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

Article abstract of DOI:10.1021/jm701052u

We designed and synthesized a classical antifolate N-{4-[(2-amino-6-methyl- 4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)methyl]benzoyl}-L-glutamic acid 4 and 11 nonclassical analogues 5-15 as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors. The key intermediate in the synthesis was N-(4-chloro-6-methyl-5H-pyrrolo[3,2-d]pyrimidin-2-yl)-2,2- dimethylpropanamide, 29, to which various 5-benzyl substituents were attached. For the classical analogue 4, the ester obtained from the N-benzylation reaction was deprotected and coupled with diethyl L-glutamate followed by saponification. Compound 4 was a potent dual inhibitor of human TS (IC ₅₀ = 46 nM, about 206-fold more potent than pemetrexed) and DHFR (IC₅₀ = 120 nM, about 55-fold more potent than pemetrexed). The nonclassical analogues were marginal inhibitors of human TS, but four analogues showed potent T. gondii DHFR inhibition along with >100-fold selectivity compared to human DHFR.

Full text of DOI:10.1021/jm701052u

68
J. Med. Chem. 2008, 51, 68–76
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
Aleem Gangjee,*,† Wei Li,† Jie Yang,† 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 August 24, 2007
We designed and synthesized a classical antifolate N-{4-[(2-amino-6-methyl-4-oxo-3,4-dihydro-5H-
pyrrolo[3,2-d]pyrimidin-5-yl)methyl]benzoyl}-L-glutamic acid 4 and 11 nonclassical analogues 5–15 as
potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors. The key intermediate
in the synthesis was N-(4-chloro-6-methyl-5H-pyrrolo[3,2-d]pyrimidin-2-yl)-2,2-dimethylpropanamide, 29,
to which various 5-benzyl substituents were attached. For the classical analogue 4, the ester obtained from
the N-benzylation reaction was deprotected and coupled with diethyl L-glutamate followed by saponification.
Compound 4 was a potent dual inhibitor of human TS (IC50 ) 46 nM, about 206-fold more potent than
pemetrexed) and DHFR (IC50 ) 120 nM, about 55-fold more potent than pemetrexed). The nonclassical
analogues were marginal inhibitors of human TS, but four analogues showed potent T. gondii DHFR inhibition
along with >100-fold selectivity compared to human DHFR.
Introduction
Folate metabolism has long been recognized as an attractive
target for chemotherapy because of its crucial role in the
biosynthesis of nucleic acid precursors.1,2 Inhibitors of folate-
dependent enzymes in cancer, microbial, and protozoan cells
provide compounds that have found clinical utility as antitumor,
antimicrobial, and antiprotozoal agents.3,4 Thymidylate synthase
(TSa),which catalyzes the reductive methylation of deoxyuridy-
late (dUMP) to deoxythymidylate (dTMP), has been of particular
interest.5 This reaction affords 7,8-dihydrofolate (7,8-DHF),
which is reduced by dihydrofolate reductase (DHFR)6 to
tetrahydrofolate (THF).7 Thus, TS and DHFR are crucial for
the synthesis of dTMP in dividing cells. Several TS and DHFR
inhibitors, as separate entities, have found clinical utility as
antitumor agents. Usually a 2,4-diamino-substituted pyrimidine
ring is considered important for potent DHFR inhibitory activity,
while a 2-amino-4-oxopyrimidine or 2-methyl-4-oxopyrimidine
ring is considered important for potent TS inhibitory activity.
Examples of clinically used TS and DHFR inhibitors are
raltitrexed8 (ZD1694), pemetrexed9 (Alimta, LY231514), and
methotrexate10 (MTX) illustrated in Figure 1. Raltitrexed is a
quinazoline analogue that can be transported into cells via the
Figure 1
reduced folate carrier (RFC)11 and undergoes rapid poly-
glutamylation by the enzyme folylpolyglutamate synthetase
methylene group replaces the N10 nitrogen in the bridge.
(FPGS).12–15 Raltitrexed is approved as a first-line agent for
Pemetrexed contains a 6-5 pyrrolo[2,3-d]pyrimidine system and
advanced colorectal cancer in several European countries,
is designated a multitargeted antifolate (MTA). Pemetrexed and
Australia, Canada, and Japan. Pemetrexed is an antifolate in
its polyglutamylated metabolites are reported to be inhibitors
which a pyrrole ring replaces the pyrazine of folic acid and a
of several important folate-dependent enzymes including TS,
DHFR, glycinamide ribonucleotide formyltransferase (GARFT),
* To whom correspondence should be addressed. Phone: 412-396-6070.
and aminoimidazole carboxamideribonucleotide formyltrans-
Fax: 412-396-5593. E-mail: gangjee@duq.edu.
ferase (AICARFT).16,17
†
Duquesne University.
Tufts University School of Medicine.
‡
However, the primary locus of action of pemetrexed is the
inhibition of TS, and this is responsible for its cytotoxic effects.
aAbbreviations: TS, thymidylate synthase; dUMP, deoxyuridylate; dTMP,
deoxythymidylate; DHFR, dihydrofolate reductase; THF, tetrahydrofolate;
MTX, methotrexate; RFC, reduced folate carrier; FPGS, folylpolyglutamate
synthetase; GARFT, glycinamide ribonucleotide formyltransferase; AICARFT,
aminoimidazole carboxamideribonucleotide formyltransferase; 5-FU, fluo-
rouracil; E. coli, Escherichia coli; tg, Toxoplasma gondii; NCI, National
Cancer Institute.
Inhibition of other folate-dependent enzymes may also be
important, since pemetrexed is cytotoxic to human cancer
cell lines that are resistant to raltitrexed and 5-FU as a result of
TS amplification.18 Pemetrexed, in combination with cisplatin,
10.1021/jm701052u CCC: $40.75
2008 American Chemical Society
Published on Web 12/12/2007

Pyrimidines as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 69
has been recently approved for the treatment of malignant pleural
mesothelioma and also for non-small-cell lung cancer.19
Val115 is absent in pemetrexed, which does not have a 6-methyl
group. Thus, compound 1 is a promising lead compound that
can be further structurally modified to optimize dual human TS
and human DHFR inhibitory activities. Though compound 1
was a dual TS/DHFR inhibitor, its human DHFR inhibitory
activity was marginal. One of the principal goals of this study
was to maintain (or improve) human TS inhibitory activity of
1 while substantially increasing the human DHFR inhibitory
activity.
Classical antifolates that have an N-benzoyl-L-glutamic acid
side chain such as raltitrexed and pemetrexed are able to function
as substrates for FPGS. FPGS catalyzes the formation of poly-
γ-glutamates, which leads to high intracellular concentrations
of these antitumor agents and increases TS inhibitory activity
for some antifolates (raltitrexed 60-fold8 and pemetrexed 130-
fold9) compared to their monoglutamate forms. Although
polyglutamylation of certain antifolates is necessary for their
cytotoxic activity, it has also been implicated in toxicity to host
cells because of cellular retention of the polyanionic poly-γ-
glutamate metabolites which also do not efflux from normal
cells.20 Additionally, tumor cells develop resistance to antifolates
that depend on polyglutamylation for their antitumor effects by
producing low or defective FPGS and thereby limiting their
utility. Another problem associated with classical antifolates is
their dependence on the carrier systems for their uptake into
tumor cells. The RFC is the most important carrier, and
impairment of the RFC system in tumor cells also leads to drug
resistance.21–25
In addition to the inhibition of human TS and DHFR we are
also interested in selective inhibition of TS and/or DHFR from
pathogens that cause opportunistic infections in AIDS patients
such as Toxoplasma gondii (tg).37
The pyrrolo[3,2-d]pyrimidine scaffold is a regioisomer of the
pyrrolo[2,3-d]pyrimidine found in pemetrexed. This pyrrolo[3,2-
d]pyrimidine modification maintains the overall size of the
molecule intact but removes a potential hydrogen bond donor
from the N7 position. In addition, quinazolines such as ralitrexed
and nolatrexed, which have a carbon atom in the 8-position
rather than a nitrogen, are more potent inhibitors of human TS
than the corresponding pyrido[2,3-d]pyrimidines and pteridines
perhaps because of a more favorable hydrophobic interaction
with Trp109 in human TS.26
In general, the problem of resistance in tumors, due to low
or defective FPGS, has placed limitations on the use of classical
antifolates, which depend on polyglutamylation for their anti-
tumor effects. To overcome these problems associated with
classical antifolates, lipophilic nonclassical antifolates were also
designed and synthesized. These lipophilic nonclassical anti-
folates lack the polar glutamate found in classical antifolates
and hence do not depend on FPGS for their inhibitory activity
of the target enzymes. Additionally, they also do not require
the RFC system for active uptake into the cell because they are
lipophilic and are passively transported into cells. Nolatrexed
(Figure 1) is the first antitumor nonclassical TS inhibitor to reach
clinical trials.26,27
Molecular modeling using SYBYL 7.035 and superimposing
4 on raltitrexed crystallized with human TS (PDB code 1KMV)
indicated that, like compound 1, the 6-methyl moiety of
compound 4 interacts with Trp109 as do the C6 and C7 atoms
of 4. Similarly molecular modeling (SYBYL 7.035) and super-
imposing the pyrimidine ring of compound 4 onto the 2,4-
diaminopyrimidine ring of SRI 9662 in human DHFR X-ray
crystal structure (PDB 1KMV38) indicate that the C7 of the
pyrrolo[3,2-d]pyrimidine ring of 4 makes hydrophobic contact
with Phe31 of human DHFR, as shown in Figure 2. This
hydrophobic interaction would be absent with the N7 of 1 and
could translate into a better DHFR inhibitory potency for 4
compared to 1. The Glu30 that makes contact with the 2-NH2
and N1 positions of the pyrimidine ring and the second Phe34
that makes contact with the side chain phenyl ring of 4 are also
indicated in Figure 2. Thus, it was anticipated that compound
4 would have better human DHFR inhibitory activity compared
to 1. It was therefore of interest to synthesize the classical
analogue N-{4-[(2-amino-4-oxo-6-methylpyrrolo[3,2-d]pyrimi-
din-5-ylmethyl)benzoyl}-L-glutamic acid, 4, to explore the
effects of N5 substitution of the pyrrolo[3,2-d]pyrimidine
scaffold on human TS and human DHFR inhibitory activity.
Gangjee et al.39 showed that nonclassical analogues of 1 with
electron-withdrawing groups in the phenyl ring of the side chain
also enhance human TS inhibitory activity. SAR studies
indicated that analogues with electron-withdrawing groups at
the 3- and/or 4-position of the phenyl side chain provide
optimum inhibitory potency against human TS. In addition,
nonclassical analogues such as 2 (IC50 ) 0.15 µM) and 3 (IC50
) 0.13 µM) were more potent inhibitors of human TS than the
clinically used raltitrexed and pemetrexed. In contrast to the
requirements for human TS inhibition, electron-donating sub-
stituents such as methoxy and methyl and bulky substituents
such as naphthyl are conducive for DHFR inhibition;39 hence,
analogues containing these substituents were also synthesized.
As indicated above for the classical analogue 4, it was
It has been demonstrated with both clinically used antifolates
as well as in preclinical studies that TS inhibitors and DHFR
inhibitors, when used separately or in combination with other
agents, can provide successful antitumor therapy. In addition,
synergism of two separate antifolates that inhibit TS and DHFR
has been demonstrated in growth inhibitory studies against
Lactobacillus casei,28,29 rat hepatoma cells,30,31 and human
lymphoma cells.32,33 Thus, one of our goals has been to design
potent dual TS and DHFR inhibitory activity in single agents.
Such dual inhibitors could act at two different sites (TS and
DHFR) and might be capable of providing “combination
chemotherapy” in single agents without the pharmacokinetic
disadvantages of two separate agents. This strategy may also
lead to an improved toxicity profile. Gangjee et al.34 reported 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 1), as a potent inhibitor of human TS (IC50 ) 54 nM)
and a marginal inhibitor of human DHFR (IC50 ) 2.2 µM) in
its monoglutamate form, thus providing a dual inhibitor of
human TS and human DHFR. Compound 1 was not a substrate
for human FPGS at concentrations up to 1045 µM, indicating
that 1 does not require polyglutamylation for its potent inhibition
of human TS. Using molecular modeling (SYBYL 6.91),35
Gangjee et al.36 suggested that the 6-methyl group in 1 makes
important hydrophobic contacts with Trp109 in human TS and
also sterically restricts the rotation of the 5-position side chain
so that it adopts a favorable conformation for binding to human
TS. In addition, molecular modeling indicated that the 6-methyl
group of 1 also makes hydrophobic contact with Val115 in
human DHFR. This potential hydrophobic interaction with

70 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Gangjee et al.
Figure 2. Stereoview of compound 4 superimposed on SRI 9662 in human DHFR (PDB code 1KMV).38 The hydrophobic interaction between C7
and Phe31 is shown.
Scheme 1a
a Reagents and conditions: (a) NaOEt, EtOH, 0 °C, 1 h; (b) AcOH, diethyl aminomalonate hydrochloride, room temp, 24 h; (c) MeOH, room temp, 5 h;
(d) NaOEt, EtOH, 60 °C, 6 h; (e) NaH, DMF, benzyl bromides, room temp, 4 h; (f) (1) 1,3-bis(methoxycarbonyl)-2-methylthiopseudourea, AcOH, MeOH,
room temp, 12 h; (2) NaOMe, MeOH, room temp, 12 h; (g) 1 N NaOH, 55 °C, 3 h.
anticipated that the nonclassical analogues 5–15 would also
provide dual inhibitory activity against human TS and human
DHFR.
synthesis of the corresponding desmethyl analogue of 21 from
diethyl[(2-cyanovinyl)amino]malonate, the desmethyl analogue
of 20, were reported.
Thus, it appeared attractive to adopt the reported methodology
for the synthesis of the intermediates 20 and 21. The conversion
of 5-methylisoxazole 18 to 3-oxobutanenitrile 19 was catalyzed
by sodium ethoxide. Compound 19 was condensed with diethyl
aminomalonate to afford 20, which was further cyclized under
basic conditions at room temperature to afford the desired
pyrrole 21. The total yield, over three steps, from 18 to 21 was
a mere 12%. In addition, the purification of the desired product
21 was difficult because the byproduct had close Rf values (TLC)
to 21. Therefore, it was necessary to explore alternative
Chemistry
Our initial methods for the synthesis of the classical com-
pound 4 and the nonclassical compounds 5–7 are outlined in
Schemes 1 and 2. The key intermediate in the synthesis of 4–7
was the ethyl 3-amino-5-methyl-1H-pyrrole-2-carboxylate, 21
(Scheme 1), which could be further condensed with a guany-
lating agent to form the pyrrolo[3,2-d]pyrimidine ring system.
A search of the literature revealed that there was no synthesis
or other report for 21. However, two methods40,41 for the

Pyrimidines as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 71
Scheme 2a
a Reagents and conditions: (h) L-glutamic acid diethyl ester hydrochloride, 2-chloro-4,6-dimethoxy-1,3,5-triazine, N-methylmorpholine, DMF, room temp,
5 h; (i) (1) 1 N NaOH, room temp, 4 h; (2) 3 N HCl.
procedures for the synthesis of 21. A further search of the
literature afforded a method that allowed a conjugate addition–
elimination of 16 (Scheme 1) with a variety of nucleophiles.42
Initial attempts to react 16 with diethyl aminomalonate free base
with variation of time (up to 8 h) and temperature (at reflux)
were unsuccessful. However, the crucial intermediate 20 (E/Z-
mixture) was readily obtained from 16 (E/Z-mixture) and diethyl
aminomalonate hydrochloride, 17 (rather than the free base),
in methanol at room temperature for 5 h. A possible explanation
is that the imine is a stronger base than the amine. Once the
hydrochloride salt was added, the imine is protonated and the
carbon of the imine is more susceptible to nucleophilic attack
by the amine. In the absence of the amine salt, 3-aminobut-2-
enenitrile 16 exists predominantly as the enamine and is less
susceptible to nucleophilic attack by the amino group. With 20
in hand, use of reaction conditions similar to those reported by
Furneaux and Tyler,40 which involved heating a mixture of 20
and sodium ethoxide in ethanol under N2 for 12 h at 55–60 °C,
afforded the pyrrole 21 in 65% yield following chromatographic
separation (Scheme 1). This reaction was incomplete (TLC) even
after 12 h. Longer reaction times resulted in decomposition of
the product (TLC). However, intermediate 21 could be isolated
and different aryl substituents were introduced at the N1 position
using appropriate benzyl bromides under basic conditions to
afford 22a-c. The isolation of pure 22a-c was tedious and
required extensive column chromatography. Longer reaction
times gave additional products (TLC), further complicating the
purification. Thus, the crude benzylic substituted pyrroles 22a-c
were directly subjected to condensation with 1,3-bis(methoxy-
carbonyl)-2-methylthiopseudourea with 5 equiv of acetic acid
as catalyst and MeOH to afford 23a-c. Compounds 23a-c were
used for the next step without further purification. Hydrolysis
of the carbamate group with aqueous sodium hydroxide at 55
°C afforded the 2-amino-4-oxo-6-methylpyrrolo[3,2-d]pyrim-
idines 5 and 6 and the corresponding acid 24 in yields that
ranged from 30% to 41% (over three steps). The yield for the
4-fluorophenyl substituted compound 7 was 20% (over three
steps).
synthesis of pyrrolo[3,2-d]pyrimidines. This method was modi-
fied to afford intermediate 27 via cyclocondensation of the
pyrrole 21 with 1,3-bis(methoxycarbonyl)-2-methyl-2-thi-
opseudourea followed by removal of the carbamate moiety with
aqueous NaOH. This afforded an efficient synthesis of the key
intermediate 27 in 63% yields (over two steps).
We initially attempted a direct N-benzylation in the N5
position of pyrrolo[3,2-d]pyrimidine 27 using the appropriate
benzyl bromides under basic conditions. Unfortunately direct
benzylation of 27 gave a mixture of the desired product
contaminated with a several side products (TLC). Chromato-
graphic and recrystallization attempts to purify the product were
fruitless. Failure of the above method led us to explore a new
alternative strategy where the pyrrolo[3,2-d]pyrimidine 27 could
be converted to its 2-protected 4-chloro derivative 29, which
could undergo N-benzylation and depivaloylation to afford target
compounds 7–15. The 2-amino group of 27 was successfully
protected with pivaloyl chloride to afford 28. Compound 28
was converted to the 4-chloro derivative 29 by treatment with
phosphorus oxychloride in 86% yield. The N-benzylated pyr-
rolo[3,2-d]pyrimidines 30–38 were synthesized from 29 with
appropriately substituted benzyl bromides in yields ranging from
65% to 74%. Hydrolysis of the benzylic substituted pyrrolo[3,2-
d]pyrimidines 30–38 under basic conditions gave the target
compounds 7–15 in 68-75% yields. The structures of target
1
compounds 4–15 and all intermediates were confirmed by H
NMR and elemental analysis. The total yield for 7 in Scheme
3 from 21 to 7 was 19% (over six steps), which is comparable
to 20% (over three steps) in Scheme 1. Though there is no
difference in the total yield, there are three additional steps in
Scheme 3, which allow the purification and characterization of
all intermediates, involved in the synthesis of target compounds
7–15.
Biological Evaluation and Discussion
The classical and nonclassical analogues 4–15 were evaluated
as inhibitors of human, Escherichia coli (E. coli) and Toxo-
plasma gondii (T. gondii) DHFR45 and TS.46 The inhibitory
potencies (IC50) are listed in Table 1 and compared with PDDF,
MTX, and trimethoprim. The classical analogue 4 was a potent
dual inhibitor of human TS (IC50 ) 46 nM) and human DHFR
(IC50 ) 120 nM). Against human TS, 4 was similar in potency
to the previously reported 1 and about 2-fold more potent than
PDDF and 206-fold more potent than pemetrexed. This result
indicates that isomeric structural modification of the pyrrolo[2,3-
d]pyrimidine scaffold to a pyrrolo[3,2-d]pyrimidine maintains
high potency against human TS and increases human DHFR
inhibition by 18-fold for 4 compared with lead compound 1.
Thus the principal goal to increase the human DHFR inhibitory
activity of 1 without compromising the potent human TS
inhibitory activity was accomplished.
Conversion of free acid 24 (Scheme 2) to the corresponding
L-glutamic acid diethyl ester 25 involved conventional peptide
coupling with L-glutamic acid diethyl ester hydrochloride using
2-chloro-4,6-dimethoxy-l,3,5-triazine43 followed by chromato-
graphic purification to afford the coupled product 25 in 73%
1
yield. The H NMR of 25 revealed the newly formed peptide
NH proton at 8.40 ppm as a doublet, which exchanged on
addition of D2O. Hydrolysis of 25 with aqueous NaOH at room
temperature, followed by acidification with 3 N HCl in the cold,
afforded compound 4 in 60% yield. Once again, because of the
tedious separation and the inability to characterize intermediates
22a-d and 23a-d (Scheme 1), it was necessary to devise an
alternative synthetic strategy for the target compounds 7–15,
and this is shown in Scheme 3. Elliott et al. reported the

72 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Gangjee et al.
Scheme 3a
a Reagents and conditions: (j) (1) 1,3-bis(methoxycarbonyl)-2-methylthiopseudourea, AcOH, MeOH, room temp, 12 h; (2) NaOMe, MeOH, room temp,
2 h; (k) 1 N NaOH, 55 °C, 3 h; (l) PivCl, DMAP, TEA, dichloroethane, 50 °C, 12 h; (m) POCl3, reflux, 3 h; (n) NaH, benzyl bromides, DMF, 0 °C, 3 h;
(o) 2 N NaOH, 1,4-dioxane, reflux, 24 h.
Table 1. Inhibitory Concentrations (IC50 in µM) against TS and DHFRa
TS (µM)
E. colib
DHFR (µM)
E. colid
21
nd
nd
>23 (23)
>39
39
>37 (0)
>35 (0)
>30 (10)
>33 (0)
>35 (0)
>35 (0)
>31 (0)
>24 (0)
>32 (0)
230
compd
humanb
T. gondiie
humanc
2.1
T. gondiie
rh/tgj
nd
nd
nd
1
0.054
0.13
0.15
0.046
20
2.3
3.7
2.9
1.3
1.4
1.5
1.3
>26 (0)
10
5.4
9.5
0.27
45
13
0.069
>39 (27)
12
31
1.5
7.5
14
15
7.5
>26 (15)
>20 (36)
27
nd
nd
nd
0.23
>39 (0)
15
22
17
18
28
17
13
>26 (13)
>20 (24)
27
nd
nd
nd
0.023
3.9
20
1.8
0.35
0.27
0.33
3.5
0.3
15
24
1.6
0.43
0.22
0.033
6.8
2f
3f
4
>54
>62 (0)g
0.12
5.2
5
6
7
8
>39
>37
>10
>1.8
>20.5
>100
>129.6
100
>10
>116.6
>2.1
>1
>20
15.35
8.6
0.6
>50
>37 (18)
>35 (32)
>35 (31)
33
>35 (10)
>35 (16)
>31 (0)
>24 (0)
>32 (0)
6.6
9
10
11
12
13
14
15
pemetrexedh
PDDFi
76
2.8
0.085
nd
nd
0.019
nd
nd
0.43
nd
nd
1.9
0.02
>340 (22)
23
0.0088
0.01
MTX
trimethoprimf
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. nd ) not determined. b Kindly provided by Dr. Frank Maley, New York State Department of Health.
c Kindly provided by Dr. Andre Rosowsky, Dana-Farber Cancer Institute, Boston, MA. d Kindly provided by Dr. R. L. Blakley, St. Jude Children’s Hospital,
Memphis, TN. e Kindly provided by Dr. Karen Anderson, Yale Univerisy, New Haven, CT. f Data derived from ref 39. g Numbers in parentheses indicate
the percent inhibition at the stated concentration. h Kindly provided by Dr. Chuan Shih, Eli Lilly and Co. i Kindly provided by Dr. M. G. Nair, University
of South Alabama. j rh/tg is the selectivity ratio for T. gondii DHFR and is the (IC50 against rhDHFR)/(IC50 against T. gondii DHFR).
Usually a 2-amino-4-oxo substitution on the pyrimidine ring
of bicyclic systems such as quinazolines and pyrrolo[2,3-
d]pyrimidines is considered to be important for potent TS
inhibitory activity. Compound 4 is a 2-amino-4-oxo system, and
its TS inhibitory activity is as anticipated. However, the human
DHFR and T. gondii DHFR inhibitory activities of 4 are not as
easily explained. 2,4-Diamino substitution on the pyrimidine
ring of bicyclic systems is usually important for DHFR
inhibitory activity. Compound 4 is not a 2,4-diamino substituted
system. The DHFR inhibitory activity of 2-amino-4-oxo-
pyrrolo[2,3-d]pyrimidines such as 1 can be explained by rotating
the NH2-C2 bond by 180° such that the N7 functions as the
4-amino moiety.47 Since analogue 4 is a pyrrolo[3,2-d]pyrimi-
dine, we cannot explain the increased DHFR inhibitory activity
on the basis of a NH2-C2 bond rotation of 180°. The increase
in activity against human DHFR of 4 over 1 can be explained
on the basis of our molecular modeling where the C7 of 4
interacts with Phe31 of human DHFR and the N7 of 1 would
not have the same interaction. Similar modeling with E. coli
DHFR in which a Leu28 replaces Phe31 in human DHFR
indicates that the C7 of 4 is >4.0 Å away from the Leu28 and
would not have a productive interaction. This lack of a
hydrophobic interaction would explain the poor E. coli DHFR
inhibitory result with 4. There are no X-ray crystal structures
available for T. gondii DHFR/TS. However, recent homology
modeling48 indicates that T. gondii DHFR contains two phe-
nylalanine residues, Phe32 and Phe35, in its active site, similar
to Phe31 and Phe34 in the active site of human DHFR. Thus,

Pyrimidines as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 73
Ethyl 3-Amino-5-methyl-1H-pyrrole-2-carboxylate (21). A
solution of NaOEt in EtOH (0.5 M, 120 mL) was added slowly to
a stirred solution of 20 (1 g, 4 mmol) in 20 mL of EtOH. The
reaction mixture was stirred for 6 h at 60 °C and cooled to room
temperature. The solvent was evaporated under reduced pressure.
The crude product was purified by column chromatography on silica
gel with 10% ethyl acetate/n-hexane as the eluent to yield 21 (0.45
g, 65%) as an off-white solid: mp 85–87 °C; Rf ) 0.36 (ethyl
the potent inhibitory activity of 4 and the nonclassical analogues
against T. gondii could be due, in part, to the interaction of the
C7 of the pyrrolo[3,2-d]pyrimidine with Phe32 of T. gondii
DHFR. Nonclassical compounds 8–10 and 12 are also reason-
ably potent against T. gondii DHFR and have 100- to >100-
fold selectivity over human DHFR. The IC50 values against T.
gondii DHFR are about 20-fold better than the clinically used
trimethoprim.
1
acetate/n-hexane, 1:1); H NMR (DMSO-d6) δ 1.24 (t, 3 H, J )
In summary, the 2-amino-4-oxo-5,6-disubstituted pyrrolo[3,2-d]-
pyrimidine classical antifolate 4 and 11 nonclassical analogues
5–15 were synthesized as potential dual TS and DHFR
inhibitors. Biological studies indicated that 4 was a potent dual
TS-DHFR inhibitor with nanomolar inhibition for both en-
zymes. Compound 4 maintained the TS inhibitory activity of 1
but increased the human DHFR inhibition by 18-fold. To our
knowledge this is the most potent dual TS-DHFR inhibitor
reported. Currently, classical analogue 4 is under further
evaluation by the National Cancer Institute (NCI) as an
antitumor agent. The nonclassical analogues were marginally
active against TS and less active against DHFR except
compounds 8–10 and 12, which showed high potency and
selectivity against T. gondii DHFR comparable to trimethoprim.
6.4 Hz, CH3), 2.03 (s, 3 H, CH3), 4.12 (q, 2 H, J ) 6.4 Hz, CH2),
4.91 (s, 2 H, NH2), 5.26 (s, 1 H, CH), 10.21 (s, 1 H, NH). Anal.
(C8H12N2O2) C, H, N.
2-Amino-5-benzyl-6-methyl-3,5-dihydro-4H-pyrrolo[3,2-d]py-
rimidin-4-one (5). To a solution of ethyl 3-amino-5-methyl-1H-
pyrrole-2-carboxylate 21 (0.5 g, 3 mmol) in dry DMF (10 mL)
was added slowly NaH (0.09 g, 3.6 mmol) under nitrogen at room
temperature. The resulting mixture was stirred for about 15 min
when there was no more gas produced, and then (bromomethyl)-
benzene (0.52 g, 3 mmol) in THF (3 mL) was added dropwise.
The reaction mixture was stirred at room temperature for 4 h, and
20 mL of water was added carefully to quench the reaction. The
sample was extracted with CHCl3 (25 mL × 4), and the organic
phases were combined and dried (Na2SO4). Evaporation of the
solvent offered a gummy residue, which was used for the next step
without purification. The gummy residue (∼3 mmol) was dissolved
in MeOH (10 mL), and 1,3-bis(methoxycarbonyl)-2-methyl-2-
thiopseudourea (0.7 g, 3.3 mmol) was added followed by AcOH
(1.0 g, 15 mmol). The mixture was stirred at room temperature
overnight and became a thick paste. NaOMe in MeOH (25%) (7
mL, 22 mmol) was added, and stirring was continued at room
temperature overnight. The mixture was neutralized with AcOH,
and the methanol was removed under reduced pressure. To the
residue was added water (20 mL), and the pH value was adjusted
to 10–11 by adding NH3 ·H2O. The solid was collected by filtration
and washed well with water. The resulting solid was added to 1 N
NaOH (2 mL), and the mixture was heated at 55 °C for 3 h. The
mixture was cooled and neutralized with acetic acid, and then the
pH of the mixture was adjusted to 10–11 by adding NH3 ·H2O.
The solid was collected by filtration. The solid was dissolved in
MeOH and CHCl3 (1:1, 5 mL), and 1 g of silica gel was added.
The solvent was removed under reduced pressure to form a plug,
which was loaded at the top of a silica gel column (15 mm × 150
mm) and eluted with 5% MeOH in CHCl3 (containing 0.5%
NH3 ·H2O). Fractions containing the desired compound (TLC) were
pooled and evaporated to afford 5 (35% for three steps) as an off-
white solid: mp >250 °C; Rf ) 0.46 (MeOH/CHCl3, 1:10); 1H
NMR (DMSO-d6) δ 2.14 (s, 3 H, CH3), 5.54 (s, 2 H, CH2), 5.75
(br s, 2 H, NH2), 5.80 (s, 1 H, CH), 7.00–7.32 (m, 5 H, C6H5),
10.35 (s, 1 H, NH). Anal. (C14H14N4O) C, H, N.
Experimental Section
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 a
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. Thin-layer
chromatography (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
1
were confirmed where possible by their presence in the H NMR
spectra. All solvents and chemicals were purchased from Aldrich
Chemical Co. or Fisher Scientific and were used as received.
Diethyl {[(E/Z)-2-Cyano-1-methylvinyl]amino}malonate (20).
To a suspension of an E/Z mixture of 3-aminobut-2-enenitrile 16
(3 g, 35.1 mmol) in MeOH (60 mL) was added diethyl aminoma-
lonate hydrochloride 17 (7.9 g, 36.8 mmol). The resulting mixture
was stirred at room temperature for 5 h. TLC showed the
disappearance of the starting materials and the formation of one
major spot at Rf ) 0.26 (ethyl acetate/n-hexane, 1:2). The reaction
solvent was diluted with ethyl acetate (50 mL), washed with brine
(30 mL × 2), and dried over MgSO4. To the organic solvent 15 g
of silica gel was added, and the mixture was evaporated to dryness
under reduced pressure. This silica gel plug was loaded on a dry
silica gel column (2 cm × 15 cm) and flash-chromatographed
initially with n-hexane (200 mL) and then sequentially with 500
mL of 5% ethyl acetate in n-hexane, 500 mL of 10% ethyl acetate
in n-hexane, and 500 mL of 15% ethyl acetate in n-hexane.
Fractions containing the desired product (TLC) were pooled and
evaporated to afford 6.74 g (80%) of 20 as an off-white solid: mp
2-Amino-6-methyl-5-(pyridin-4-ylmethyl)-3,5-dihydro-4H-
pyrrolo[3,2-d]pyrimidin-4-one (6). Compound 6 was synthesized
as described for 5: yield 31%, mp >250 °C; Rf ) 0.31 (MeOH/
CHCl3, 1:10); 1H NMR (DMSO-d6) δ 2.13 (s, 3 H, CH3), 5.57 (s,
2 H, CH2), 5.79 (br s, 2 H, NH2), 5.85 (s, 1 H, CH), 6.94 (d, 2 H,
J ) 7.2 Hz, C5NH4), 8.49 (d, 2 H, J ) 7.2 Hz, C5NH4), 10.39 (s,
1 H, NH). Anal. (C13H13N5O·0.2H2O) C, H, N.
2-Amino-5-(4-fluorobenzyl)-6-methyl-3,5-dihydro-4H-pyrro-
lo[3,2-d]pyrimidin-4-one (7). Compound 7 was synthesized as
described for 5: yield 20%, mp 185–186 °C; Rf ) 0.40 (MeOH/
1
CHCl3, 1:5); H NMR (DMSO-d6) δ 2.14 (s, 3 H, CH3), 5.50 (s,
2 H, CH2), 5.75 (s, 1 H, CH), 5.60 (s, 2 H, NH2), 7.07–7.10 (m, 4
H, C6H4), 10.36 (s, 1 H, NH). Anal. (C14H13FN4O·0.11CH3COOH)
C, H, N, F.
4-[(2-Amino-6-methyl-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]py-
rimidin-5-yl)methyl]benzoic Acid (24). Compound 24 was syn-
1
1
50–52 °C; H NMR (DMSO-d6) δ 1.18–1.23 (t, 3 H, J ) 6.9 Hz,
thesized as described for 5: yield 41%; mp >250 °C; H NMR
CH3), 2.05 (s, 6 H, CH3), E isomer 3.94 (s, 1 H, CH), 4.18–4.24
(m, 4 H, CH2), 4.96 (d, 1 H, J ) 7.8 Hz, CH), Z isomer 5.32 (s, 1
H, CH), 7.51–7.56 (d, 2 H, J ) 7.8 Hz, NH). Anal. (C11H16N2O4)
C, H, N.
(DMSO-d6) δ 2.15 (s, 3 H, CH3), 5.62 (s, 2 H, CH2), 5.87 (s, 1 H,
CH), 6.05 (br s, 2 H, NH2), 7.12 (d, 2 H, J ) 6.9 Hz, C6H4), 7.89
(d, 2 H, J ) 6.9 Hz, C6H4), 11.15 (s, 1 H, NH). Anal. (C15H14N4O3)
C, H, N.

74 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Gangjee et al.
Diethyl N-{4-[(2-Amino-6-methyl-4-oxo-3,4-dihydro-5H-pyr-
rolo[3,2-d]pyrimidin-5-yl)methyl]benzoyl}-L-glutamate (25). To
a suspension of benzoic acid 24 (150 mg, 0.5 mmol) in DMF (10
mL) at 25 °C was added N-methylmorpholine (NMM, 0.062 mL,
0.57 mmol) followed by 2-chloro-4,6-dimethoxy-1,3,5-triazine (100
mg, 0.57 mmol), and the resulting solution was stirred at 25 °C for
2 h. NMM (0.062 mL, 0.57 mmol) was added to the solution
followed by L-glutamic acid diethyl ester hydrochloride (140 mg,
0.59 mmol). The resulting mixture was stirred at 25 °C for 4 h and
concentrated in vacuo (0.5 mmHg), and the residue was dissolved
in CH2Cl2 (50 mL). The CH2Cl2 solution was washed with 5%
NaHCO3, dried (Na2SO4), and concentrated in vacuo. The residue
was chromatographed on silica gel with 5% MeOH in CH2Cl2 as
the eluent. Fractions containing the product (TLC) were combined
and evaporated to give 25 as a white solid (73%): mp 86–88 °C;
Rf ) 0.75 (MeOH/CHCl3, 1:10); lH NMR (DMSO-d6) δ 1.10–1.21
(m, 6 H, 2 × OCH2CH3), 1.90–2.15 (m, 2 H, CH2), 2.15 (s, 3 H,
CH3), 2.40 (t, 2 H, CH2), 4.00–4.18 (m, 4 H, 2 × OCH2CH3), 4.40
(m, 1 H, CH), 5.60 (s, 2 H, CH2), 5.78 (br s, 2 H, NH2), 5.82 (s,
1 H, CH), 7.12 (d, 2 H,, J ) 6.9 Hz, C6H4), 7.78 (d, 2 H,, J ) 6.9
Hz, C6H4), 8.40 (d, 1 H, J ) 7.4 Hz, NH), 10.38 (s, 1 H, NH).
Anal. (C24H29N5O6 ·0.1CH3OH) C, H, N.
N-{4-[(2-Amino-6-methyl-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-
d]pyrimidin-5-yl)methyl]benzoyl}-L-glutamic Acid (4). To a
solution of 25 (193 mg, 0.4 mmol) in methanol (4 mL) was added
1 N NaOH (2 mL) at 0 °C. After the reaction mixture was stirred
at room temperature for 3–4 h, the TLC showed the disappearance
of the starting material and a new spot appeared at the original
position (MeOH/CHCl3, 1:10). The methanol was evaporated under
reduced pressure, the residue was dissolved in water (4 mL), and
then the solution was cooled to 0 °C and acidified carefully to pH
4 with dropwise addition of 3 N HCl. The suspension was left at
5 °C for 24 h and filtered. The residue was washed well with water
and ether and dried to offer 4 (102 mg, 60%) as an off-white solid:
mp >250 °C; 1H NMR (DMSO-d6) δ 1.82–2.14 (m, 2 H, -CH2),
2.14 (s, 3 H, CH3), 2.38 (t, 2 H, J ) 6.9 Hz, CH2), 4.38 (m, 1 H,
CH), 5.61 (s, 2 H, CH2), 5.80 (br s, 2 H, NH2), 5.82 (s, 1 H, CH),
7.16 (d, 2 H, J ) 6.9 Hz, C6H4), 7.82 (d, 2 H, J ) 6.9 Hz, C6H4),
8.54 (d, 1 H, J ) 7.4 Hz, NH), 10.50 (s, 1 H, NH), 12.40 (s, br, 2
H, 2 × COOH). Anal. (C20H21N5O6 ·0.6H2O) C, H, N.
was cooled, diluted with dichloromethane (50 mL), washed with
brine (40 mL × 2), dried over Na2SO4, and concentrated in vacuo.
To this solution were added methylene chloride (30 mL) and silica
gel (5 g), and the solvent evaporated. The silica gel plug obtained
was loaded onto a silica gel column and eluted with 9:1 ethyl
acetate/n-hexane The fractions containing the product (TLC) were
pooled and the solvent was evaporated to afford 1.33 g (67%) of
28 as a white solid: TLC Rf ) 0.47 (MeOH/CHCl3, 1:10); mp
1
156–157 °C; H NMR (DMSO-d6) δ 1.19 (s, 9 H, CH3), 2.29 (s,
3 H, CH3), 5.97 (s, 1 H, CH), 10.72 (s, 1 H, NH), 11.78 (s, 1 H,
NH), 11.87 (s, 1H, NH). Anal. (C12H16N4O2 ·0.15CH3COCH3) C,
H, N.
N-(4-Chloro-6-methyl-5H-pyrrolo[3,2-d]pyrimidin-2-yl)-2,2-
dimethylpropanamide (29). To a 100mL round-bottomed flask
was added 28 (1.16 g, 4.67 mmol) suspended in 30 mL of
phosphorus oxychloride. The reaction mixture was heated at reflux
with stirring in an anhydrous atmosphere for 3 h. The dark-orange
solution was allowed to cool to room temperature and concentrated
in vacuo. Water (20 mL) was then added to the residue at 0 °C
with vigorous stirring to give an exothermic reaction. Concentrated
aqueous ammonium hydroxide was added to attain pH 5 to give a
precipitate, which was collected by filtration, washed with water
(3 × 5 mL), and dried in vacuo. The crude product was purified
by silica gel column chromatography with 2% MeOH/CHCl3.
Recrystallization from MeOH afforded 1.07 g (86%) of 29 as a
white solid: TLC Rf ) 0.35 (MeOH/CHCl3, 1:10); mp 162–163
°C; 1H NMR (DMSO-d6) δ 1.19 (s, 9 H, CH3), 2.47 (s, 3 H, CH3),
6.33 (s, 1 H, CH), 9.85 (s, 1 H, NH), 12.07 (s, 1 H, NH). Anal.
(C12H15ClN4O) C, H, N, Cl.
N-[4-Chloro-5-(4-fluorobenzyl)-6-methyl-5H-pyrrolo[3,2-d]py-
rimidin-2-yl]-2,2-dimethylpropanamide (30). To a mixture of
NaH (33 mg, 1.3 mmol) and anhydrous DMF (10 mL) at 0 °C was
added dropwise 29 (0.31 g, 1.08 mmol) in DMF (5 mL), and the
mixture was stirred for 20 min. 1-(Bromomethyl)-4-fluorobenzene
(0.27 g, 1.40 mmol) was added, and the mixture was stirred at 0
°C for 3 h. Analysis (TLC) of the product mixture revealed a
complete conversion of starting material to product(s). The reaction
mixture was removed under reduced pressure, and the crude product
was purified by column chromatography on silica gel with 15%
ethyl acetate/n-hexane as the eluent to afford 0.28 g (70%) of 30
as a white power: TLC Rf ) 0.30 (MeOH/CHCl3, 1:10); mp
Methyl (6-Methyl-4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyri-
midin-2-yl)carbamate (26). The pyrrole 21 (2.68 g, 16 mmol)
was dissolved in MeOH (40 mL), and 1,3-bis(methoxycarbonyl)-
2-methyl-2-thiopseudourea (3.74 g, 18 mmol) was added fol-
lowed by AcOH (4.6 mL). The mixture was stirred at room
temperature overnight and became a thick paste. To the reaction
mixture was added 45 mL of NaOMe in MeOH (25%), and
stirring was continued at room temperature for 2 h. The mixture
was neutralized with AcOH, and the solid was collected by
filtration and washed well with water. After drying, 26 (2.44 g,
69%) was obtained as an off-white powder: mp 234–236 °C;
TLC Rf ) 0.22 (MeOH/CHCl3, 1:5); 1H NMR (DMSO-d6) δ
2.28 (s, 3 H, CH3), 3.73 (s, 3 H, OCH3), 5.95 (s, 1 H, CH),
10.90 (s, 1 H, NH), 11.10 (s, 1 H, NH), 11.76 (s, 1 H, NH).
Anal. (C9H10N4O3 · 0.79C6H6 · 0.55C7H8O3S) C, H, N.
2-Amino-6-methyl-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-
4-one (27). To a 200 mL round-bottomed flask was added 26 (1 g,
4.5 mmol) suspended in 1 N NaOH (35 mL). The reaction mixture
was heated at 55 °C for 3 h. The resulting solution was cooled in
an ice bath and neutralized with AcOH. The precipitated solid was
collected by filtration, washed with brine, and dried in vacuo to
afford 0.67 g (92%) of 27 as a white solid: mp 252–254 °C; TLC
Rf ) 0.15 (MeOH/CHCl3, 1:5); 1H NMR (DMSO-d6) δ 2.20 (s, 3
H, CH3), 5.65 (s, 1 H, CH), 5.65 (s, 2 H, NH2), 10.21 (s, 1 H,
NH), 11.15 (s, 1 H, NH). Anal. (C7H8N4O·0.73H2O) C, H, N.
2,2-Dimethyl-N-(6-methyl-4-oxo-4,5-dihydro-3H-pyrrolo[3,2-
d]pyrimidin-2-yl)propanamide (28). To a 250 mL round-bottomed
flask was added 27 (1.37 g, 8 mmol) suspended in 40 mL of
dichloroethane. Then trimethylacetyl chloride (1.99 mL, 16 mmol),
DMAP (0.13 g, 1 mmol), and triethylamine (2.68 mL) were added.
The mixture was stirred overnight at 50 °C. The resulting mixture
1
175–176 °C; H NMR (DMSO-d6) δ 1.20 (s, 9 H, CH3), 2.42 (s,
3 H, CH3), 5.68 (s, 2 H, CH2), 6.54 (s, 1 H, CH), 6.94–7.14 (m, 4
H, C6H4), 9.94 (s, 1 H, NH). Anal. (C19H20ClFN4O·0.06H2O) C,
H, N, F, Cl.
N-[4-Chloro-5-(4-chlorobenzyl)-6-methyl-5H-pyrrolo[3,2-d]py-
rimidin-2-yl]-2,2-dimethylpropanamide (31). Compound 31 was
synthesized as described for 30: yield 65%; TLC Rf ) 0.35 (MeOH/
1
CHCl3, 1:10); mp 175–176 °C; H NMR (DMSO-d6) δ 1.19 (s, 9
H, CH3), 2.40 (s, 3 H, CH3), 5.67 (s, 2 H, CH2), 6.53 (s, 1 H, CH),
6.88–7.35 (m,
4 H, C6H4), 9.94 (s, 1H, NH). Anal.
(C19H20Cl2N4O·0.12C6H14) C, H, N, Cl.
N-[5-(4-Bromobenzyl)-4-chloro-6-methyl-5H-pyrrolo[3,2-d]py-
rimidin-2-yl]-2,2-dimethylpropanamide (32). Compound 32 was
synthesized as described for 30: yield 72%; TLC Rf ) 0.38 (MeOH/
1
CHCl3, 1:10); mp 182–183 °C; H NMR (DMSO-d6) δ 1.18 (s, 9
H, CH3), 2.40 (s, 3 H, CH3), 5.65 (s, 2 H, CH2), 6.53 (s, 1 H, CH),
6.82–7.53 (m,
4 H, C6H4), 9.93 (s, 1 H, NH). Anal.
(C19H20BrClN4O·0.01H2O) C, H, N, Br, Cl.
N-[4-Chloro-6-methyl-5-(4-nitrobenzyl)-5H-pyrrolo[3,2-d]py-
rimidin-2-yl]-2,2-dimethylpropanamide (33). Compound 33 was
synthesized as described for 30: yield 71%; TLC Rf ) 0.37
1
(MeOH/CHCl3, 1:10); mp 175–176 °C; H NMR (DMSO-d6) δ
1.11 (s, 9 H, CH3), 2.33 (s, 3 H, CH3), 5.75 (s, 2 H, CH2), 6.48
(s, 1 H, CH), 7.07–8.09 (m, 4 H, C6H4), 9.92 (s, 1 H, NH).
Anal. (C19H20ClN5O3 · 0.25CH3COCH3) C, H, N, Cl.
N-[4-Chloro-5-(4-methoxybenzyl)-6-methyl-5H-pyrrolo[3,2-
d]pyrimidin-2-yl]-2,2-dimethylpropanamide (34). Compound 34
was synthesized as described for 30: yield 72%; TLC Rf ) 0.40
1
(MeOH/CHCl3, 1:10); mp 178–179 °C; H NMR (DMSO-d6) δ
1.21 (s, 9 H, CH3), 2.43 (s, 3 H, CH3), 5.63 (s, 2 H, CH2), 6.53 (s,

Pyrimidines as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 75
1 H, CH), 6.86 (m, 4 H, C6H4), 9.94 (s, 1 H, NH). HRMS calcd
for C20H23N4O2NaCl 409.1407, found 409.1416.
2-Amino-6-methyl-5-[4-(trifluoromethoxy)benzyl]-3,5-dihydro-
4H-pyrrolo[3,2-d]pyrimidin-4-one (12). Compound 12 was syn-
thesized as described for 7: yield 75%; TLC Rf ) 0.45 (MeOH/
N-{4-Chloro-6-methyl-5-[4-(trifluoromethoxy)benzyl]-5H-
pyrrolo[3,2-d]pyrimidin-2-yl}-2,2-dimethylpropanamide(35).Com-
pound 35 was synthesized as described for 30: yield 70%; TLC Rf
) 0.40 (MeOH/CHCl3, 1:10); mp 182–183 °C; 1H NMR (DMSO-
d6) δ 1.21 (s, 9 H, CH3), 2.43 (s, 3 H, CH3), 5.81 (s, 2 H, CH2),
6.58 (s, 1 H, CH), 7.10–7.71 (m, 4 H, C6H4), 9.96 (s, 1 H, NH).
Anal. (C20H20ClF3N4O2) C, H, N, F, Cl.
1
CHCl3, 1:5); mp 148–149 °C; H NMR (DMSO-d6) δ 2.14 (s, 3
H, CH3), 5.63 (s, 2 H, CH2), 5.83 (s, 2 H, NH2), 5.83 (s, 2 H, CH),
7.17–7.69 (m, 4 H, C6H4), 10.42 (s, 1 H, NH). Anal. (C15H13F3N4O2)
C, H, N, F.
2-Amino-5-(3,5-dichlorobenzyl)-6-methyl-3,5-dihydro-4H-
pyrrolo[3,2-d]pyrimidin-4-one (13). Compound 13 was synthe-
sized as described for 7: yield 74%; TLC Rf ) 0.42 (MeOH/CHCl3,
1:5); mp 188–189 °C; 1H NMR (DMSO-d6) δ 2.16 (s, 3 H, CH3),
5.53 (s, 2 H, CH2), 5.81 (s, 2 H, NH2), 5.84 (s, 2 H, CH), 7.02–7.49
N-[4-Chloro-5-(3,5-dichlorobenzyl)-6-methyl-5H-pyrrolo[3,2-
d]pyrimidin-2-yl]-2,2-dimethylpropanamide (36). Compound 36
was synthesized as described for 30: yield 72%; TLC Rf ) 0.36
1
(m,
4
H, C6H4), 10.42 (s,
1
H, NH). Anal.
(MeOH/CHCl3, 1:10); mp 182–183 °C; H NMR (DMSO-d6) δ
(C14H12Cl2N4O·0.4CH3COOH) C, H, N, Cl.
1.20 (s, 9 H, CH3), 2.42 (s, 3 H, CH3), 5.71 (s, 2 H, CH2), 6.56 (s,
1 H, CH), 6.90–7.53 (m, 3 H, C6H3), 9.96 (s, 1 H, NH). Anal.
(C19H19Cl3N4O) C, H, N, Cl.
2-Amino-5-(3,5-dibromobenzyl)-6-methyl-3,5-dihydro-4H-
pyrrolo[3,2-d]pyrimidin-4-one (14). Compound 14 was synthe-
sized as described for 7: yield 72%; TLC Rf ) 0.41 (MeOH/CHCl3,
1:5); mp 188–189 °C; 1H NMR (DMSO-d6) δ 2.16 (s, 3 H, CH3),
5.53 (s, 2 H, CH2), 5.83 (s, 2 H, NH2), 5.83 (s, 2 H, CH), 7.20–7.72
(m, 3 H, C6H3), 10.44 (s, 1 H, NH). Anal. (C14H12Br2N4O·0.1C6H14)
C, H, N, Br.
N-[4-Chloro-5-(3,5-dibromobenzyl)-6-methyl-5H-pyrrolo[3,2-
d]pyrimidin-2-yl]-2,2-dimethylpropanamide (37). Compound 37
was synthesized as described for 30: yield 74%; TLC Rf ) 0.35
1
(MeOH/CHCl3, 1:10); mp 182–183 °C; H NMR (DMSO-d6) δ
1.21 (s, 9 H, CH3), 2.43 (s, 3 H, CH3), 5.72 (s, 2 H, CH2), 6.58 (s,
1 H, CH), 7.08–7.77 (m, 3 H, C6H3), 9.97 (s, 1 H, NH). Anal.
(C19H19 Br2ClN4O·0.10C6H14) C, H, N, Br, Cl.
2-Amino-5-(3,4-dichlorobenzyl)-6-methyl-3,5-dihydro-4H-
pyrrolo[3,2-d]pyrimidin-4-one (15). Compound 15 was synthe-
sized as described for 7: yield 68%; TLC Rf ) 0.38 (MeOH/CHCl3,
1:5); mp 195–196 °C; 1H NMR (DMSO-d6) δ 2.17 (s, 3 H, CH3),
5.54 (s, 2 H, CH2), 5.82 (s, 2 H, NH2), 5.85 (s, 2 H, CH), 7.03 (s,
1 H, C6H3), 7.51 (d, 2 H, J ) 6.9 Hz, C6H3), 10.40 (s, 1 H, NH).
HRMS calcd for C14H12ClN4O m/z ) 322.0382, found m/z )
322.0388.
N-[4-Chloro-5-(3,4-dichlorobenzyl)-6-methyl-5H-pyrrolo[3,2-
d]pyrimidin-2-yl]-2,2-dimethylpropanamide (38). Compound 38
was synthesized as described for 30: yield 72%; TLC Rf ) 0.31
1
(MeOH/CHCl3, 1:10); mp 162–164 °C; H NMR (DMSO-d6) δ
1.21 (s, 9 H, CH3), 2.44 (s, 3 H, CH3), 5.73 (s, 2 H, CH2), 6.58 (s,
1 H, CH), 6.92 (s, 1 H, CH), 7.36–7.54 (m, 3 H, C6H3), 9.97 (s, 1
H, NH). HRMS calcd for C19H20N4OCl3 425.0703, found 425.0671.
2-Amino-5-(4-fluorobenzyl)-6-methyl-3,5-dihydro-4H-pyrro-
lo[3,2-d]pyrimidin-4-one (7). To a 100 mL round-bottomed flask
was added 30 (50 mg, 0.13 mmol) suspended in 2 N NaOH (10
mL) and 1,4-dioxane (5 mL). The reaction mixture was stirred under
reflux for 24 h. The solution was cooled to room temperature and
neutralized with glacial AcOH. The resulting precipitate was
collected by filtration and dried in vacuo. The crude product was
purified by silica gel column chromatography, eluting with 5%
MeOH/CHCl3 to yield 25 mg (73%) of 7 as a white powder: TLC
Rf ) 0.40 (MeOH/CHCl3, 1:5); mp 185–186 °C; 1H NMR (DMSO-
d6) δ 2.14 (s, 3 H, CH3), 5.50 (s, 2 H, CH2), 5.75 (s, 1 H, CH),
5.60 (s, 2 H, NH2), 7.07–7.10 (m, 4 H, C6H4), 10.36 (s, 1 H, NH).
Anal. (C14H13FN4O·0.11CH3COOH) C, H, N, F.
Acknowledgment. This work was supported in part by a
grant from the National Institutes of Health, NIAID, Grant
AI069966 (A.G.).
Supporting Information Available: Results from elemental
analysis. This material is available free of charge via the Internet
at http://pubs.acs.org.
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