The effect of 5-alkyl modification on the biological activity of pyrrolo[2,3-d]pyrimidine containing classical and nonclassical antifolates as inhibitors of dihydrofolate reductase and as antitumor and/or antiopportunistic infection agents

Article abstract of DOI:10.1021/jm800244v

Novel classical antifolates (3 and 4) and 17 nonclassical antifolates (11-27) were synthesized as antitumor and/or antiopportunistic infection agents. Intermediates for the synthesis of 3, 4, and 11-27 were 2,4-diamino-5- alkylsubstituted-7H-pyrrolo[2,3-d]pyrimidines, 31 and 38, prepared by a ring transformation/ring annulation sequence of 2-amino-3-cyano-4-alkyl furans to which various aryl thiols were attached at the 6-position via an oxidative addition reaction using I₂. The condensation of α-hydroxy ketones with malonodinitrile afforded the furans. For the classical analogues 3 and 4, the ester precursors were deprotected, coupled with diethyl-L-glutamate, and saponified. Compounds 3 (IC₅₀ = 60 nM) and 4 (IC₅₀ = 90 nM) were potent inhibitors of human DHFR. Compound 3 inhibited tumor cells in culture with GI₅₀ ≤ 10^-7 M. Nonclassical 17 (IC ₅₀ = 58 nM) was a potent inhibitor of Toxoplasma gondii (T. gondii) DHFR with > 500-fold selectivity over human DHFR. Analogue 17 was 50-fold more potent than trimethoprim and about twice as selective against T. gondii DHFR.

Full text of DOI:10.1021/jm800244v

J. Med. Chem. 2008, 51, 4589–4600
4589
The Effect of 5-Alkyl Modification on the Biological Activity of Pyrrolo[2,3-d]pyrimidine
Containing Classical and Nonclassical Antifolates as Inhibitors of Dihydrofolate Reductase and
as Antitumor and/or Antiopportunistic Infection Agents1a–e
Aleem Gangjee,*,† Hiteshkumar D. Jain,|,† Sherry F. Queener,‡ and Roy L. Kisliuk§
DiVision of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne UniVersity, Pittsburgh, PennsylVania 15282,
Department of Pharmacology and Toxicology, School of Medicine, Indiana UniVersity, Indianapolis, Indiana 46202, Department of
Biochemistry, Tufts UniVersity School of Medicine, Boston, Massachusetts 02111
ReceiVed March 6, 2008
Novel classical antifolates (3 and 4) and 17 nonclassical antifolates (11-27) were synthesized as antitumor
and/or antiopportunistic infection agents. Intermediates for the synthesis of 3, 4, and 11-27 were 2,4-
diamino-5-alkylsubstituted-7H-pyrrolo[2,3-d]pyrimidines, 31 and 38, prepared by a ring transformation/
ring annulation sequence of 2-amino-3-cyano-4-alkyl furans to which various aryl thiols were attached at
the 6-position via an oxidative addition reaction using I2. The condensation of R-hydroxy ketones with
malonodinitrile afforded the furans. For the classical analogues 3 and 4, the ester precursors were deprotected,
coupled with diethyl-L-glutamate, and saponified. Compounds 3 (IC50 ) 60 nM) and 4 (IC50 ) 90 nM)
were potent inhibitors of human DHFR. Compound 3 inhibited tumor cells in culture with GI50 e 10-7 M.
Nonclassical 17 (IC50 ) 58 nM) was a potent inhibitor of Toxoplasma gondii (T. gondii) DHFR with >500-
fold selectivity over human DHFR. Analogue 17 was 50-fold more potent than trimethoprim and about
twice as selective against T. gondii DHFR.
Introduction
methotrexate6 (MTX) (Figure 1), raltitrexed,7 and pemetrexed8
are clinically used as antitumor agents. Nonclassical antifolates
like trimetrexate (TMQ), pyrimethamine, and trimethoprim
(TMP) are clinically used as antiopportunistic infection agents.4
Dihydrofolate reductase (DHFR) along with thymidylate
synthase (TS) forms part of the system responsible for the
synthesis of 2′-deoxythymidine-5′-monophosphate (dTMP), a
key component in DNA biosynthesis and cell replication. TS
catalyzes the de novo synthesis of dTMP from 2′-deoxyuridine-
5′-monophosphate (dUMP). The cofactor, N5,N10-methylene-
tetrahydrofolate (N5,N10-CH2-THF), serves as the donor of the
methyl group as well as the reductant for this step and is itself
oxidized to 7,8-dihydrofolate (7,8-DHF). The recyclization of
7,8-DHF to 5,6,7,8-tetrahydrofolate (5,6,7,8-THF) is catalyzed
by DHFR2 for which NADPH acts as the source of the reductant.
Thus inhibition of DHFR and/or TS leads to “thymineless
death”.a
Pneumocystis jiroVecii (P. jiroVecii) previously known as
Pneumocystis carinii (P. carinii)3,4 [Note: P. jiroVecii is the
strain that infects humans, while P. carinii refers to the strain
that infects rats] and Toxoplasma gondii (T. gondii)3,4 are often
fatal opportunistic infections in AIDS patients. Mycobacterium
aVium (M. aVium) complex (MAC),3,4 a group of organisms
that is responsible for disseminated infections in AIDS patients,
additionally decreases the quality of life of patients with AIDS.
Several DHFR and TS inhibitors have found clinical utility as
antitumor and antiopportunistic agents.5 Classical antifolates like
The combination of a weak DHFR inhibitor (TMP, py-
rimethamine), along with a potent dihydropteroate synthase
(DHPS) inhibitor (sulfamethoxazole), is currently used to treat
infections caused by opportunistic pathogens in AIDS patients.9
However, the combination therapy is successful in only 50-75%
of the AIDS population; up to 60% are unable to tolerate the
combination therapy due to severe, adverse drug reactions.10
Trimetrexate is coadministered with leucovorin, the classical
folate cofactor (6R,6S)-5-formyl-5,6,7,8-THF, which selectively
rescues the host cell from the toxicity caused by nonselective
TMQ.11
Gangjee et al.12 recently reported 1 (Figure 2) as a dual
inhibitor of human DHFR (IC50 ) 0.21 µM) and human TS
(IC50 ) 0.54 µM). The 5-CH3 moiety of 1 was incorporated to
provide hydrophobic interaction with Val115 in human DHFR.
Compound 1 was designed as a nonpolyglutamylatable DHFR
inhibitor. However, unexpectedly, 1 had reasonable folyl poly-
γ-glutamate synthetase (FPGS) substrate activity. Molecular
modeling using SYBYL 6.813 suggested that 1 binds to human
DHFR in the normal 2,4-diamino mode (Figure 3) while it could
bind to human TS in the flipped mode. In addition, molecular
modeling also indicated that the 5-CH3 group in 1 could provide
hydrophobic interaction with Trp109 in human TS. Compound
1 was a reasonably potent inhibitor of the growth of human
CCRF-CEM leukemia cells in culture with an EC50 value of
190 nM as compared with MTX (EC50 ) 12.5 nM). In 11 of
the 60 tumor cell lines evaluated at the National Cancer Institute
(NCI) preclinical screening program, compound 1 showed GI50
values of e10-7 M (Table 3). Homologation of the 5-methyl
group in 1 to a 5-ethyl group as in 2 (Figure 2) afforded a 3-fold
more potent human DHFR inhibitor (IC50 ) 0.066 µM).14
Surprisingly, compound 2 was devoid of any significant TS
* To whom correspondence should be addressed. Phone: 412-396-6070.
Fax: 412-396-5593. E-mail: gangjee@duq.edu.
†
Division of Medicinal Chemistry, Graduate School of Pharmaceutical
Sciences, Duquesne University.
‡
Department of Pharmacology and Toxicology, School of Medicine,
Indiana University.
§
Department of Biochemistry, Tufts University School of Medicine.
Current address: Department of Chemistry, University of Wisconsins
|
Milwaukee, Milwaukee, WI 53211.
a Abbreviations: T. gondii, Toxoplasma gondii; DHFR, Dihydrofolate
reductase; TS, thymidylate synthase; P. jiroVecii, Pneumocystis jiroVecii;
M. aVium, Mycobacterium aVium; MTX, methotrexate; TMQ, trimetrexate;
TMP, trimethoprim; DHPS, dihydropteroate synthase; FPGS, folyl poly-
γ-glutamate synthetase.
10.1021/jm800244v CCC: $40.75
2008 American Chemical Society
Published on Web 07/08/2008

4590 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Gangjee et al.
Figure 1
Figure 2
pyrrolo[2,3-d]pyrimidine scaffold dictates the activity against
DHFR and/or TS as well as tumor cell growth inhibitory
potency.
Molecular modeling (SYBYL 6.91)13 further indicated that
the 5-alkyl group could be homologated to a 5-propyl or
5-isopropyl. This homologation could further enhance the van
der Waals interaction with Val115 in human DHFR (Figure 4)
in the normal DHFR binding mode. To determine the optimum
size of the 5-alkyl group for DHFR and/or TS inhibitory activity
as well as tumor cell growth inhibitory potency, the 2,4-diamino-
5-propyl-6-arylthio-7H-pyrrolo[2,3-d]pyrimidine (3) and 2,4-
diamino-5-isopropyl-6-arylthio-7H-pyrrolo[2,3-d]pyrimidine (4)
classical analogues (Figure 2) were designed and synthesized.
Figure 3. The “Normal” and “Flipped” modes of pyrrolo[2,3-d]pyr-
imidine.
inhibitory activity (37% inhibition @ >17 µM). However,
compound 2 demonstrated increased tumor cell growth inhibi-
tory activities against certain tumor cell lines compared to 1 in
the NCI preclinical screening program (Table 3). Thus the size
of the alkyl group attached to the 5-position of the 2,4-diamino
The existing regimen used to treat opportunistic infections
in AIDS and other immunocompromised patients is suppressive
rather than curative and the therapy must be continued
indefinitely.3,4 Thus, it is of considerable interest to design single
agents that have both the desired selectivity of TMP and the

Pyrrolo[2,3-d]pyrimidine Containing Antifolates
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4591
Table 1. Inhibitory Concentration (IC50, µM) and Selectivity Ratiosj against Isolated TS and DHFRa
TS
E. colib
DHFR
E. colie
Selectivity ratiosj
h/ecj h/tgj
cmpd
humanb
0.54
T. gondiic
humand
T. gondiic
1f
>180
1.8
0.21
0.066
2.6
3.0
0.016
0.002
1.3
1.5
1.3
0.61
1.4
0.5
1.5
0.60
2.1
0.17
13
33
2
2
1.2
2g
>17 (37)h
>22 (0)
>24 (0)
>14 (0)
>13 (0)
>12 (0)
>11 (0)
>12 (0)
>12 (0)
>22 (18)
>13 (0)
>27 (0)
>25 (0)
>23 (0)
>23 (0)
>12 (0)
>24 (39)
>23 (14)
29
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
>22 (0)
>24 (0)
>14 (0)
>13 (0)
>12 (0)
>11 (0)
>12 (0)
>12 (0)
>2.2 (0)
>13 (0)
>2.7 (0)
>2.5 (0)
>2.3 (0)
>2.3 (0)
>12 (0)
>2.4 (0)
>2.3 (0)
90
ND
ND
ND
ND
0.12
0.23
1.4
>1.4 (0)
>13 (12)
>12 (0)
>11 (16)
>12 (16)
>15 (12)
>2.2 (0)
>13 (0)
>2.7 (0)
>2.5 (0)
>2.3 (0)
>2.3 (0)
>12 (0)
>2.4 (0)
>2.3 (0)
18
33
25
275
>30 (15)
>28 (13)
>26 (35)
>29 (17)
>15 (30)
>26 (37)
>30 (10)
16
30
>28 (0)
27
>29 (27)
>29 (36)
>27 (0)
0.022
1.5
680
>49
>20
>52
>19
>25
>12
>10
123
200
>254
245
>193
>100
>193
3.3
>130
>20
>26
>500
>100
>50
>100
250
50
>20
10
>241
>19
>19
2
3.26
234
1.0
0.058
0.15
0.52
0.30
0.064
0.60
1.4
2.7
0.12
1.5
3.0
0.13
0.15
0.11
0.11
0.15
0.29
0.14
0.0066
230
1.4
MTX
0.011
0.46
2.9
pemetrexedi
TMP
9.5
76
2.8
0.006
34000
3
0.020
2.0
pyrimethamine
6.0
0.080
75
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, Albany, NY. c Kindly
provided by Dr. K. Anderson, Yale University. 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 Data taken from ref 12. g Data taken from ref 14. h Number in parenthesis indicated % inhibition
at that concentration. i Kindly provided by Dr. Chuan Shih, Eli Lilly & Co., Indianapolis IN. j Selectivity ratios, h/ec ) IC50 human dihydrofolate reductase/
IC50 E. coli dihydrofolate reductase; h/tg ) IC50 human dihydrofolate reductase/IC50 T. gondii dihydrofolate reductase.
Figure 4. Stereo view of compound 4 in human DHFR (PDB code 1U72).30 The hydrophobic interaction between 5-isopropyl and Val 115 is
shown.
potency of TMQ. Such agents could be used as single agents
to treat opportunistic infections in immunocompromised patients
to decrease cost and increase patient compliance. Because
patients with AIDS are often infected with multiple opportunistic
infections, it is highly desirable to develop single agents that
simultaneously target two or more opportunistic pathogen
DHFR.
afford hydrophobic interaction with Ile123 in P. carinii DHFR,
Val151 in T. gondii DHFR, and Ile102 in M. aVium DHFR on
the basis of X-ray crystal structure,16,17 multiple sequence
alignment,18,19 and molecular modeling (SYBYL 6.813) studies,
respectively. The 5-CH3 group was also suggested to influence
the conformations of the 6-arylthio side chain in these inhibitors,
thus limiting its flexibility and contributing to the potency of
these compounds. Several compounds, including 5 and 6 (Table
2), displayed 10-fold or higher selectivity ratios for T. gondii
DHFR and/or M. aVium DHFR compared to rat liver (rl)
Gangjee et al.15 also reported nonclassical analogues of 1,
including 5 and 6 (Figure 2) as inhibitors of DHFR from
opportunistic pathogens. The 5-CH3 moiety was designed to

4592 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Gangjee et al.
Table 2. Inhibitory Concentration (IC50, µM) against Isolated DHFRa
Rosowsky et al.,20 using a different approach, reported
compounds 9 and 10 (Figure 2) with a single carbon atom bridge
that displayed fair T. gondii DHFR potency and good selectivity.
Analogue 9 (IC50 ) 0.07 µM) was the most potent in this series
against T. gondii DHFR, while analogue 10 was the most
selective for T. gondii DHFR compared to rlDHFR with a
selectivity ratio of 81.
and Selectivity Ratiosb
cmpd
P. carinii
rat liver
rl/pcb
M. aVium
rl/mab
3
4
5
6
7
8
0.00311
0.0048
37.3
14.6
21.8
6.04
5.35
5.32
4.21
0.06
0.186
4.57
7.8
19.3
38.8
0.12
0.5
0.3
0.7
1.1
0.5
0.3
0.3
0.9
0.4
0.8
ND
NDd
0.5
0.2
ND
1.4
0.2
1.3
0.3
1.2
0.07
15
0.000737
0.000391
0.7
81.4
475.7
6.5
0.1
78
5.6
0.88
0.1043
12.3
10.4
1.31
0.63
0.6
0.21
4.1
7.8
6.36
38.8
0.5
0.23
1.0
A sulfur atom was incorporated in compounds 5 and 6 rather
than a carbon atom, as in compounds 9 and 10, to increase the
proximity of the 6-arylthio ring to the hydrophobic residues on
the pathogen DHFR due to the increased atomic size of the
sulfur atom as well as a decrease in the C-S-C angle (98°)
compared to a C-C-C angle (109°).15 Compound 6 was 19-
fold more potent and nearly one-half as selective as the most
selective compound (10) of the 6-carbon-bridged analogues. The
biological activity of compounds 5 and 6 supported the
hypothesis that the 6-arylthio side chain of these compounds
indeed interacts more favorably with Phe91 in T. gondii DHFR
and Val158 in M. aVium DHFR and that the sulfur bridge
increased activity and selectivity. Gangjee et al.14 have also
synthesized the ethyl homologues of 5 and 6 with the goal of
further increasing the potency and selectivity. Compound 8
(Figure 2), the ethyl homologue of 6, was found to have
increased potency and/or selectivity against P. carinii and T.
gondii DHFR compared to rlDHFR (Table 2). Similar to their
methyl counterparts, the ethyl homologues including 7 and 8
were found to have increased potency and/or selectivity against
T. gondii and/or M. aVium DHFR. In most instances, the ethyl
homologues tested were found to be more active and/or selective
against two or more pathogen DHFR. In an attempt to optimize
the size of the 5-alkyl substitution on the potency and selectivity
for P. carinii DHFR, T. gondii DHFR, and M. aVium DHFR
compounds 11-27 (Figure 2) were also designed and synthe-
sized. Compounds 11-18 contain a 5-propyl group, while
compounds 19-27 contain a 5-isopropyl group.
4.05
5.95
2.43
1.25
1.25
8.9
3.45
6.5
3.8
2.4
14.8
2
1.9
50.3
4.6
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
TMQ
TMP
4.21
10.4
8.45
8.6
2.0
14.8
16.4
1.59
0.5
3.5(16%)c
22(14%)
27.2
2.7
0.9
28.6
0.3687
ND
2.1
3.4
0.24
2.54
1.14
0.0015
0.3
0.52
5.42
ND
24
1.3
3.8
0.83
11.84
2.0
11.2
ND
35.6
28.2
0.68
7.1
10.9
0.042
12
0.91
2.1
13.5
0.003
180
600
a These assays were carried out at 37 °C under conditions of substrate
(90 µM dihydrofolic acid) and cofactor (119 µM NADPH) in the presence
of 150 mM KCl. b Selectivity ratios, rl/pc ) IC50 rat liver dihydrofolate
reductase/IC50 P. carinii dihydrofolate reductase; rl/ma ) IC50 rat liver
dihydrofolate reductase/ IC50 M. aVium dihydrofolate reductase. c Number
in parenthesis indicate the percentage inhibition at the given concentration.
d ND ) not determined.
Table 3. Cytotoxicity Evaluation (GI50, M) of Compounds 1-4 against
Selected Tumor Cell Lines29
i
cell line
R ) Me (1) R ) Et (2)
Leukemia
R ) Pr (3) R ) Pr (4)
CCRF-CEM
HL-60 × (TB)
K-562
MOLT-4
RPMI-8226
2.21 × 10-7
5.36 × 10-7
1.28 × 10-6 5.65 × 10-6
9.52 × 10-7 9.32 × 10-7
1.94 × 10-5 1.31 × 10-6
7.74 × 10-6
4.35 × 10-8
2.95 × 10-8
3.61 × 10-8
1.4 × 10-7
Chemistry
<1.00 × 10-8
<1.00 × 10-8
The syntheses of compounds 3 and 11-18 required the
synthesis of 2,4-diamino-5-propyl-7H-pyrrolo[2,3-d]pyrimidine,
31 (Scheme 1), while the synthesis of 4 and 19-27 required
the synthesis of 2,4-diamino-5-isopropyl-7H-pyrrolo[2,3-d]py-
rimidine, 38 (Scheme 2). Taylor et al.21 have reported the
synthesis of various 2,4-diamino-5-alkyl-7H-pyrrolo[2,3-d]py-
rimidines by a ring transformation/ring annulation sequence of
2-amino-3-cyano-4-alkyl furans. These furans were in turn
obtained by the condensation of suitable R-hydroxy ketones with
malonodinitrile in the presence of a suitable base such as
triethylamine. Gangjee et al.12,14,15,22 and Rosowsky et al.23,24
have also successfully adopted this methodology in their
synthesis of pyrrolo[2,3-d]pyrimidine containing antifolates.
Extending this general methodology to the synthesis of 31
required the synthesis of 1-hydroxy-2-pentanone, 29 (Scheme
1). Compound 29 was in turn obtained from the commercially
available 1,2-pentanediol, 28, by regiospecific oxidation of the
secondary alcohol using hexabutyldistannoxane (HBD) and Br2
(Scheme 1).25 Two methods were attempted for the separation
of the 1-hydroxy-2-pentanone, 29, from the reaction mixture.
The first involved silica gel chromatography on the crude
reaction mixture and the second was a direct distillation of the
crude reaction mixture. In general, distillation was found to be
superior to column chromatography. Condensation of 29 with
malonodinitrile using triethylamine as base afforded the 2-amino-
4-propyl-furan-3-carbonitrile, 30, in 60% yield. Further, con-
densation of 30 with guanidine (liberated from guanidine
Nonsmall Cell Lung Cancer
NCI-H460
6.31 × 10-7
2.44 × 10-7
2.67 × 10-7 6.45 × 10-6
Colon Cancer
HT29
SW-620
HCT 15
1.60 × 10-7
1.24 × 10-7
5.95 × 10-5
5.4 × 10-8
9.58 × 10-6 5.76 × 10-6
1.21 × 10-6 4.71 × 10-6
9.0 × 10-8
6.7 × 10-7 >1.0 × 10-4 3.48 × 10-5
Central Nervous System Cancer
U251
SF268
8.24 × 10-7
1.6 × 10-6
7.24 × 10-6
7.47 × 10-5
2.2 × 10-7
1.61 × 10-6 1.78 × 10-5
Melanoma
LOX IMVI
PC-3
1.71 × 10-7
Prostrate Cancer
4.36 × 10-8 >1.0 × 10-4 >1.0 × 10-4
4.9 × 10-8 <1.00 × 10-8 1.14 × 10-6
Breast Cancer
MCF-7
>1 × 10-4
5.9 × 10-7
1.64 × 10-7
Renal Cancer
ACHN
CAKI-1
786-0
2.42 × 10-5
>1 × 10-4
4.5 × 10-7
1.52 × 10-5 7.39 × 10-6
3.76 × 10-7 4.25 × 10-6
1.28 × 10-8 5.58 × 10-6
1.25 × 10-7
DHFR.15 Compound 6 with a 2′,5′-(OCH3)2 substitution was
16-fold more potent and equally selective compared to TMP
against T. gondii DHFR.

Pyrrolo[2,3-d]pyrimidine Containing Antifolates
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4593
Scheme 1a
a Conditions: (a) O(SnBu3)2, Br2, CH2Cl2; (b) malonodinitrile, NEt3, MeOH, 24 h; (c) guanidine hydrochloride, NaOMe, overnight; (d) ArSH, I2, EtOH/
H2O (2:1), 100-110 °C; (e) 1N NaOH, 80 °C, 24 h; (f) 2-chloro-4,6-dimethoxy-1,3,5-triazine, N-methylmorpholine, diethyl-L-glutamate hydrochloride, 0
°C to r.t.; (g) 1N NaOH, 0 °C to r.t.
Scheme 2a
a Conditions: (a) HCHO, 3-ethylbenzothiazolium bromide, Et3N, EtOH, 60 °C, 72 h; (b) malonodinitrile, NEt3, MeOH, 24 h; (c) guanidine hydrochloride,
NaOMe, 96 h; (d) ArSH, I2, EtOH/H2O (2:1), 100-110 °C; (e) 1N NaOH, 80 °C, 24 h. (f) 2-chloro-4,6-dimethoxy-1,3,5-triazine, N-methylmorpholine,
diethyl-L-glutamate hydrochloride, 0 °C to r.t.; (g) 1N NaOH, 0 °C to r.t.
hydrochloride and NaOMe) afforded 31 in 50% yield. Reaction
of 2,4-diamino-5-propyl-7H-pyrrolo[2,3-d]pyrimidine, 31, and
ethyl 4,4′-bismercaptobenzoate in EtOH/H2O followed by the
addition of I2 at reflux afforded compound 32 in 28% yield.26,27
The disappearance of the 6-vinyl proton at δ 6.38 and the
appearance of the characteristic AA′XX′ pattern for the 6-aryl
protons in the 1H NMR spectrum of 32 in DMSO (d6) indicated
the success of the oxidative addition reaction.
Hydrolysis of the ester 32 with aqueous 1N NaOH at 80 °C
(24 h) followed by acidification gave the required acid, 33, in
83% yield. Peptide coupling21 of the acid 33 with diethyl-L-
glutamate using 2,6-dimethoxy-4-chlorotriazine and N-methyl

4594 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Gangjee et al.
morpholine, followed by chromatographic purification afforded
the coupled product 34 in 62% yield. The H NMR spectrum
11-27 were all poor inhibitors of all three TS tested. They were
however reasonably potent inhibitors of E. coli DHFR and T.
gondii DHFR. Most of the analogues were weak or poor
inhibitors of hDHFR.
1
of 34 in DMSO (d6) revealed the newly formed amide NH
proton at δ 8.64-8.67 ppm as a doublet. Hydrolysis of the
diester 34 with aqueous NaOH at 0 °C (4 h) and then at room
temperature (24 h), followed by acidification, gave the desired
compound 3 in 86% yield.
Similarly, reaction of 31 with appropriately substituted aryl
thiols in a mixture of EtOH/H2O (2:1) followed by addition of
I2 at reflux as reported previously27 afforded 11-18 in
45%-70% yields. The yields reveal no apparent correlation
between the extent of pyrrolo[2,3-d]pyrimidine substitution and
the electron-donating or -withdrawing effects of substituents in
the thiophenol.
Analogous to 31, the synthesis of 37 (Scheme 2) required
the synthesis of 1-hydroxy-3-methyl-2-butanone, 36. Thiazolium
salt-catalyzed benzoin condensation of isopropyl aldehyde 35
with paraformaldehyde catalyzed by N-ethylbenzothiazolium
bromide and triethylamine afforded the R-hydroxy ketone, 36,
after distillation, in 35% yield.28 Compounds 4 and 19-27 were
synthesized as shown in Scheme 2 starting with 36 in essentially
the same way as described for 3 and 11-18 in Scheme 1.
The yields in Scheme 2, as before for Scheme 1, reveal no
apparent correlation between the extent of pyrrolo[2,3-d]pyri-
midine substitution, and the electronic nature of the substituents
in the thiophenol. The lower yields of 18 (Scheme 1) and 19
(Scheme 2) may be a result of unfavorable steric interactions
between the bulky 5-isopropyl group in 37 and the 2′,6′-
disubstitution present on the thiophenols, which makes 6-sub-
stitution more difficult.
E. coli DHFR (Table 1): In general the 5-isopropyl com-
pounds (19-27) with the exceptions of 19 and 20 were more
potent and selective against E. coli DHFR than the correspond-
ing 5-propyl compounds (11-18). In the 5-propyl series,
analogue 16 with a 3,4-dimethoxyphenyl side chain was the
most potent and selective compound against E. coli DHFR. In
the 5-isopropyl series, analogues 21-27 were potent against
E. coli DHFR. A number of the nonclassical compounds in the
5-isopropyl series showed good selectivity for E. coli DHFR
as compared to hDHFR. Compounds 21-27 were 100-fold to
254-fold more selective for E. coli DHFR than hDHFR. Thus
the 5-isopropyl-6-substituted phenyl analogues were reasonably
selective for bacterial DHFR.
T. gondii DHFR (Table 1): In general the 5-propyl compounds
(13-18) with the exception of 13 were more potent and selective
against T. gondii DHFR than the corresponding 5-isopropyl
compounds (21-26). In the 5-propyl series, analogue 17 with
a 1-naphthyl side chain was the most potent compound against
T. gondii DHFR and was 50-fold more potent than TMP, 5-fold
less potent than MTX and equipotent with pyrimethamine.
Compound 17 was also the most selective compound against
T. gondii DHFR with >500-fold selectivity over human DHFR.
Thus 17 is 50-fold more potent than TMP and about twice as
selective against T. gondii DHFR. Compound 13 with a phenyl
side chain was 20-fold more potent than TMP and equally
selective against T. gondii DHFR compared with human DHFR.
In the 5-isopropyl series, analogue 21 with an unsubstituted
phenyl side chain was the most potent analogue against T. gondii
DHFR and was 45-fold more potent than TMP and equally
selective. Analogue 25 with a 1-naphthyl side chain was 24-
fold more potent than TMP and equally selective.
Biological Evaluation and Discussion
Compounds 3, 4, and 11-27 were evaluated as inhibitors of
human (h), Escherichia coli (E. coli), and T. gondii DHFR and
TS. The inhibitory potency (IC50) values are compared with
MTX, pemetrexed, TMP, pyrimethamine, and the previously
synthesized 1 and 2 (Table 1). Compounds 3 and 4 are good
inhibitors of hDHFR with nanomolar IC50 values and were about
3-fold and 4-fold less potent as hDHFR inhibitors, respectively,
compared with MTX and about 25-fold and 17-fold more potent
respectively than pemetrexed. Compound 3 was equipotent with
the previously synthesized 2 and about 3.5-fold more potent
than 1. The biological data of 1-4 indicate that an ethyl, propyl,
or isopropyl group at the 5-position are all conducive for potent
hDHFR inhibition. The potent hDHFR activity of 2-4 compared
to 1 could be attributed to increased hydrophobic interaction of
the bulkier alkyl groups in 2-4 with Val115 in hDHFR. The
increased activity of 2-4 may also result from favorable
orientation of the 6-position thioaryl side chain when bound to
hDHFR. Against hTS, 3 and 4 had similar inhibitory potency
as MTX but were 4-fold less inhibitory than pemetrexed.
Compounds 3 and 4 were about 63-74-fold less potent than 1
as inhibitors of hTS. These results indicate that homologation
of the 5-methyl group in 1 to larger alkyl groups as in 2-4 is
detrimental to hTS inhibition. This decrease in potency may be
due to steric hindrance between the larger alkyl groups in 2-4
and Trp109 in hTS and/or due to unfavorable orientation of
the 6-position side chains for interaction with the hTS in the
presence of the bulkier 5-alkyl moiety.
Compounds 3, 4, 11-27, MTX, PYR, and TMP were also
assayed against T. gondii DHFR using protocols described for
Table 2 (data for T. gondii DHFR not shown), as well as under
the conditions described for Table 1. Of the 20 compounds
jointly assayed, MTX, 3, and 4 were identified as the three most
potent compounds by both protocols; both laboratories also
placed TMP, 23, 24, and 27 as among the six least potent
compounds. Selectivity could only be directly compared for the
compounds in Table 1 that had defined selectivity values. Assays
under both sets of conditions identified TMP, 13, and 21 as the
most selective compounds in this set of nine compounds and
MTX as the least selective. Considering all compounds inde-
pendently assayed as described in Table 2 for T. gondii, DHFR,
the most selective compounds are TMP, 21, 15, 20, 13, 17, and
23; this list is consistent with the results in Table 1, except that
the selectivity of compounds 15 and 20 is artificially depressed
in Table 1 by the inability to generate full inhibition curves for
the human reference enzyme.
Compounds 3, 4, and 11-27 were also evaluated as inhibitors
of P. carinii DHFR, M. aVium DHFR, and rlDHFR, which
served as the mammalian surrogate under slightly different assay
conditions. The inhibitory potency (IC50) values are compared
with TMQ, TMP, and the previously synthesized 5-8 (Table
2). Several compounds displayed 10-fold or higher selectivity
ratios for M. aVium DHFR. Against P. carinii DHFR, in general,
the 5-propyl nonclassical analogues (11-18) were more potent
and selective than the corresponding 5-isopropyl analogues
(19-27). Against M. aVium DHFR, the nature of the phenyl
Compound 3 was a poor inhibitor of hTS and E. coli TS
(Table 1) but showed moderate inhibition against T. gondii TS
(equipotent to pemetrexed). Compound 3 was a good inhibitor
of all three DHFR tested. In addition, 3 is also a dual inhibitor
of T. gondii DHFR and T. gondii TS. The nonclassical analogues

Pyrrolo[2,3-d]pyrimidine Containing Antifolates
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4595
substitution along with the 5-alkyl group determined the potency
and selectivity of the compound.
concentration required to inhibit the growth of tumor cells in
culture by 50% compared to a control. In 6 of the 60 tumor
cell lines evaluated, compound 3 showed GI50 values of <1 ×
10-6 M (Table 3). While in only 2 of the 60 tumor cell lines
evaluated, compound 4 showed GI50 values of <1 × 10-6 M.
It is noteworthy that compound 3 was not a general cell poison
but showed selectivity both within a type of tumor cell line
and across different tumor cell lines, with inhibitory values
which in some instances differed by 10000-fold. In the
melanoma LOX IMVI cell line and the renal cancer cell line
786-0, compound 3 displayed GI50 values of e1 × 10-8 M. It
can be seen from the tumor cell growth inhibitory activity (Table
3) of compounds 1-4 that the tumor cell growth inhibitory
potency, in certain instances, were more potent than either their
human DHFR and/or human TS inhibitory activity alone (Table
1) and could be the result of a synergistic effect of dual
inhibitory activities against TS and DHFR and/or that poly-
glutamylation increases inhibitory activity against TS and/or
DHFR in tumor cell systems. Against the outgrowth of tumor
cells in culture compound, 2 was in general the most potent
compound followed by 3 then 1, and the least potent is com-
pound 4. Though a strict structure-activity relationship cannot
be considered for tumor cells in culture the 5-ethyl is clearly
superior to a methyl, propyl or isopropyl.
In summary, homologation of a 5-methyl (compound 1) to a
5-propyl (compound 3) or 5-isopropyl (compound 4) in 2,4-
diamino-6-thiobenzoyl-5-alkylpyrrolo[2,3-d]pyrimidines in-
creases the human DHFR inhibitory activity but is detrimental
to the human TS inhibitory activity. We have found that in
classical N-[4-[(2,4-diamino-5-alkyl-7H-pyrrolo[2,3-d]pyrimi-
din-6-yl)-thio]-benzoyl]-L-glutamic acid containing analogues
the size of the alkyl group at the 5-position dictates inhibition
of TS and/or DHFR activity as well as tumor cell growth
inhibitory activity. The fact that homologated 5-alkyl substit-
uents such as ethyl, propyl, and isopropyl are not tolerated by
human TS indicates that homologation of the 5-alkyl group
beyond a methyl is not conducive for dual human TS-DHFR
inhibition in classical 5-alkyl-6-arylthiosubstituted pyrrolo[2,3-
d]pyrimidines. Homologation however maintains dual DHFR-
TS inhibitory activity against the bifunctional enzyme derived
from T. gondii. In the nonclassical series, homologation of the
5-alkyl group is highly conducive for potent inhibition of P.
carinii DHFR; however, it does not improve the selectivity of
the analogues. Homologation of the 5-alkyl group to a propyl
or isopropyl is highly conducive for potent and selective
inhibition of T. gondii DHFR compared to human DHFR. The
size of the alkyl group present at the 5-position of the
pyrrolo[2,3-d]pyrimidine ring system along with the nature of
the lipophilic substituents present on the 6-arylthio side chain
determines the potency and selectivity against T. gondii DHFR
and M. aVium DHFR.
P. carinii DHFR (Table 2): Against P. carinii DHFR the most
potent analogues bore an unsubstituted phenyl (in 13) or a 4′-
methoxyphenyl substitution (in 14) in the 5-propyl (11-18)
series. Other substitutions such as a 2,6-dichloro 11, 2,6-
dimethyl 12, 3,4-dimethoxy 16, or 2,5-dimethoxy 15 caused a
slight drop in activity. The 1-naphthyl substitution in 17 was
found to display moderate DHFR inhibitory activity, while a
2-naphthyl substitution in 18 was found to be slightly detrimental
for activity compared to 13. In the 5-isopropyl (19-27) series,
the most potent analogue contained a 1-naphthyl side chain 25
and displayed submicromolar inhibitory potency. All other
substitutions in the side chains as in 19-27, with the exception
of 25, displayed micromolar or higher inhibitory potency.
Compounds 11-27 were not selective against P. carinii DHFR
and increasing the size of the 5-alkyl group did not improve
the selectivity, however, it increased the potency of the
compounds against P. carinii DHFR compared to the corre-
sponding methyl (compare 5 with 26 or 6 with 15) and ethyl
(compare 7 with 13 or 21) analogues. The biological data of
analogues 11-21 indicate that the homologation to a 5-propyl
or 5-isopropyl group is conducive for potent inhibition of P.
carinii DHFR, however, it does not improve the selectivity.
Mycobacterium aVium DHFR (Table 2): Analogue 16 con-
taining a 3′,4′-dimethoxy substitution in the phenyl ring was
the most potent and selective analogue in the 5-propyl series.
The second best analogue 15 had a 2′,5′-dimethoxy substitution
in the side chain. Substitution of the phenyl ring with other
substitutions as in 11-14, 17, and 18 resulted in analogues that
were considerably less potent and selective. In the 5-isopropyl
series, the most potent analogue 25 contains a 1-naphthyl side
chain. The 2-naphthyl substituted analogue 26 was 10-fold less
potent than 25. The most selective analogue was 23, with a
2′,5′-dimethoxy substitution in the side chain. The electron
donating 3,4-dimethoxy substituted analogue 24 was found to
be devoid of any selectivity. In sharp contrast, the analogue
with electron withdrawing 3,4-dichloro substitution 27 had the
second best selectivity. Again, the biological data of 11-27
indicate that both the alkyl group present at the 5-position as
well as the substituents present on the 6-position thioaryl side
chain play a role in determining the potency and selectivity of
the analogues against M. aVium DHFR.
Rat liver DHFR (Table 2): In the 5-propyl series, analogues
13 and 14 with an unsubstituted phenyl and a 4′-methoxy
substitution, respectively, were the most potent. Dimethoxy
substituted analogues 15 and 16 were 2-fold less potent than
the monomethoxy 14. Substitution of the phenyl ring with bulky
groups such as 1-naphthyl 17 and 2-naphthyl 18 also resulted
in 2-fold less potent compounds compared to 13. However,
substitution of the phenyl ring with either a 2′,6′-dichloro 11
or 2′,6′-dimethyl 12 substitution maintained activity compared
to 13. In the 5-isopropyl series, the 1-naphthyl substituted
analogue 25 was the most potent. The 2-naphthyl substituted
analogue 26 was 10-fold less potent than 25. Replacing the
1-naphthyl substituent with an unsubstituted phenyl 21 or
substitution of the phenyl ring with various electron donating
methoxy (23, 24), methyl (20) or electron withdrawing chloro
(19, 27) substitution also afforded analogues that were consider-
ably less potent.
Experimental Section
All evaporations were carried out in vacuum with a rotary
evaporator. Analytical samples were dried in vacuum (0.2 mmHg)
in an Abderhalden drying apparatus over P2O5 at 70 °C. Thin-layer
chromatography (TLC) was performed on silica gel plates (What-
man 250 µM PE SiLG/UV) with fluorescent indicator. Spots were
visualized by UV light (254 and 365 nm) or by staining with a
solution of KMnO4 in EtOH. All analytical samples were homo-
geneous on TLC in at least two different solvent systems.
Purification by column and flash chromatography was carried out
using Merck silica gel 60 (200-400 mesh). The amount (weight)
of silica gel for column chromatography was in the range of 50-100
times the amount (weight) of the crude compounds being separated.
Compounds 3 and 4 were selected by the National Cancer
Institute (NCI) for evaluation in its in vitro preclinical antitumor
screening program.29 The ability of compounds 3 and 4 to inhibit
the growth of tumor cells was measured as GI50 values, the

4596 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Gangjee et al.
Columns were dry-packed unless specified otherwise. Solvent
systems are reported as volume percent of mixture. Melting points
were determined on a Mel-Temp II melting point apparatus and
are uncorrected. Proton nuclear magnetic resonance (1H NMR)
spectra were recorded on a Bruker WH-300 (300 MHz) spectrom-
eter. The chemical shift (δ) values are reported as parts per million
(ppm) relative to tetramethylsilane as internal standard; s ) singlet,
d ) doublet, t ) triplet, q ) quartet, m ) multiplet, br ) broad
singlet. Elemental analyses were performed by Atlantic Microlab,
Inc., Norcross, GA. Elemental compositions were 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 removed despite 24 h of drying in vacuum and were
confirmed, where possible, by their presence in the 1H NMR
spectrum. High-resolution mass spectra (HRMS), using electron
impact (EI), were recorded on a VG Autospec (Fisons Instruments)
micromass (EBE Geometry) double-focusing mass spectrometer.
All solvents and chemicals were used as received.
1-Hydroxy-2-pentanone (29). To a solution of 1,2 pentanediol,
28 (2.1 g, 20 mmol) and hexabutyldistannoxane (HBD) (15.5 g,
26 mmol) in anhydrous CH2Cl2 (100 mL), Br2 (4.16 g, 26 mmol)
solution in CH2Cl2 (10 mL) was added dropwise at room temper-
ature with stirring under N2 atmosphere. The mixture was stirred
for 3 h at room temperature. The solvent was evaporated under
reduced pressure, and the resulting oil was distilled under reduced
pressure to give 29 as a colorless oil, bp ) 68-70° (16 mmHg)
[lit.28 bp ) 70° (20 mmHg)].
2-Amino-4-propyl-furan-3-carbonitrile (30). A mixture of
malonodinitrile (2.65 g, 40 mmol) and N(C2H5)3 (5.58 mL, 40
mmol) in anhydrous MeOH (120 mL) was added dropwise to a
solution of the R-hydroxy ketone 29 (4 g, 40 mmol) in MeOH,
and the resulting dark-red solution was stirred at room temperature
for 24 h. To this solution was added silica gel (10 g), and the solvent
was evaporated to dryness under reduced pressure to afford a dry
silica gel plug, which was loaded on top of a wet (hexane) silica
gel column and eluted first with hexane and then with 2:1 hexane/
EtOAc to afford 1.88 g (60%) of the furan 30 as a red-cream solid:
mp 58.8-62.5 °C; TLC Rf ) 0.51 (hexane/EtOAc, 2:1). 1H NMR
(DMSO-d6): δ 0.85-0.94 (t, 3 H, 4-CH2CH2CH3), 1.47-1.56 (m,
2 H, 4-CH2CH2CH3), 2.21-2.26 (t, 2 H, 4-CH2CH2CH3), 6.74 (s,
1 H, 5-CH), 7.20 (s, 2 H, 2-NH2). Anal. calcd for (C8H10N2O) C,
H, N.
2,4-Diamino-5-propyl-7H-pyrrolo[2,3-d]pyrimidine (31). Amino
nitrile furan 30 (1.8 g, 12 mmol) was added to a solution of
guanidine hydrochloride (2.5 g, 26 mmol) and NaOMe (1.4 g, 26
mmol) in anhydrous EtOH (100 mL). The resulting dark-red
solution was stirred under reflux overnight, during which time it
became dark brown. To this solution was added silica gel (5 g),
and the solvent was evaporated to dryness under reduced pressure
to afford a dry silica gel plug, which was loaded on top of a wet
(CHCl3) column and eluted first with CHCl3 and then with a
gradient of 1-5% MeOH in CHCl3 to afford 1.15 g (50%) of 31
as a dark-brown solid: mp 215-220 °C; TLC Rf ) 0.41 (CHCl3/
MeOH, 5:1, with 2 drops of conc NH4OH). 1H NMR (DMSO-d6):
δ 0.83-0.89 (t, 3 H, 5-CH2CH2CH3), 1.50-1.60 (m, 2 H,
CH2CH2CH3), 2.57-2.62 (t, 2 H, CH2CH2CH3), 5.36 (s, 2 H, 2/4-
NH2), 5.91 (s, 2 H, 2/4-NH2), 6.38 (s, 1 H, 6-CH), 10.36 (s, 1 H,
7-NH). Anal. calcd for (C9H13N5 ·0.1H2O) C, H, N.
and then with a gradient of 1-5% MeOH in CHCl3. Fractions
containing the desired spot (TLC) were pooled and evaporated to
dryness to afford 610 mg (28%) of 32 as a white solid: mp )
260.6-261 °C; TLC Rf ) 0.54 (CHCl3/MeOH, 5:1). 1H NMR
(DMSO-d6): δ 0.82 (t, 3 H, CH2CH2CH3), 1.29 (t, 3 H, CH2CH3),
1.41-1.43 (m, 2 H, CH2CH2CH3), 2.76 (t, 2 H, CH2CH2CH3),
4.27-4.29 (q, 2 H, CH2CH3), 7.09 (s, 2 H, 2/4-NH2), 7.74 (s, 2 H,
2/4-NH2), 7.13-7.16 (d, 2 H, C6H4), 7.84-7.87 (d, 2 H, C6H4),
12.08 (s, 1 H, 7NH). Anal. calcd for (C18H21N5O2S) C, H, N, S.
4-[2,4-Diamino-5-propyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)sul-
fanyl]benzoic acid (33). To a suspension of 32 (330 mg, 0.9 mmol)
in EtOH (30 mL) was added aqueous 1N NaOH (12 mL) and the
reaction mixture was stirred at 80 °C for 24 h. At this time, TLC
indicated the disappearance of the starting ester at Rf ) 0.54 (CHCl3/
MeOH, 5:l) and formation of one major spot at the origin. The
solvent was evaporated to dryness under reduced pressure, and the
resulting sodium salt (yellow oil) was dissolved in water (15 mL)
and carefully acidified to pH 4 by dropwise addition of 3N HCl.
The resulting suspension was filtered and washed carefully with
cold water and dried over P2O5 to afford 295 mg (83%) of 33 as a
white solid. This was directly used in the next step without further
characterization.
Diethyl N-[4-[(2,4-Diamino-5-propyl-7H-pyrrolo[2,3-d]pyri-
midin-6-yl)sulfanyl]-benzoyl]-L-glutamate (34). To a suspension
of the acid 33 (344 mg, 1 mmol) in anhydrous DMF (15 mL) under
N2 was added N-methyl morpholine (145 µL, 1.33 mmol) and the
resulting suspension was cooled to 0 °C. At this point, 2-chloro-
4,6-dimethoxy-1,3,5-benzotriazine (235 mg, 1.33 mmol) was added
and the suspension was stirred for 2 h at 0 °C; during this time, it
formed a solution. The reaction mixture was again cooled to 0 °C
and diethyl-L-glutamic acid (317 mg, 1.33 mmol) was added
followed by N-methyl morpholine (145 µL, 1.33 mmol). The
solution was slowly allowed to warm to room temperature with
stirring and left at room temperature for a total of 24 h. At this
time, TLC indicated the formation of one major spot at Rf ) 0.58
(CHCl3/MeOH, 5:l). To the resulting solution was added silica gel
(5 g), and the DMF was evaporated to dryness at room temperature
using an oil pump. The silica gel plug was loaded on a wet (CHCl3)
silica gel column and eluted with a gradient of 1-3% MeOH in
CHCl3. Fractions containing the desired spot (TLC) were pooled
and evaporated to dryness under vacuum to afford 330 mg (62%)
of 34 as a white solid: mp 260-260.5 °C; TLC Rf ) 0.58 (CHCl3/
MeOH, 5:1). 1H NMR (DMSO-d6): δ 0.80-0.84 (t, 3 H,
CH2CH2CH3), 1.13-1.19 (m, 6 H, CH2CH3), 1.43-1.45 (m, 2 H,
CH2CH2CH3), 1.98-2.09 (m, 2 H, Glu ꢀ-CH2), 2.42-2.50 (t, 2 H,
Glu γ-CH2), 2.72 (t, 2 H, CH2CH2CH3), 4.00-4.10 (m, 4 H,
CH2CH3), 4.40 (m, 1 H, Glu R-CH), 5.64 (s, 2 H, 2/4-NH2), 6.22
(s, 2 H, 2/4-NH2), 7.04-7.07 (d, 2 H, C6H4), 7.75-7.77 (d, 2 H,
C6H4), 8.64-8.67 (d, 1 H, CONH), 11.06 (s, 1 H, 7-NH). Anal.
calcd for (C25H32N6SO5) C, H, N, S.
N-[4-[(2,4-Diamino-5-propyl-7H-pyrrolo[2,3-d]pyrimidin-6-
yl)sulfanyl]benzoyl]-L-glutamic Acid (3). To a suspension of 34
(200 mg, 0.4 mmol) in EtOH (15 mL) was added 1N NaOH (6
mL) at 0 °C and the resulting suspension was stirred at 0 °C (4 h)
and then at room temperature for 24 h. At this point, TLC showed
the disappearance of the starting ester at Rf ) 0.58 (CHCl3/MeOH,
5:l) and formation of one major spot at the origin. The solvent was
evaporated to dryness under reduced pressure, and the sodium salt
(yellow oil) was dissolved in water (5 mL) and the solution was
cooled in an ice bath and acidified carefully to pH 4.0 with dropwise
addition of 3N HCl. The resulting suspension was frozen using
dry ice/acetone and the reaction flask was kept at 5 °C for 24 h
and filtered. The residue was washed carefully with cold water and
dried over P2O5 to afford 160 mg (86%) of 3 as a white solid: mp
259.5-260 °C. 1H NMR (DMSO-d6): δ 0.74-0.82 (t, 3 H,
CH2CH2CH3), 1.43-1.45 (m, 2 H, CH2CH2CH3), 1.93-2.08 (m,
2 H, Glu ꢀ-CH2), 2.51 (t, 2 H, Glu γ-CH2), 2.99 (t, 2 H,
CH2CH2CH3), 4.37 (m, 1 H, Glu R-CH), 5.67 (s, 2 H, 2/4-NH2),
6.26 (s, 2 H, 2/4-NH2), 7.04-7.07 (d, 2 H, C6H4), 7.76-7.78 (d, 2
Ethyl 4-[2,4-Diamino-5-propyl-7H-pyrrolo[2,3-d]pyrimidin-
6-yl)sulfanyl]benzoate (32). To a suspension of 31 (1.1 g, 5.7
mmol) in a mixture of EtOH/H2O (2:1, 75 mL) was added diethyl
4,4′-dithiobis(benzoate) (2.2 g, 6 mmol) and the suspension was
heated to 100-110 °C, then I2 (3 g, 12 mmol) was added and the
reaction was monitored (TLC) for completion (3 h). To this solution
was added excess sodium thiosulfate and the solution was evapo-
rated to dryness under reduced pressure and the resulting residue
was washed with water and air-dried. This residue was then
dissolved in MeOH (100 mL) and to this was added silica gel (15
g) and the resulting suspension was evaporated to dryness under
reduced pressure to afford a dry silica gel plug that was loaded on
top of a wet silica gel (CHCl3) column and eluted first with CHCl3

Pyrrolo[2,3-d]pyrimidine Containing Antifolates
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4597
H, C6H4), 8.51-8.53 (d, 1 H, CONH), 11.09 (s, 1 H, 7-NH), 12.37
(bs, 2 H, COOH). Anal. calcd for (C21H24N6SO5 ·1.0 H2O) C, H,
N, S.
4-[2,4-Diamino-5-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-
sulfanyl]-benzoic acid (40). To a suspension of 39 (530 mg, 1.43
mmol) in EtOH (50 mL) was added aqueous 1N NaOH (20 mL)
and the reaction mixture was stirred at 80 °C for 24 h. At this point,
TLC indicated the disappearance of the starting ester at Rf ) 0.54
(CHCl3/MeOH, 5:l) and formation of one major spot at the origin.
The solvent was evaporated to dryness, and the resulting sodium
salt (yellow oil) was dissolved in water (15 mL) and carefully
acidified to pH 4 by dropwise addition of 3N HCl. The resulting
suspension was filtered and washed carefully with cold water and
dried over P2O5 to afford 436 mg (90%) of 40 as a white solid. 1H
NMR (DMSO-d6): δ 1.24-1.25 (d, 6 H, CH(CH3)2), 3.35 (m, 1
H, CH(CH3)2), 5.60 (s, 2 H, 2/4-NH2), 6.07 (s, 2 H, 2/4-NH2),
6.93-6.95 (d, 2 H, C6H4), 7.75-7.76 (d, 2 H, C6H4), 10.98 (s, 1
H, 7-NH). Anal. calcd for (C16H17N5O2S) MS (EI) calcd m/z )
343.110297; found m/z ) 343.109307 (M+).
Diethyl N-[4-[(2,4-Diamino-5-isopropyl-7H-pyrrolo[2,3-d]py-
rimidin-6-yl)sulfanyl]-benzoyl]-L-glutamate (41). To a suspension
of the acid 40 (300 mg, 0.87 mmol) in anhydrous DMF (25 mL)
under N2 was added N-methylmorpholine (145 µL, 1.33 mmol) and
the resulting suspension was cooled to 0 °C. At this point, 2-chloro-
4,6-dimethoxy-1,3,5-triazine (235 mg, 1.34 mmol) was added and
the suspension was stirred for 2 h, during which time it formed a
solution. The reaction mixture was again cooled to 0 °C and diethyl-
L-glutamate (317 mg, 1.33 mmol) was added followed by N-
methylmorpholine (145 µL, 1.33 mmol). The solution was slowly
allowed to warm to room temperature with stirring and left at room
temperature for a total of 24 h. To the resulting solution was added
silica gel (5 g) and the DMF was evaporated using an oil pump.
The silica gel plug was loaded on a wet (CHCl3) silica gel column
and eluted with a gradient of 1-3% MeOH in CHCl3. Fractions
containing the desired spot (TLC) were pooled and evaporated to
dryness under vacuum to give 330 mg (70%) of 41 as a white solid:
mp 217.6-218 °C; TLC Rf ) 0.53 (CHCl3/MeOH, 5:1). 1H NMR
(DMSO-d6): δ 1.12-1.19 (m, 6 H, CH2CH3), 1.24-1.27 (d, 6 H,
CH(CH3)2), 1.97-2.07 (m, 2 H, Glu ꢀ-CH2), 2.39-2.44 (t, 2 H,
Glu γ-CH2), 3.99-4.12 (m, 4 H, CH2CH3), 4.40 (m, 1 H, Glu
R-CH), 5.63 (s, 2 H, 2/4-NH2), 6.12 (s, 2 H, 2/4-NH2), 7.02-7.05
(d, 2 H, C6H4), 7.74-7.77 (d, 2 H, C6H4), 8.63-8.65 (d, 1 H,
CONH), 11.01 (s, 1 H, 7-NH). Anal. calcd for (C25H32N6O5-
S·0.5H2O) C, H, N, S.
N-[4-[(2,4-Diamino-5-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-
6-yl)sulfanyl]-benzoyl]-L-glutamate (4). To a suspension of 41
(200 mg, 0.37 mmol) in EtOH (15 mL) was added 1N NaOH (6
mL) and the suspension stirred at 0 °C (4 h) and then at room
temperature for 24 h. The EtOH was evaporated to dryness under
reduced pressure, the yellow oil was dissolved in water (5 mL),
and the solution was cooled in an ice-bath and acidified carefully
to pH 4.0 with dropwise addition of 3N HCl. This suspension was
left at 5 °C for 24 h and filtered. The residue was washed well
with water and O(C2H5)2 and then dried over P2O5/vacuum to afford
165 mg (80%) of 4 as a white solid: mp 206.5-207 °C. 1H NMR
(DMSO-d6): δ 1.25-1.27 (d, 6 H, CH(CH3)2), 1.93-2.06 (m, 2
H, Glu ꢀ-CH2), 2.31-2.33 (t, 2 H, Glu γ-CH2), 4.36 (m, 1 H, Glu
R-CH), 5.78 (s, 2 H, 2/4-NH2), 6.29 (s, 2 H, 2/4-NH2), 7.03-7.06
(d, 2 H, C6H4), 7.76-7.79 (d, 2 H, C6H4), 8.51-8.54 (d, 1 H,
CONH), 11.13 (s, 1 H, 7-NH), 12.4 (bs, 2 H, COOH). Anal. calcd
for (C21H24N6O5S·0.2H2O·1.8C4H10O) C, H, N, S.
2,4-Diamino-5-propyl-6-(2′,6′-dichlorophenylsulfanyl)-7H-
pyrrolo[2,3-d]pyrimidine (11). To a solution of 31 (300 mg, 1.57
mmol) in a mixture of EtOH/water (2:1, 30 mL) was added 2,6-
dichlorophenylthiol (540 mg, 3.00 mmol) and the reaction mixture
was heated to 100-110 °C, then I2 (750 mg, 3.00 mmol) was added
and the heating continued with stirring for a total of 3 h. To this
mixture was added an excess of sodium thiosulfate and the reaction
mixture concentrated under reduced pressure. To the resulting
residue was added silica gel (10 g) and MeOH (50 mL) and the
solution evaporated to dryness under reduced pressure to afford a
dry silica gel plug, which was loaded on top of a wet (CHCl3)
silica gel column and eluted with a gradient of 1-3% MeOH in
CHCl3. Fractions containing the desired spot (TLC) were pooled
1-Hydroxy-3-methyl-2-butanone (36). In a 1000 mL flask were
placed paraformaldehyde (9.45 g, 0.3 mol), 3-ethylbenzothiazolium
bromide (7.32 g, 0.03 mol), isobutraldehyde, 35, (27.5 mL, 0.3 mol),
anhydrous EtOH (300 mL), and Et3N (4.2 mL, 0.03 mol), and then
dry N2 gas was bubbled into the reaction mixture. The mixture
was then heated in an oil bath at 60 °C for 72 h, during which time
the color of the reaction mixture changed to dark reddish-brown.
The solvent was evaporated to dryness under reduced pressure, and
to the resulting residue was added EtOAc (20 mL). The resulting
suspension was filtered, and the solid was washed repeatedly with
EtOAc. The filtrate was evaporated under reduced pressure to a
dark-brown oil, which was distilled under low pressure to afford
10.7 g (35%) of 36 as a colorless oil: bp ) 65-68 °C (16 mmHg)
[lit.28 bp ) 65 °C (20 mmHg)]. 1H NMR (CDCl3-d): δ 1.15-1.17
(d, 6 H, CH(CH3)2), 2.60-2.69 (m, 1 H, CH(CH3)2), 3.13 (bs, 1
H, OH), 4.32 (s, 2 H, CH2).
2-Amino-4-isopropyl-furan-3-carbonitrile (37). A mixture of
malonodinitrile (12.55 g, 190 mmol) and Et3N (19.19 g, 190 mmol)
in MeOH (220 mL) was added dropwise to a solution of the
R-hydroxy ketone 36 (19.4 g, 190 mmol) in MeOH (10 mL) and
the resulting solution was stirred at room temperature for 24 h. To
this solution was added silica gel (50 g), and the solvent was
evaporated under reduced pressure to afford a dry silica gel plug,
which was loaded on top of a wet (hexane) silica gel column and
eluted first with hexane and then with 2:1 hexane/EtOAc to afford
the furan 37 (11.1 g, 73%) as a reddish-brown solid: mp 62-62.5
°C; TLC Rf ) 0.64 (hexane/EtOAc, 2:1). 1H NMR (DMSO-d6): δ
1.13-1.15 (d, 6 H, CH(CH3)2), 2.59-2.65 (m, 1 H, CH(CH3)2),
6.71 (s, 1 H, 5-CH), 7.23 (s, 2 H, 2-NH2) Anal. calcd for
(C8H10N2O) C, H, N.
2,4-Diamino-5-isopropyl-7H-pyrrolo[2,3-d]pyrimidine (38).
Amino nitrile furan 37 (1.5 g, 10 mmol) was added to a solution
prepared from guanidine hydrochloride (1.43 g, 15 mmol) and
NaOMe (0.81 g, 15 mmol) in anhydrous EtOH (100 mL). The
resulting dark-red reaction mixture was stirred under reflux for 96 h,
during which time it turned dark-brown. To this solution was added
silica gel (15 g), and the solvent was evaporated to dryness under
reduced pressure to afford a silica gel plug, which was loaded on
top of a wet (CHCl3) silica gel column and eluted first with CHCl3
and then with a gradient of 1-5% MeOH in CHCl3 to give 1.15 g
(60%) of 38 as a white solid: mp 221-222 °C; TLC Rf ) 0.38
1
(CHCl3/MeOH, 5:1). H NMR (DMSO-d6): δ 1.17-1.19 (d, 6 H,
CH(CH3)2), 3.17 (m, 1 H, CH(CH3)2), 5.33 (s, 2 H, 2/4-NH2), 5.90
(s, 2 H, 2/4-NH2), 6.39 (s, 1 H, 6-CH), 10.35 (s, 1 H, 7-NH). Anal.
calcd for (C9H13N5) C, H, N.
Ethy l4-[2,4-Diamino-5-isopropyl-7H-pyrrolo[2,3-d]pyrimi-
din-6-yl)sulfanyl]-benzoate (39). To a suspension of 38 (1.0 g,
5.2 mmol) in a mixture of EtOH/H2O (2:1, 75 mL) was added
diethyl 4,4′-dithiobis(benzoate) (2.2 g, 6 mmol) and the suspension
was heated to 100-110 °C, then I2 (3 g, 12 mmol) was added and
the reaction was monitored for completion (3 h). To this solution
was added excess sodium thiosulfate and the solution was evapo-
rated to dryness under reduced pressure and the resulting residue
was washed with water and air-dried. This residue was then
dissolved in MeOH (100 mL) and to this was added silica gel (15
g), and the resulting suspension was evaporated to dryness under
reduced pressure to afford a dry silica gel plug, which was loaded
on top of a wet (CHCl3) silica gel column and eluted first with
CHCl3 and then with a gradient of 1-5% MeOH in CHCl3.
Fractions containing the desired spot (TLC) were pooled and
evaporated to dryness to afford 890 mg (45%) of 39 as a white
solid: mp ) 263-264.7 °C; TLC Rf ) 0.61 (CHCl3/MeOH, 5:1).
1H NMR (DMSO-d6): δ 1.23-1.29 (m, 9 H, CH2CH3 and
CH(CH3)2), 3.33-3.41 (m, 1 H, CH(CH3)2), 4.23-4.29 (q, 2 H,
CH2CH3), 5.65 (s, 2 H, 2/4-NH2), 6.15 (s, 2 H, 2/4-NH2), 7.05-7.08
(d, 2 H, C6H4), 7.82-7.84 (d, 2 H, C6H4), 11.03 (s, 1 H, 7-NH).
Anal. calcd for (C18H21N5O2S·0.4H2O) C, H, N, S.

4598 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Gangjee et al.
and evaporated to dryness. The resulting residue was washed with
MeOH, filtered, and dried to yield 385 mg (67%) of 11: mp
238-240 °C; TLC Rf ) 0.51 (CHCl3/MeOH, 5:1, with 2 drops of
2,4-Diamino-5-propyl-6-(2′-napthylsulfanyl)-7H-pyrrolo[2,3-
d]pyrimidine (18). Compound 18 was synthesized as described
for 11 using 2-naphthylthiol (480 mg, 3.00 mmol) and 31 (300
mg, 1.57 mmol): yield 70%; mp > 250 °C (dec); TLC Rf ) 0.56
1
NH4OH). H NMR (DMSO-d6): δ 0.76 (t, 3 H, 5-CH2CH2CH3),
1
1.15 (m, 2 H, 5-CH2CH2CH3), 2.64 (t, 2 H, 5-CH2CH2CH3), 5.54
(s, 2 H, 2/4-NH2), 6.08 (s, 2 H, 2/4-NH2), 7.32-7.35 (m, 1 H,
C6H3), 7.48-7.51 (d, 2 H, C6H3), 10.93 (s, 1 H, 7-NH). Anal. calcd
for (C15H15N5Cl2S·0.17CHCl3) C, H, N, Cl, S.
(CHCl3/MeOH, 5:1, with 2 drops of NH4OH). H NMR (DMSO-
d6): δ 0.80-0.85 (t, 3 H, 5-CH2CH2CH3), 1.42-1.49 (m, 2 H,
5-CH2CH2CH3), 2.74-2.79 (t, 2 H, 5-CH2CH2CH3), 5.60 (s, 2 H,
2/4-NH2), 6.18 (s, 2 H, 2/4-NH2), 7.17-7.20 (d, 1 H, C10H7),
7.40-7.50 (m, 3 H, C10H7), 7.73-7.76 (d, 1 H, C10H7), 7.81 (s, 1
H, C10H7), 7.84-7.85 (d, 1 H, C10H7), 11.06 (s, 1 H, 7-NH). Anal.
calcd for (C19H19N5S·0.5H2O) C, H, N, S.
2,4-Diamino-5-propyl-6-(2′,6′-dimethylphenylsulfanyl)-7H-
pyrrolo[2,3-d] pyrimidine (12). Compound 12 was synthesized
as described for 11 using 2,6-dimethylphenylthiol (420 mg, 3.00
mmol) and 31 (300 mg, 1.57 mmol): yield 52%; mp 227-230 °C;
2,4-Diamino-5-isopropyl-6-(2′,6′-dichlorophenylsulfanyl)-7H-
pyrrolo[2,3-d]pyrimidine (19). To a solution of 38 (300 mg, 1.57
mmol) in a mixture of EtOH/water (2:1, 30 mL) was added 2,6-
dichlorophenylthiol (540 mg, 3.00 mmol) and the reaction mixture
was heated to 100-110 °C, then I2 (750 mg, 3.0 mmol) was added
and the heating continued with stirring for a total of 2 h. To this
mixture was added an excess of sodium thiosulfate and the reaction
mixture concentrated under reduced pressure. To the resulting
residue was added silica gel (10 g) and MeOH (50 mL) and the
solution evaporated to dryness under reduced pressure to afford a
dry silica gel plug, which was loaded on top of a wet (CHCl3)
silica gel column and eluted with a gradient of 1-3% MeOH in
CHCl3. Fractions containing the desired spot (TLC) were pooled
and evaporated to dryness. The resulting residue was washed with
MeOH, filtered, and dried to yield 70 mg (12%) of 19: mp 230-231
°C; TLC Rf ) 0.60 (CHCl3/MeOH, 5:1, with 2 drops of NH4OH).
1H NMR (DMSO-d6): δ 1.10-1.12 (d, 6 H, CH(CH3)2), 5.54 (s, 2
H, 2/4-NH2), 5.87 (s, 2 H, 2/4-NH2), 7.31-7.33 (m, 1 H, C6H3),
7.46-7.49 (d, 2 H, C6H3), 10.90 (s, 1 H, 7-NH). Anal. calcd for
(C15H15N5Cl2S·0.3H2O) C, H, N, Cl, S.
2,4-Diamino-5-isopropyl-6-(2′,6′-dimethylphenylsulfanyl)-7H-
pyrrolo[2,3-d] pyrimidine (20). Compound 20 was synthesized
as described for 19 using 2,6-dimethylphenylthiol (420 mg, 3.00
mmol) and 38 (300 mg, 1.57 mmol): yield 22%; mp 248-248.3
°C; TLC Rf ) 0.63 (CHCl3/MeOH, 5:1, with 2 drops of NH4OH).
1H NMR (DMSO-d6): δ 1.11-1.14 (d, 6 H, 5-CH(CH3)2), 2.30 (s,
6 H, 2′,6′-diCH3), 5.46 (s, 2 H, 2/4-NH2), 5.79 (s, 2 H, 2/4-NH2),
7.08 (m, 3 H, C6H3), 10.66 (s, 1 H, 7-NH). Anal. calcd for
(C17H21N5S·0.2H2O) C, H, N, S.
2,4-Diamino-5-isopropyl-6-(phenylsulfanyl)-7H-pyrrolo[2,3-
d]pyrimidine (21). Compound 21 was synthesized as described
for 19 using phenylthiol (330 mg, 3.00 mmol) and 38 (300 mg,
1.57 mmol) except that the compound was washed with hexane
and dried. Yield: 45%; mp 228.5-229 °C; TLC Rf ) 0.53 (CHCl3/
MeOH, 5:1, with 2 drops of NH4OH). 1H NMR (DMSO-d6): δ
1.25-1.27 (d, 6 H, 5-CH(CH3)2), 5.39 (bs, 2 H, 2/4-NH2), 5.89
(bs, 2 H, 2/4-NH2), 6.97-7.29 (m, 5 H, C6H5), 10.97 (s, 1 H, 7-NH).
Anal. calcd for (C15H17N5S·0.1C6H14) C, H, N, S.
2,4-Diamino-5-isopropyl-6-(4′-methoxyphenylsulfanyl)-7H-
pyrrolo[2,3-d]pyrimidine (22). Compound 22 was synthesized as
described for 19 using 4-methoxyphenylthiol (280 mg, 2.00 mmol)
and 38 (200 mg, 1.04 mmol): yield 40%; mp 288-289 °C; TLC
Rf ) 0.58 (CHCl3/MeOH, 5:1, with 2 drops of NH4OH). 1H NMR
(DMSO-d6): δ 1.26-1.28 (d, 6 H, 5-CH(CH3)2), 3.41-3.48 (m, 1
H, 5-CH(CH3)2), 3.70 (s, 3 H, 4-OCH3), 5.56 (bs, 2 H, 2/4-NH2),
6.02 (bs, 2 H, 2/4-NH2), 6.86-6.89 (d, 2 H, C6H4), 7.02-7.04 (d,
2 H, C6H4), 10.93 (s, 1 H, 7-NH). Anal. calcd for (C16H19N5-
SO·0.1H2O) C, H, N, S.
1
TLC Rf ) 0.55 (CHCl3/MeOH, 5:1, with 2 drops of NH4OH). H
NMR (DMSO-d6): δ 0.74-0.78 (t, 3 H, 5-CH2CH2CH3), 1.16-1.25
(m, 2 H, 5-CH2CH2CH3), 2.19-2.25 (t, 2 H, 5-CH2CH2CH3), 2.35
(s, 6 H, 2′,6′-diCH3), 5.46 (s, 2 H, 2/4-NH2), 6.00 (s, 2 H, 2/4-
NH2), 7.07 (m, 3 H, C6H3), 10.75 (s, 1 H, 7-NH). Anal. calcd for
(C17H21N5S·0.6CH3OH) C, H, N, S.
2,4-Diamino-5-propyl-6-(phenylsulfanyl)-7H-pyrrolo[2,3-d]py-
rimidine (13). Compound 13 was synthesized as described for 11
using phenylthiol (280 mg, 2.00 mmol) and 31 (200 mg, 1.04
mmol): yield 65%; mp 252.2-252.7 °C; TLC Rf ) 0.53 (CHCl3/
MeOH, 5:1, with 2 drops of NH4OH). 1H NMR (DMSO-d6): δ
0.81-0.86 (t, 3 H, 5-CH2CH2CH3), 1.43-1.45 (m, 2 H,
5-CH2CH2CH3), 2.70-2.75 (t, 2 H, 5-CH2CH2CH3), 5.59 (s, 2 H,
2/4-NH2), 6.15 (s, 2 H, 2/4-NH2), 7.00-7.02 (d, 2 H, C6H5),
7.12-7.14 (m, 1 H, C6H5), 7.24-7.29 (m, 2 H, C6H5), 10.98 (s, 1
H, 7-NH). Anal. calcd for (C15H17N5S·0.2H2O) C, H, N, S.
2,4-Diamino-5-propyl-6-(4′-methoxyphenylsulfanyl)-7H-pyr-
rolo[2,3-d]pyrimidine (14). Compound 14 was synthesized as
described for 11 using 4-methoxyphenylthiol (280 mg, 2.00 mmol)
and 31 (200 mg, 1.04 mmol): yield 50%; mp 247.9-248.2 °C;
1
TLC Rf ) 0.63 (CHCl3/MeOH, 5:1, with 2 drops of NH4OH). H
NMR (DMSO-d6): δ 0.82-0.87 (t, 3 H, 5-CH2CH2CH3), 1.41 (m,
2 H, 5-CH2CH2CH3), 2.74 (t, 2 H, 5-CH2CH2CH3), 3.69 (s, 3 H,
4′-OCH3), 5.55 (s, 2 H, 2/4-NH2), 6.11 (s, 2 H, 2/4-NH2), 6.85-6.88
(d, 2 H, C6H4), 7.04-7.07 (d, 2 H, C6H4), 10.95 (s, 1 H, 7-NH).
Anal. calcd for (C16H19N5OS·0.5H2O) C, H, N, S.
2,4-Diamino-5-propyl-6-(2′,5′-dimethoxyphenylsulfanyl)-7H-
pyrrolo[2,3-d]pyrimidine (15). Compound 15 was synthesized as
described for 11 using 2,5-dimethoxyphenylthiol (525 mg, 3.00
mmol) and 31 (300 mg, 1.57 mmol): yield 45%; mp 217-218 °C;
1
TLC Rf ) 0.56 (CHCl3/MeOH, 5:1, with 2 drops of NH4OH). H
NMR (DMSO-d6): δ 0.81-0.86 (t, 3 H, 5-CH2CH2CH3), 1.40-1.47
(m, 2 H, 5-CH2CH2CH3), 2.66-2.71 (t, 2 H, 5-CH2CH2CH3), 3.53
(s, 3 H, 2′/5′-OCH3), 3.80 (s, 3 H, 2′/5′-OCH3), 5.59 (s, 2 H, 2/4-
NH2), 6.17 (s, 2 H, 2/4-NH2), 5.96 (s, 1 H, C6H3), 6.64-6.67 (d,
1 H, C6H3), 6.89-6.92 (d, 1 H, C6H3), 10.91 (s, 1 H, 7-NH). Anal.
calcd for (C17H21N5O2S) C, H, N, S.
2,4-Diamino-5-propyl-6-(3′,4′-dimethoxyphenylsulfanyl)-7H-
pyrrolo[2,3-d]pyrimidine (16). Compound 16 was synthesized as
described for 11 using 3,4-dimethoxyphenylthiol (350 mg, 2.00 mmol)
and 31 (200 mg, 1.04 mmol): yield 65%; mp >230 °C (dec); TLC Rf
) 0.53 (CHCl3/MeOH, 5:1, with 2 drops of NH4OH). 1H NMR
(DMSO-d6): δ 0.83-0.88 (t, 3 H, 5-CH2CH2CH3), 1.43-1.45 (m, 2
H, 5-CH2CH2CH3), 2.73-2.75 (t, 2 H, 5-CH2CH2CH3), 3.68 (s, 6 H,
3′,4′-diOCH3), 5.59 (s, 2 H, 2/4-NH2), 6.15 (s, 2 H, 2/4-NH2),
6.59-6.62 (d, 1 H, C6H3), 6.82-6.88 (m, 2 H, C6H3), 10.98 (s, 1 H,
7-NH). Anal. calcd for (C17H21N5O2S·0.2CHCl3) C, H, N, S.
2,4-Diamino-5-propyl-6-(1′-napthylsulfanyl)-7H-pyrrolo[2,3-
d]pyrimidine (17). Compound 17 was synthesized as described
for 11 using 1-naphthylthiol (320 mg, 2.00 mmol) and 31 (200
mg, 1.04 mmol): yield 58%; mp > 255 °C dec; TLC Rf ) 0.63
2,4-Diamino-5-isopropyl-6-(2′,5′-dimethoxyphenylsulfanyl)-
7H-pyrrolo[2,3-d]pyrimidine (23). Compound 23 was synthesized
as described for 19 using 2,5-dimethoxyphenylthiol (800 mg, 4.00
mmol) and 38 (350 mg, 2.00 mmol): yield 53%; mp 243-244 °C;
1
(CHCl3/MeOH, 5:1, with 2 drops of NH4OH). H NMR (DMSO-
1
TLC Rf ) 0.60 (CHCl3/MeOH, 5:1, with 2 drops of NH4OH). H
d6): δ 0.80-0.84 (t, 3 H, 5-CH2CH2CH3), 1.43-1.45 (m, 2 H,
5-CH2CH2CH3), 2.73-2.75 (t, 2 H, 5-CH2CH2CH3), 5.62 (s, 2 H,
2/4-NH2), 6.19 (s, 2 H, 2/4-NH2), 6.86 (d, 1 H, C10H7), 7.37 (t, 1
H, C10H7), 7.58-7.61 (m, 3 H, C10H7), 7.70 (d, 1 H, C10H7), 7.94
(d, 1 H, C10H7), 11.05 (s, 1 H, 7-NH). Anal. calcd for (C19H19N5S)
C, H, N, S.
NMR (DMSO-d6): δ 1.24-1.26 (d, 6 H, 5-CH(CH3)2), 3.53 (s, 3
H, 2′/5′-OCH3), 3.80 (s, 3 H, 2′/5′-OCH3), 5.59 (bs, 2 H, 2/4-NH2),
5.93 (s, 1 H, C6H3), 6.07 (bs, 2 H, 2/4-NH2), 6.63-6.66 (d, 1 H,
C6H3), 6.89-6.92 (d, 1 H, C6H3), 10.87 (s, 1 H, 7-NH). Anal. calcd
for (C17H21N5SO2) C, H, N, S.

Pyrrolo[2,3-d]pyrimidine Containing Antifolates
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Queener, S. F. Design and Synthesis of Classical and Nonclassical
6-Arylthio-2,4-diamino-5-ethylpyrrolo[2,3-d]pyrimidines as Anti-
folates. J. Med. Chem. 2007, 50, 3046–3053.
(15) Gangjee, A.; Lin, X.; Queener, S. F. Design, Synthesis, and Biological
Evaluation of 2,4-Diamino-5-methyl-6-substituted-pyrrolo[2,3-d]py-
rimidines as Dihydrofolate Reductase Inhibitors. J. Med. Chem. 2004,
47, 3689–3692.
2,4-Diamino-5-isopropyl-6-(3′,4′-dimethoxyphenylsulfanyl)-
7H-pyrrolo[2,3-d]pyrimidine (24). Compound 24 was synthesized
as described for 19 using 3,4-dimethoxyphenylthiol (520 mg, 3.00
mmol) and 38 (300 mg, 1.57 mmol): yield 58%; mp 293-293.5
°C; TLC Rf ) 0.58 (CHCl3/MeOH, 5:1, with 2 drops of NH4OH).
1H NMR (DMSO-d6): δ 1.27-1.29 (d, 6 H, 5-CH(CH3)2), 3.67 (s,
3 H, 3′/4′-OCH3), 3.69 (s, 3 H, 3′/4′-OCH3), 5.56 (bs, 2 H, 2/4-
NH2), 6.01 (bs, 2 H, 2/4-NH2), 6.56-6.59 (d, 1 H, C6H3), 6.79 (s,
1 H, C6H3), 6.87-6.89 (d, 1 H, C6H3), 10.93 (s, 1 H, 7-NH). Anal.
calcd for (C17H21N5SO2 ·0.5H2O) C, H, N, S.
2,4-Diamino-5-isopropyl-6-(1′-napthylsulfanyl)-7H-pyrrolo[2,3-
d]pyrimidine (25). Compound 25 was synthesized as described
for 19 using 1-naphthylthiol (320 mg, 2.00 mmol) and 38 (200
mg, 1.04 mmol): yield 45%; mp 267-267.5 °C; TLC Rf ) 0.60
1
(CHCl3/MeOH, 5:1, with 2 drops of NH4OH). H NMR (DMSO-
d6): δ 1.26-1.28 (d, 6 H, 5-CH(CH3)2), 5.63 (s, 2 H, 2/4-NH2),
6.11 (s, 2 H, 2/4-NH2), 6.76-6.78 (d, 1 H, C10H7), 7.37 (t, 1 H,
C10H7), 7.59-7.72 (m, 3 H, C10H7), 7.94 (d, 1 H, C10H7), 8.2 (d, 1
H, C10H7), 11.02 (s, 1 H, 7-NH). Anal. calcd for (C19H19N5S) C,
H, N, S.
2,4-Diamino-5-isopropyl-6-(2′-napthylsulfanyl)-7H-pyrrolo[2,3-
d]pyrimidine (26). Compound 26 was synthesized as described
for 19 using 2-naphthylthiol (640 mg, 4.00 mmol) and 38 (350
mg, 1.83 mmol): yield 40%; mp 247-247.5 °C; TLC Rf ) 0.58
1
(CHCl3/MeOH, 5:1, with 2 drops of NH4OH). H NMR (DMSO-
d6): δ 1.27-1.29 (d, 6 H, 5-CH(CH3)2), 5.63 (s, 2 H, 2/4-NH2),
6.12 (s, 2 H, 2/4-NH2), 7.15-7.18 (d, 1 H, C10H7), 7.44-7.47 (m,
3 H, C10H7), 7.74-7.76 (d, 1 H, C10H7), 7.82-7.85 (d, 2 H, C10H7),
11.05 (s, 1 H, 7-NH). Anal. calcd for (C19H19N5S) C, H, N, S.
2,4-Diamino-5-isopropyl-6-(3′,4′-dichlorophenylsulfanyl)-7H-
pyrrolo[2,3-d]pyrimidine (27). Compound 27 was synthesized as
described for 19 using 3,4-dichlorophenylthiol (540 mg, 3.00 mmol)
and 38 (300 mg, 1.57 mmol): yield 37%; mp 244-244.5 °C; TLC
Rf ) 0.58 (CHCl3/MeOH, 5:1, with 2 drops of NH4OH). 1H NMR
(DMSO-d6): δ 1.24-1.27 (d, 6 H, 5-CH(CH3)2), 5.67 (bs, 2 H,
2/4-NH2), 6.17 (bs, 2 H, 2/4-NH2), 6.96 (s, 2 H, C6H3), 7.37 (s, 1
H, C6H3), 11.04 (s, 1 H, 7-NH). Anal. calcd for (C15H15N5SCl2) C,
H, N, S, Cl.
(16) Cody, V.; Wojtczak, A.; Kalman, T. I.; Friesheim, J. H.; Blakley, R. L.
Conformational Analysis of Human Dihydrofolate Reductase Inhibitor
Complexes: Crystal Structure Determination of Wild Type and F31
Mutant Binary and Ternary Inhibitor Complexes. AdV. Exp. Med. Biol.
1993, 338, 481–486.
(17) Champness, J. N.; Achari, A.; Ballantine, S. P.; Bryant, P. K.; Delves,
C. J.; Stammers, D. K. The Structure of Pneumocystis carinii
Dihydrofolate Reductase to 1.9 Å Resolution. Structure 1994, 2, 915–
924.
Acknowledgment. This work was supported in part by the
National Institutes of Health, grants AI41743 (A.G.); AI44661
(A.G.), CA89300 (A.G.), and AI47759 (A.G.) from the National
Institute of Allergy and Infectious Diseases.
Supporting Information Available: Elemental analysis. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(18) Roos, D. S. Primary Structure of the Dihydrofolate Reductase-
Thymidylate Synthase Gene from Toxoplasma gondii. J. Biol. Chem.
1993, 268, 6269–6280.
(19) Ginkel, S. Z.; Dooley, T. P.; Suling, W. J.; Barrow, W. W.
Identification and Cloning of the Mycobacterium aVium FolA Gene,
Required for Dihydrofolate Reductase Activity. FEMS Microbiol. Lett.
1997, 156, 69–78.
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