Design and synthesis of classical and nonclassical 6-arylthio-2,4-diamino- 5-ethylpyrrolo[2,3-d]pyrimidines as antifolates

Article abstract of DOI:10.1021/jm070165j

The classical antifolate N-{4-[(2,4-diamino-5-ethyl-7H-pyrrolo[2,3-d] pyrimidin-6-yl)sulfanyl]benzoyl}-L-glutamic acid (2) and 15 nonclassical analogues (3-17) were synthesized as potential dihydrofolate reductase (DHFR) inhibitors and as antitumor agents

Full text of DOI:10.1021/jm070165j

3046
J. Med. Chem. 2007, 50, 3046-3053
Design and Synthesis of Classical and Nonclassical
6-Arylthio-2,4-diamino-5-ethylpyrrolo[2,3-d]pyrimidines as Antifolates
Aleem Gangjee,*,† Yibin Zeng,†,‡ Tina Talreja,† John J. McGuire,§ Roy L. Kisliuk,| and Sherry F. Queener
DiVision of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne UniVersity, Pittsburgh, PennsylVania 15282,
Grace Cancer Drug Center, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York 14263, Department of Biochemistry,
Tufts UniVersity School of Medicine, Boston, Massachusetts 02111, and Department of Pharmacology and Toxicology, School of Medicine,
Indiana UniVersity, 635 Barnhill DriVe, Indianapolis, Indiana 46202
ReceiVed February 13, 2007
The classical antifolate N-{4-[(2,4-diamino-5-ethyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)sulfanyl]benzoyl}-L-
glutamic acid (2) and 15 nonclassical analogues (3-17) were synthesized as potential dihydrofolate reductase
(DHFR) inhibitors and as antitumor agents. 5-Ethyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (20) served
as the key intermediate to which various aryl thiols and a heteroaryl thiol were appended at the 6-position
via an oxidative addition reaction. The classical analogue 2 was synthesized by coupling the benzoic acid
derivative 18 with diethyl L-glutamate followed by saponification. The classical compound 2 was an excellent
inhibitor of human DHFR (IC50 ) 66 nM) as well as a two digit nanomolar (<100 nM) inhibitor of the
growth of several tumor cells in culture. Some of the nonclassical analogues were potent and selective
inhibitors of DHFR from two pathogens (Toxoplasma gondii and Mycobacterium aVium) that cause
opportunistic infections in patients with compromised immune systems.
Introduction
interaction with human DHFR.7,8 Molecular modeling of 1a also
suggested that homologation of the C5-methyl to an ethyl could
further enhance the hydrophobic interaction with human DHFR
at Val115 and perhaps the antitumor activity as well. Figure 3
shows the superimposition of the homologated 5-ethyl classical
compound 2 (Figure 1) onto MTX (not shown) in the X-ray
crystal structure of MTX in human DHFR (PDB 1U72). The
van der Waals distance of the methyl moiety of the 5-ethyl group
from Val115 is about 3.5 Å, which is much closer than that for
the 5-methyl group of 1a. Thus, compound 2, the C5-ethyl
homologue of 1a, was designed and synthesized to explore this
possibility.
Folate metabolism is an attractive target for chemotherapy
as a result of its crucial role in the biosynthesis of nucleic acid
precursors.1 Inhibition of folate-dependent enzymes in cancer
cells, in microbial cells, and protozoan cells provides compounds
that have found clinical utility as antitumor, antimicrobial, and
antiprotozoal agents.2,3
As part of a continuing effort to develop novel classical
antifolates as antitumor agents, Gangjee et al.4 reported the
synthesis of N-{4-[(2,4-diamino-5-methyl-7H-pyrrolo[2,3-d]-
pyrimidin-6-yl)sulfanyl]benzoyl}-L-glutamic acid (1a; Figure 1)
as a potent dual inhibitor of dihydrofolate reductase (DHFRa)
A disadvantage of classical antifolates as antitumor agents
is that they require an active transport mechanism to enter cells,
which, when impaired, causes tumor resistance.9 In addition,
cells that lack these transport mechanisms, including many
bacterial and protozoan cells, are not susceptible to the action
of classical antifolates. In an attempt to overcome these potential
drawbacks, nonclassical lipophilic antifolates have been devel-
oped as antitumor agents that do not require the folate transport
system(s) but enter cells via diffusion.10
and thymidylate synthase (TS) with IC50 values in the 10-7
M
range. Compound 1a also demonstrated potent inhibitory activity
against the growth of several human tumor cell lines in the
National Cancer Institute (NCI)5 preclinical in vitro screen, with
GI50 values of 10-7 to 10-8 M or lower. The DHFR inhibitory
potency and tumor cell inhibitory activities of 1a were in part
attributed to its C5-methyl group. Molecular modeling (Figure
2) using Sybyl 7.06 indicated that the C5-substituent of 6-5
ring-fused bicyclic pyrrolo[2,3-d]pyrimidines (red) could mimic
the C5-substituent of 6-6 ring-fused bicyclic pteridines or
pyrido[2,3-d]pyrimidines (green) such as 5-methyl-5-deaza
methotrexate or 5-methyl-5-deaza aminopterin in which the
5-methyl group was reported to have a conducive hydrophobic
An additional aspect of nonclassical antifolates involves their
utility in opportunistic infections. The principal causes of death
in patients with acquired immunodeficiency syndrome (AIDS)
are opportunistic infections caused by Pneumocystis jiroVecii
(previously Pneumocystis carinii, pc) and Toxoplasma gondii
(tg).11 Current therapeutic regimens include the use of selective
but weak inhibitors of protozoal DHFR such as trimethoprim
(TMP) and pyrimethamine (Figure 1), in combination with
sulfonamides to enhance potency.11 Toxicity of the sulfa drug
component of these combinations is often a serious problem.
In addition, the potent but toxic nonclassical antifolates trime-
trexate (TMQ) and piritrexim (PTX; Figure 1), coadministered
with leucovorin for host rescue, are also used.11 Serious toxicities
associated with the use of TMQ and PTX often force the
cessation of treatment.11 Mycobacterium aVium (M. aVium)
complex or MAC infection also adversely affects the quality
of life of AIDS patients.11 Thus, it is of considerable interest to
* To whom correspondence should be addressed. Tel.: (412) 396-6070.
Fax: (412) 396-5593. E-mail: gangjee@duq.edu.
† Duquesne University.
‡ Current address: ChemoCentryx, 850 Maude Avenue, Mountain View,
California 94043.
§ Roswell Park Cancer Institute.
| Tufts University School of Medicine.
Indiana University.
a Abbreviations: DHFR, dihydrofolate reductase; TS, thymidylate syn-
thase; NCI, National Cancer Institute; AIDS, acquired immunodeficiency
syndrome; pc, Pneumocystis carinii; tg, Toxoplasma gondii; TMP, trime-
thoprim; TMQ, trimetrexate; PTX, piritrexim; M. aVium, Mycobacterium
aVium; ec, Escherichia coli; rh, recombinant human; SAR, structure-activity
relationship; FPGS, folylpolyglutamate synthetase; TdR, thymidine; Hx,
hypoxanthine.
10.1021/jm070165j CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/07/2007

6-Arylthio-2,4-diamino-5-ethylpyrrolo[2,3-d]pyrimidine
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13 3047
Figure 1.
(pcDHFR), Val151 (tgDHFR), and Ile102 (maDHFR). The
5-methyl group also affects the conformations of the 6-side chain
in these molecules and could contribute to the potency and/or
selectivity. Molecular modeling (Sybyl 7.0)6 suggested that an
extension of the 5-methyl group to an ethyl group might enhance
the potency and selectivity against some pathogenic DHFR.
Thus, a series of nonclassical 5-ethyl-6-[(substituted-phenyl)-
sulfanyl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines (3-17) were
also synthesized.
Figure 2. Superimposition of a 6-5 pyrrolo[2,3-d]pyrimidine ring
system (in red) on to a 6-6 pyrido[2,3-d]pyrimidine ring system (in
green) illustrating that the 5-Me moiety of the 6-5 system mimics the
5-Me of the 6-6 system.
Chemistry
Syntheses of 3-18 (Scheme 1) were achieved by oxidative
thiolation of intermediate 20 with the appropriate thiophenol
as previously reported.13 Intermediate 20 was in turn obtained
from the reaction of malononitrile with 1-hydroxy-2-butanone
(21) via a two-step procedure reported by Taylor et al.14
Coupling of 18 with diethyl-L-glutamate using isobutyl chlo-
roformate as the activating agent15 afforded the diester 19. Final
saponification of the diester gave the desired diacid 2 in 70%
over two steps.
incorporate selectivity and potency into a single nonclassical
antifolate that can be used alone to treat these infections.
Gangjee et al.12 recently described the design and synthesis
of several nonclassical 5-methyl-6-[(substituted-phenyl)sulfa-
nyl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines (1b-i) as DHFR
inhibitors against opportunistic pathogens that infect AIDS
patients. Some of these compounds were potent and selective
against DHFR from both tg and M. aVium compared to
mammalian DHFR. Compound 1b with a 1-naphthyl substituent
is 16-fold more potent and equally selective against tgDHFR
as the clinically used TMP. The enhanced selectivity and
potency of these analogues were in part attributed to the
hydrophobic interaction between the 6-hydrophobic phenyl ring
with Phe69 in pcDHFR, Phe91 in tgDHFR, and Val58 in
maDHFR (vs the lack of interaction with hydrophilic Asn64 in
hDHFR in the same region). Hydrophobic interactions at this
region of the molecule with DHFR is complemented with the
hydrophobic interaction of the 5-methyl moiety with Ile123
Biological Evaluation and Discussion
DHFR Inhibition. Compounds 2-17 were evaluated as
inhibitors of Escherichia coli (ec) and recombinant human (rh)
DHFR.4 The inhibitory potency (IC50) values are listed in Table
1 and compared with MTX, TMP, and the previously reported
values for 1a.4 The most potent inhibitor against both ecDHFR
and hDHFR in the C5-ethyl series was the classical analogue
2. Against human DHFR, 2 was 3-fold more potent than the
5-methyl substituted parent compound 1a, thus supporting our
hypothesis that homologation of the C5-methyl of 1a to an ethyl
would enhance its interaction with DHFR.

3048 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13
Gangjee et al.
Figure 3. Stereoview. Compound 2 superimposed on to methotrexate (not shown) in the X-ray crystal structure of MTX and human DHFR. The
van der Waals surface of the methyl moiety (in purple) of the 5-ethyl group interacts with the van der Waals surface of the methyl and methylene
carbon of Val115 (in yellow) in hDHFR. This interaction affords better hDHFR inhibitory activity for 2 than the 5-methyl moiety in 1a. The
hDHFR Phe31 and Glu30 are also labeled.
Table 1. Inhibitory Concentrations (IC50 in µM) and Selectivity Ratios
for 2-17 Against Isolated Human and ecDHFRa
Scheme 1
human
DHFRc
selectivity ratios
human DHFR/ecDHFR
cmpd
ecDHFRb
1ad
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
0.016
0.002
0.15
0.15
0.09
0.14
0.16
nde
0.28
0.14
nde
0.32
0.87
0.32
0.17
0.16
3.2
0.21
0.066
2.9
6.0
5.8
2.7
>31 (30%)
>31 (36%)
2.8
1.4
>28 (30%)
2.9
5.8
>32 (35%)
18
13
33
20
40
64.4
19.3
>194
nde
10
10
nde
9.1
6.7
>100
106
8.75
10
1.4
32
methotrexate
TMP
0.0066
0.02
0.022
680
3.33
34 000
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. R. L. Blakely, St. Jude Children’s Hospital, Memphis TN.
c Kindly provided by Dr. J. H. Friesheim, Medical College of Ohio, Toledo,
OH. d Data derived from ref 4. e nd ) not determined.
In the nonclassical analogues, compound 5 with a 4-nitro-
phenyl substitution was the most potent compound in this series
against ecDHFR, but it was 45-fold less potent than the classical
2. A number of the nonclassical compounds in this series showed
good selectivity for ecDHFR as compared to human DHFR.
Compounds 5, 7, 14, and 15 were 64- to 194-fold more selective
for ecDHFR than human DHFR. Thus the 6-substituted phenyl
analogs were reasonably selective for bacterial DHFR. The
structure-activity relationship (SAR) among these analogues
suggests that selectivity for ecDHFR over hDHFR is indepen-
dent of the electronic nature of the substituent but that mono
meta-substitution is most favorable to ecDHFR selectivity.
Compounds 3-17 were also evaluated as inhibitors against
pcDHFR, tgDHFR, maDHFR, and rlDHFR (which served as
the mammalian standard) and compared with TMP, TMQ, and
the previously reported C5-methyl analogues 1b-i where
applicable. The results (IC50) are reported in Table 2.
None of the analogues were potent or selective inhibitors of
pcDHFR.
For tgDHFR in the 6-phenyl substituted analogues 5-15 and
17, compounds 13 and 15 were most potent followed by 5, 7,
and 10. Compounds with a single 4-halogen (6 and 8), 3,4-
diCl (9), or 2,6-disubstituted compounds (11 and 17) were
comparatively poor inhibitors. Among the 6-bicyclic compounds
3, 4, and 16, only the quinoline 16 has activity comparable to
13 and 15. The naphthyl analogues were 10-fold less potent.
Selectivity for tgDHFR was the highest for 13 with 14, 15, and
16 in the same range. Thus, 13 with a 2,5-diOMePh was the
most potent and selective. For maDHFR, the most potent
analogues were again 13 and 16, with 10 a close third. These
were followed by 5, 7, 8, 14, and 15.
With the exception of 7, the trends for potency against
maDHFR was quite similar to tgDHFR. Selectivity for maDHFR
was dependent on the substitution with 13 and 14 being the

6-Arylthio-2,4-diamino-5-ethylpyrrolo[2,3-d]pyrimidine
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13 3049
Table 2. Inhibitory Concentrations (IC50) in µM and Selectivity Ratios
for 3-17 against P. carinii (pc), T. gondii (tg), M. aVium (ma), and Rat
Liver (rl) DHFRa
Table 3. Growth Inhibition of Parental CCRF-CEM Human Leukemia
Cells and Sub-lines with Single, Defined Mechanisms of MTX
Resistance during Continuous (0-120 h) Exposure to MTX, 1a, or 2.
Values Presented Are Average ( Range for n ) 2 and Average ( S.D.
for n > 2.
inhibition concn
(IC50 mM)
selectivity ratio
(IC50/IC50
)
EC50, nM
cmpd
pcDHFR
tgDHFR maDHFR rlDHFR rl/pc rl/tg rl/ma
R1a
(v DHFR)
R2b
(V uptake)
R30dmc
(V Glun)
3
1b
4
5
6
1c
7
1d
8
1h
9
10
11
1g
12
1i
13
14
15
16
17
18.4(20%)b
37.3
13.88
8.68
26.6
42.9
3.15
0.16
5.12
0.32
19.8
0.70
40.1
4.57
8.62
3.2
nd
12.7
28.5
1.7
10.0
0.9
6.9
12.6
2.1
3.6
5.5
3.0
5.3
2.0
6.5
1.3
8.6
1.4
10.8
15.1
4.8
7.8
5.8
4.8
3.3
drug
CCRF-CEM
0.1
0.6
0.4
0.4
1.1
1.6
1.1
0.5
9.1
3.9
0.2
0.2
6.52
0.37
7.40
4.40
0.42
4.00
0.70
19.0
16.9
0.35
12.0
0.69
3.07
0.1
0.10
0.77
0.88
0.11
MTX
13.3 ( 1.5
(n ) 4)
190 ( 10
(n ) 2)
68 ( 11
(n ) 2)
410 ( 130
(n ) 4)
1950 ( 150
(n ) 2)
15.7 ( 0.9
(n ) 3)
515 ( 15
(n ) 2)
200 ( 25
(n ) 2)
11.6
10.1
47.7
6.33
19
5.48
110
81.6
1.17
4.0
31.3
31.8
7.8
4.05
31
5.6
3.55
10.1
1ad
2
8300 ( 800
(n ) 2)
7700 ( 300
(n ) 2)
6.94
0.50
8.93
1.54
20.0
27.6
0.22
3.80
2.69
4.62
0.17
0.12
1.09
0.18
0.16
7.10
0.010
2.8
4.02
16.7
11.9
5700 ( 200
(n ) 2)
6700 ( 300
(n ) 2)
20(14%)b
20.7
a CCRF-CEM subline resistant to MTX solely as a result of a 20-fold
increase in wild-type DHFR protein and activity.16 b CCRF-CEM subline
resistant as a result of decreased uptake of MTX.17 c CCRF-CEM subline
resistant to MTX solely as a result of decreased polyglutamylation; this
cell line has 1% of the FPGS specific activity (measured with MTX as the
folate substrate) of parental CCRF-CEM.18 d Data from ref 4.
7.19
20.6
112
53
14.6
6.04
28.3
21.8
1.1
0.33
0.28 11.6
45.4
10.4
78
40.5
40.5
6.36
32.3
0.55
0.6
0.5
0.7
1.1
0.3
0.4
0.3
6.9
45.9
33.8
28.4
31.1
22.2
1.4
Table 4. Protection of CCRF-CEM Human Leukemia Cells against the
Growth Inhibitory Effects of MTX and 2 by 10 µM Hx, 5 µM TdR, and
Their Combinationa
9.58
30.2
relative growthb (%)
18.4
0.0015
0.3
TMQ 0.042
TMP 12
0.003 0.07
180 15
0.3
64
2
600
drug
no addition
5 µM TdR
10 µM Hx
Hx+TdR
a These assays were carried out at 37 °C under conditions of saturating
substrate (90 µM dihydrofolic acid) and cofactor (119 µM NADPH) in the
presence of 150 mM KCl. b Value in parentheses is the percent inhibition
at the concentration indicated.
MTX (40 nM)
2 (5000 nM)
11 ( 0
12 ( 0
13 ( 1
15 ( 0
14 ( 1
12 ( 0
74 ( 1
74 ( 2
a Growth is expressed relative to quadruplicate cultures not treated with
either drug or metabolite and is the average range for duplicate-treated
(
samples. The experiment was repeated with similar results. b Deoxycytidine
(dCyd; 10 µM) was present in all the above cultures to prevent the inhibition
of growth caused in T-cell leukemias like CCRF-CEM by TdR (see
Experimental Section). In typical results, dCyd alone had no effect on
CCRF-CEM growth (99 ( 3% of control) and did not protect against growth
inhibition by either drug (data not shown). Hx alone (97 ( 1% of control)
or in the presence of dCyd (98 ( 1% of control) did not affect CCRF-
CEM growth and did not protect against MTX-induced growth inhibition
(table above). TdR alone inhibited growth of CCRF-CEM (31 ( 1% of
control), but TdR+dCyd was essentially not growth inhibitory (95 ( 2%
of control) and neither protected against MTX-induced growth inhibition
(table above). Similarly, Hx+TdR+dCyd did not appreciably inhibit growth
of CCRF-CEM (96 ( 4% of control).
most selective, followed by 16. Once again, as with tgDHFR,
13 was the most potent and selective analogue of the series.
The 5-ethyl compounds were also compared with the corre-
sponding 5-methyl analogues from the previous study.12 This
comparison afforded variable results. Compound 13, the most
potent and selective analogue for both tgDHFR and maDHFR,
was not much different in both potency and selectivity from its
5-methyl analogue 1i. Similarly, compounds 9 and 1h were not
very different. However, 4 and 1b, 7 and 1c, 8 and 1d, and 12
and 1g (for maDHFR) were quite different with respect to
potency and selectivity for one or both pathogen DHFR. For
the 2,5-diOMePh analogues and the 3,4-diCl analogues, the
5-substitution of methyl or ethyl is immaterial; however, for
the 2-naphthyl analogue, there is a significant difference in the
5-methyl and 5-ethyl analogues. Clearly these results reflect the
ease (or difficulty) with which the particular enzyme simulta-
neously accommodates both the 5- and the 6-substitutions.
Compound 13 emerges as the best analogue being 35-fold
and 39-fold more selective for tgDHFR and maDHFR, respec-
tively. In addition, it was 23-fold more potent against tgDHFR
than TMP and 3-fold more potent as TMP against maDHFR.
In Vitro Human Tumor Cell Growth Inhibition. The
classical analogue 2 was also evaluated as inhibitors of the in
vitro growth of CCRF-CEM human leukemia cells and of three
CCRF-CEM sublines (Table 3) with single defined mechanisms
of resistance to MTX during continuous exposure. The results
(Table 3) indicate that the classical 6-ethyl analogue 2 was
highly cytotoxic against the growth of CCRF-CEM cells in
culture and had an EC50 about 3-fold more potent than the parent
5-methyl compound 1a. This indicates that homologation of the
C5-methyl to an ethyl for the classical 5-substituted 2,4-diamino
pyrolo[2,3-d]pyrimindine is highly conducive to tumor growth
inhibition in culture and may indicate better transport and more
potent hDHFR inhibition.
MTX (31-fold), indicating that DHFR is likely the primary target
of this 2,4-diamino-pyrrolo[2,3-d]pyrimidine. The MTX-
transport resistant subline R2,17 which does not express func-
tional reduced folate carrier (RFC), is 99-fold cross-resistant to
2, while it is 147-fold cross-resistant to MTX. The data suggest
that 2 utilizes the RFC as its primary means of transport. The
R30dm subline18 expressing low levels of folylpolyglutamate
synthetase (FPGS) is 3-fold cross-resistant to 2 under continuous
exposure conditions, suggesting that polyglutamates of 2 must
be considered as part of its mechanism of action.
Metabolite protection studies were performed to further
elucidate the mechanism of action of 2. At concentrations of
drug that inhibited the growth of CCRF-CEM cells by g90%,
leucovorin at g0.1 µM was able to fully protect against the
effects of MTX and 2 (data not shown). This is consistent with
an antifolate mechanism of action for 2. Further studies in
CCRF-CEM examined the ability of thymidine (TdR) and
hypoxanthine (Hx) to protect against growth inhibition of 2
(Table 4). These metabolites can be salvaged to produce dTTP
and the purine dNTPs required for DNA synthesis and, thus,
bypass the MTX blockade.19 As described in Experimental
Section, in T-lymphoblast cell lines like CCRF-CEM, TdR can
only be tested in the presence of dCyd, which reverses its toxic
effects; however, dCyd has no protective effect on MTX either
alone or in paired combination with either Hx or TdR (Table
DHFR overexpressing line R116 was 84-fold cross-resistant
to 2, which is slightly higher than the cross-resistance of R1 to

3050 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13
Gangjee et al.
Table 5. Cytotoxic Evaluation (GI50, M) of Compounds 1a4 and 2
inhibitory potency, selectivity, and antitumor activity. The
biological results indicate that elongation of the C5-methyl
classical, 5,6-disubstituted pyrrolo[2,3-d]pyrimidine (1a) to an
ethyl group (2) increases the inhibitory activity against both
DHFR and the growth of tumor cells in culture and significantly
increases the spectrum of tumor inhibition in culture compared
to the C5-methyl parent compound 1a. The DHFR inhibitory
activity of the 15 nonclassical analogues indicates that these
compounds are potent inhibitors of ecDHFR, tgDHFR, and
maDHFR, with moderate to good selectivity compared to
mammalian DHFR. SAR for the nonclassical analogues indi-
cates that the 2,5-dimethoxy substitution on the phenyl ring
provides the most potent and selective inhibitor for tgDHFR
and maDHFR. This 2,5-dimethoxy phenyl substitution occurs
in several other potent and selective DHFR inhibitors, such as
PTX. Potency and selectivity was also found with the unsub-
stituted phenyl analogue and to some extent with electron
withdrawing chloro and nitro substitutions. Homologation of
the 5-methyl group to an ethyl had variable effects on the
inhibition of pathogenic DHFR and mammalian DHFR and are
dependent on the nature of the 6-position substituent.
against Selected Tumor Cell Lines5
cell line
GI50 of 1a (M)
GI50 of 2 (M)
Leukemia
CCRF-CEM
K-562
MOLT-4
RPMI-8226
SR
2.2 × 10-7
2.7 × 10-8
<1.0 × 10-8
<1.0 × 10-8
1.7 × 10-8
1.2 × 10-8
4.8 × 10-7
5.9 × 10-7
<1.0 × 10-8
a
-
Non-Small Cell Lung Cancer
NCI-H460
6.3 × 10-7
1.8 × 10-7
Colon Cancer
-
HCT-116
HT29
KM12
2.3 × 10-8
9.8 × 10-8
5.6 × 10-8
2.6 × 10-8
1.6 × 10-7
-
SW-620
1.2 × 10-7
Central Nervous System Cancer
SF-268
SF-295
SF-539
U-251
-
4.3 × 10-7
6.6 × 10-7
2.4 × 10-7
5.3 × 10-7
-
-
8.2 × 10-7
Melanoma
1.7 × 10-7
-
LOX IMVI
UACC-62
4.4 × 10-8
8.8 × 10-7
Experimental Section
Ovarian Cancer
7.4 × 10-7
All evaporations were carried out in vacuo with a rotary
evaporator. Analytical samples were dried in vacuo (0.2 mmHg)
in a CHEM-DRY drying apparatus over P2O5 at 80 °C. Melting
points were determined on a MEL-TEMP II melting point apparatus
with FLUKE 51 K/J electronic thermometer and are uncorrected.
Nuclear magnetic resonance spectra for proton (1H NMR) were
recorded on a Bruker WH-300 (300 MHz) spectrometer. Chemical
shift values are expressed in ppm (parts per million) relative to
tetramethylsilane as internal standard; s ) singlet, d ) doublet, t
) triplet, q ) quartet, m ) mutiplet, br ) broad singlet. The relative
integrals of peak areas agreed with those expected for the assigned
structures. Thin-layer chromatography (TLC) was performed on
Polygram Sil G/UV254 silica gel plates with 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 230-400 mesh silica gel
purchased from Aldrich, Milwaukee, WI. LCMS analysis was
performed with an HP 1100 series LC/MSD equipped with a Waters
Nova-Pak C18 column and with a Finnigan LCQ equipped with a
Phenomenox Luna C18 column. Elemental analyses were performed
by Atlantic Microlab, Inc., Norcross, GA. Element compositions
are within (0.4% of the calculated values. Fractional moles of water
or organic solvents frequently found in some analytical samples of
antifolates could not be prevented in spite of 24-48 h of drying in
vacuo and were confirmed where possible by their presence in the
1H NMR spectra. All solvents and chemicals were purchased from
Aldrich Chemical Co. or Fisher Scientific and were used as
received.
General Procedure for the Synthesis of Compounds 3-18.
To a solution of 5-ethyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine
(20;14 2-3 mmol) in EtOH/H2O (1:1) was added two equivalents
of the appropriately substituted aromatic thiol, and the reaction
mixture heated to reflux. At reflux, two equivalents of I2 was added
and the reaction continued for 2 h. when TLC showed the
disappearance of starting material 20. At this point, the reaction
was stopped and the reaction mixture was allowed to cool. The
precipitated disulfide was filtered out and the filtrate was evaporated
to dryness under reduced pressure. To the resulting residue was
added methanol and silica gel, and this suspension was evaporated
to dryness. The silica gel plug was loaded on a dry silica gel column
and flushed with CHCl3 (500 mL) and eluted with a gradient of
1% to 5% MeOH in CHCl3. Fractions containing the product were
pooled and evaporated to afford pure 5-ethyl-6-[(substituted-
phenyl)sulfanyl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines 3-19.
5-Ethyl-6-(1-naphthylsulfanyl)-7H-pyrrolo[2,3-d]pyrimidine-
2,4-diamine (3). Compound 3 was synthesized from 20 (0.35 g, 2
OVCAR-8
-
Renal Cancer
ACHN
CAKI-1
SN12C
UO-31
-
-
-
-
4.4 × 10-7
1.7 × 10-7
6.0 × 10-7
3.0 × 10-7
Prostate Cancer
4.4 × 10-7
-
PC-3
DU-145
4.0 × 10-7
3.7 × 10-7
Breast Cancer
-
-
MCF7
MDA-MB-435
1.7 × 10-7
6.6 × 10-7
a - ) concn > 1.0 × 10-7 M.
4; footnote). The data (Table 4) show that for 2, neither TdR
nor Hx alone protected to any significant extent; both metabo-
lites were required to achieve significant protection. This
indicates that both purine and thymidylate syntheses are inhibited
and is consistent with DHFR being the primary target of 2.
Compound 2 was selected by the NCI for evaluation in its in
vitro preclinical antitumor screening program.5 The ability of
compound 2 to inhibit the growth of tumor cell lines was
measured as GI50 values, the concentration required to inhibit
the growth of tumor cells in culture by 50% as compared to a
control. In 25 of the 59 cell lines, compound 2 showed GI50
values of e1 × 10-7 M (Table 5). In three of the leukemia cell
lines, four of the colon cancer cell lines, and one melanoma,
the GI50 values for 2 were two digit nanomolar (<100 nM) and
paralleled the hDHFR IC50 value. In addition, the spectrum of
tumor inhibition (GI50 e 1 × 10-7 M) was increased from 11
for 1a to 25 for 2, indicating that homologation of the 5-methyl
to the 5-ethyl was instrumental in increasing the spectrum of
tumor inhibition in culture. It was also interesting to note that
compound 2 was not a general cell poison but showed selectivity
both within a type of tumor cell line as well as across different
tumor cell lines with inhibitory values that in some instances
differed by 10 000-fold (data not shown).
In summary, diethyl N-{4-[(2,4-diamino-5-ethyl-7H-pyrrolo-
[2,3-d]pyrimidin-6-yl)sulfanyl]benzoyl}-L-glutamic acid 2, the
C5-ethyl homologue of compound 1a, and its nonclassical
analogues 3-17 were designed and synthesized to investigate
the effect of homologation of the 5-methyl substituent on DHFR

6-Arylthio-2,4-diamino-5-ethylpyrrolo[2,3-d]pyrimidine
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13 3051
mmol), 1-naphthalene thiol (0.66 g, 4 mmol), and iodine (1.02 g,
4 mmol) using the general procedure described above to afford
159 mg (24%) of 3 as a white solid: mp 291.7 °C (dec); TLC Rf
6-[(2,4-Dichlorophenyl)sulfanyl]-5-ethyl-7H-pyrrolo[2,3-d]py-
rimidine-2,4-diamine (10). Compound 10 was synthesized from
20 (0.35 g, 2 mmol), 2,4-dichlorobenzenethiol (0.71 g, 4 mmol),
and iodine (1.02 g, 4 mmol) using the general procedure described
above to afford 91 mg (13%) of 10 as a white solid: mp 291.6 °C
(dec); TLC Rf 0.56 (CHCl3/MeOH, 5:1, silica gel); 1H NMR
(DMSO-d6) 0.98-1.03 (t, 3 H, 5-CH2CH3), 2.68-2.71 (q, 2 H,
CH2CH3), 5.65 (s, 2 H, 2- or 4-NH2 exch with D2O), 6.26 (s, 2 H,
2- or 4-NH2 exch with D2O), 7.30-7.62 (m, 3 H, C6H3), 11.01 (s,
1 H, 7-H). Anal. (C14H13N5SCl2‚0.02CHCl3) C, H, N, S, Cl.
6-[(2,6-Dichlorophenyl)sulfanyl]-5-ethyl-7H-pyrrolo[2,3-d]py-
rimidine-2,4-diamine (11). Compound 11 was synthesized from
20 (0.35 g, 2 mmol), 2,6-dichlorobenzenethiol (0.71 g, 4 mmol),
and iodine (1.02 g, 4 mmol) using the general procedure described
above to afford 126 mg (18%) of 11 as a white solid: mp 295.2
1
0.57 (CHCl3/MeOH, 5:1, silica gel); H NMR (DMSO-d6) 0.99-
1.07 (t, 3 H, 5-CH2CH3), 2.76-2.78 (q, 2 H, CH2CH3), 5.60 (s, 2
H, 2- or 4-NH2 exch with D2O), 6.21 (s, 2 H, 2- or 4-NH2 exch
with D2O), 6.81-8.25 (m, 7H, C10H7), 11.01 (s, 1 H, 7-H). Anal.
(C18H17N5S) C, H, N, S.
5-Ethyl-6-(2-naphthylsulfanyl)-7H-pyrrolo[2,3-d]pyrimidine-
2,4-diamine (4). Compound 4 was synthesized from 20 (0.35 g, 2
mmol), 2-naphthalene thiol (0.66 g, 4 mmol), and iodine (1.02 g,
4 mmol) using the general procedure described above to afford
298 mg (45%) of 4 as a white solid: mp 231 °C (dec); TLC Rf
1
0.57 (CHCl3/MeOH, 5:1, silica gel); H NMR (DMSO-d6) 0.99-
1.07 (t, 3 H, 5-CH2CH3), 2.76-2.78 (q, 2 H, CH2CH3), 5.60 (s, 2
H, 2- or 4-NH2 exch with D2O), 6.21 (s, 2 H, 2- or 4-NH2 exch
with D2O), 6.81-8.25 (m, 7H, C10H7), 11.01 (s, 1 H, 7-H). Anal.
(C18H17N5S.0.20H2O) C, H, N, S.
1
°C (dec); TLC Rf 0.56 (CHCl3/MeOH, 5:1, silica gel); H NMR
(DMSO-d6) 0.75-0.80 (t, 3 H, 5-CH2CH3), 2.69-2.71 (q, 2 H,
CH2CH3), 5.50 (s, 2 H, 2- or 4-NH2 exch with D2O), 6.07 (s, 2 H,
2- or 4-NH2 exch with D2O), 7.29-7.50 (m, 3 H, C6H3), 10.87 (s,
1 H, 7-H). Anal. (C14H13N5SCl2) C, H, N, S, Cl.
5-Ethyl-6-[(4-nitrophenyl)sulfanyl]-7H-pyrrolo[2,3-d]pyrimi-
dine-2,4-diamine (5). Compound 5 was synthesized from 20 (0.35
g, 2 mmol), 4-nitrobenzenethiol (0.62 g, 4 mmol), and iodine (1.02
g, 4 mmol) using the general procedure described above to afford
228 mg (35%) of 5 as an orange solid: mp 286.4 °C (dec); TLC
Rf 0.55 (CHCl3/MeOH, 5:1, silica gel); 1H NMR (DMSO-d6) 0.99-
1.04 (t, 3 H, 5-CH2CH3), 2.72-2.75 (q, 2 H, CH2CH3), 5.67 (s, 2
H, 2- or 4-NH2 exch with D2O), 6.29 (s, 2 H, 2- or 4-NH2 exch
with D2O), 7.16-7.18 (d, 2 H, 3′,5′-CH), 8.11-8.14 (d, 2 H, 2′,
6′-CH) 11.09 (s, 1 H, 7-H). Anal. (C14H14N6O2S‚0.40H2O) C, H,
N, S.
6-[(4-Bromophenyl)sulfanyl]-5-ethyl-7H-pyrrolo[2,3-d]pyri-
midine-2,4-diamine (6). Compound 6 was synthesized from 20
(0.35 g, 2 mmol), 4-bromobenzenethiol (0.75 g, 4 mmol), and iodine
(1.02 g, 4 mmol) using the general procedure described above to
afford 273 mg (38%) of 6 as a white solid: mp 272.5 °C (dec);
TLC Rf 0.55 (CHCl3/MeOH, 5:1, silica gel); 1H NMR (DMSO-d6)
0.98-1.03 (t, 3 H, 5-CH2CH3), 2.72-2.74 (q, 2 H, CH2CH3), 5.58
(s, 2 H, 2- or 4-NH2 exch with D2O), 6.19 (s, 2 H, 2- or 4-NH2
exch with D2O), 6.91-6.94 (d, 2 H, 3′,5′-CH), 7.43-7.45 (d, 2 H,
2′, 6′-CH) 10.97 (s, 1 H, 7-H). Anal. (C14H14N5SBr‚0.30H2O) C,
H, N, S, Br.
5-Ethyl-6-[(4-methoxyphenyl)sulfanyl]-7H-pyrrolo[2,3-d]py-
rimidine-2,4-diamine (12). Compound 12 was synthesized from
20 (0.35 g, 2 mmol), 4-methoxybenzenethiol (0.56 g, 4 mmol),
and iodine (1.02 g, 4 mmol) using the general procedure described
above to afford 143 mg (23%) of 12 as an off-white solid: mp
1
276.7 °C (dec); TLC Rf 0.55 (CHCl3/MeOH, 5:1, silica gel); H
NMR (DMSO-d6) 1.06-1.11 (t, 3 H, 5-CH2CH3), 2.89-2.91 (q, 2
H, CH2CH3), 3.79 (s, 3 H, 4-OCH3), 5.58 (s, 2 H, 2- or 4-NH2
exch with D2O), 6.19 (s, 2 H, 2- or 4-NH2 exch with D2O), 6.91-
6.94 (d, 2 H, 3′,5′-CH), 7.43-7.45 (d, 2 H, 2′, 6′-CH) 10.97 (s, 1
H, 7-H). Anal. (C15H17N5SO) C, H, N, S.
6-[(2,5-Dimethoxyphenyl)sulfanyl]-5-ethyl-7H-pyrrolo[2,3-d]-
pyrimidine-2,4-diamine (13). Compound 13 was synthesized from
20 (0.35 g, 2 mmol), 2,5-dimethoxybenzenethiol (0.68 g, 4 mmol),
and iodine (1.02 g, 4 mmol) using the general procedure described
above to afford 204 mg (30%) of 13 as a white solid: mp 239.5
°C, TLC Rf 0.55 (CHCl3/MeOH, 5:1, silica gel); 1H NMR (DMSO-
d6) 0.98-1.03 (t, 3 H, 5-CH2CH3), 2.55-2.69 (q, 2 H, CH2CH3),
3.52 (s, 3 H, OCH3), 3.85 (s, 3 H, OCH3), 5.59 (s, 2 H, 2- or 4-NH2
exch with D2O), 6.21 (s, 2 H, 2- or 4-NH2 exch with D2O), 6.62-
6.91 (m, 3 H, C6H3), 10.89 (s, 1 H, 7-H). Anal. (C16H19N5SO2) C,
H, N, S.
6-[(3-Chlorophenyl)sulfanyl]-5-ethyl-7H-pyrrolo[2,3-d]pyri-
midine-2,4-diamine (7). Compound 7 was synthesized from 20
(0.35 g, 2 mmol), 3-chlorobenzenethiol (0.57 g, 4 mmol), and iodine
(1.02 g, 4 mmol) using the general procedure described above to
afford 88 mg (14%) of 7 as a white solid: mp 244.5 °C TLC Rf
5-Ethyl-6-[(3-methoxyphenyl)sulfanyl]-7H-pyrrolo[2,3-d]py-
rimidine-2,4-diamine (14). Compound 14 was synthesized from
20 (0.35 g, 2 mmol), 3-methoxybenzenethiol (0.56 g, 4 mmol),
and iodine (1.02 g, 4 mmol) using the general procedure described
above to afford 155 mg (25%) of 14 as an off-white solid: mp
1
0.56 (CHCl3/MeOH, 5:1, silica gel); H NMR (DMSO-d6) 0.99-
1.03 (t, 3 H, 5-CH2CH3), 2.73-2.76 (q, 2 H, CH2CH3), 5.62 (s, 2
H, 2- or 4-NH2 exch with D2O), 6.23 (s, 2 H, 2- or 4-NH2 exch
with D2O), 6.95-7.29 (m, 4 H, C6H4), 11.01 (s, 1 H, 7-H). Anal.
(C14H14N5SCl) C, H, N, S, Cl.
1
182.2 °C, TLC Rf 0.55 (CHCl3/MeOH, 5:1, silica gel); H NMR
(DMSO-d6) 0.98-1.03 (t, 3 H, 5-CH2CH3), 2.72-2.74 (q, 2 H,
CH2CH3), 3.66 (s, 3 H, 3-OCH3), 5.58 (s, 2 H, 2- or 4-NH2 exch
with D2O), 6.19 (s, 2 H, 2- or 4-NH2 exch with D2O), 6.91-6.94
(d, 2 H, 3′,5′-CH), 7.43-7.45 (d, 2 H, 2′, 6′-CH) 10.97 (s, 1 H,
7-H). Anal. (C15H17N5SO) C, H, N, S.
6-[(4-Chlorophenyl)sulfanyl]-5-ethyl-7H-pyrrolo[2,3-d]pyri-
midine-2,4-diamine (8). Compound 8 was synthesized from 20
(0.35 g, 2 mmol), 4-chlorobenzenethiol (0.57 g, 4 mmol), and iodine
(1.02 g, 4 mmol) using the general procedure described above to
afford 95 mg (15%) of 8 as an off-white solid: mp 245.1 °C (dec);
TLC Rf 0.56 (CHCl3/MeOH, 5:1, silica gel); 1H NMR (DMSO-d6)
0.99-1.03 (t, 3 H, 5-CH2CH3), 2.73-2.75 (q, 2 H, CH2CH3), 5.66
(s, 2 H, 2- or 4-NH2 exch with D2O), 6.27 (s, 2 H, 2- or 4-NH2
exch with D2O), 6.98-7.01 (d, 2 H, 3′,5′-CH), 7.31-7.34 (d, 2 H,
2′, 6′-CH) 11.02 (s, 1 H, 7-H). Anal. (C14H14N5SCl‚0.40H2O) C,
H, N, S, Cl.
6-[(3,4-Dichlorophenyl)sulfanyl]-5-ethyl-7H-pyrrolo[2,3-d]py-
rimidine-2,4-diamine (9). Compound 9 was synthesized from 20
(0.35 g, 2 mmol), 3,4-dichlorobenzenethiol (0.71 g, 4 mmol), and
iodine (1.02 g, 4 mmol) using the general procedure described above
to afford 168 mg (24%) of 9 as a white solid: mp 300.7 °C (dec);
TLC Rf 0.56 (CHCl3/MeOH, 5:1, silica gel); 1H NMR (DMSO-d6)
0.98-1.02 (t, 3 H, 5-CH2CH3), 2.74-2.76 (q, 2 H, CH2CH3), 5.53
(s, 2 H, 2- or 4-NH2 exch with D2O), 6.13 (s, 2 H, 2- or 4-NH2
exch with D2O), 6.80-7.03 (m, 3 H, C6H3), 11.02 (s, 1 H, 7-H).
Anal. (C14H13N5SCl2) C, H, N, S, Cl.
5-Ethyl-6-(phenylsulfanyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-
diamine (15). Compound 15 was synthesized from 20 (0.35 g, 2
mmol), thiophenol (0.44 g, 4 mmol), and iodine (1.02 g, 4 mmol)
using the general procedure described above to afford 180 mg (32%)
of 15 as an off-white solid: mp 275 °C (dec); TLC Rf 0.55 (CHCl3/
1
MeOH, 5:1, silica gel); H NMR (DMSO-d6) 0.99-1.03 (t, 3 H,
5-CH2CH3), 2.74-2.76 (q, 2 H, CH2CH3), 5.63 (s, 2 H, 2- or 4-NH2
exch with D2O), 6.24 (s, 2 H, 2- or 4-NH2 exch with D2O), 6.98-
7.28 (m, 5 H, C6H5), 10.99 (s, 1 H, 7-H). Anal. (C14H15N5S‚
0.20H2O) C, H, N, S.
5-Ethyl-6-(8-quinolinylsulfanyl)-7H-pyrrolo[2,3-d]pyrimidine-
2,4-diamine (16). Compound 16 was synthesized from 20 (0.35 g,
2 mmol), 2-quinolinethiol (0.64 g, 4 mmol), and iodine (1.02 g, 4
mmol) using the general procedure described above to afford 292
mg (44%) of 16 as a light brown solid: mp 260.5 °C (dec); TLC
Rf 0.57 (CHCl3/MeOH, 5:1, silica gel); 1H NMR (DMSO-d6) 0.96-
1.08 (t, 3 H, 5-CH2CH3), 2.76-2.79 (q, 2 H, CH2CH3), 5.65 (s, 2
H, 2- or 4-NH2 exch with D2O), 6.28 (s, 2 H, 2- or 4-NH2 exch

3052 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13
Gangjee et al.
with D2O), 7.47-8.21 (m, 6 H, C9H6) 11.10 (s, 1 H, 7-H). Anal.
(C17H16N6S‚1.10H2O) C, H, N, S.
HCl. The resulting suspension was frozen in a dry ice-acetone
bath and thawed in the refrigerator to 4-5 °C and filtered. The
residue was washed with a small amount of cold water and ethyl
acetate and dried in vacuo using P2O5 to afford 120 mg (88%) of
6-[(2,6-Dimethylphenyl)sulfanyl]-5-ethyl-7H-pyrrolo[2,3-d]py-
rimidine-2,4-diamine (17). Compound 17 was synthesized from
20 (0.35 g, 2 mmol), 2,6-dimethylbenzenethiol (0.55 g, 4 mmol),
and iodine (1.02 g, 4 mmol) using the general procedure described
above to afford 346 mg (56%) of 17 as a white solid: mp 290.5
1
2 as a yellow powder: mp 213-215 °C (dec); H NMR (DMSO-
d6) δ 0.98-1.03 (t, 3 H, C6-CH2CH3), 1.91-2.08 (m, 2 H, Glu
â-CH2), 2.30-2.35 (t, 2 H, Glu γ-CH2), 2.71-2.77 (q, 2 H, C6-
CH2CH3), 4.35 (m, 1 H, Glu R-CH), 5.72 (s, 2 H, 2-NH2), 6.58 (s,
2 H, 2-NH2), 7.05-7.07 (d, 2 H, Ph-CH), 7.76-7.78 (d, 2 H,
Ph-CH), 8.53-8.55 (d, 1 H, -CONH), 11.24 (s, 1 H, 7-NH). Anal.
(C20H22N6O5S‚1.8H2O) C, H, N, S.
1
°C (dec); TLC Rf 0.55 (CHCl3/MeOH, 5:1, silica gel); H NMR
(DMSO-d6) 1.01 (t, 3 H, 5-CH2CH3), 2.78 (q, 2 H, CH2CH3), 2.08
(s, 6 H, 2- and 6-CH3), 5.58 (s, 2 H, 2- or 4-NH2 exch with D2O),
6.19 (s, 2 H, 2- or 4-NH2 exch with D2O), 6.91-6.94 (d, 2 H,
3′,5′-CH), 7.43-7.45 (d, 2 H, 2′, 6′-CH) 10.97 (s, 1 H, 7-H). Anal.
(C16H19N5S) C, H, N, S.
Cell Lines and Methods for Measuring Growth Inhibitory
Potency (Table 3). Solutions used in cell culture studies were
standardized using extinction coefficients. Extinction coefficients
were determined for 2 [pH 1, λmax 236 nm (29 300) and λmax 277
nm (23 400); pH 7, λmax 274 nm (22 700) and λmax 297 nm (20 200);
pH 13, λmax 222 nm (30 200) and λmax 274 nm (22 400)]. Extinction
coefficients for methotrexate (MTX), a gift from Immunex (Seattle,
WA), were from the literature.20 Aminopterin, Hx, TdR, and
deoxycytidine (dCyd) were purchased from Sigma Chemical Co.
(St. Louis, MO). Other chemicals and reagents were reagent grade
or higher.
4-[(2,4-Diamino-5-ethyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)sul-
fanyl]benzoic acid (18). To a mixture of 20 (500 mg, 2.8 mmol)
and 4-mercaptobenzoic acid (900 mg, 5.8 mmol) in EtOH/H2O (3:
2) at reflux was added 2 equiv of iodine. The resulting mixture
turned into a suspension within 1 min, and the precipitate formed
dissolved to form a dark solution within half an hour. The reflux
was continued for another 1.5 to 2 h, until TLC showed the
disappearance of the starting materials and the formation of desired
product. The reaction mixture was filtered to remove the excess
disulfide, and the filtrate was evaporated to dryness under reduced
pressure. EtOAc was added to the resulting residue, the mixture
was refluxed for 15 min, and the precipitate formed was collected
through filtration and recrystallized from MeOH to afforded 0.93
g (quantitative) of 18 as white crystals: mp >250 °C (dec); Rf )
0.45 (CHCl3/MeOH, 5:1); 1H NMR (DMSO-d6) δ 0.98-1.03 (t, 3
H, CH2CH3), 2.78-2.90 (q, 2 H, CH2CH3), 7.08-7.11 (d, 2 H,
C6H4), 7.29 (s, 2 H, 2- or 4-NH2), 7.82-7.85 (d, 2 H, C6H4), 8.02
(s, 2 H, 4- or 2-NH2), 11.51 (br, 1 H, 7-NH), 12.20 (s, 1 H, COOH).
HRMS (m/z) calcd for C15H15 N5O2S, 329.0946; found, 329.0959.
Diethyl N-{4-[(5-Ethyl-2-amino-3,4-dihydro-4-oxo-7H-pyr-
rolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamate (19). To a
solution of 18 (125 mg, 0.38 mmol) in anhydrous DMF (9 mL)
was added triethylamine (130 µL), and the mixture was stirred under
nitrogen at room temperature for 5 min. The resulting solution was
cooled to 0 °C, isobutyl chloroformate (130 µL 1.0 mmol) was
added, and the mixture was stirred at 0 °C for 30 min. At this time.
TLC (MeOH/CHCl3, 1:5) indicated the formation of the activated
intermediate at Rf 0.55 and the disappearance of the starting acid
at Rf 0.18. Diethyl-L-glutamate hydrochloride (240 mg, 1.0 mmol)
was added to the reaction mixture followed immediately by
triethylamine (130 µL, 1.0 mmol). The reaction mixture was slowly
allowed to warm to room temperature and stirred under nitrogen
for 24 h. TLC showed the formation of one major spot at Rf )
0.61 (MeOH/CHCl3, 1:5). The reaction mixture was evaporated to
dryness under reduced pressure. The residue was dissolved in a
minimum amount of CH3Cl/MeOH, 4:1, and chromatographed on
a silica gel column (3 × 10 cm) with 4% MeOH in CHCl3 as the
eluent. Fractions that showed the desired single spot at Rf 0.61 were
pooled and evaporated to dryness and crystallized from ethyl ether
to afford 165 mg (85%) of 19 as white crystals: mp 197.6-200
°C; 1H NMR (DMSO-d6) δ 0.98-1.03 (t, 3 H, C6-CH2CH3), 1.08-
1.25 (m, 6 H, OEt), 1.90-2.08 (m, 2 H, Glu â-CH2), 2.32-2.35
(t, 2 H, Glu γ-CH2), 2.78-2.90 (q, 2 H, C6-CH2CH3), 4.00-4.13
(m, 4 H, OEt), 4.33-4.40 (m, 1 H, Glu R-CH), 6.25 (br, 2 H, 2-
or 4-NH2), 6.90 (s, 2 H, 2- or 4-NH2), 7.06-7.08 (d, 2 H, Ph-
CH), 7.76-7.78 (d, 2 H, Ph-CH), 8.69 (d, 1 H, -CONH), 11.45
(s, 1 H, 7-NH). HRMS (m/z) calcd for C24H30N6O5S, 514.1998;
found, 514.2010.
Cell Lines and Methods for Measuring Growth Inhibitory
Potency. Cell lines were verified to be negative for Mycoplasma
contamination (Mycoplasma Plus PCR primers, Stratagene, La Jolla,
CA). The human % lymphoblastic leukemia cell line CCRF-CEM21
and its MTX-resistant sublines R1,16 R2,17 and R30dm18 were
cultured as described.18 R1 expresses 20-fold elevated levels of
DHFR, the target enzyme of MTX. R2 has dramatically reduced
MTX uptake but normal levels of MTX-sensitive DHFR. R30dm
expresses 1% of the FPGS activity of CCRF-CEM and is resistant
to short-term, but not continuous, MTX exposure; however, R30dm
is cross-resistant in continuous exposure to antifolates that require
polyglutamylation to form potent inhibitors. Growth inhibition of
all cell lines by continuous drug exposure was assayed as
described.18 EC50 values (drug concentration effective at inhibiting
cell growth by 50%) were determined visually from plots of percent
growth relative to a solvent-treated control culture versus the
logarithm of drug concentration.
Protection against growth inhibition of CCRF-CEM cells was
assayed by including leucovorin ((6R,S)-5-formyltetrahydrofolate)
at 0.1-10 µM with a concentration of drug previously determined
to inhibit growth by 90-95%; the reminder of the assay was as
described. Growth inhibition was measured relative to the appropri-
ate leucovorin-treated control; leucovorin, even at 10 µM, caused
no growth inhibition in the absence of drug, however. Protection
against growth inhibition of CCRF-CEM cells was assayed by
including Hx (10 µM), TdR (5 µM), or dCyd (10 µM) individually,
in pairs (Hx+dCyd, TdR+dCyd), or all together (Hx+TdR+dCyd),
with concentrations of MTX or 2 that would inhibit growth by
≈90-95% over a growth period of ≈72 h. The growth period was
limited because beyond 72 h, CCRF-CEM cells deplete TdR in
the growth media and drug effects are no longer protected. dCyd
is added only to alleviate the growth inhibitory effects of 5 µM
TdR against CCRF-CEM cells.20 Controls with metabolites alone
(no drug) in the combinations described above (in duplicate),
controls with drug alone with no metabolites (in duplicate), and
untreated controls with neither drugs nor metabolites (in quadru-
plicate) were performed. Growth inhibition was measured as percent
growth relative to untreated control cells (absence of drugs and
metabolites).
N-{4-[(5-Ethyl-2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]-
pyrimidin-6-yl)thio]-benzoyl}-L-glutamic Acid (2). To a solution
of the diester 19 (150 mg, 0.3 mmol) in MeOH (10 mL) was added
1 N NaOH (6 mL), and the mixture was stirred under nitrogen at
room temperature for 16 h. TLC showed the disappearance of the
starting material (Rf ) 0.55) and formation of one major spot at
the origin (MeOH/CHCl3, 1:5). The reaction mixture was evaporated
to dryness under reduced pressure. The residue was dissolved in
water (10 mL), the resulting solution was cooled in an ice bath,
and the pH was adjusted to 3-4 with dropwise addition of 1 N
Acknowledgment. This work was supported in part by
grants from the National Institutes of Health CA89300 (A.G.),
AI44661 (A.G.), and CA43500 (J.J.M.) and the Roswell Park
Cancer Institute CCSG CA16065 and CA10914 (R.L.K.). The
authors thank Mr. William H. Haite for performing growth
inhibition and metabolic protection studies.

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Supporting Information Available: Results from elemental
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