Dual inhibitors of thymidylate synthase and dihydrofolate reductase as antitumor agents: Design, synthesis, and biological evaluation of classical and nonclassical pyrrolo[2,3-d]pyrimidine antifolates

Article abstract of DOI:10.1021/jm058276a

We designed and synthesized a classical analogue N-[4-[(2-amino-6-ethyl-3, 4-dihydro-4-oxo-7H-pyrrolo-[2,3-d]pyrimidin-5-yl)thio]benzoyl]-L-glutamic acid (4) and thirteen nonclassical analogues 5-17 as potential dual thymidylate synthase (TS) and dihydrof

Full text of DOI:10.1021/jm058276a

J. Med. Chem. 2006, 49, 1055-1065
1055
Dual Inhibitors of Thymidylate Synthase and Dihydrofolate Reductase as Antitumor Agents:
Design, Synthesis, and Biological Evaluation of Classical and Nonclassical
Pyrrolo[2,3-d]pyrimidine Antifolates1
Aleem Gangjee,*,† Hiteshkumar D. Jain,† Jaclyn Phan,† Xin Lin,† Xiaohong Song,†,| John J. McGuire,‡ and Roy L. Kisliuk§
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, and
Department of Biochemistry, Tufts UniVersity School of Medicine, Boston, Massachusetts 02111
ReceiVed October 20, 2005
We designed and synthesized a classical analogue N-[4-[(2-amino-6-ethyl-3,4-dihydro-4-oxo-7H-pyrrolo-
[2,3-d]pyrimidin-5-yl)thio]benzoyl]-L-glutamic acid (4) and thirteen nonclassical analogues 5-17 as potential
dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors and as antitumor agents.
The key intermediate in their synthesis was 2-amino-6-ethyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidine,
22, to which various aryl thiols were conveniently attached at the 5-position via an oxidative addition reaction
using iodine. For the classical analogue 4, the ester obtained from the reaction was deprotected and coupled
with diethyl L-glutamate followed by saponification. Compound 4 was a potent dual inhibitor of human TS
(IC50 ) 90 nM) and human DHFR (IC50 ) 420 nM). Compound 4 was not a substrate for human FPGS.
Metabolite protection studies established TS as its principal target. Most of the nonclassical analogues were
only inhibitors of human TS with IC50 values of 0.23-26 µM.
Introduction
Folate metabolism has long been recognized as an attractive
target for cancer chemotherapy because of its indispensable role
in the biosynthesis of nucleic acid precursors.2,3 Within folate
metabolism, thymidylate synthase (TS), which catalyzes the
reductive methylation of 2′-deoxyuridine-5′-monophosphate
(dUMP) to 2′-deoxythymidine-5′-monophosphate (dTMP), has
been of particular interest.4 For this biochemical transformation,
5,10-methylenetetrahydrofolate serves as the source of the
methyl group as well as the reductant and is oxidized to 7,8-
dihydrofolate (7,8-DHF).4 For continuous production of dTMP
in dividing cells, the oxidized 7,8-DHF must be converted back
to 5,10-methylenetetrahydrofolate. Dihydrofolate reductase (DH-
FR)5 catalyzes the first of the two steps in this biotransformation
in which NADPH acts as the source of the reductant.6 Thus TS
and DHFR are crucial for the synthesis of dTMP in dividing
cells, and hence inhibition of either leads to “thymineless death”.
Thus both TS and DHFR represent attractive targets for
developing antitumor agents. Several TS and DHFR inhibitors,
as separate entities, have found clinical utility as antitumor
agents.7 Figure 1 illustrates important examples of clinically
used TS inhibitors such as raltitrexed8 and pemetrexed9 (Figure
1). Similarly, methotrexate (MTX), a DHFR inhibitor, has been
used clinically for more than 50 years.10 Generally 2,4-
diaminopyrimidine containing antifolates are inhibitors of DHFR
while 2-amino-4-oxopyrimidine containing antifolates are in-
Figure 1.
hibitors of TS.11,12 Raltitrexed is a quinazoline analogue
containing a 6-6 ring-fused system similar to natural folates.
DHFR, glycinamideribonucleotide formyltransferase (GARFT),
and aminoimidazole carboxamideribonucleotide formyltrans-
ferase (AICARFT).13,14 The primary locus of action of peme-
trexed is the inhibition of TS which is also responsible for its
cytotoxic effects.14 However, inhibition of other folate-dependent
enzymes may also be important, since pemetrexed is cytotoxic
to human cancer cell lines that are resistant to raltitrexed and
5-FU as a result of TS amplification.15 Using molecular
modeling, we16 suggested that pemetrexed might bind to DHFR
in a “2,4-diamino mode” (Figure 2). This binding mode is
Pemetrexed contains a 6-5 ring-fused pyrrolo[2,3-d]pyrimidine
system and is designated a multitargeted antifolate (MTA).
Pemetrexed and its polyglutamylated metabolites are inhibitors
of several important folate-dependent enzymes including TS,
* To whom correspondence should be addressed. E-mail: gangjee@
duq.edu. Phone: 412-396-6070. Fax: 412-396-5593.
† Duquesne University.
‡ Roswell Park Cancer Institute.
§ Tufts University School of Medicine.
| Present address: Abbott Laboratories, Abbott Park, IL 60064.
10.1021/jm058276a CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/12/2006

1056 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Gangjee et al.
pyrimidine, 1, as a potent inhibitor of human TS (IC50 ) 42
nM) and a marginal inhibitor of human DHFR (IC50 ) 2.2 µM)
in its monoglutamate form thus providing a dual inhibitor of
human TS and human DHFR. Additionally, compound 1 was
not a substrate for human FPGS at concentrations up to 1045
µM thus indicating that 1 does not require polyglutamylation
for its potent inhibition of the target enzymes. The lack of FPGS
substrate activity of 1 was attributed, in part, to the presence of
the 6-methyl group on the pyrrolo[2,3-d]pyrimidine of 1.29 Thus
compound 1 is a promising lead compound which can be further
modified to enhance human TS, human DHFR, and tumor cell
inhibitory activity. Molecular modeling using SYBYL 6.9130
indicated that compound 1 should bind to human DHFR in the
“2,4-diamino mode” (Figure 2) similar to that proposed for
pemetrexed,16 thereby allowing for moderate human DHFR
inhibition. Additionally, molecular modeling indicated that the
6-methyl group of 1 makes hydrophobic contact with Val115
in human DHFR. This potential hydrophobic interaction with
Val115 is absent in pemetrexed which does not have the
6-methyl group. We31,32 have long advocated and recently16,33
shown that dual TS-DHFR inhibitory activity in 6-5 antifolates
can occur in two different binding modes one to TS and a flipped
mode to DHFR. For 2-amino-4-oxo-5-substituted-6-alkyl pyr-
rolo[2,3-d]pyrimidines the two modes are shown in Figure 2.
The 2-amino-4-oxo mode is proposed for TS binding and
rotation about the NH2-C2 bond by 180° affords the flipped
2,4-diamino mode proposed for DHFR binding.
Figure 2.
obtained on rotation of 180° about the 2-NH2-C2 bond in which
the pyrrole nitrogen mimics the 4-amino group of MTX.16 The
fact that X-ray crystal structures show that this flipped mode
of binding actually occurs with 2-amino-4-methyl antifolates16,17
lends credence to our hypothesis.
Raltitrexed and pemetrexed are good substrates for the
enzyme folylpoly-γ-glutamate synthetase (FPGS).8,9 FPGS
catalyzes the production of poly-γ-glutamates which leads to
high intracellular concentrations of folates and antifolates and
in some cases increases TS inhibitory activity for antifolates
such as raltitrexed8 (60-fold) and pemetrexed9 (130-fold)
compared to their monoglutamate forms.18-21 Although poly-
glutamylation of certain antifolates is necessary for cytotoxicity,
it has also been implicated in toxicity to normal host cells due
to cellular retention of the polyanionic poly-γ-glutamate me-
tabolites which do not efflux from normal cells.22 Additionally,
tumor cells develop resistance to antifolates which depend on
polyglutamylation for their antitumor effects by producing low
Gangjee et al.34 using molecular modeling (SYBYL 6.91)30
suggested that the 6-methyl group in 1 makes important
hydrophobic contacts with Trp109 in human TS and also
sterically restricts the rotation of the 5-position side-chain so
that it adopts a favorable conformation for binding to TS. Above
all, compound 1 (EC50 ) 0.45 µM) was a better inhibitor of
the growth of CCRF-CEM human leukemic cells in culture than
was N-[4-[N-[(2-amino-3,4-dihydro-4-oxo-6-quinazolinyl)methyl]-
or defective FPGS and thereby limiting their use.36,23-26
A
potential approach to circumvent drawbacks associated with
FPGS, including resistance, is to design compounds that are
not substrates for FPGS and yet have high antitumor activity
in their monoglutamate forms. A compound not subject to
activation by polyglutamylation needs to have high intrinsic
potency as a TS inhibitor. One such nonpolyglutamylatable,
potent TS inhibitor plevitrexed (Figure 1), is currently under-
going phase I/II clinical trial as an antitumor agent.27,28
N-prop-2-ynylamino]benzoyl]-L-glutamic acid (PDDF) (EC50
)
It has been our long standing goal to design and synthesize
compounds that are potent dual inhibitors of TS and DHFR and
that do not depend on FPGS for inhibitory potency. Such
compounds would be capable of providing “combination
therapy” as a single agent without the pharmacokinetic dis-
advantages of two separate agents. Gangjee et al.29 reported a
classical 2-amino-4-oxo-6-methyl-5-substituted pyrrolo[2,3-d]-
0.72 µM), a standard TS inhibitor. Molecular modeling (SYBYL
6.91)30 further indicated that the 6-methyl can be homologated
to a 6-ethyl which further enhances the van der Waals interaction
with Trp109 in human TS (Figure 3) in the 2-amino-4-oxo mode
and Val115 in human DHFR in the flipped 2,4-diamino mode.
We were aware of a minor steric hindrance between the 6-ethyl
group of compound 4 and Trp109 in human TS; however, it
Figure 3. Stereoview compound 4 superimposed on LY231514 (not shown) in human TS (PDB code 1JU6).64 The hydrophobic interaction between
6-ethyl and Trp109 is shown.

Pyrrolo[2,3-d]pyrimidine Antifolates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1057
Scheme 1a
a Conditions: (a) EtI, NaHCO3, rt, 120 h; (b) DMF, 40-50 °C, 72 h; (c) NaBH4, EtOH, rt, 30 min; (d) I2, EtOH/H2O (2:1), 100-110 °C; (e) 1N NaOH,
i
80 °C, 24 h; (f) BuOCOCl, NEt3, diethyl L-glutamate hydrochloride, 0 °C to room temperature; (g) 1 N NaOH, 0 °C, 4 h, then rt, 20 h; (h) ArSH, I2,
EtOH/H2O (2:1), 100-110 °C for 5-7, 9, 11, 12, 14, and 15; (i) ArSH, PIFA, (CF3)2CHOH, rt, N2 for 8, 10, 13, 16, and 17.
was anticipated that this would not be deleterious for the binding
of 4 to human TS since the molecule could tilt sufficiently in
its binding to alleviate the mild clash of the 6-ethyl group with
Trp109. Thus the 6-ethyl group might decrease the TS inhibitory
activity compared to 1 but was expected to enhance the DHFR
inhibitory activity compared to 1. Thus N-[4-[(2-amino-6-ethyl-
3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)thio]-ben-
zoyl]-L-glutamic acid, 4, the 6-ethyl homologue of compound
1 was designed and synthesized as a potential dual inhibitor of
human TS and human DHFR.
An additional problem associated with classical antifolates
is their dependence on the carrier systems for their uptake into
the cell. The reduced folate carrier (RFC) is most important in
this regard.35 Impairment of either FPGS36 and/or RFC37-40
system(s) can lead to drug resistance. To overcome the
aforementioned problems associated with classical antifolates,
lipophilic nonclassical antifolates were also designed and
synthesized. These lipophilic nonclassical antifolates lack the
polar glutamate found in classical antifolates and hence do not
have to depend on FPGS for their inhibitory activity of the target
enzymes. Additionally, they do not have to depend on the RFC
system for active uptake into the cell system since they are
lipophilic and are passively taken up into the cells. Nolatrexed
(Figure 1) is the first nonclassical TS inhibitor to reach clinical
trials.41,42
Gangjee et al.43 have shown that nonclassical analogues of 1
with electron-withdrawing groups in the phenyl ring of the side
chain enhance human TS inhibitory activity. Extensive SAR
studies indicated that analogues with electron withdrawing
groups at both the 3′- and/or 4′-positions of the phenyl side
chain provide optimum inhibitory potency against human TS.
Nonclassical analogues such as 2 (IC50 ) 0.15 µM) and 3 (IC50
) 0.13 µM) were much more potent than the clinically used
raltitrexed and pemetrexed against human TS.43 Electron-
donating substituents such as methoxy, methyl, and bulky
substituents such as naphthyl are conducive for DHFR inhibi-
tion;11,12 hence analogues containing these substituents were also
synthesized. As indicated above for the classical 4 it was
anticipated that the 6-ethyl nonclassical analogues of 1 would
also provide dual inhibitory activity. To determine the effect
of 6-substituent homologation on human TS and human DHFR
inhibitory activity in the nonclassical analogues, compounds
5-17 were also synthesized. The synthesis and biological
activities of analogues 4-17 are presented in this report.
Chemistry
The key intermediate in the synthesis of analogues 4-17 was
2-amino-6-ethyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimi-
dine, 22 (Scheme 1), to which various side chains were
conveniently attached at the 5-position via an oxidative addition
reaction using iodine as reported previously.29,34,43 A search
revealed that compound 22 was not reported in the literature.
Gangjee et al.44 had previously reported that R-bromoketones
on cyclization with 2,6-diamino-4-oxopyrimidine, 20, give
2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine in reason-
ably good yield. Thus we envisioned that compound 22 could
be obtained in a single step from commercially available
1-bromo-2-butanone, 21, and 2,6-diamino-4-oxopyrimidine, 20.
Thus44 heating a mixture of 20 and 21 in DMF under N2 for a
period of 72 h at 40-50 °C gave 22 in a moderate 40% yield.
The reaction was found to be incomplete (TLC) even after 72
h and longer reaction times resulted in decomposition of the
product (TLC). Separation of pure 22 was tedious and difficult
and required extensive column chromatography. Higher reaction
temperatures were also found to give additional products (TLC)
further complicating the purification. Reaction of 22 with
appropriately substituted aryl thiols in a mixture of ethanol/
water (2:1) followed by addition of I2 at reflux as reported
previously29,34,43 afforded the desired compounds 5, 6, 7, 9, 11,
12, 14, 15 in 35-55% yields. In an attempt to circumvent the
harsh reaction conditions (reflux) and to further improve the
overall yield for the thiolation procedure that we have utilized
thus far, an alternative thiolation procedure was tried. This
method45 involved direct nucleophilic sulfenylation of pyrrolo-
[2,3-d]pyrimidines using hypervalent iodine reagent, phenyl-
iodine(III)bis(trifluoroacetate) (PIFA) in 1,1,1,3,3,3-hexafluoro-

1058 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Table 1. Inhibition of Isolated TS and DHFR
Gangjee et al.
of rhTS and rh DHFR. Against rhTS, analogue 4 was similar
in potency to PDDF and about 2-fold less potent than the
previously reported compound 1. This result indicates that
homologation of a 6-methyl to a 6-ethyl in pyrrolo[2,3-d]-
pyrimidines is marginally detrimental to rhTS inhibitory activity.
This decrease in potency may be due to steric hindrance between
the 6-ethyl group of 4 and Trp109 in human TS as predicted
from molecular modeling and/or due to unfavorable orientation
of the 5-position side chain for interaction with the human TS
enzyme in the presence of the 6-ethyl moiety. Interestingly 4
was 4-fold more potent than raltitrexed and a remarkable 106-
fold more than pemetrexed against rhTS. Similar results were
obtained with E. coli TS. Analogue 4 was 6-fold more potent
against human TS than E. coli TS, indicating a significant
species difference. Compound 4 was 8-fold less potent against
E. coli TS than PDDF but was more than 140-fold as potent as
pemetrexed. Compound 4 was also a potent inhibitor of human
DHFR (Table 1) and was >40-fold more potent in inhibiting
human DHFR than E. coli DHFR. Compound 4 was 5-fold more
potent than 1 and 16-fold more potent than pemetrexed as an
inhibitor of rhDHFR. This increase in activity against rhDHFR,
of 4 over 1, may be attributed to increased hydrophobic
interaction of the 6-ethyl moiety of 4 and Val115 in human
DHFR as predicted by molecular modeling. The increased
activity may also result from favorable orientation of the
5-position thioaryl side chain that is more conducive for binding
to human DHFR. Interestingly 4 was only 19-fold less potent
than MTX as an inhibitor of rhDHFR. These data suggest that
homologation of a 6-methyl to a 6-ethyl is highly conducive to
rhDHFR inhibitory activity and maintains the TS inhibitory
potency, thus affording an improved dual TS-DHFR inhibitor
over 1. The nonclassical analogues 5-17 were also evaluated
as inhibitors of TS and DHFR (Table 1). Except for 8, 13, and
14, all of the nonclassical analogues were inhibitors of human
TS with IC50 values of 0.23-26 µM. The nonclassical analogues
5 and 6 were comparable in potency to the 6-methyl homologues
, 3 and 2, respectively, and were more potent against human
TS than the clinically used classical antifolates raltitrexed and
pemetrexed. Most of the nonclassical analogues were also more
potent than pemetrexed as inhibitors of human TS. This result
indicates that homologation of a 6-methyl to a 6-ethyl in
nonclassical 6-alkylpyrrolo[2,3-d]pyrimidine maintains potent
human TS inhibitory activity. The SAR against human TS
indicates that electron withdrawing substituents (except 8 and
14) on the side-chain phenyl ring are more conducive for human
TS inhibition than electron-donating substituents. In addition
bulky substituents such as 1-naphthyl (16) and 2-naphthyl (17)
afford marginal activity against human TS. These data is
consistent with SAR studies in the previously reported 6-methyl
series. Analogues 5-17 were in general poor inhibitors of
human DHFR, E. coli TS and E. coli DHFR (Table 1).
TS IC50 (nM)
DHFR IC50 (µM)
compd
humana
E. colia
humanb
E. colic
1
54
270
nd
nd
2.1
nd
nd
0.42
21
nd
nd
>21
nd
2d
3d
4
150
130
90
230
240
2700
540
5
6
7
8
>2.3 × 10-5 (41)e >27 (0)
24000
>29 (33) nd
>32 (0)
nd
>26 (14) nd
nd
>31 (0)
>27 (0)
nd
>24 (14) nd
>31 (0)
nd
nd
nd
nd
6.6
>2.7 × 10-5 (0)
nd
nd
>3.1 × 10-5 (25) >3.1 × 10-5 (27)
9
1000
16000
2600
1100
>2.2 × 10-5 (15)
>3.2 × 10-5 (22)
>2.6 × 10-5 (13)
>2.3 × 10-5 (10)
10
11
12
13
14
15
16
17
nd
nd
nd
nd
>3.2 × 10-5 (15) >3.2 × 10-5 (5)
>2.2 × 10-5 (18) >2.0 × 10-5 (0)
26000
5600
5600
72
>2.6 × 10-5 (0)
nd
nd
nd
nd
nd
230
0.0066
>3.0 × 10-5 (15)
3.0 × 10-5
PDDFf
72
raltitrexedg
380
5700
76000
nd
pemetrexedh 9500
MTX
nd
0.022
a Kindly provided by Dr. Frank Maley, New York State Department of
Health. b Kindly provided by Dr. J. H. Freisheim, Medical College of Ohio,
Toledo, OH. c Kindly provided by Dr. R. L. Blakley, St. Jude Children’s
hospital, Memphis TN. d Data derived from ref 43; nd ) not determined.
e Numbers in parentheses indicate the % inhibition at the stated concentra-
tion. f Kindly provided by Dr. M. G. Nair, University of South Alabama.
g Kindly provided by Dr. Ann Jackman, Institute of Cancer Research, Sutton,
Surrey, UK. h Kindly provided by Dr. Chuan Shih, Eli Lilly and Co.
2-propanol [(CF3)2CHOH], a poorly nucleophilic and polar
solvent. This reaction could be carried out under mild conditions
(rt) without the necessity of heating. Thus a solution of 22 (1
equivalent), the appropriate arylthiols (2 equivalents), and PIFA
(1.5 equivalents) in (CF3)2CHOH was stirred under N2 at room
temperature for 24 h to afford target compounds 8, 10, 13, 16,
and 17 in 9-17% yields. The low yield of this reaction
discouraged further utilization of this procedure. For the
synthesis of the classical analogue 4, the required disulfide, 19,
was prepared as reported previously29 from commercially
available 4,4′-dimercaptobis(benzoic acid), 18. Reduction of 19
to the corresponding sodium salt of ethyl 4-mercaptobenzoate
was achieved using NaBH4 in EtOH.29,34 Reaction of the sodium
salt of the mercaptan with the pyrrolo[2,3-d]pyrimidine, 22, gave
ester 23 (Scheme 1) in 44% yield.29,34 The structures of 5-17
1
and 23 were established by H NMR where the disappearance
of the 5-aryl proton at 5.87 ppm, present in 22, and the presence
of the requisite protons of the aryl side chain confirmed the
required structures. Ester hydrolysis of 23 with 1N NaOH at
80 °C afforded the corresponding acid 24 in 75% yield.29,34
Peptide coupling29,34 of the acid 24 with diethyl L-glutamate
using the mixed anhydride method with isobutyl chloroformate
1
and triethylamine afforded 25 in 65% yield. The H NMR
spectrum of 25 in deuterated dimethyl sulfoxide revealed the
expected peptide NH doublet at 8.44-8.47 ppm which ex-
changed on addition of D2O. Hydrolysis of the diester 25 with
aq NaOH at room temperature, followed by acidification with
3 N HCl at lowered temperatures, afforded the desired target 4
in 80% yield.
Growth inhibitory potency of 4 was compared to that of MTX
in continuous exposure against CCRF-CEM human lympho-
blastic leukemia and a series of MTX-resistant sublines (Table
2). Compound 4 was nearly 200-fold less potent than MTX at
inhibiting the growth of CCRF-CEM cells in culture. The DHFR
overexpressing cell line R1 was <3-fold cross-resistant to 4,
suggesting that DHFR is probably not the primary target of this
analogue. The MTX-resistant transport-deficient subline R2, that
does not express functional reduced folate carrier48 (RFC), is
≈5-fold cross-resistant to 4, while it is 120-fold cross-resistant
to MTX. These data suggest that 4 utilizes the RFC as its
primary means of transport into the cell, but at high extracellular
levels it may be able to diffuse through the plasma membrane.
Biological Evaluation and Discussion
The classical and nonclassical analogues 4-17 were evaluated
as inhibitors of recombinant human (rh) and Escherichia coli
(E. coli) TS46 and DHFR47 (Table 1) and compared with PDDF
(a standard TS inhibitor), raltitrexed, pemetrexed, and meth-
otrexate (MTX). The classical analogue 4 was a potent inhibitor

Pyrrolo[2,3-d]pyrimidine Antifolates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1059
Table 2. Growth Inhibition of Parental CCRF-CEM Human Leukemia
Cells and Sublines with Single, Defined Mechanisms of MTX
Resistance during Continuous Exposure (0-120 h) Exposure to MTX
and 4d
Table 4. Activity of Folate Analogue as Substrates for Recombinant
Human FPGSa
b
substrate
Km, µM
6.5 ( 0.6
inactive e 100 µM
Vmax,rel
V
max,rel/Km
n
AMT
4
1.00
0
0.15
0
2
2
EC50, (nM)
R1a
R2b
R30dmc
a FPGS substrate activity was determined as described in Experimental
drug
CCRF-CEM
(V DHFR)
(V uptake)
(V Glun)
b
Section. Values presented are the average ( SD. Vmax,rel is calculated based
MTX
1e
4
14 ( 0
450 ( 100
2700 ( 200
565 ( 65
nd
7050 ( 250
1700 ( 100
nd
12500 ( 500
17 ( 1
810 ( 200
2900 ( 100
on the apparent Vmax of a substrate relative to the apparent Vmax of AMT
within the same experiment.
a CCRF-CEM subline resistant to MTX solely as a result of a 20-fold
increase in wild-type DHFR protein and activity.37 b CCRF-CEM subline
resistant as a result of decrease uptake of MTX.38 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.16 d Values presented are average
( range for n ) 2. e Data derived from ref 29.
or TdR (Table 3; footnote). The data (Table 3) show that for 4,
TdR alone protects against growth inhibition, while Hx alone
does not; addition of Hx to TdR does not affect the protection.
These data suggest that 4 inhibits only thymidylate synthesis,
indicating that TS is the target of this drug. Thus the one-carbon
homologated analogue has the same mechanism of action as
its methylated parent 1.
The substrate activity of 4 with recombinant human FPGS
was evaluated in vitro and, compared to that of AMT, a good
substrate for FPGS. The data (Table 4) show that 4 is a very
poor substrate for human FPGS at up to 100 µM (at 100 µM,
4 has e3% of the activity exhibited by 40 µM AMT). The lack
of FPGS substrate activity is consistent with the lack of cross-
resistance of the FPGS-deficient subline R30dm (above) to 4
and underscores the absence of a requirement for polyglutamy-
lation for the cytotoxicity of 4. These data also further
substantiate the idea that methyl and ethyl substituents in the
6-position of classical pyrrolo[2,3-d]pyrimidines and in the
7-position of quinazolines52 prevent FPGS substrate activity as
a consequence of bulk and/or hydrophobicity which is not
tolerated by human FPGS.
Table 3. Protection of CCRF-CEM Human Leukemia Cells against the
Growth Inhibitory Effects of MTX or 4 by 10 µM Hypoxanthine (Hx),
5 µM Thymidine (TdR), and Their Combination
relative growth (%)a,b
drug
no addition
5 µM TdR
10 µM Hx
Hx + TdR
MTX (40 nM)
4 (15000 nM)
10 ( 1
18 ( 0
10 ( 0
106 ( 3
11 ( 0
16 ( 1
99 ( 1
105 ( 3
a Deoxycytidine (dCyd; 10 µM) was present in all the above cultures to
prevent the inhibition of growth caused in T-cell leukemias such as CCRF-
CEM by TdR (see experimental). In typical results, dCyd alone had no
effect on CCRF-CEM growth (100 ( 3% of control) and did not protect
against growth inhibition by either drug (data not shown). Hx alone (101
( 3% of control) or in the presence of dCyd (102 ( 3% 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 (44 ( 2% of control), but TdR+dCyd was essentially not growth
inhibitory (101 ( 2% of control) and neither protected against MTX-induced
growth inhibition (Table above). Similarly, Hx+TdR+dCyd was only
slightly inhibitory to growth of CCRF-CEM (87 ( 1% of control). b 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.
Molecular Modeling
In an attempt to explain the superior human DHFR inhibitory
activity of compound 4 over its congener 1, a docking study
53-55
using GOLD 2.2
was performed. In this study, the 1.9 Å
X-ray crystal structure of human DHFR complexed with MTX
and NADPH (PDB entry 1U72)56 was chosen as the human
DHFR standard. The binding site was defined by the cocrys-
tallized ligand MTX with the default setting, which for the active
site definition, is all protein atoms within 5.0 Å of each ligand
atom plus the atoms of their associated residues.
Thus the reason for the decreased CCRF-CEM inhibitory
activity of compound 4 relative to 1 could be that it is more
poorly transported into CCRF-CEM cells. A subline (R30dm)
expressing low levels of folylpoly-γ-glutamate synthetase
(FPGS) is not cross-resistant to 4 under continuous exposure
conditions suggesting that polyglutamate forms of 4 are not
essential to its mechanisms of action.
The Goldscore-CS docking protocol53 was adopted in this
study. In this protocol, the poses obtained with the original
Goldscore function are rescored and reranked with the GOLD
implementation of the Chemscore function.53,57 To perform a
thorough and unbiased search of the conformation space, each
docking run was allowed to produce 30 poses without the option
of early termination.
To test the validity of this protocol for the human DHFR
system, MTX, the cocrystallized ligand, was first docked back
into its binding site. In this docking run, the 30 poses produced
by GOLD could be divided into two clusters on the basis of
their conformations: twelve of the poses closely resembled the
cocrystallized conformation with a heavy atom root-mean-square
deviation (RMSD) ranging from 0.68 to 1.34 Å, while the
remaining eighteen poses roughly adopted the “flipped confor-
mation” with a heavy atom RMSD varying between 1.8 and
2.1 Å. Chemscore was able to rank eight out of the twelve poses
from the first cluster as the highest ranked eight poses (Figure
4). Thus this docking protocol was considered to be suitable
for the subsequent docking runs for compounds 1 and 4.
Both compounds 1 and 4 were docked as described above
for MTX. In both cases, 29 of 30 poses, which included the
highest ranked pose, adopted the “flipped conformation”. The
Metabolite protection studies were performed to further
elucidate the mechanism of action of 4 and are shown in Table
3. At a concentration of MTX that inhibited growth of CCRF-
CEM cells by 95%, leucovorin at 0.1 µM was able to fully
protect against the effects of MTX (10 ( 4% growth inhibition).
In contrast, at a concentration of 4 that inhibited growth by 95%,
even 10 µM leucovorin only afforded marginal protection (75
( 0% growth inhibition). Although this might suggest that 4 is
not an antifolate, some validated antifolates (e.g., BW1843U8949)
are poorly protected by leucovorin. These data do suggest,
however, that leucovorin “rescue”, as used clinically with high-
dose MTX,50 would not be successful with 4. Further studies
in CCRF-CEM cells examined the ability of thymidine (TdR)
and/or hypoxanthine (Hx) to protect against growth inhibition.
These metabolites can be salvaged to produce dTTP and the
purine dNTPs required for DNA synthesis and thus bypass the
MTX blockade.51 As described in the Experimental Section, in
T-lymphoblast cell lines such as CCRF-CEM, TdR can only
be tested in the presence of deoxycytidine (dCyd), which
reverses its toxic effects; however, dCyd has no protective effect
on MTX either alone or in paired combination with either Hx

1060 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Gangjee et al.
Figure 4. Comparison of the crystallized MTX (red) and the docked MTX (green) in the binding site of human DHFR (PDB code 1U72).56
Table 5. Binding Energy Calculated by Chemscore
ently due to the extra carbon at the 6-position, which according
to the docking study interacts with the alkyl side chain of Val115
(Figure 6). Finally, since compound 4 has one extra carbon, it
has a higher degree of freedom than compound 1; thus it will
also suffer a greater entropy loss upon binding.
∆Gbinding ∆Ghbond
∆Glipo
∆S
∆Eclash
∆Eint
compd (kJ/mol) (kJ/mol) (kJ/mol) (kJ/mol) (kJ/mol) (kJ/mol)
1
4
-25.51
-27.39
-14.73 -19.82
-13.56 -22.03
10.74
11.10
2.58
0.89
1.2
1.69
In summary, homologation of a 6-methyl (compound 1) to a
6-ethyl (compound 4) in 2-amino-4-oxo-5-thiobenzoyl-6-alkyl-
pyrrolo[2,3-d]pyrimidine increases the human DHFR inhibitory
activity while maintaining the human TS inhibitory activity, thus
providing an improved dual human TS and human DHFR
inhibitor. Thus we have shown that in classical N-[4-[(2-amino-
6-alkyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-
thio]-benzoyl]-L-glutamic acid containing analogues dual TS-
DHFR activity can be obtained by 6-alkyl substitution. The fact
that homologated 6-alkyl substituents such as ethyl are well
tolerated by human TS coupled with increased DHFR inhibitory
activity indicates that further homologation of the 6-alkyl group
may be possible and will be the subject of future communica-
tions. Docking study revealed that both compounds 1 and 4
will most possibly adopt the “flipped” binding mode when
complexed with human DHFR. It also suggested that the 6-ethyl
group not only interacts with Val115 but may also control the
conformation of the 5-side chain. Compound 4 is 6-fold less
potent than the parent analogue 1 as an inhibitor of CCRF-
CEM cell growth. Cross-resistance and metabolite protection
data corroborate a similar mechanism of action for 1 and 4.
Both compounds 1 and 4 target TS directly. Interestingly
leucovorin does not afford complete protection against 1 or 4.
Neither analogue is a substrate for human FPGS, and hence
polyglutamates are not critical to their mechanism of action.
This lack of substrate activity of compound 4 can be an
important attribute to overcome potential resistance to classical
TS inhibitors such as raltitrexed and pemetrexed which depend
on intracellular polyglutamylation to exert their cytotoxic effects.
Nonclassical homologated ethyl analogues 5 and 6 were also
potent inhibitors of human TS similar to the methyl analogues
2 and 3. These nonclassical analogues were also more potent
inhibitors of human TS than pemetrexed or raltitrexed in their
monoglutamate forms.
free energy of binding for the highest ranked pose for compound
1 and 4 according to Chemscore53 is listed in Table 5. The
highest ranked poses of MTX, 1 and 4 were compared in Figure
5.
Chemscore calculates the free energy of binding with the
following formula:53
∆Gbinding ) -5.48 + ∆Ghbond + ∆Glipo + ∆S + ∆Eclash
+
∆Eint
where ∆Ghbond, ∆Glipo are hydrogen-bonding and lipophilic
interaction respectively, ∆S represents the loss of conformational
entropy of the ligand upon binding to the receptor, and ∆Eclash
and ∆Eint are the protein-ligand clash-energy and ligand-
internal-energy, respectively. Chemscore also considers accep-
tor-metal and covalently bound ligand energy terms, these terms
are not applicable to the human DHFR system and are omitted
from this formula.
According to this calculation, by homologating the 6-methyl
group to an ethyl, compound 4 lost 1.17 kJ/mol in hydrogen-
bonding, 0.36 kJ/mol in entropy, 0.49 kJ/mol in internal energy
but gained 2.2 kJ/mol in lipophilic interaction and 1.69 kJ/mol
in protein-ligand clashing energy. Overall, compound 4 gained
1.87 kJ/mol in the free energy of binding, which corresponds
to 2.12-fold difference in affinity compared to 1.
A possible explanation for the loss of hydrogen-bonding and
gain in protein-ligand clashing energy for compound 4 is that
the bulkier 6-ethyl group might restrain the 5-side-chain to some
extent and provide a conformation which affords less hydrogen-
bonding but also fewer steric clashes between compound 4 and
the active site. The fact that compound 4 has a slightly higher
ligand internal energy (0.49 kJ/mol higher) supports the above
hypothesis. The difference in hydrophilic interaction is appar-

Pyrrolo[2,3-d]pyrimidine Antifolates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1061
Figure 5. A comparison of the highest ranked poses of MTX (red), compound 1 (yellow), and 4 (green).
Figure 6. Possible interaction between compound 4 and human DHFR based on its docking result. Arg70 and Glu30 interact with the R-carboxy
group of the glutamate moiety and 2-NH2/3-NH group, respectively, and are shown in the figure as reference residues. The hydrophobic interaction
between 6-ethyl and Val115 is shown.
2-Amino-6-ethyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimi-
dine (22). A mixture of 2,6-diamino-4-oxopyrimidine (20) (4.18
g, 33.1 mmol), 1-bromo-2-butanone (21) (5.00 g, 33.1 mmol), and
DMF (20 mL) was heated between 40 and 50 °C under N2 for 72
h. At this time silica gel (30 g) was added and the solvent was
evaporated to dryness (oil pump) to afford a silica gel plug, which
was loaded on a wet (CHCl3) silica gel column and eluted initially
with CHCl3 (500 mL) and then sequentially with 500 mL of 5%,
10%, 15%, and 20% MeOH in CHCl3. Fractions that showed the
major spot at Rf 0.37 were pooled and evaporated to dryness. EtOAc
was added to the resulting residue and the mixture filtered. The
collected solid was recrystallized using methanol to afford 2.6 g
(40%) of 22 as a light pink solid; mp 251-258 °C; TLC Rf 0.37
Experimental Section
All evaporations were carried out in vacuo with a rotary
evaporator. Analytical samples were dried in vacuo (0.2 mmHg)
in an Chem-Dry vacuum-drying oven apparatus over P2O5. Melting
points were determined on a MEL-TEMP II melting point apparatus
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 the internal standard; s
) singlet, d ) doublet, dd ) doublet of doublets, t ) triplet, q )
quartet, m ) multiplet, bs ) broad singlet. The relative integrals
of peak areas agreed with those expected for the assigned structures.
Mass spectra were recorded on a VG-7070 double-focusing mass
spectrometer or in a LKB-9000 instrument in the electron ionization
(EI) mode. 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. Elemental
analyses were performed by Atlantic Microlabs Inc., Norcoss, GA.
Analytical results indicated by element symbols are within (0.4%
of calculated values. Fractional moles of water or organic solvents
frequently found in some analytical samples of antifolates could
not be removed despite 24-48 h of drying in vacuo and were
confirmed where possible by their presence in the 1H NMR
spectrum. All solvents and chemicals were purchased from Aldrich
Chemical Co. and Fisher Scientific and were used as received.
1
(CHCl3/MeOH, 5:1, with 2 drops of concentrated NH4OH); H
NMR (DMSO-d6) δ 1.13-1.17 (t, 3 H, 6-CH2CH3), 2.45-2.50 (q,
2 H, 2-CH2CH3), 5.87 (s, 1 H, 5-CH), 6.17 (s, 2 H, 2-NH2), 10.29
(s, 1 H, 3-NH), 10.90 (s, 1 H, 7-NH).
2-Amino-6-ethyl-5-[(3′,4′-dichlorophenyl)sulfanyl]-3,4-dihy-
dro-4-oxo-7H-pyrrolo[2,3-d]pyrimidine (5). To a solution of 22
(0.7 g, 3.93 mmol) in a mixture of ethanol/water (3:2, 40 mL) was
added 3,4-dichlorothiophenol (1.4 g, 7.86 mmol), and the reaction
mixture was heated to 100-110 °C. At this time I2 (2.0 g, 8.0 mmol)
was added and the heating continued with stirring for a total of 3
h. To this mixture was added excess Na2S2O7, and the mixture was
concentrated under reduced pressure. To the resulting residue was
added 5 g of silica gel and MeOH, and the solution was evaporated

1062 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Gangjee et al.
to dryness 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
1-5% MeOH in CHCl3. Fractions containing the desired spot
(TLC) were pooled and evaporated to dryness. The resulting residue
was recrystallized from MeOH, filtered, and dried to yield 0.670 g
(48%) of 5 as a pale white solid: mp 252-257 °C; TLC Rf 0.49
7.11 (s, 1 H, C6H4), 7.21-7.26 (t, 1 H, C6H4), 10.26 (s, 1 H, 3-NH),
11.50 (s, 1 H, 7-NH). Anal. Calcd for (C14H13N4OSCl‚0.2H2O) C,
H, N, S, Cl.
2-Amino-6-ethyl-5-[(3′-bromophenyl)sulfanyl]-3,4-dihydro-4-
oxo-7H-pyrrolo[2,3-d]p-yrimidine (12). Compound 12 (synthe-
sized as described for 5): yield 40%; mp 249-255 °C; TLC Rf
0.56 (CHCl3/MeOH, 5:1, with 2 drops of concentrated NH4OH);
1H NMR (DMSO-d6) δ 1.06-1.11 (t, 3 H, 6-CH2CH3), 2.56-2.61
(q, 2 H, 2-CH2CH3), 6.15 (s, 2 H, 2-NH2), 7.00-7.02 (d, 1 H, C6H4),
7.10 (s, 1 H, C6H4), 7.14-7.25 (m, 2 H, C6H4), 10.26 (s, 1 H,
3-NH), 11.50 (s, 1 H, 7-NH). Anal. Calcd for (C14H13N4OSBr‚
0.2H2O) C, H, N, S, Br.
1
(CHCl3/MeOH, 5:1, with 2 drops of concentrated NH4OH); H
NMR (DMSO-d6) δ 1.06-1.11 (t, 3 H, 6-CH2CH3), 2.56-2.63 (q,
2 H, 2-CH2CH3), 6.16 (s, 2 H, 2-NH2), 6.95-6.98 (d, 1 H, C6H3),
7.17 (s, 1 H, C6H3), 7.43-7.46 (d, 1 H, C6H3), 10.26 (s, 1 H, 3-NH),
11.53 (s, 1 H, 7-NH). Anal. Calcd for (C14H12N4SOCl2‚0.5H2O)
C, H, N, S, Cl.
2-Amino-6-ethyl-5-[(4′-nitrophenyl)sulfanyl]-3,4-dihydro-4-
oxo-7H-pyrrolo[2,3-d]pyrimidine (6). Compound 6 (synthesized
as described for 5): yield 55%; mp 215-220 °C; TLC Rf 0.45
2-Amino-6-ethyl-5-[(3′-methoxyphenyl)sulfanyl]-3,4-dihydro-
4-oxo-7H-pyrrolo[2,3-d]pyrimidine (13). Compound 13 (synthe-
sized as described for 8): yield 16%; mp 249-255 °C; TLC Rf
0.56 (CHCl3/MeOH, 5:1, with 2 drops of concentrated NH4OH);
1H NMR (DMSO-d6) δ 1.06-1.11 (t, 3 H, 6-CH2CH3), 2.56-2.61
(q, 2 H, 2-CH2CH3), 6.15 (s, 2 H, 2-NH2), 7.00-7.02 (d, 1 H, C6H4),
7.10 (s, 1 H, C6H4), 7.14-7.25 (m, 2 H, C6H4), 10.26 (s, 1 H,
3-NH), 11.50 (s, 1 H, 7-NH). Anal. Calcd for (C15H16N4O2S) MS
(EI) Calc m/z ) 316.0994 Found m/z ) 316.0990 (M+)
2-Amino-6-ethyl-5-[(3′,5′-trifluoromethylphenyl)sulfanyl]-3,4-
dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidine (14). Compound 14
(synthesized as described for 5): yield 35%; mp 290-294.5 °C;
TLC Rf 0.47 (CHCl3/MeOH, 5:1, with 2 drops of concentrated NH4-
OH); 1H NMR (DMSO-d6) δ 1.06-1.10 (t, 3 H, 6-CH2CH3), 2.57-
2.65 (q, 2 H, 2-CH2CH3), 6.19 (s, 2 H, 2-NH2), 7.61 (s, 2 H, C6H3),
7.75 (s, 1 H, C6H3), 10.34 (s, 1 H, 3-NH), 11.62 (s, 1 H, 7-NH).
Anal. Calcd for (C16H12N4OSF6) C, H, N, S, F.
2-Amino-6-ethyl-5-[(2′,6′-dimethylphenyl)sulfanyl]-3,4-dihy-
dro-4-oxo-7H-pyrrolo[2,3-d]pyrimidine (15). Compound 15 (syn-
thesized as described for 5): yield 35%; mp 290-294.5 °C; TLC
Rf 0.47 (CHCl3/MeOH, 5:1, with 2 drops of concentrated NH4-
OH); 1H NMR (DMSO-d6) δ 1.06-1.10 (t, 3 H, 6-CH2CH3), 2.57-
2.65 (q, 2 H, 2-CH2CH3), 6.19 (s, 2 H, 2-NH2), 7.61 (s, 2 H, C6H3),
7.75 (s, 1 H, C6H3), 10.34 (s, 1 H, 3-NH), 11.62 (s, 1 H, 7-NH).
Anal. Calcd for (C16H12N4OSF6) C, H, N, S, F.
2-Amino-6-ethyl-5-[(1′-naphthyl)sulfanyl]-3,4-dihydro-4-oxo-
7H-pyrrolo[2,3-d]pyrimidine (16). Compound 16 (synthesized as
described for 8): yield 10%; mp 249-255 °C; TLC Rf 0.56 (CHCl3/
MeOH, 5:1, with 2 drops of concentrated NH4OH); 1H NMR
(DMSO-d6) δ 1.04-1.09 (t, 3 H, 6-CH2CH3), 2.57-2.64 (q, 2 H,
2-CH2CH3), 6.12 (s, 2 H, 2-NH2), 6.83-6.86 (d, 1 H, C10H7), 7.29-
7.32 (d, 1 H, C10H7), 7.55-7.66 (m, 3 H, C10H7), 7.90-7.93 (d, 1
H, C10H7), 8.24-8.26 (d, 1 H, C10H7), 10.24 (s, 1 H, 3-NH), 11.51
(s, 1 H, 7-NH). Anal. Calcd for (C18H16N4OS) MS (EI) Calc m/z
) 336.1045 Found m/z ) 336.1054 (M+)
1
(CHCl3/MeOH, 5:1, with 2 drops of concentrated NH4OH); H
NMR (DMSO-d6) δ 1.06-1.10 (t, 3 H, 6-CH2CH3), 2.56-2.64 (q,
2 H, 6-CH2CH3), 5.61 (s, 2 H, 2-NH2), 7.17-7.20 (d, 2 H, C6H4),
8.03-8.06 (d, 2 H, C6H4), 10.78 (s, 1 H, 7-NH), 11.89 (s, 1 H,
3-NH). Anal. Calcd for (C14H13N5O3S‚1.0H2O) C, H, N, S.
2-Amino-6-ethyl-5-[(4′-fluorophenyl)sulfanyl]-3,4-dihydro-4-
oxo-7H-pyrrolo[2,3-d]pyrimidine (7). Compound 7 (synthesized
as described for 5): yield 50%; mp 300-304.7 °C; TLC Rf 0.44
1
(CHCl3/MeOH, 5:1, with 2 drops of concentrated NH4OH); H
NMR (DMSO-d6) δ 1.05-1.10 (t, 3 H, 6-CH2CH3), 2.57-2.64 (q,
2 H, 2-CH2CH3), 6.10 (s, 2 H, 2-NH2), 7.04-7.07 (d, 4 H, C6H4),
10.20 (s, 1 H, 3-NH), 11.41 (s, 1 H, 7-NH). Anal. Calcd for
(C14H13N4OSF‚0.5H2O) C, H, N, S, F.
2-Amino-6-ethyl-5-[(4′-chlorophenyl)sulfanyl]-3,4-dihydro-4-
oxo-7H-pyrrolo[2,3-d]pyrimidine (8). To a stirred solution of 22
(154.3 mg, 0.87 mmol) in (CF3)2CHOH (2 mL) was added 6 drops
of 4-chlorothiophenol and PIFA (613.4 mg, 1.43 mmol), and the
reaction mixture was stirred under N2 at room temperature for 24
h. Solvent was removed under reduced pressure. To the resulting
residue was added 0.5 g silica gel and MeOH (10 mL), and the
solution was evaporated to dryness 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. The resulting residue was recrystallized
from MeOH, filtered, and dried to yield 24 mg (9%) of 8 as a pale
white solid: mp 252-257 °C; TLC Rf 0.49 (CHCl3/MeOH, 5:1,
1
with 2 drops of concentrated NH4OH); H NMR (DMSO-d6) δ
1.04-1.09 (t, 3 H, 6-CH2CH3), 2.57-2.60 (q, 2 H, 2-CH2CH3),
6.14 (s, 2 H, 2-NH2), 6.98-7.01 (d, 2 H, C6H4), 7.23-7.26 (d, 2
H, C6H4), 10.24 (s, 1 H, 3-NH), 11.47 (s, 1 H, 7-NH). Anal. Calcd
for (C14H13N4OSCl) MS (EI) Calc m/z ) 320.0499 Found m/z )
320.0501 (M+)
2-Amino-6-ethyl-5-[(2′-naphthyl)sulfanyl]-3,4-dihydro-4-oxo-
7H-pyrrolo[2,3-d]pyrimidine (17). Compound 17 (synthesized as
described for 8): yield 17%; mp 249-255 °C; TLC Rf 0.56 (CHCl3/
MeOH, 5:1, with 2 drops of concentrated NH4OH); 1H NMR
(DMSO-d6) δ 1.07-1.11 (t, 3 H, 6-CH2CH3), 2.62-2.65 (q, 2 H,
2-CH2CH3), 6.13 (s, 2 H, 2-NH2), 7.22 (d, 1 H, C10H7), 7.41 (m, 2
H, C10H7), 7.68-7.81 (m, 3 H, C10H7), 8.05 (s, 1 H, C10H7), 10.22
(s, 1 H, 3-NH), 11.48 (s, 1 H, 7-NH). Anal. Calcd for (C18H16N4-
OS) MS (EI) Calc m/z ) 336.1045 Found m/z ) 336.1049 (M+)
Ethyl 4-[(2-Amino-6-ethyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-
d]pyrimidine-5-yl)sulfanyl]benzoate (23). To a solution of 4,4′-
dithiobis(benzoic acid), 18 (4.59 g, 15 mmol), in anhydrous N,N-
dimethylacetamide (25 mL) was added powdered NaHCO3 (5.04
g, 60 mmol) followed by EtI (9.36 g, 60 mmol), and the reaction
mixture was stirred under N2 for 120 h. The mixture was then
diluted with water (50 mL) and extracted with EtOAc (3 × 100
mL). The combined organic layer was washed with water (50 mL)
and brine (50 mL) and then dried (MgSO4) and filtered. The filtrate
was evaporated to a dark brown oil under reduced pressure. This
oil was chromatographed on a wet (CH2Cl2) silica gel column and
eluted with CH2Cl2. Fractions containing the desired spot (TLC)
were pooled and evaporated to dryness to afford 19 as a tan oil
that was used immediately in the next step. To a solution of 19
(2.2 g, 6 mmol) in absolute EtOH (20 mL) was added NaBH4 (0.23
2-Amino-6-ethyl-5-[(4′-bromophenyl)sulfanyl]-3,4-dihydro-4-
oxo-7H-pyrrolo[2,3-d]pyrimidine (9). Compound 9 (synthesized
as described for 5): yield 40%; mp 301-306 °C; TLC Rf 0.45
1
(CHCl3/MeOH, 5:1, with 2 drops of concentrated NH4OH); H
NMR (DMSO-d6) δ 1.07 (t, 3 H, 6-CH2CH3), 2.57 (q, 2 H, 6-CH2-
CH3), 6.11 (s, 2 H, 2-NH2), 6.91-6.94 (d, 2 H, C6H4), 7.36-7.38
(d, 2 H, C6H4), 10.22 (s, 1 H, 7-NH), 11.46 (s, 1 H, 3-NH). Anal.
Calcd for (C14H13N4OSBr‚1.0H2O) C, H, N, S,Br.
2-Amino-6-ethyl-5-[(4′-methoxyphenyl)sulfanyl]-3,4-dihydro-
4-oxo-7H-pyrrolo[2,3-d]pyrimidine (10). Compound 10 (synthe-
sized as described for 8): yield 12%; mp 215-220 °C; TLC Rf
0.45 (CHCl3/MeOH, 5:1, with 2 drops of concentrated NH4OH);
1H NMR (DMSO-d6) δ 1.07 (t, 3 H, 6-CH2CH3), 2.63 (q, 2 H,
6-CH2CH3), 3.68 (s, 3 H, 4′-OCH3), 6.08 (s, 2 H, 2-NH2), 6.81 (d,
2 H, C6H4), 7.05 (d, 2 H, C6H4), 10.18 (s, 1 H, 7-NH), 11.32 (s, 1
H, 3-NH). Anal. Calcd for (C15H16N4O2S) MS (EI) Calc m/z )
316.0994 Found m/z ) 316.0981 (M+)
2-Amino-6-ethyl-5-[(3′-chlorophenyl)sulfanyl]-3,4-dihydro-4-
oxo-7H-pyrrolo[2,3-d]pyrimidine (11). Compound 11 (synthesized
as described for 5): yield 46%; mp 282-288 °C; TLC Rf 0.51
1
(CHCl3/MeOH, 5:1, with 2 drops of concentrated NH4OH); H
NMR (DMSO-d6) δ 1.06-1.10 (t, 3 H, 6-CH2CH3), 2.59-2.61 (q,
2 H, 2-CH2CH3), 6.14 (s, 2 H, 2-NH2), 6.95-6.99 (m, 2 H, C6H4),

Pyrrolo[2,3-d]pyrimidine Antifolates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1063
g, 6 mmol) all at once and the mixture stirred under N2 for 30 min.
To this ethanolic solution of the sodium salt of ethyl 4-mercapto-
benzoate was added 22 (0.53 g, 3 mmol), the ratio of ethanol/water
was adjusted to 3/2 (40 mL), and the resulting suspension was
heated to 100 °C. At this time I2 (1.5 g, 6 mmol) was added and
the resulting solution was refluxed for 4 h, when TLC indicated
the disappearance of starting material at Rf 0.37 and the formation
of a major spot at Rf 0.50 (CHCl3/MeOH 5:1, with 2 drops of
concentrated NH4OH). To the resulting solution was added excess
Na2S2O3, and the reaction mixture was evaporated to dryness. To
the resulting residue was added silica gel (10 g) and MeOH, and
the solvent was evaporated under low pressure to give a plug which
was chromatographed on silica gel using a gradient of 1-5% MeOH
in CHCl3. Fractions containing the desired spot (TLC) were pooled
and evaporated to dryness to afford 470 mg (44%) of 23 as a light
cream solid: mp 228-234 °C; TLC Rf ) 0.50 (CHCl3/MeOH 5:1,
7.02-7.05 (d, 2 H, C6H4), 7.68-7.71 (d, 2 H, C6H4), 8.44-8.47
(d, 1 H, CONH), 10.26 (s, 1 H, 7NH), 11.50 (s, 1 H, 3NH), 12.42
(bs, 2 H, COOH). Anal. Calcd for (C20H21N5SO6‚1.5H2O) C, H,
N, S
Drugs and Chemicals. Drug solutions were standardized using
extinction coefficients. The extinction coefficients were determined
for 4 (pH 1, λmax 280 nm (23600); pH 7, λmax 274 nm (24000); pH
13, λmax 280 nm (26400)). Extinction coefficients for methotrexate
(MTX), a gift of Immunex (Seattle, WA), were from the literature.58
Aminopterin, hypoxanthine (Hx), thymidine (TdR), and deoxy-
cytidine (dCyd) were purchased from Sigma Chemical Co. (St.
Louis, MO). Calcium leucovorin (LV) was purchased from Schircks
Laboratoties (Jona, Switzerland). Other chemicals and reagents were
reagent grade or higher.
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 T-lymphoblastic leukemia cell line CCRF-CEM59
and its MTX-resistant sublines R1,60 R2,61 and R30dm25 were
cultured as described.25 R1 expresses 20-fold elevated levels of
dihydrofolate reductase (DHFR), the target enzyme of MTX. R2
has dramatically reduced MTX uptake, but normal levels of MTX-
sensitive DHFR. R30dm expresses 1% of the folylpolyglutamate
synthetase (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 poly-
glutamylation to form potent inhibitors. Growth inhibition of all
cell lines by continuous drug exposure was assayed as described.25,
49 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 remainder 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 also 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 4 that would
inhibit growth by 80-90% over a growth period of ≈72 h; horse
serum was decreased to 5% (normally 10%) in these studies to
reduce its contribution of metabolites. 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.62 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).
1
with 2 drops of concentrated NH4OH); H NMR (DMSO-d6) δ
1.05-1.10 (t, 3 H, 6-CH2CH3), 1.26-1.30 (t, 3 H, COOCH2CH3),
2.53-2.59 (q, 2 H, 6-CH2CH3), 4.25-4.29 (q, 2 H, COOCH2CH3),
6.15 (s, 2 H, 2-NH2), 7.05-7.08 (d, 2 H, C6H4), 7.75-7.78 (d, 2
H, C6H4), 10.26 (s, 1 H, 7-NH), 11.53 (s, 1 H, 7-NH).
Diethyl N-[4-[(2-Amino-6-ethyl-3,4-dihydro-4-oxo-7H-pyrrolo-
[2,3-d]pyrimidin-5-yl) thio]benzoyl]-L-glutamate (25). To a solu-
tion of 23 (400 mg, 1.1 mmol) in ethanol (40 mL) was added
aqueous 1 N NaOH, and the reaction mixture was heated at 80 °C
for 16 h. The solution was evaporated to dryness, and the sodium
salt was dissolved in water (20 mL) and carefully acidified to pH
4 by dropwise addition of 3 N HCl. The resulting suspension was
left at 0 °C for 24 h and filtered. The residue was washed with
water, ethyl acetate, and diethyl ether and dried over P2O5 at 78
°C to afford 277 mg (75%) of the free acid 24 as a light brown
solid. To a suspension of the acid 24 (250 mg, 0.75 mmol) in
anhydrous DMF (15 mL) under N2 was added triethylamine (270
µL, 1.9 mmol), and the suspension was stirred under N2 at room
temperature to form a solution. This solution was cooled to 0 °C
and isobutyl chloroformate (270 µL, 2.0 mmol) was added, followed
15 min later by diethyl L-glutamate hydrochloride (0.48 g, 2.0
mmol) and immediately followed by triethylamine (270 µL, 2.0
mmol). The reaction mixture was slowly allowed to warm to room
temperature and stirred for 24 h. The DMF was evaporated using
an oil pump at room temperature. To the resulting residue was added
25 mL of methanol and silica gel (2 g) and the suspension was
evaporated to dryness. The silica gel plug was loaded on a wet
(CHCl3) silica gel column and eluted with a gradient of 1-5%
MeOH in CHCl3. Fractions containing the desired spot (TLC) were
pooled and evaporated to dryness under vaccum to give 260 mg
(65%) of 25 as a light cream solid: mp 198-202 °C; TLC Rf )
0.52 (CHCl3/MeOH 5:1, with 2 drops of concentrated NH4OH);
1H NMR (DMSO-d6) δ 1.07-1.10 (t, 3 H, 6-CH2CH3), 1.13-1.17
(t, 6 H, COOCH2CH3), 1.14-1.21 (m, 6 H, CH2CH3), 1.97-2.08
(m, 2 H, Glu â-CH2), 2.38-2.41 (t, 2 H, Glu γ-CH2), 2.57-2.59
(q, 2 H, 6-CH2CH3), 4.00-4.10 (q, 4 H, COOCH2CH3), 4.39 (m,
1 H, Glu R-CH), 6.11 (s, 2 H, 2-NH2), 7.02-7.04 (d, 2 H, C6H4),
7.67-7.70 (d, 2 H, C6H4), 8.55-8.58 (d, 1 H, CONH), 10.22 (s, 1
H, 7NH), 11.48 (s, 1 H, 3NH). Anal. Calcd for (C24H29N5SO6‚
0.5H2O) C, H, N, S
N-[4-[(2-Amino-6-ethyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]-
pyrimidin-5-yl)thio]- benzoyl]-L-glutamic Acid (4). To a solution
of 25 (200 mg, 0.38 mmol) in ethanol (5 mL) was added 1 N NaOH
(1 mL), and the solution was stirred at 0 °C (4 h) and then at room
temperature for 24 h. The ethanol was evaporated under reduced
pressure, the residue was dissolved in water (5 mL), and the solution
was stirred for a further 24 h. The solution was then cooled in an
ice-bath and acidified carefully to pH 4.0 with dropwise addition
of 3 N HCl. This suspension was left at 5 °C for 24 h and filtered.
The residue was washed well with water and ether and dried over
P2O5 /vacuum to afford 140 mg (80%) of 4 as a light cream solid:
mp 214-220 °C; TLC Rf ) 0.60 (CHCl3/MeOH/NH4OH 3:9:1);
1H NMR (DMSO-d6) δ 1.05-1.10 (t, 3 H, 6-CH2CH3), 1.93-2.08
(m, 2 H, Glu â-CH2), 2.31-2.35 (t, 2 H, Glu γ-CH2), 2.55-2.60
(q, 2 H, 6-CH2CH3), 4.35 (m, 1 H, Glu R-CH), 6.15 (s, 2 H, 2-NH2),
Folylpolyglutamate Synthetase (FPGS) Purification and As-
say. Recombinant human cytosolic FPGS was purified and assayed
as described previously.63 Analogue 4 (88% recovery) was itself
nearly quantitatively recovered during the assay procedure, thus
ensuring that its polyglutamate products would also be quantitatively
recovered. Kinetic constants for substrates were determined by the
hyperbolic curve fitting subroutine of SigmaPlot (Jandel) or
Kaleidagraph (Synergy Software) using a g10-fold range of
substrate concentration. Activity was linear with respect to time at
the highest and lowest AMT concentrations tested. Assays contained
≈400 units of FPGS activity; one unit of FPGS catalyzes
incorporation of 1 pmol of [3H]glutamate/h.
Acknowledgment. This work was supported, in part, by
grants from the National Institutes of Health, NCI (CA89300

1064 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Gangjee et al.
(19) Jackman, A. L.; Newell, D. R.; Gibson, W.; Jodrell, D. I.; Taylor,
G. A.; Bishop, J. A.; Hughes, L. R.; Calvert, A. H. The Biochemical
Pharmacology of the Thymidylate Synthase Inhibitor 2-Desamino-
2-methyl-N10-propargyl-5,8-dideazafolic Acid (ICI 198583). Biochem.
Pharmacol. 1991, 41, 1885-1895.
(A.G.); CA43500 (JJM); CA10914 (R.L.K.)) and by a Roswell
Park Cancer Institute Core Grant CA16065. The authors thank
Mr. William Haile for performing growth inhibition studies and
FPGS activity assays.
(20) Nair, M. G.; Abraham, A.; McGuire, J. J.; Kisliuk, R. L.; Galivan,
J. Polyglutamylation as a Determinant of Cytotoxicity of Classical
Folate Analogue Inhibitors of Thymidylate Synthase and Glycinamide
Ribonucleotide Formyltransferase. Cell. Pharmacol. 1994, 1, 245-
249.
Supporting Information Available: Results from elemental
analyses. This material is available free of charge via the Internet
at http://pubs.acs.org.
(21) Bisset, G. M. F.; Pawelczak, K.; Jackman, A. L.; Calvert, A. H.;
Hughes, L. R. Syntheses and Thymidylate Synthase Inhibitory
Activity of the Poly-γ-glutamyl Conjugates of N-[5-[N-(3,4-Dihydro-
2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]- 2-thienoyl]-
L-glutamic Acid (ICI D1694) and Other Quinazoline Antifolates. J.
Med. Chem. 1992, 35, 859-866.
(22) Bisset, G. M. F.; Bavetsias, V.; Thornton, T. J.; Pawelczak, K.;
Calvert, A. H.; Hughes, L. R.; Jackman, A. L. The Synthesis and
Thymidylate Synthase Inhibitory Activity of L-γ-L-Linked Dipeptide
and L-γ-Amide Analogues of 2-Desamino-2-methy1-N10-propargyl-
5,8-dideazafolic acid (IC1 198583). J. Med. Chem. 1994, 37, 3294-
3302.
References
(1) Presented in part at the 227th American Chemical Society National
Meeting, Anaheim, CA, March 28-April 1, 2004; Abstr. MEDI-76.
(2) Kisliuk, R. L. The Biochemistry of Folates. In Folate Antagonists
as Therapeutic Agents; Sirotnak, F. M., Burchall, J. J., Ensminger,
W. D., Montgomery, J. A., Eds.; Academic Press: New York, 1984;
pp 1-68.
(3) Berman, E. M.; Werbel, L. M. The Renewed Potential for Folate
Antagonists in Contemporary Cancer Chemotherapy. J. Med. Chem.
1991, 34, 479-485.
(23) Jackman, A. L.; Kelland, L. R.; Kimbell, R.; Brown, M.; Gibson,
W.; Aherne, W.; Hardcastle, A.; Boyle, F. T. Mechanisms of
Acquired Resistance to the Quinazoline Thymidylate Synthase
Inhibitor ZD1694 (Tomudex) in One Mouse and Three Human Cell
Lines. Br. J. Cancer. 1995, 71, 914-924.
(24) Barakat, R. R.; Li, W. W.; Lovelace, C.; Bertino, J. R. Intrinsic
Resistance of Squamous Cell Carcinoma Cell Lines as a Result of
Decreased Accumulation of Intracellular Methotrexate Polyglutamates.
Gynecol. Oncol. 1993, 51, 54-60.
(25) McCloskey, D. E.; McGuire, J. J.; Russell, C. A.; Rowan, B. G.;
Bertino, J. R.; Pizzorno, G.; Mini, E. Decreased Folylpolyglutamate
Synthetase Activity as a Mechanism of Methotrexate Resistance in
CCRF-CEM Human Leukemia Sublines. J. Biol. Chem. 1991, 266,
6181-6187.
(26) Braakhuis, B. J.; Jansen, G.; Noordhius, P.; Kegel, A.; Peters, G. J.
Importance of Pharmacodynamics in the In Vitro Antiproliferative
Activity of the Antifolates Methotrexate and 10-EDAM Against Head
and Neck Squamous Cell Carcinoma. Biochem. Pharmacol. 1993,
46, 2155-2161.
(27) Jackman, A. L.; Kimbell, R.; Aherne, G. W.; Brunton, L.; Jansen,
G.; Stephens, T. C.; Smith, M. N.; Wardleworth, J. M.; Boyle, F. T.
Cellular Pharmacology and In ViVo Activity of a New Anticancer
Agent, ZD9331: A Water-soluble, Nonpolyglutamatable, Quinazo-
line-Based Inhibitor of Thymidylate Synthase. Clin. Cancer Res.
1997, 3, 911-921.
(28) Benepal, T. S.; Judson, I. ZD9331: Discovery to Clinical Develop-
ment. Anti-Cancer Drugs 2005, 16, 1-9.
(29) Gangjee, A.; Devraj, R.; McGuire, J. J.; Kisliuk, R. L. 5-Arylthio
Substituted 2-Amino-4-oxo-6-methylpyrrolo[2,3-d]pyrimidine Anti-
folates as Thymidylate Synthase Inhibitors and Antitumor Agents.
J. Med. Chem. 1995, 38, 4495-4502.
(4) Carreras, C. W.; Santi, D. V. Catalytic Mechanism and Structure of
Thymidylate Synthase. Ann. ReV. Biochem. 1995, 64, 721-762.
(5) Blakley, R. L. Eukaryotic Dihydrofolate Reductase. AdV. Enzymol.
Mol. Biol. 1995, 70, 23-102.
(6) MacKenzie, R. E. Biogenesis and Interconversion of Substituted
Tetrahydrofolates. In Folates and Pterins Chemistry and Biochem-
istry; Blakley, R. L., Benkovic, S. J., Eds.; Wiley: New York, 1984;
Vol. I, pp 255-306.
(7) Takimoto, C. H. Antifolates in Clinical Development. Semin. Oncol.
1997, 24, S18-40-S18-51.
(8) Jackman, A. L.; Taylor, G. A.; Gibson, W.; Kimbell, R.; Brown,
M.; Calvert, A. H.; Judson, I. R.; Hughes, L. R. ICI D1694, A
Quinazoline Antifolate Thymidylate Synthase Inhibitor That is a
Potent Inhibitor of L1210 Tumour Cell Growth In Vitro and In
ViVo: A New Agent for Clinical Study. Cancer Res. 1991, 51, 5579-
5586.
(9) Taylor, E. C.; Kuhnt, D.; Shih, C.; Rinzel, S. M.; Grindey, G. B.;
Barredo, J.; Jannatipour, M.; Moran, R. A Dideazatetrahydrofolate
Analogue Lacking a Chiral Center at C-6, N-[4-[2-(2-Amino-3,4-
dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-y1)ethylbenzoyl]-L-
glutamic Acid, Is an Inhibitor of Thymidylate Synthase. J. Med.
Chem. 1992, 35, 4450-4454.
(10) Bertino, J. R.; Kamen, B.; Romanini, A. Folate Antagonists. In Cancer
Medicine; Holland, J. F., Frei, E., Bast, R. C., Kufe, D. W., Morton,
D. L., Weichselbaum, R. R., Eds.; Williams & Wilkins; Baltimore,
MD, 1997; Vol. 1, pp 907-921.
(11) Rosowsky, A. Chemistry and Biological Activity of Antifolates. In
Progress in Medicinal Chemistry; Ellis, G. P., West, G. B., Eds.;
Elsevier Science: New York, 1989; Vol. 26, pp 1-252.
(12) Gangjee, A.; Elzein, E.; Kothare, M.; Vasudevan, A. Classical and
Nonclassical Antifolates as Potential Antitumor, Antipneumocystis
and Antitoxoplasma Agents. Curr. Pharm. Des. 1996, 2, 263-280.
(13) Mendelsohn, L. G.; Shih, C.; Chen, V. J.; Habeck, L. L.; Gates, S.
B.; Shackelford, K. A. Enzyme Inhibition, Polyglutamation, and the
Effect of LY231514 (MTA) on Purine Biosynthesis. Semin. Oncol.
1999, 26, 42-47.
(30) Tripos Inc., 1699 South Hanley Road, St. Louis, MO, 63144.
(31) Gangjee, A.; Devraj, R.; McGuire, J. J.; Kisliuk, R. L.; Queener, S.
F.; Barrows, L. R. Classical and Nonclassical Furo[2,3-d]pyrimidines
as Novel Antifolates: Synthesis and Biological Activities. J. Med.
Chem. 1994, 37, 1169-1176.
(32) Gangjee, A.; Devraj, R.; McGuire, J. J.; Kisliuk, R. L. Effect of Bridge
Region Variation on Antifolate and Antitumor Activity of Classical
5-substituted 2,4-diaminofuro[2,3-d]pyrimidines. J. Med. Chem. 1995,
38, 3798-3805.
(33) Gangjee, A.; Lin, X.; McGuire, J. J.; Kisliuk, R. L. Synthesis of
N-{4-[(2,4-diamino-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimi-
din-6-yl)thio]benzoyl}-L-glutamic Acid and N-{4-[(2-amino-4-oxo-
5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-
L-glutamic Acid as Dual Inhibitors of Dihydrofolate Reductase and
Thymidylate Synthase, and as Potential Antitumor Agents. J. Med.
Chem. 2005, 48, 7215-7222.
(34) Gangjee, A.; Jain, H. D.; McGuire, J. J.; Kisliuk, R. L. Benzoyl Ring
Halogenated Classical 2-Amino-6-methyl-3,4-dihydro-4-oxo-5-sub-
stitutedthiobenzoyl-7H-pyrrolo[2,3-d]pyrimidine Antifolates as In-
hibitors of Thymidylate Synthase and as Antitumor Agents. J. Med.
Chem. 2004, 47, 6730-6739.
(14) Shih, C.; Chen, V. J.; Gossett, L. S.; Gates, S. B.; Mackellar, W. C.;
Habeck, L. L.; Shackelford, K. A.; Mendelsohn, L. G.; Soose, D. J.;
et al. LY231514, a Pyrrolo[2,3-d]pyrimidine-based Antifolate that
Inhibits Multiple Folate-requiring Enzymes N-[4-[2-(2-amino-3,4-
dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-ethyl]-benzoyl]-L-
glutamic Acid (LY231514) is a novel Pyrrolo[2,3-d]pyrimidine-based
Antifolate Currently Undergoing Extensive Phase II Clinical Trials.
Cancer Res. 1997, 57, 1116-1123.
(15) Schultz, R. M.; Patel, V. F.; Worzalla, J. F.; Shih, C. Role of
Thymidylate Synthase in the Antitumor Activity of the Multitargeted
Antifolate, LY231514. Anticancer Res. 1999, 19, 437-443.
(16) Gangjee, A.; Yu, J.; McGuire, J. J.; Cody, V.; Galitsky, N.; Kisliuk,
R. L.; Queener, S. F. Design, Synthesis, and X-ray Crystal Structure
of a Potent Dual Inhibitor of Thymidylate Synthase and Dihydrofolate
Reductase as an Antitumor Agent. J. Med. Chem. 2000, 43, 3837-
3851.
(17) Kuyper, L. F.; Garvey, J. M.; Baccanari, D. P.; Champness, J. N.;
Stammers, D. K.; Beddell, C. R. Pyrrolo[2,3-d]pyrimidines and
Pyrido[2,3-d]pyrimidines as Conformationally Restricted Analogs of
the Antibacterial Agent Trimethoprim. Bioorg. Med. Chem. 1996,
4, 593-602.
(18) Sikora, E.; Jackman, A. L.; Newell, D. F.; Calvert, A. H. Formation
and Retention and Biological Activity of N10-Propargyl-5,8-dide-
azafolic Acid (CB 3717) Polyglutamates in L1210 Cells. In Vitro.
Biochem. Pharmacol. 1988, 37, 4047-4054.
(35) Cao, W.; Matherly, L. H. Structural Determinants of Folate and
Antifolate Membrane Transport by the Reduced Folate Carrier. In
Drug Metabolism and Transport; Lash, L. H., Ed.; Humana Press:
Totowa, NJ, 2005; pp 291-318.
(36) Liani, E.; Rothem, L.; Bunni, M. A.; Smith, C. A.; Jansen, G.;
Assaraf, Y. G. Loss of Folylpoly-γ-glutamate Synthetase Activity is
a Dominant Mechanism of Resistance to Polyglutamylation-depend-
ent Novel Antifolates in Multiple Human Leukemia Sublines. Int. J.
Cancer 2003, 103, 587-599.

Pyrrolo[2,3-d]pyrimidine Antifolates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1065
(37) Fry, D. W.; Jackson, R. C. Membrane Transport Alterations as a
Mechanism of Resistance to Anticancer Agents. Cancer SurV. 1986,
5, 47-49.
(38) Schornagel, J. H.; Pinard, M. F.; Westerhof, G. R.; Kathmann, I.;
Molthoff, C. F. M.; Jolivet, J.; Jansen, G. Functional Aspects of
Membrane Folate Receptors expressed in Human Breast Cancer Lines
with Inherent and Acquired Transport-Related Resistance to Metho-
trexate. Proc. Am. Assoc. Cancer Res. 1994, 35, 302.
(39) Nair, M. G.; Galivan, J.; Maley, F.; Kisliuk, R. L.; Ferone, R.
Transport, Inhibition of Tumor Cell Growth and Unambiguous
Synthesis of 2-Desamino-2-methyl-N10-propargyl-5,8-dideazafolate
(DMPDDF) and Related Compounds. Proc. Am. Assoc. Cancer Res.
1989, 30, 476.
(40) Jackman, A. L.; Gibson, W.; Brown, M.; Kimbell, R.; Boyle, F. T.
The Role of the Reduced Folate Carrier and Metabolism to Intra-
cellular Polyglutamates for the Activity of ICI D1694. AdV. Exp.
Med. Biol. 1993, 339, 265-276.
(41) Webber, S. E.; Bleckman, T. M.; Attard, J.; Deal, J. G.; Katherdekar,
V.; Welsh, K. M.; Webber, S.; Janson, C. A.; Matthews, D. A.; Smith,
W. W.; Freer, S. T.; Jordan, S. R.; Bacquet, R. J.; Howland, E. F.;
Booth, C. J. L.; Ward, R. W.; Hermann, S. M.; White, J.; Morse, C.
A.; Hilliard, J. A.; Bartlett, C. A. Design of Thymidylate Synthase
Inhibitors Using Protein Crystal Structures: The Synthesis and
Biological Evaluation of a Novel Class of 5-Substituted Quinazolines.
J. Med. Chem. 1993, 36, 733-746.
(42) Hughes, A.; Calvert, A. H. Preclinical and Clinical Studies with the
Novel Thymidylate Synthase Inhibitor Nolatrexed Dihydrochloride
(Thymitaq, AG337). In Antifolate Drugs in Cancer Therapy; Jack-
man, A. L., Ed.; Humana Press: Totowa, NJ, 1999; pp 229-241.
(43) Gangjee A.; Mavandadi F.; Kisliuk R. L.; McGuire J. J.; Queener S.
F. 2-Amino-4-oxo-5-substituted-pyrrolo[2,3-d]pyrimidines as Non-
classical Antifolate Inhibitors of Thymidylate Synthase. J. Med.
Chem. 1996, 39, 4563-4568.
(44) Gangjee, A.; Yang, J.; Ihnat, M. A.; Kamat, S. Antiangiogenic and
antitumor agents. Design, synthesis, and evaluation of novel 2-amino-
4-(3-bromoanilino)-6-benzyl substituted pyrrolo[2,3-d]pyrimidines as
inhibitors of receptor tyrosine kinases. Bioorg. Med. Chem. 2003,
11, 5155-5170.
(45) Kita, Y.; Takada, T.; Mihara, S.; Whelan, B. A.; Tohma, H. Novel
and Direct Nucleophilic Sulfenylation and Thiocyanation of Phenol
Ethers Using a Hypervalent Iodine(III) Reagent. J. Org. Chem. 1995,
60, 7144-7148.
(51) Hakala, M. T.; Taylor, E. The Ability of Purine and Thymine
Derivatives and of Glycine to Support the Growth of Mammalian
Cells in Culture. J. Biol. Chem. 1959, 234, 126-128.
(52) Marsham, P. R.; Jackman, A. L.; Barker, A. J.; Boyle, F. T.; Pegg,
S. J.; Wardleworth, J. M.; Kimbell, R.; O’Connor, B. M.; Calvert,
A. H.; Hughes, L. R. Quinazoline Antifolate Thymidylate Synthase
Inhibitors: Replacement of Glutamic Acid in the C2-Methyl Series.
J. Med. Chem. 1995, 38, 994-1004.
(53) Verdonk, M. L.; Cole, J. C.; Hartshorn, M. J.; Murray, C. W.; Taylor,
R. D. Improved Protein-Ligand Docking Using GOLD. Proteins:
Struct., Funct., Genet. 2003, 52, 609-623.
(54) Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R.
Development and Validation of a Genetic Algorithm for Flexible
Docking. J. Mol. Biol. 1997, 267, 727-748.
(55) Jones, G.; Willett, P.; Glen, R. C. Molecular Recognition of a
Receptor Sites Using a Genetic Algorithm with a Description of
Desolvation. J. Mol. Biol. 1995, 245, 43-53.
(56) Cody, V.; Luft, J. R.; Pangborn, W. Understanding the Role of Leu22
Variants in Methotrexate Resistance: Comparison of Wild-Type and
Leu22Arg Variant Mouse and Human Dihydrofolate Reductase
Ternary Crystal Complexes with Methotrexate and NADPH. Acta
Crystallogr., Sect. D 2005, 61, 147-155.
(57) Eldridge, M. D.; Murray, C. W.; Auton, T. R.; Paolini, G. V.; Mee,
R. P. Empirical Scoring Functions: I. The Development of a Fast
Empirical Scoring Function to Estimate the Binding Affinity of
Ligands in Receptor Complexes. J. Comput.-Aided Mol. Des. 1997,
11, 425-445.
(58) Blakley, R. L. The Biochemistry of Folic acid and Related Pteridines;
Elsevier: Amsterdam, 1969; p 569.
(59) Foley, G. F.; Lazarus, H.; Farber, S.; Uzman, B. G.; Boone, B. A.;
McCarthy, R. E. Continuous Culture of Lymphoblasts from Peripheral
Blood of a Child with Acute Leukemia. Cancer 1965, 18, 522-529.
(60) Mini, E.; Srimatkandada, S.; Medina, W. D.; Moroson, B. A.;
Carman, M. D.; Bertino, J. R. Molecular and Karyological Analysis
of Methotrxate-Resistant and -Sensitive Human Leukemic CCRF-
CEM Cells. Cancer Res. 1985, 45, 317-325.
(61) Rosowsky, A.; Lazarus, H.; Yuan, G. C.; Beltz, W. R.; Mangini, L.;
Abelson, H. T.; Modest, E. J.; Frei, E.; III Effects of Methotrexate
Esters and Other Lipophilic Antifolates on Methotrexate-Resistant
Human Leukemic Lymphoblasts. Biochem. Pharmacol. 1980, 29,
648-652.
(62) Grindey, G. B.; Wang, M. C.; Kinahan, J. J. Thymidine Induced
Perturbations in Ribonucleoside Triphosphate Pools in Human
Leukemic CCRF-CEM Cells. Mol. Pharmacol. 1979, 16, 601-606
(63) Gangjee, A.; Yu, J.; Kisliuk, R. L.; Haile, W. H.; Sobrero, G.;
McGuire, J. J. Design, Synthesis, and Biological Activities of
Classical N-{4-[2-(2-Amino-4-ethylpyrrolo[2,3-d]pyrimidin-5-yl)eth-
yl]benzoyl}-L-glutamic Acid and Its 6-Methyl Derivative as Potential
Dual Inhibitors of Thymidylate Synthase and Dihydrofolate Reductase
and as Potential Antitumor Agents. J. Med. Chem. 2003, 46, 591-
600.
(46) Wahba, A. J.; Friedkin, M. The Enzymatic Synthesis of Thymidylate.
Early Steps in the Purification of Thymidylate Synthetase of
Escherichia coli. J. Biol. Chem. 1962, 237, 3794-3801.
(47) Kisliuk, R. L.; Strumpf, D.; Gaumont, Y.; Leary, R. P.; Plante, L.
Diastereoisomers of 5,10-Methylene-5,6,7,8-tetrahydropteroyl- D-
glutamic Acid. J. Med. Chem. 1977, 20, 1531-1533.
(48) Wong, S. C.; Zhang, L.; Witt, T. L.; Proefke, S. A.; Bhushan, A.;
Matherly, L. H. Impaired Membrane Transport in Methotrexate-
Resistant CCRF-CEM Cells Involves Early Translation Termination
and Increased Turnover of a Mutant Reduced Folate Carrier. J. Biol.
Chem. 1999, 274, 10388-10394
(49) McGuire, J. J.; Magee, K. J.; Russell, C. A.; Canestrari, J. M.
Thymidylate Synthase as a Target for Growth Inhibition in Metho-
trexate Sensitive and -Resistant Human Head and Neck Cancer and
Human Leukemia Cell Lines. Oncol. Res. 1997, 9, 139-147.
(50) Bertino, J. R. Rescue Techniques in Cancer Chemotherapy: Use of
Leucovorin and Other Rescue Agents after Methotrexate Treatment.
Semin. Oncol. 1977, 4, 203-216.
(64) Sayre, P. H.; Finer-Moore, J. S.; Fritz, T. A.; Biermann, D.; Gates,
S. B.; MacKellar, W. C.; Patel, V. F.; Stroud, R. M. Multi-targeted
Antifolates Aimed at Avoiding Drug Resistance Form Covalent
Closed Inhibitory Complexes with Human and Escherichia coli
Thymidylate Synthases. J. Mol. Biol. 2001, 313, 813-829.
JM058276A