines. J. Biol. Chem. 1991, 266,
6181–6187.
(36) Webber, S. E.; Bleckman, T. M.; Attard, J.; Deal, J. G.; Katherdekar,
V.; Welsh, K. M.; Webber, S.; Janson, C. A.; Matthews, D. A.; Smith,
W. W.; Freer, S. T.; Jordan, S. R.; Bacquet, R. J.; Howland, E. F.;
Booth, C. J. L.; Ward, R. W.; Hermann, S. M.; White, J.; Morse,
C. A.; Hilliard, J. A.; Bartlett, C. A. Design of Thymidylate Synthase
Inhibitors Using Protein Crystal Structures: The Synthesis and
Biological Evaluation of a Novel Class of 5-Substituted Quinazolines.
J. Med. Chem. 1993, 36, 733–746.
(37) Hughes, A.; Calvert, A. H. Preclinical and Clinical Studies with the
Novel Thymidylate Synthase Inhibitor Nolatrexed Dihydrochloride
(Thymitaq, AG337). In Antifolate Drugs in Cancer Therapy; Jackman,
A. L. Ed.; Humana Press: Totowa, NJ, 1999; pp 229-241.
(38) Gangjee, A.; Mavandadi, F.; Kisliuk, R. L.; McGuire, J. J.; Queener,
S. F. 2-Amino-4-oxo-5-substituted-pyrrolo[2,3-d]pyrimidines as Non-
classical Antifolate Inhibitors of Thymidylate Synthase. J. Med. Chem.
1996, 39, 4563–4568.
(39) Masur, H. Problems in the Management of Opportunistic Infections
in Patients Infected with Human Immunodeficiency Virus. J. Infect.
Dis. 1990, 161, 858–864.
(40) Kovace, J. A.; Hiemenz, J. W.; Macher, A. M.; Stover, D.; Murray,
H. W.; Shelhamer, J.; Lane, H. C.; Urmacher, U.; Honig, C.; Longo,
D. L.; Parker, M. M.; Nataneon, J. E.; Parrillo, J. E.; Fauci, A. S.;
Pizzo, P. A.; Maeur, H. Pneumocystis carinii Pneumonia: A Com-
parison between Patients with the Acquired Immunodeficiency Syn-
drome and Patients with Other Immunodeficiencies. Ann. Intern. Med.
1984, 100, 68–71.
(41) Klepser, M. E.; Klepser, T. B. Drug Treatment of HIV-Related
Opportunistic Infections. Drugs 1997, 53, 40–73.
(42) Rosowsky, A.; Chaykovsky, M.; Chen, K. K. N.; Lin, M.; Modest,
E. J. 2,4-Diaminothieno[2,3-dlpyrimidines as Antifolates and Anti-
malarials. 1. Synthesis of 2,4-Diamino-5,6,7,8-tetrhydrothianaph-
theno[2,3-d]pyrimidines and Related Compounds. J. Med. Chem. 1973,
16, 185–188.
(43) Rosowsky, A.; Chen, K. K. N.; Lin, M. 2,4-Diaminothieno[2,3-
d]pyrimidines as Antifolates and Antimalarials. 3. Synthesis of 5,6-
Disubstituted Derivatives and Related Tricyclic Analogs. J. Med.
Chem. 1973, 16, 191–194.
(44) Elslager, E. F.; Jacob, P.; Werbel, L. M. Folate Antagonists. 6.
Synthesis and Antimalarial Effect of Fused 2,4-diaminothieno[2,3-
d]pyrimidines. J. Heterocycl. Chem. 1972, 9, 775–782.
(45) Rosowsky, A.; Mota, C. E.; Wright, J. E.; Freisheim, J. H.; Heusner,
J. J.; McCormack, J. J.; Queener, S. F. 2,4-Diaminothieno[2,3-
d]pyrimidine Analogues of Trimetrexate and Piritrexim as Potential
Inhibitors of Pneumocystis carinii and Toxoplasma gondii Dihydro-
folate Reductase. J. Med. Chem. 1993, 36, 3103–3112.
(46) Gangjee, A.; Qiu, Y.; Kisliuk, R. L. Synthesis Classical and Nonclas-
sical 2-Amino-4-oxo-6-benzyl-thieno[2,3-d]pyrimidines as Potential
Thymidylate Synthase Inhibitors. J. Heterocycl. Chem. 2004, 41, 941–
946.
(47) Gewald, K. Heterocyclen aus CH-aciden nitrilen. VII. 2-Ami-
nothiophene aus R-Oxo-mercaptanen und Methylenaktiven Nitrilen.
Chem. Ber. 1966, 98, 3571–3577.
(48) Gangjee, A.; Li, W.; Yang, J.; Kisliuk, R. L. Design, Synthesis, and
Biological Evaluation of Classical and Nonclassical 2-Amino-4-oxo-
5-substituted-6-methylpyrrolo[3,2-d]pyrimidines as Dual Thymidylate
Synthase and Dihydrofolate Reductase Inhibitors. J. Med. Chem. 2008,
51, 68–76.
(49) Kisliuk, R. L.; Strumpf, D.; Gaumont, Y.; Leary, R. P.; Plante, L.
Diastereoisomers of 5,10-Methylene-5,6,7,8-tetrahydropteroyl-D-
glutamic Acid. J. Med. Chem. 1977, 20, 1531–1533.
(29) Cao, W.; Matherly, L. H. Structural Determinants of Folate and
Antifolate Membrane Transport by the Reduced Folate Carrier. In Drug
Metabolism and Transport; Lash, L. H. Ed.; Humana Press: Totowa,
NJ, 2005; pp 291-318.
(30) Fry, D. W.; Jackson, R. C. Membrane Transport Alterations as a
Mechanism of Resistance to Anticancer Agents. Cancer SurV. 1986,
5, 47–49.
(31) Schornagel, J. H.; Pinard, M. F.; Westerhof, G. R.; Kathmann, I.;
Molthoff, C. F. M.; Jolivet, J.; Jansen, G. Functional Aspects of
Membrane Folate Receptors Expressed in Human Breast Cancer Lines
with Inherent and Acquired Transport-Related Resistance to Meth-
otrexate. Proc. Am. Assoc. Cancer Res. 1994, 35, 302.
(32) Wong, S. C.; Zhang, L.; Witt, T. L.; Proefke, S. A.; Bhushan, A.;
Matherly, L. H. Impaired Membrane Transport in Methotrexate-
Resistant CCRF-CEM Cells Involves Early Translation Termination
and Increased Turnover of a Mutant Reduced Folate Carrier. J. Biol.
Chem. 1999, 274, 10388–10394.
(33) Gangjee, A.; Elzein, E.; Kothare, M.; Vasudevan, A. Classical and
Nonclassical Antifolates as Potential Antitumor, Antipneumocystis and
Antitoxoplasma Agents. Curr. Pharm. Des. 1996, 2, 263–280.
(34) Gangjee, A.; Jain, H. D.; Kurup, S. Recent Advances in Classical and
Non-Classical Antifolates as Antitumor and Antiopportunistic Infection
Agents: Part I. Anti-Cancer Agents Med. Chem. 2007, 7, 524–542.
(35) Gangjee, A.; Jain, H. D.;.; Kurup, S. Recent Advances in Classical
and Non-Classical Antifolates as Antitumor and Antiopportunistic
Infection Agents: Part II. Anti-Cancer Agents Med. Chem. 2008, 8,
205–231.
(50) Wahba, A. J.; Friedkin, M. The Enzymatic Synthesis of Thymidylate.
Early Steps in the Purification of Thymidylate Synthetase of Escheri-
chia coli. J. Biol. Chem. 1962, 237, 3794–3801.
JM8006933