Synthesis, antifolate, and antitumor activities of classical and nonclassical 2-amino-4-oxo-5-substituted-pyrrolo[2,3-d]pyrimidines

Article abstract of DOI:10.1021/jm0100382

Classical and nonclassical isosteric C8-N9 bridged analogues of the multitargeted antifolate LY231514 were synthesized as inhibitors of thymidylate synthase (TS), dihydrofolate reductase (DHFR), and as antitumor and antiopportunistic infection agents. The

Full text of DOI:10.1021/jm0100382

J . Med. Chem. 2001, 44, 1993-2003
1993
Syn th esis, An tifola te, a n d An titu m or Activities of Cla ssica l a n d Non cla ssica l
2-Am in o-4-oxo-5-su bstitu ted -p yr r olo[2,3-d ]p yr im id in es
Aleem Gangjee,*,† Anup Vidwans,†,‡ Elfatih Elzein,†,§ J ohn J . McGuire,|| Sherry F. Queener, and Roy L. Kisliuk#
Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, Pennsylvania,
DuPont Company, Wilmington, Delaware, CV Therapeutics, Palo Alto, California, Roswell Park Cancer Institute, Grace Cancer
Drug Center, Buffalo, New York, Department of Pharmacology and Toxicology, School of Medicine, Indiana University,
Indianapolis, Indiana, and Department of Biochemistry, Tufts University, Boston, Massachusetts
Received J anuary 25, 2001
Classical and nonclassical isosteric C8-N9 bridged analogues of the multitargeted antifolate
LY231514 were synthesized as inhibitors of thymidylate synthase (TS), dihydrofolate reductase
(DHFR), and as antitumor and antiopportunistic infection agents. The syntheses of the
analogues were accomplished by reductive amination of the appropriate anilines with 2-amino-
4-oxo-5-cyanopyrrolo[2,3-d]pyrimidine (28) followed by saponification of the ethyl esters, for
the classical analogue 6. The N9-methyl analogues were obtained from the N9-H precursors
by reductive methylation. In general, the nonclassical compounds 7-17 were similar in potency
to TMP against Toxoplasma gondii DHFR, with selectivity ratios greater than 38 and 21 for
11 and 16, respectively. These compounds were poor inhibitors of Pneumocystis carinii DHFR
and rat liver DHFR. The nonclassical analogues were also inactive against TS. The classical
analogue 6 was a marginal inhibitor of isolated human TS (IC50 ) 46 µM) and of human DHFR
(IC50 ) 10 µM), however, it was a potent inhibitor of the growth of two human head and neck
squamous cell carcinoma cell lines and of CCRF-CEM human lymphoblastic leukemia cells
in culture and was similar to LY231514 against ZR-75-1 human breast carcinoma cell line.
Evaluation of 6 against MTX-resistant sublines indicated that DHFR is not the major target
of 6. Metabolite protection studies of the growth inhibitory activity of 6 suggest that TS is a
major target of this drug and that polyglutamyl forms of 6 may serve as the intracellular TS
inhibitors. These studies also suggest that 6 has a site of action in addition to sites in the
folate pathway.
Thymidylate synthase (TS)1 catalyzes a two-step
conversion of deoxyuridine monophosphate (dUMP) to
deoxythymidine monophosphate (dTMP) utilizing the
cofactor 5,10-methylenetetrahydrofolate (5,10-CH2FH4).
This enzyme is unique among those which utilize
tetrahydrofolate (FH4) cofactors in that 5,10-CH2FH4
serves the dual function of both a one-carbon donor and
a reductant, by concomitant transfer of its methylene
group and the 6H atom to the 5-position of dUMP. In
the process, the cofactor is oxidized to 7,8-dihydrofolate
(FH2). The enzyme dihydrofolate reductase (DHFR)
catalyzes the NADPH-dependent reduction of 7,8-dihy-
drofolate to regenerate FH4.2 This is the sole de novo
source of dTMP, and hence, TS and DHFR play a pivotal
role in DNA biosynthesis and cell replication. Antifolate
inhibitors of TS and DHFR have found clinical utility
as antitumor, antibacterial, and antiprotozoan agents.3
Usually a 2,4-diamino-substituted pyrimidine ring is
considered important for potent DHFR inhibitory activ-
ity, while a 2-substituted-4-oxopyrimidine ring is con-
sidered important for potent TS inhibitory activity.3-5
Several 5-substituted-2,4-diaminopyrrolo[2,3-d]pyrim-
idines,5-7 including 1, 2, and the 2-amino-4-oxopyrrolo-
[2,3-d]pyrimidine8 (3, LY231514) have shown excellent
antitumor activity; compound 3 has shown significant
promise as an antitumor agent in phase III clinical
trials. The strong DHFR inhibitory potency of 1 and 2
was in accord with that reported for 2,4-diamino 6-6
fused ring system analogues such as methotrexate
(MTX, 4). However, the potent DHFR inhibitory activity
of 3 (which contains a 2-amino-4-oxopyrimidine ring)
in addition to its TS inhibition was unusual, except if
one considers a possible alternate mode of DHFR
binding, in which the pyrrole NH of compound 3 mimics
the 4-NH2 of the 2,4-diaminopyrimidine ring system of
MTX and the pyrrolo[2,3-d]pyrimidine ring binds in this
alternate mode to DHFR as shown in Figure 1. Gangjee
et al.9 have recently demonstrated that a 4-methyl
analogue of 3 does bind in the alternate mode to DHFR.
5-Deazafolic acid 5 synthesized by Piper et al.10 is a
2-amino-4-oxo classical antifolate that contains a C9-
N10 bridge and has appreciable DHFR inhibitory
potency. It was thus of interest to determine if replace-
ment of the C8-C9 bridge of LY231514 with a C8-N9
bridge would afford a useful antitumor agent with dual
TS-DHFR inhibitory effects. Thus, we synthesized the
classical analogue 6. Dual inhibitors could act at two
different sites (TS and DHFR) and could combine dual
mechanisms of action in a single agent without the
* Corresponding author. Tel: (412) 396-6070. Fax: (412) 396-5593.
E-mail: gangjee@duq.edu.
† Duquesne University.
‡ DuPont Company.
§ CV Therapeutics.
|| Grace Cancer Drug Center.
Indiana University.
#
Tufts University.
10.1021/jm0100382 CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/28/2001

1994 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Gangjee et al.
The problem of tumor resistance to classical antifolates,
which is, in part, a result of low or defective FPGS
activity, is also a limitation of those classical antifolates
that depend on polyglutamylation for their antitumor
effects.23,24 To overcome these disadvantages associated
with classical antifolates, lipophilic nonclassical anti-
folates that lack the glutamate moiety have been
developed.25-28 Thus, in addition to the antitumor
activity of 6, we were also interested in the antitumor
activity of the nonclassical analogues 7-17, which could
also function as dual inhibitors.
Another facet of our interest in nonclassical dual TS-
DHFR inhibitors is the prevalence of opportunistic
infections in immune-compromized patients such as
those receiving organ transplants or cancer chemo-
therapy and those with AIDS.29,30 Two of the most
prevalent opportunistic infections which are the major
cause of mortality in patients with AIDS are caused by
Pneumocystis carinii (pc)31 and Toxoplasma gondii
(tg).32 Several nonclassical 2,4-diaminopyrido[2,3-d]-
pyrimidines,33 quinazolines,34 pyrrolo[2,3-d]pyrimi-
dines,5,8,33-35 and furo[2,3-d]pyrimidines36 have been
evaluated as inhibitors of P. carinii and T. gondii
DHFR. Some of these have been found to be selective
for DHFR from these pathogens. Thus, we were also
interested in evaluating the nonclassical compounds
7-17 as inhibitors of pcDHFR and tgDHFR. The phenyl
ring substitutions in 7-17 are based on similar substi-
tutions that have provided potent and/or selective
agents.33-36 The nonclassical analogues 7-17 were
anticipated to inhibit DHFR and/or TS from P. carinii
and/or T. gondii, and perhaps provide selective inhibi-
tors against these pathogens. P. carinii and T. gondii
lack the transport system(s) required for classical anti-
folates; however, the lipophilic nonclassical compounds
7-17 were anticipated to gain access to the pathogenic
cells by passive diffusion.
Ch em istr y
F igu r e 1.
The syntheses of analogues 6-17 were initially
envisioned through the formation of the key intermedi-
ate ester 2137 (Scheme 1). Reaction of methyl formate
18 with methyl chloroacetate 19 in toluene using
NaOCH3 as a base afforded the chloroaldehyde 20 in
quantitative yield. Compound 20 was cyclocondensed
with 2,4-diamino-6-hydroxypyrimidine 27 and sodium
acetate in water at 100 °C.38 1H NMR indicated the
regiospecific formation of the methyl 2-amino-4-oxopyr-
rolo[2,3-d]pyrimidine-5-carboxylate (21) with no detect-
able formation of the corresponding 6-substituted-
furo[2,3-d]pyrimidine 22. The 1H NMR spectrum showed
the characteristic downfield lactam NH signal at 11.64
ppm and the pyrrole NH signal at 10.36 ppm. In
addition, the presence of only one amino group at 6.22
ppm and the absence of the pyrimidine 5-proton be-
tween 5 and 6 ppm confirmed the structure of the
desired intermediate 21. The ester 21 was insoluble in
DMF, methanol, or ethanol at room temperature. To
improve the solubility of 21 and to enhance the carbonyl
character of the ester (hence improving the chances of
its reduction), the pyrrole nitrogen was protected with
a Boc group38 to afford 23. Attempts to reduce 23 to the
alcohol using LiAlH4 or to the aldehyde using DIBAL
were unsuccessful.
pharmacokinetic disadvantages of two separate agents.
Synergism of the two separate antifolates which inhibit
TS and DHFR has been demonstrated in growth inhibi-
tory studies against Lactobacillus casei,11,12 rat hepato-
ma cells,13,14 and human lymphoma cells.12,15,16
A major drawback of classical antifolates is that they
require transport into cells via the reduced folate uptake
systems, which when impaired can lead to drug resis-
tance.17,18 In addition, the antitumor activities of several
classical antifolates, particularly TS inhibitors, are in
part, determined by their ability to function as sub-
strates for the enzyme folylpolyglutamate synthetase
(FPGS).19,20 Polyglutamylation catalyzed by FPGS pro-
duces long-acting, noneffluxing poly-γ-glutamates of
classical antifolates that lead to high intracellular
concentrations of these antitumor agents. Further,
polyglutamylation often provides a significant increase
in inhibitory activity against TS compared to the mono-
glutamate, as has been shown for 3 (LY231514).21
Though polyglutamylation for many antifolates is nec-
essary for cytotoxcicity to tumor cells, it has also been
implicated in adverse effects and toxicity to normal
tissues due to retention of polyglutamate metabolites.22

2-Amino-4-oxo-5-substituted-pyrrolo[2,3-d]pyrimidines
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1995
Sch em e 1a
a
(a) toluene, NaOMe; (b) water, sodium acetate, reflux; (c) DMF, triethylamine, (Boc)2O; (d) 15% acetic acid/DMF, Ni/H2/50 psi.
Sch em e 2a
a
(a) formic acid, Ni/H2/50 psi; (b) NaOH/MeOH.
Since the reduction of ester 21 was unsuccessful, an
alternate route to compounds 6-17 was designed. The
use of the 2-amino-4-oxo-5-cyanopyrrolo[2,3-d]pyrimi-
dine 28 (Scheme 1) instead of the ester 21 appeared to
be an attractive method, since similar nitriles have been
reductively aminated with a variety of anilines in 6-6
ring systems. Reductive amination of 28 with the
appropriate anilines was expected to afford the target
analogues. Thus formylation37 of chloroacetonitrile 25
with methyl formate in toluene using NaOCH3 as base
afforded chloro(formyl)acetonitrile 26, which was used
without further purification. Condensation38 of 26 with
27 in water and sodium acetate at 100 °C afforded
regiospecifically the key 5-cyanopyrrolo[2,3-d]pyrimi-
dine (28), without the formation of the corresponding
6-cyanofuro[2,3-d]pyrimidine (29). The 1H NMR in
DMSO-d6 of 28 exhibited the 2-amino signal at 6.37 ppm
(exchangeable with D2O), the lactam proton signal at
11.97 ppm, the pyrrole NH signal at 10.69 ppm and the
6-H signal as a doublet at 7.59 ppm, which confirmed
the structure of 28.
Reductive amination of 28 (Scheme 2) in formic acid
with ethyl p-aminobenzoyl-L-glutamate in the presence
of Raney nickel and hydrogen at 50 psi afforded, after
1
saponification, the target 6. The H NMR spectrum of
6 in DMSO-d6 showed the â- and γ-CH2 signals at 1.92
and 2.29 ppm, respectively, the 8-CH2 and the 9-NH
signals at 4.31 ppm as a multiplet, the 2-amino signal
(exchanges with D2O) at 6.07 ppm, and the R-CH signal
of the glutamic acid at 6.45 ppm. The spectrum also
showed the 6-CH signal at 6.49 ppm, the aromatic
proton signals as two sets of doublets at 6.65 and 7.65
ppm, and the characteristic lactam NH and pyrrole NH
at 10.81 and 10.28 ppm, respectively. For the nonclas-
sical target compounds, reductive amination (Scheme
1) of 28 in 15% acetic acid/DMF with the appropriately

1996 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Gangjee et al.
Sch em e 3a
Ta ble 2. Inhibitory Concentration (IC50, M) against Isolated
DHFR39 and TS40
DHFR
compound
reca
rtgb
rhc
6
2.3 × 10-4
2.3 × 10-4
1.0 × 10-8
1.8 × 10-8
4.4 × 10-9
8.0 × 10-7
2.3 × 10-7
3.7 × 10-6
5.4 × 10-9
1.1 × 10-8
1.0 × 10-5
2.3 × 10-6
2.0 × 10-4
9.0 × 10-8
6.6 × 10-8
LY231514f
trimethoprim
trimetrexate
methotrexate
a
(a) HCHO, NaCNBH3, CH3CN, HCl.
TS
compound
rece
rpcd
rhd
Ta ble 1. Inhibitory Concentrations (IC50 µM) and Selectivity
Ratios of Analogues 7-17 against P. carinii (pc) DHFR, T.
gondii (tg) DHFR, Rat Liver (rl) DHFR
6
>1.2 × 10-4 (23)i
5.8 × 10-5
4.6 × 10-5
5.7 × 10-6
1.0 × 10-6
1.5 × 10-7
3.6 × 10-5
LY231514f
Tomudexg
PDDFh
1.1 × 10-4
5.7 × 10-6
8.0 × 10-6
compd
pcDHFR
rlDHFR
tgDHFR
rl/tg
5.8 × 10-8
5.0 × 10-8
1.8 × 10-4
7
8
9
47 (10%)a
20 (16%)
16 (14%)
22 (11%)
25 (42%)
66.2
15 (15%)
26 (9%)
56 (0%)
87 (8%)
45 (2%)
12.0
47 (17%)
20 (7%)
16 (10%)
22 (3%)
25 (19%)
63 (28%)
15 (4%)
26 (3%)
56 (20%)
87 (27%)
45 (14%)
133.0
2.2
2.6
7.1
9.3
0.66
4.1
2.2
3.5
10.3
4.2
37
>21
>8
methotrexate
a
Kindly provided by Dr. R. L. Balkley, St. J ude Children’s
>2.3
>2.4
>38
>15.4
>7
b
Research Hospital, Memphis, TN. Kindly provided by Dr. D.
10
11
12
13
14
15
16
17
TMP
Borhani, Southern Research Institute, Birmingham, AL. c Kindly
provided by Dr. J . H. Freisheim, Medical University of Ohio,
d
Toledo, OH. Kindly provided by Dr. D. V. Santi, University of
California, San Francisco, CA. e Kindly provided by Dr. D. A.
Matthews, Agouron Pharmaceuticals, San Diego, CA. f MTA, a
multitargeted inhibitor, kindly provided by Dr. Chuan Shih, Eli
>7
>6
>21
>2
g
Lilly & Co., Indianapolis, IN. ZD 1694, Raltitrexed, Kindly
provided by Dr. Ann J ackman, Institute of Cancer Research,
Sutton, Surrey, UK. 5,8-Dideaza-10-propargylfolic acid, CB3717,
kindly provided by Dr. M. G. Nair, University of South Alabama,
Mobil, AL. i Number in parentheses indicates the level of inhibition
at the indicated concentration.
2.7
49.0
h
a
Values in parentheses are the percent inhibition at the highest
concentration tested.
substituted anilines in the presence of Raney nickel and
hydrogen at 50 psi for 6 h afforded the desired com-
pounds 7-14 in 20-62% yield. The N9-methyl ana-
logues 15-17 were synthesized (Scheme 3) from the
N9-H precursors 7, 8, and 10, respectively, by reductive
methylation using formaldehyde and sodium cyanoboro-
hydride in acetonitrile.
electron-withdrawing chloro-substituted analogues 11-
14 were similar in potency (except 11) against tgDHFR.
With the exception of compound 11, the 2,5-dichloro
analogue, the position and number of chloro substitu-
tion(s) were immaterial to potency against tgDHFR.
Clearly the 2,5-dichloro-substituted analogue was the
most potent of all of the analogues tested. In addition,
similar to the 2,5-dichloro analogue 11, the 2,5-
dimethoxy substitution (compound 7) in the methoxy
series was most conducive to potency against tgDHFR.
Since IC50 values against pcDHFR and rlDHFR were
not obtained (except compound 12 against pcDHFR),
construction of a structure-selectivity relationship was
not possible for these analogues against pcDHFR or
rlDHFR. However, from the data in Table 1, it is clear
that the 2-amino-4-oxo-5-substituted-pyrrolo[2,3-d]py-
rimidines are selective against tgDHFR, and compound
11 could serve as a lead compound for further structural
modification to improve selectivity and potency, par-
ticularly since rlDHFR, the mammalian standard, was
inhibited only 19% at 25 µM by this compound.
Biologica l Activity a n d Discu ssion
The nonclassical analogues 7-17 were evaluated as
inhibitors of pcDHFR, tgDHFR, and rat liver (rl) DHFR,
and the results (IC50) are reported in Table 1. Selectivity
ratios (IC50 rlDHFR/IC50 tgDHFR) for tgDHFR were
determined using rlDHFR as the mammalian standard
and are also reported in Table 1. The compounds were
more potent against tgDHFR than against pcDHFR or
rlDHFR. The most potent analogue, the 2,5-dichloro-
substituted compound 11, was 4 times more potent than
TMP against tgDHFR. All the analogues were also more
selective against tgDHFR than against pcDHFR. In the
N9-H series (compounds 7-14), the potency against
tgDHFR varied from an IC50 of 2.2 to 7.1 µM, with the
exception of 11 (IC50 ) 0.66 µM). In the dimethoxy series
(7-9), the position of the methoxy moieties had little
effect on the potency. The addition of a third methoxy
group (compound 10) did not change the potency to any
significant extent. N9-methylation of 7, 8, and 10 to
afford 15, 16, and 17, respectively, decreased potency
against tgDHFR. This decrease was 4-fold for 17
compared to 10. Thus, unlike some previously reported
heterocyclic 6-6 fused systems where bridge nitrogen
methylation substantially increased potency, in this
2-amino-4-oxo-5-substituted-pyrrolo[2,3-d]pyrimidine se-
ries bridge N-methylation decreased potency. These
results underscore the difference in SAR between dif-
ferent heterocyclic systems against different DHFR. The
Compounds 7-14 were also evaluated against TS
from Escherichia coli, Lactobacillus casei, Pneumocystis
carinii, and human recombinant TS and were es-
sentially inactive with IC50 values >160 µM. Thus,
analogues 7-17 do not exhibit dual inhibition of DHFR
and TS anticipated from their structure.
The classical compound 6 was evaluated as an inhibi-
tor of human CCRF-CEM TS, and the results indicated
that 6 (IC50 ) 94 µM) was a slightly weaker human TS
inhibitor than was MTX (IC50 ) 42 µM). Similar results
were obtained with recombinant human TS (IC50 ) 46
and 36 µM for 6 and MTX, respectively, Table 2) and
with rpc(recombinant P. carinii)TS (Table 2). Compound
6 was much less inhibitory to rec(recombinant E. coli)-

2-Amino-4-oxo-5-substituted-pyrrolo[2,3-d]pyrimidines
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1997
Ta ble 3. Inhibition of Growth of A253 and FaDu Human Head
and Neck Squamous Cell Carcinoma Cell Lines by MTX, 6, and
7 in Continuous Exposurea
Ta ble 5. Metabolite Protection of FaDu Cells by Leucovorin
(LV), Hypoxanthine (Hx), and/or Thymidine (TdR) from Growth
Inhibition Induced by MTX or 6a
EC50 (nM)
relative growth
drug
MTX
A253
FaDu
no
drug addn 10 µM LV +50 µM Hx +40 µM TdR +(Hx+TdR)
19 ( 3
(n ) 5)
17 ( 4
(n ) 5)
MTX 1 ( 0 100 ( 1
5 ( 0 64 ( 0
1 ( 0
5 ( 0
4 ( 0
74 ( 2
110 ( 2
79 ( 2
6
7
1000 ( 100
(n ) 2)
800 ( 100
(n ) 2)
6
a
Cells were exposed to drug concentrations previously shown
>10000
(n ) 2)
>10000
(n ) 2)
to inhibit growth by g95% (50 nM MTX, 9 µM 6); the indicated
concentrations of metabolites were present simultaneously with
drug. Values are the average growth relative to a control culture
treated only with metabolite (see Experimental Section); the
indicated error is the range of the two values used to determine
the average. LV and TdR/Hx protection were measured in separate
experiments that gave essentially identical growth inhibition data
(no addition); each experiment was repeated with comparable
results. MTX was fully protected by g0.1 µM LV, but 6 also showed
incomplete protection at lower levels (36 ( 1% relative growth at
0.1 µM LV and 59 ( 2% relative growth at 1 µM LV; data not
shown).
a
Growth inhibition was assayed as described in Experimental
Section. Values presented are average ( SD if n g 3 and average
( range for n ) 2.
Ta ble 4. Growth Inhibition of Parental CCRF-CEM and
Sublines with Single, Defined Mechanisms of MTX Resistance
during Continuous (0-120 h) Exposurea
EC50, nM
R1b
R2c
R30dmd
drug
MTX
CCRF-CEM
(v DHFR)
(V uptake)
(V Glun)
6-fold less potent than MTX against parental CCRF-
CEM cells; this is similar to the differential potency of
MTX and LY231514 in this cell line.21 DHFR-overex-
pressing cells (R1) were 43-fold resistant to MTX, but
only 2.5-fold resistant to 6, suggesting that DHFR is
not a major target of this agent, in contrast to
LY231514.21 This is consistent with the low level of
rhDHFR inhibition observed with 6 (Table 2). MTX
transport-defective cells (R2) are 135-fold resistant to
MTX, but less than 4-fold resistant to 6. This suggests
either that the mutation decreasing the Vmax of the
reduced folate/MTX carrier system (RFC) in this cell line
does not affect 6 in a similar manner or that 6 primarily
uses a separate transport pathway. LY231514 has been
suggested to use the RFC on the basis of similar low
(4-fold) cross-resistance data.21 Polyglutamylation of
tight-binding enzyme inhibitors, like MTX, is not re-
quired in continuous exposure; this accounts for the lack
of resistance of polyglutamylation-defective R30dm to
MTX. However, for inhibitors where polyglutamylation
is required for potent target inhibition (e.g., ZD1694),
resistance may become apparent for polyglutamylation-
defective cell lines such as R30dm, even in continuous
exposure. The low cross-resistance of R30dm to 6
suggests either that the parent drug is a potent inhibitor
of its target or that polyglutamylation of this drug is
extremely efficient. LY231514 exhibits cross-resistance
in a similar polyglutamylation-deficient cell line.21 Lipo-
philic 7 was less potent than the classical analogue at
inhibiting the growth of CCRF-CEM cells in culture
and all the sublines; however, the degree of cross-
resistance was similar for the two drugs. The source of
the low-level cross-resistance to 7 of the subline with
impaired MTX transport (R2) is unclear, since this
analogue presumably does not require active transport.
The slope of the concentration-dependence curve for 7
with all the CCRF-CEM lines was shallow; it required
a 0.3 log range of drug to decrease cell growth from
100% to <10% (compared to 3-fold change for MTX; data
not shown).
14 ( 1
600 ( 110
(n ) 5)
200 ( 20
(n ) 2)
420 ( 50
(n ) 3)
1900 ( 510
(n ) 5)
15 ( 1
(n ) 5)
120 ( 10
(n ) 2)
460 ( 115
(n ) 3)
(n ) 7)
80 ( 11
(n ) 3)
230 ( 60
(n ) 4)
6
7
310 ( 30
(n ) 2)
1530 ( 240
(n ) 3)
a
Average values are presented ( range for n ) 2 and ( SD for
b
n g 3. CCRF-CEM subline resistant to MTX solely as a result
of a 20-fold increase in wild-type DHFR protein and activity.45
c CCRF-CEM subline resistant as a result of decreased uptake
of MTX.46 CCRF-CEM subline resistant to MTX solely as a
d
result of decreased polyglutamylation; this cell line has 1% of the
FPGS specific activity (measured with MTX as the folate sub-
strate) of parental CCRF-CEM.41
TS (Table 2). In contrast, LY231514, as the mono-
glutamate, is 10 times more potent than 6 against TS
for both rh(recombinant human)TS and rpcTS (Table
2). The low inhibitory potency of 6 suggests either that
this drug does not target TS or that polyglutamylation
enhances its TS inhibitory activity (see below). Com-
pound 6 was also tested as an inhibitor of rh-, rtg-
(recombinant T. gondii)-, and recDHFR (Table 2). The
IC50 for rhDHFR was 10 µM and as with rhTS, LY231514
was 10 times more inhibitory than compound 6. tgDH-
FR was the most sensitive DHFR tested (IC50 ) 0.8 µM)
and recDHFR the least sensitive (IC50 ) 230 µM).
Classical compound 6 and the lipophilic analogue 7
were evaluated as inhibitors of in vitro human tumor
cell growth. Growth inhibition of two human head and
neck squamous cell carcinoma cell lines, A253 and
FaDu, were assessed in continuous (120 h) exposure
(Table 3). Compound 6 was ≈0.50-fold less potent than
MTX as a growth inhibitor of each cell line. However,
the potency of 6 is similar to that observed for LY231514
against the ZR-75-1 human breast carcinoma cell line.21
The lipophilic analogue 7 was not cytotoxic at 10 µM,
the highest concentration that could be tested without
solvent (DMSO) toxicity.
The growth inhibitory potency of classical compound
6 and lipophilic 7 was also compared to that of MTX in
continuous exposure against CCRF-CEM human lym-
phoblastic leukemia cells in culture (Table 4). In addi-
tion, the compounds 6 and 7 were also evaluated against
a series of MTX-resistant sublines to determine the
mechanism of action (Table 4). Compound 6 was only
Metabolite protection studies of the growth inhibition
of compound 6 were carried out in order to further
elucidate its mechanism of action. FaDu cells were fully
protected by leucovorin (LV) against growth inhibition
by MTX (Table 5), even at LV levels as low as 0.1 µM

1998 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Gangjee et al.
with 100 mL of toluene and ethyl ether. The aqueous layer
was separated, cooled to 0 °C, and acidified to pH 4 using 5 N
HCl. The aqueous layer was then extracted with 2 × 100 mL
of EtOAc. The combined organic layer was dried over MgSO4
and concentrated to afford 3.6 g (72%) of a white foam that
was used without further purification. TLC on silica gel
indicated two spots (cis and trans enol forms) Rf ) 0.36 and
0.38, Hex:EtOAc 1:1.
Meth yl 2-Am in o-4-oxop yr r olo[2,3-d ]p yr im id in e-5-ca r -
boxyla te (21). 2,4-Diamino-6-hydroxypyrimidine (27) (1.0 g,
7.9 mmol) was suspended in water along with NaOAc (0.33 g,
3.9 mmol). The mixture was heated to 100 °C and at that time
all the material dissolved. A suspension of methyl chloro-
(formyl)acetate (20) (1.7 g, 11.5 mmol) in water was added
followed by the addition of NaOAc (0.17 g, 2.0 mmol) in 5 mL
of water. The reaction mixture was stirred at 100 °C for 2 h,
during which time a white precipitate was formed. The
reaction mixture was cooled to room temperature and filtered.
The precipitate was washed with water, acetone, and ethyl
ether to afford 4.7 g (62%) of a white solid: mp >295 °C; TLC,
silica gel Rf 0.21 CHCl3:MeOH 3:1; 1H NMR (DMSO-d6) δ 3.72
(s, 3 H, OCH3), 6.22 (s, 2 H, 2-NH2), 7.43 (d, 1 H, 6-H), 10.36
(s, 1 H, 7-H), 11.64 (s, 1 H, 3-H). Anal. Calcd for (C8H8N4O3):
C, H, N.
(data not shown). In contrast, although LV provided
some protection against 6, complete protection could not
be achieved even at 10 µM. This is in contrast to
LY231514, against which LV is fully protective.41 Pro-
tection by the purine hypoxanthine (Hx) and by thymi-
dine (TdR) was also assessed (Table 4). TdR alone, but
not Hx alone, protected against MTX to a low degree,
but full protection required both Hx and TdR. Hx alone
offered no protection against 6, while TdR alone was
only partially protective; the combination of Hx and TdR
typically offered only a slightly higher level of protection
against 6 than TdR alone. It is to be noted that the lack
of full protection cannot be a result of insufficient
metabolite concentration, since their levels are sufficient
to fully protect against MTX. This suggests that 6 has
a site of action in addition to sites in the folate pathway.
Our cellular and biochemical studies offer some clues
as to the mechanism of action of compound 6. The low
level of cross-resistance of the DHFR-amplified cell line,
along with the lack of enhanced protection by the
combination of TdR + Hx relative to TdR alone, is
inconsistent with DHFR as a target of this drug.
Similarly, the lack of protection by Hx alone suggests
that purine synthesis is not a target. Substantial
protection against 6 by TdR alone suggests that thymidy-
late synthesis, and hence TS itself, is a major target of
this drug. In contrast to these results with whole cells,
6 is a weak inhibitor of isolated TS. Since TS shows
substantial increase in inhibitor potency for poly-
glutamyl relative to monoglutamyl forms,42 these ob-
servations suggest that polyglutamyl forms of 6 may
serve as the actual intracellular TS inhibitors. Since the
polyglutamylation-deficient subline R30dm is not cross-
resistant to 6 in continuous exposure, the data further
suggest that polyglutamylation may be a very efficient
process for 6. This hypothesis will be evaluated in
further studies. We have already shown that 7 is, as
expected, not a human FPGS substrate or inhibitor up
to 50 µM. Compound 7 did not inhibit human FPGS at
50 µM.
Meth yl 2-Am in o-4-oxo-7-(ter t-bu toxyca r bon yl)p yr r olo-
[2,3-d ]p yr im id in e-5-ca r boxyla te (23). Compound 21 (1.0 g,
4.8 mmol) was dissolved in 50 mL of DMF at 100 °C. The
solution was cooled to room temperature and treated with
triethylamine (0.48 g, 4.8 mmol) and (BOC)2O (1.65 g, 7.6
mmol). The reaction was stirred at room temperature for 24
h. The solvent was removed under reduced pressure. The
residue was dissolve in 2 mL of MeOH and placed on a dry
silica gel column (1.5 × 10 cm) and eluted with 10:1 CHCl3:
MeOH to afford 1.2 g (93%) of 23: mp >250 °C; TLC, silica gel
1
Rf 0.61 CHCl3:MeOH 3:1; H NMR (DMSO-d6) δ 1.56 (s, 9 H,
Boc), 3.72 (s, 3 H, OCH3), 6.58 (s, 2 H, 2-NH2), 7.53 (s, 1 H,
6-H), 10.75 (s, 1 H, 3-H). Anal. Calcd for (C13H16N405): C, H,
N.
Ch lor o(for m yl)a ceton itr ile (26). While the temperature
was maintained between 0 and 5 °C, methyl formate 18 (4.3
g, 77.0 mmol) was added to a stirred mixture of NaOCH3 (3.57
g, 66.0 mmol) in 100 mL of toluene. This was followed by
dropwise addition of chloroacetonitrile 25 (5.0 g, 66.0 mmol)
over a period of 1 h. The mixture was allowed to stir for an
additional 3 h, 100 mL of water was added, and the mixture
was extracted with 2 × 100 mL of toluene. The aqueous layer
was cooled to 0 °C and acidified to pH 4 using 5 N HCl. The
aqueous layer was extracted with 3 × 100 mL EtOAc, and the
combined organic layer was dried over MgSO4 and concen-
trated under reduced pressure to afford 5.1 g (65%) of 26,
which was used without further purification.
2-Am in o-5-cya n op yr r olo[2,3-d ]p yr im id in -4-on e (28).
NaOAc (1.4 g, 11.0 mmol) was dissolved in 20 mL of distilled
water. 2-Diamino-6-hydroxypyrimidine (27) (0.66 g, 5.28 mmol)
was added, and the mixture was heated to 100 °C, at which
time 0.65 g (6.3 mmol) of 26 in 10 mL of water was added.
The reaction mixture was refluxed for 5 h and cooled to room
temperature, and the precipitate that formed was filtered and
washed with copious amounts of water and acetone to yield
0.65 g (72%) of 28 as the only product: mp >360 °C; 1H NMR
(DMSO-d6) δ 6.37 (s, 2 H, 2-NH2), 7.59 (d,1 H, 6-H), 10.69 (s,
1 H, 7-H), 11.97 (s, 1 H, 3-H).
N-[4-[N-[(2-Am in o-4-oxop yr r olo[2,3-d ]p yr im id in -5-yl)-
m eth yl]a m in o]ben zoyl]-L-glu ta m ic Acid (6). 2-Amino-5-
cyanopyrrolo[2,3-d]pyrimidin-4-one (28, 0.3 g, 1.7 mmol) was
dissolved in 10 mL of formic acid in a hydrogenator bottle. To
the solution was added N-(4-aminobenzoyl)-L-glutamic acid
diethyl ester (30) (0.640 g, 2.0 mmol) and Raney Ni. The
reaction mixture was hydrogenated at 50 psi for 3 h. The
reaction mixture was filtered and concentrated under reduced
pressure. The residue was dissolve in 2 mL of MeOH and
placed on a dry silica gel column (1.5 × 10 cm) and eluted with
10:1 CHCl3:MeOH to afford 0.63 g (75%) of the diester 31 as
a white solid: mp >250 °C; TLC, silica gel Rf 0.51 CHCl3:MeOH
Exp er im en ta l Section
Melting points were determined on a Fisher-J ohns melting
point apparatus or a Mel Temp apparatus and are uncorrected.
Infrared spectra (IR) were recorded with a Perkin-Elmer Model
1430, in Nujol mulls. Nuclear magnetic resonance spectra for
1H NMR were recorded on a Bruker WH-300 (300 MHz). The
data were accumulated by 16K size with 0.5 s delay time and
70° tip angle with internal standard TMS; s ) singlet, br )
broad, d ) doublet, t ) triplet, q ) quartet, m ) multiplet.
Thin-layer chromatography was performed on silica gel plates
with fluorescent indicator and were visualized at 254 and 366
nm, unless indicated otherwise. Column chromatography was
performed with 230-400 mesh silica gel purchased from
Aldrich Chemical Co., Milwaukee, WI. All solvents were
purchased from Aldrich Chemical Co. and were used without
further purification. Samples for microanalysis were dried in
vacuo over P2O5 at 70 or 110 °C. Elemental analysis were
performed by Atlantic Microlabs, Norcross, GA. Despite drying,
fractions of solvents could not be removed from some of the
samples and were confirmed, where possible, from the 1H
NMR.
Meth yl Ch lor o(for m yl)a ceta te (20). Methyl chloroacetate
19 (3.2 g, 30.0 mmol) and methyl formate 18 (1.8 g, 30.0 mmol)
were dissolved in toluene volume, and the mixture was cooled
to 0 °C using an ice bath. NaOCH3 (95%) (2.0 g, 35.0 mmol)
was added and the reaction mixture was stirred at 0 °C for 5
h. Water (100 mL) was added and the mixture was extracted

2-Amino-4-oxo-5-substituted-pyrrolo[2,3-d]pyrimidines
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1999
1
3:1; H NMR (DMSO-d6) δ 1.16 (2t, 6 H, CH3), 2.03 (m, 2 H,
mmol) using the general procedure described above to afford
after purification 0.18 g (18%) as a brown solid: mp 262-268
°C; TLC Rf 0.41 (CHCl3:MeOH:NH4OH, 7:3:0.5); 1H NMR
(DMSO-d6) δ 3.49 (s, 3 H, OCH3), 3.67 (s, 6 H, OCH3), 4.18 (d,
2 H, 8-CH2), 5.71 (t, 1 H, 9-NH), 5.91 (s, 2 H, Ar), 6.05 (s, 2 H,
2-NH2), 10.31 (s, 1 H, 7-NH), 10.77 (s, 1 H, 3-NH). Anal. Calcd
for (C16H19N5O4): C, H, N.
2-Am in o-4-oxo-5-[(2′,5′-dich lor oan ilin o)m eth yl]pyr r olo-
[2,3-d ]p yr im id in e (11). Compound 11 was synthesized from
2-amino-4-oxo-5-cyanopyrrolo[2,3-d]pyrimidine (25) (0.5 g, 2.85
mmol) and 2,5-dichloroaniline (0.55 g, 3.42 mmol) using the
general procedure described above to afford after purification
0.17 g (18%) as a white solid: mp 282-285 °C; TLC Rf 0.40
(CHCl3:MeOH:NH4OH, 7:3:0.5); 1H NMR (DMSO-d6) δ 4.29 (d,
2 H, 8-CH2), 6.09 (s, 2 H, 2-NH2), 6.27 (t, 1 H, 9-NH), 6.57 (m,
2 H, Ar), 6.87 (d, 1 H, Ar), 7.22 (d, 1 H, Ar), 10.30 (s, 1 H,
7-NH), 10.82 (s, 1 H, 3-NH). Anal. Calcd for (C13H11N5OCl2):
C, H, N, Cl.
2-Am in o-4-oxo-5-[(3′,5′-dich lor oan ilin o)m eth yl]pyr r olo-
[2,3-d ]p yr im id in e (12). Compound 12 was synthesized from
2-amino-4-oxo-5-cyanopyrrolo[2,3-d]pyrimidine (25) (0.5 g, 2.85
mmol) and 3,5-dichloroaniline (0.55 g, 3.42 mmol) using the
general procedure described above to afford after purification
0.16 g (17%) as a white solid: mp 270-275 °C; TLC Rf 0.39
(CHCl3:MeOH:NH4OH, 7:3:0.5); 1H NMR (DMSO-d6) δ 4.22 (d,
2 H, 8-CH2), 5.55 (t, 1 H, 9-NH), 6.07 (t, 3 H, Ar, 2-NH2), 6.53
(s, 1 H, Ar), 6.67 (d, 1 H, Ar), 10.33 (s, 1 H, 7-NH), 10.75 (s, 1
H, 3-NH). Anal. Calcd for (C13H11N5OCl2): C, H, N. Cl.
2-Am in o-4-oxo-5-[(2′,4′-dich lor oan ilin o)m eth yl]pyr r olo-
[2,3-d ]p yr im id in e (13). Compound 13 was synthesized from
2-amino-4-oxo-5-cyanopyrrolo[2,3-d]pyrimidine (25) (0.5 g, 2.85
mmol) and 2,4-dichloroaniline (0.55 g, 3.42 mmol) using the
general procedure described above to afford after purification
0.18 g (19%) as a white solid: mp >280 °C; TLC Rf 0.41
(CHCl3:MeOH:NH4OH, 7:3:0.5); 1H NMR (DMSO-d6) δ 4.28 (d,
2 H, 8-CH2), 6.07 (s, 2 H, 2-NH2), 6.57 (t, 1 H, 9-NH), 6.85 (s,
1 H, Ar), 7.10 (d, 1 H, Ar), 7.13 (d, 1 H, Ar), 10.37(s, 1 H, 7-NH),
10.79 (s, 1 H, 3-NH). Anal. Calcd for (C13H11N5OCl2): C, H,
N, Cl.
2-Am in o-4-oxo-5-[(3′-ch lor oa n ilin o)m eth yl]p yr r olo[2,3-
d ]p yr im id in e (14). Compound 14 was synthesized from
2-amino-4-oxo-5-cyanopyrrolo[2,3-d]pyrimidine (25) (0.5 g, 2.85
mmol) and 3-chloroaniline (0.43 g, 3.42 mmol) using the
general procedure described above to afford after purification
0.19 g (23%) as a white solid: mp 267-270 °C; TLC Rf 0.43
(CHCl3:MeOH:NH4OH, 7:3:0.5); 1H NMR (DMSO-d6) δ 4.21 (d,
2 H, 8-CH2), 6.05 (s, 2 H, 2-NH2), 6.48 (t, 1 H, 9-NH), 6.53 (m,
4 H, Ar), 7.05 (m, 1 H, Ar), 10.30 (s, 1 H, 7-NH), 10.79 (s, 1 H,
3-NH). Anal. Calcd for (C13H12N5OCl): C, H, N, Cl.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
15-17.
To 2-amino-4-oxo-5-[(substituted anilino)methyl]pyrrolo[2,3-
d]pyrimidine (0.10 g) in 25 mL of acetonitrile was added
formaldehyde (1.1 equiv) with constant stirring. To this
suspension was added sodium cyanoborohydride, and then the
pH of the mixture was adjusted to 2-3 using concentrated
HCl. The starting material initially went into solution at pH
2-3, and after 5 min a bright yellow precipitate was formed.
TLC (CHCl3:CH3OH:NH4OH; 7:3:1 drop) showed the presence
of a new spot with the disappearance of the starting material.
The acetonitrile was evaporated under reduced pressure and
the residue obtained was suspended in 10 mL of water and
neutralized using NH4OH to afford the desired analogues 15-
17.
2-Am in o-4-oxo-5-[(2′,5′-d im et h oxy-N-m et h yla n ilin o)-
m eth yl]p yr r olo[2,3-d ]p yr im id in e (15). Compound 15 was
obtained as a white solid in 86% yield using the general
procedure described above: mp 250-252 °C; TLC Rf 0.43
(CHCl3:MeOH:NH4OH, 7:3:0.5); 1H NMR (DMSO-d6) δ 2.85 (s,
3 H, N-CH3), 3.61 (s, 3 H, OCH3), 3.70 (s, 3 H, -OCH3), 4.48
(s, 2 H, 8-CH2), 6.02 (s, 2 H, 2-NH2), 6.21 (m, 1 H, Ar), 6.31 (s,
1 H, 2-Ar), 6.60 (s, 1 H, Ar), 6.74 (d, 1 H, Ar) 10.77 (s, 1 H,
3-NH), 10.76 (s, 1 H, 3-NH). Anal. Calcd for (C16H19N5O3‚1.0
H2O): C, H, N.
â-CH2), 2.39 (m, 2 H, γ-CH2), 4.01 (m, 4 H, CH2CH3), 4.31 (m,
3 H, 8-CH2 and 9-NH), 6.08 (s, 2 H, 2-NH2), 6.46 (m, 1 H,
R-CH), 6.50 (d, 1 H, 6-CH), 6.0 (d, 2 H, Ph), 7.62 (d, 2 H, Ph),
8.23 (d, 1 H, glu-NH), 10.35 (s, 1 H, 7-NH), 10.81 (s, 1 H,
3-NH). Anal. Calcd for (C23H28N6O6): C, H, N.
The diester 31 (0.12 g, 0.25 mmol) was dissolved in 5 mL of
MeOH. To the solution was added 5 mL of 1 N NaOH, and
the mixture was stirred at room temperature for 24 h. The
solvent was removed under reduced pressure and the residue
was dissolved in 10 mL of water and acidified to pH 4 using
concentrated HCl. The mixture was then extracted with 2 ×
50 mL EtOAc and the combined organic layer was dried over
MgSO4 and concentrated under reduced pressure. The residue
was dissolved in 2 mL of MeOH and treated with 10 mL of
ethyl ether. The precipitate formed was collected by filtration
to afford 0.1 g (94%) of 6: 1H NMR (DMSO-d6) δ 1.92 (m, 2 H,
â-CH2), 2.29 (m, 2 H, γ-CH2), 4.31 (m, 3 H, 8-CH2 and 9-NH),
6.07 (s, 2 H, 2-NH2), 6.45 (m, 1 H, R-CH), 6.49 (d, 2 H, 6-CH),
6.65 (d, 2 H, Ph), 7.65 (d, 2 H, Ph), 8.10 (d, 1 H, glu-NH), 10.28
(s, 1 H, 7-NH), 10.81 (s, 1 H, 3-NH), 12.31 (b s, 1 H, COOH).
Anal. Calcd for (C19H20N6O6): C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
7-14. The nitrile 25 (0.5 g, 2.8 mmol) and the appropriate
aniline (3.4 mmol) were dissolved in 100 mL of 15% AcOH in
DMF with heating. To this solution was added 1.0 g of Raney
nickel and the mixture was hydrogenated at 50 psi for a period
of 6-8 h. The progress of the reaction was followed by TLC
(7:2:0.5, CHCl3:MeOH, NH4OH). After completion of the reac-
tion, the mixture was filtered through a Celite pad and washed
with DMF. Silica gel (1.0 g) was added to the filterate and the
solvent was evaporated to afford a dry plug. This plug was
placed on a top of 1.05 in. × 23 in. silica gel column and eluted
with CHCl3:MeOH (a gradient elution, 95:5 to 85:15). Fractions
containing the product were pooled and evaporated to afford
analytically pure compound.
2-Am in o-4-oxo-5-[(2′,5′-d im eth oxya n ilin o)m eth yl]p yr -
r olo[2,3-d ]p yr im id in e (7). Compound 7 was synthesized
from 2-amino-4-oxo-5-cyanopyrrolo[2,3-d]pyrimidine (25) (0.5
g, 2.85 mmol) and 2,5-dimethoxyaniline (0.52 g, 3.42 mmol)
using the general procedure described above to afford after
purification 0.3 g (33%) as a brownish solid: mp >230 °C; TLC
1
Rf 0.43 (CHCl3:MeOH:NH4OH, 7:3:0.5); H NMR (DMSO-d6)
δ 3.58 (s, 3 H, OCH3), 3.63 (s, 3 H, OCH3), 4.20 (d, 2 H, 8-CH2),
5.46 (t, 1 H, 9-NH), 6.01 (d, 1 H, 6-CH), 6.04 (s, 2 H, 2-NH2),
6.24 (d, 1 H, Ar), 6.56 (d, 1 H, Ar), 6.64 (s, 1 H, Ar), 10.31 (s,
1 H, 7-NH), 10.75 (s, 1 H, 3-NH). Anal. Calcd for (C15H17
N5O3): C, H, N.
2-Am in o-4-oxo-5-[(3′,5′-d im eth oxya n ilin o)m eth yl]p yr -
r olo[2,3-d ]p yr im id in e (8). Compound 8 was synthesized
from 2-amino-4-oxo-5-cyanopyrrolo[2,3-d]pyrimidine (25) (0.5
g, 2.85 mmol) and 3,5-dimethoxyaniline (0.52 g, 3.42 mmol)
using the general procedure described above to afford after
purification 0.18 g (20%) as a brown solid: mp 241 °C; TLC Rf
-
1
0.45 (CHCl3: MeOH: NH4OH, 7:3:0.5); H NMR (DMSO-d6) δ
3.62 (s, 6 H, 3,5-OCH3), 4.17 (d, 2 H, 8-CH2), 5.61 (s, 1 H, Ar),
5.70 (d, 2 H, Ar), 5.79 (t, 1 H, 9-NH), 6.05 (s, 2 H, 2-NH2), 6.53
(d, 1 H, Ar), 10.30 (s, 1 H, 7-NH), 10.77 (s, 1 H, 3-NH). Anal.
Calcd for (C15H17N5O3·0.2H2O): C, H, N.
2-Am in o-4-oxo-5-[(2′,4′-d im eth oxya n ilin o)m eth yl]p yr -
r olo[2,3-d ]p yr im id in e (9). Compound 9 was synthesized
from 2-amino-4-oxo-5-cyanopyrrolo[2,3-d]pyrimidine (25) (0.5
g, 2.85 mmol) and 2,4-dimethoxyaniline (0.52 g, 3.42 mmol)
using the general procedure described above to afford after
purification 0.22 g (24%) as a brown solid: mp 252-256 °C;
TLC Rf 0.43 (CHCl3:MeOH:NH4OH, 7:3:0.5); 1H NMR (DMSO-
d6) δ 3.58 (s, 3 H, OCH3), 3.66 (s, 3 H, OCH3), 4.15 (d, 2 H,
8-CH2), 5.55 (t, 1 H, 9-NH), 6.09 (t, 3 H, Ar, 2-NH2), 6.53 (s, 1
H, Ar), 6.67 (d, 1 H, Ar), 10.33 (s, 1 H, 7-NH), 10.75 (s, 1 H,
3-NH). Anal. Calcd for (C15H17N5O3): C, H, N.
2-Am in o-4-oxo-5-[(3′,4′,5′-t r im et h oxya n ilin o)m et h yl]-
p yr r olo[2,3-d ]p yr im id in e (10). Compound 10 was synthe-
sized from 2-amino-4-oxo-5-cyanopyrrolo[2,3-d]pyrimidine (25)
(0.5 g, 2.85 mmol) and 3,4,5-dimethoxyaniline (0.62 g, 3.42

2000 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Gangjee et al.
2-Am in o-4-oxo-5-[(3′,5′-d im et h oxy-N-m et h yla n ilin o)-
m eth yl]p yr r olo[2,3-d ]p yr im id in e (16). Compound 16 was
obtained as a white solid in 81% yield using the general
procedure described above: mp 250-252 °C; TLC Rf 0.46
(CHCl3:MeOH:NH4OH, 7:3:0.5); 1H NMR (DMSO-d6) δ 2.95 (s,
3 H, N-CH3), 3.45 (s, 3 H, OCH3), 3.67 (s, 3 H, -OCH3), 4.55
(d, 2 H, 8-CH2), 5.77 (s, 1 H, Ar), 5.90 (d, 2 H, Ar), 6.21 (s, 2 H,
2-NH2), 6.46 (s, 1 H, Ar), 10.30 (s, 1 H, 7-NH), 10.76 (s, 1 H,
3-NH). Anal. Calcd for (C16H19N5O3C·0.75H2O): C, H, N.
2-Am in o-4-oxo-5-[(3′,4′,5′-tr im eth oxy-N-m eth yla n ilin o)-
m eth yl]p yr r olo[2,3-d ]p yr im id in e (17). Compound 17 was
obtained as a brown solid in 86% yield using the general
procedure described above: mp 265-270 °C; TLC Rf 0.42
of enzyme activity (1 IU ) 0.005 OD units/min), and dihydro-
folic acid (0.092 mM). KCl (150 mM) was included in the assay
for T. gondii and rat liver DHFR, because it stimulated the
enzymes 1.4- and 2.6-fold, respectively. KCl was omitted from
assays of P. carinii DHFR, because no stimulation was
produced with high salt. Background activity measured with
no added dihydrofolic acid ranged from 4% of signal for the
enzyme obtained from cultured T. gondii to 15% of signal for
M. avium DHFR. All DHFR inhibitors were tested against rat
liver DHFR as well as against pathogen DHFR to allow
assessment of selectivity.
Stock solutions of inhibitors were prepared in DMSO and
then diluted in water to provide a range of concentrations
suitable for testing in the enzyme assay. Carry-over of DMSO
into the DHFR assay was kept below 1%, because higher
concentrations caused inhibition. Solutions of drug were stored
at -70 °C in the dark for short periods.
Deter m in a tion of IC50 Va lu es. DHFR was assayed with-
out inhibitor and with a series of concentrations of inhibitors
to produce 10-90% inhibition. At least three concentrations
were required for calculation. Semilogarithmic plots of the data
yielded normal sigmoidal curves for most inhibitors. The data
were converted from percent inhibition to probit values, which
were plotted versus the log of the drug concentration The
resultant straight lines were analyzed by least-squares linear
regression. The 50% inhibitory concentration (IC50) is the
concentration at which the probit value is 5.0.
1
(CHCl3: MeOH: NH4OH, 7:3:0.5); H NMR (DMSO-d6) δ 2.92
(s, 3 H, N-CH3), 3.48 (s, 3 H, OCH3), 3.70 (s, 6 H, 3,5-diOCH3),
4.53 (d, 2 H, 8-CH2), 5.19 (s, 1 H, Ar), 6.04 (s, 1 H, Ar), 6.09 (s,
2 H, 2-NH2), 6.39 (s, 1 H, Ar), 10.81 (s, 1 H, 3-NH). Anal. Calcd
for (C17H21N5O4·H2O): C, H, N.
Meth od s for Biologica l Eva lu a tion s.
Dr u gs. Drug solutions were standardized using extinction
coefficients. Extinction coefficients were determined for 6 [pH
1, λmax 252 nm (sh; 23 100); pH 7, λmax 287 nm (43 000); pH
13, λmax 282 nm (43 500)] and for 7 (pH 1, λmax 262 nm (10 300);
pH 7, λmax 289 nm (10 800); pH 13, λmax 272 nm (sh; 10 300)].
Extinction coefficients for methotrexate (MTX), a gift of
Immunex (Seattle, WA), were from the literature.56
Cell Lin es a n d Meth od s for Mea su r in g Gr ow th In -
h ibitor y P oten cy. All cell lines were verified to be negative
for mycoplasma contamination using the GenProbe (San Diego,
CA) test kit. The human T-lymphoblastic leukemia cell line
CCRF-CEM44 and its MTX-resistant sublines R1,45 R2,46 and
R30dm47 were cultured as described.47 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 folylpoly-
glutamate synthetase (FPGS) activity of CCRF-CEM and is
resistant to short-term, but not continuous, MTX exposure;
however, R30dm is generally cross-resistant in continuous
exposure to antifolates requiring polyglutamylation to form
potent inhibitors. The A253 and FaDu human head and neck
squamous cell carcinoma cell lines, obtained from ATCC
(Manassas, VA), were propagated in RPMI 1640/10% fetal calf
serum (FCS); however, growth inhibition was measured in
medium containing 5% FCS48 to minimize the levels of purine
and pyrimidines introduced from FCS. Growth inhibition of
all cell lines by continuous drug exposure was assayed as
described.47,48 Compound 7 was soluble only in neat DMSO;
appropriate vehicle controls were included in all growth
experiments, and only data where the vehicle was nontoxic
were analyzed. EC50 values were determined visually from
plots of percent control growth versus the logarithm of drug
concentration.
Sou r ce of T. gon d ii. A frozen sample of the RH strain of
T. gondii was obtained from the Centers for Disease Control,
Atlanta, GA, and inoculated intraperitoneally into female ICR
mice (Harlan Industries, Indianapolis, IN). Peritoneal exudate
was collected 7 days later and found to contain numerous T.
gondii organisms, as well as many host cells, some of which
were infected with T. gondii. Approximately 2 × 106 T. gondii
were inoculated intraperitoneally into new host mice. The
organisms grew more rapidly on second passage in mice and
were harvested within 4 days after inoculation. Harvests were
scaled up to 20 mice and peritoneal exudate was pooled and
centrifuged, and the organisms were resuspended in RPMI
medium containing 10% fetal calf serum. Frozen stocks were
prepared by adding 5% DMSO to the medium and freezing
slowly over 8-15 h. Stocks were stored in liquid nitrogen.
Four years ago we obtained a clinical isolate of T. gondii
from the Department of Pathology, Indiana University School
of Medicine. This strain was handled as described above for
the RH strain and stocks were prepared in liquid nitrogen.
The clinical isolate displays kinetics of growth in culture that
are more advantageous for production of enzymes and is now
our standard source of DHFR from T. gondii. The material is
maintained as described in the previous paragraph for the RH
strain.
Cu ltu r e of T. gon d ii for En zym e P r od u ction . T. gondii
was grown on a Chinese hamster ovary cell line that lacks
DHFR (American Type Culture Collection, 3952 CL, CHO/
dhfr-).50 Cells are maintained in Iscove’s Modified Eagle’s
Medium with 10% fetal calf serum, 1% penicillin/streptomycin,
100 mM hypoxanthine, and 10 mM thymidine. An inoculum
of approximately 107 organisms was added to each 75 cm2
tissue culture flask containing the monolayer of cells. Within
6-8 days, 4 × 108 organisms can be harvested from each flask.
Protection of FaDu cells against growth inhibition was
assayed by including metabolites simultaneously with
a
concentration of drug previously determined to inhibit growth
by g95%; the remainder of the assay was as described. The
metabolites included were leucovorin (LV, 0.1-10 µM) or
thymidine (TdR, 40 µM) or hypoxanthine (Hx, 50 µM) or a
combination of TdR and Hx at the same individual concentra-
tions. Growth inhibition was measured relative to the ap-
propriate metabolite-treated control; LV had no effect on
growth and 40 µM TdR or 50 µM Hx caused e10% growth
inhibition, while the TdR + Hx combination caused e30%
growth inhibition.
P r ep a r a tion of En zym es fr om T. gon d ii. When har-
vested from tissue culture, T. gondii organisms were minimally
contaminated with mammalian host cells, which in any case
should contain no DHFR activity. To confirm this property,
uninoculated monolayer cultures from three flasks were
harvested to prepare a 100000g supernatant from host cells;
no activity of DHFR could be detected. Organisms from culture
were washed in phosphate-buffered saline containing 10 mM
citrate and resuspended in 50 mM phosphate buffer (pH 7.0)
containing leupeptin (20 µg/mL), phenylmethylsulfonyl fluo-
ride (9 µg/mL), soybean trypsin inhibitor (50 µg/mL), aprotinin
(50 µg/mL), and 20 mM 2-mercaptoethanol. This buffer lysed
the T. gondii. The suspension was centrifuged at 100000g. The
supernate contained both DHFR and dihydropteroate synthase
En zym e Assa ys:
En zym e In h ibition . Partial purification of human CCRF-
CEM thymidylate synthase (TMPS) and measurement of its
inhibition were performed as previously described.43
Dih yd r ofola te Red u cta se (DHF R). The spectrophoto-
metric assay for DHFR was modified to optimize for temper-
ature, substrate concentration, and cofactor concentration for
each enzyme form assayed.49,50 The standard assay performed
at 37 °C contained sodium phosphate buffer pH 7.4 (40.7 mM),
2-mercaptoethanol (8.9 mM), NADPH (0.117 mM), 1-3.7 IU

2-Amino-4-oxo-5-substituted-pyrrolo[2,3-d]pyrimidines
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 2001
activity. The preparation was stored in liquid nitrogen without
appreciable loss of activity. The yield of DHFR from T. gondii
cultures has been about 40 IU per flask.
The 100000g supernates from T. gondii prepared in culture
contain both DHFR and dihydropteroate synthase (DHPS)
activity. The presence of DHPS, which is not found in mam-
malian cells, and the absence of DHFR in the specific host cell
line used for culture supports the conclusion that the DHFR
activity being measured arises from T. gondii.
tion to yield a concentrated 45-90% cut, which consistently
contains about 72% of the original activity. This material is
loaded onto a P-100 column equilibrated with 100 mM sodium
phosphate buffer pH 7.4 containing 20 mM 2-mercaptoethanol
and eluted with the same buffer. Most of the protein is eluted
in the void volume of the column, but DHFR activity elutes in
a broad peak at about 1.3 times the void volume. Fractions
containing significant DHFR activity are combined and loaded
directly onto an affinity column.
The affinity column is prepared from folate-Sepharose
(Sigma). The resin is poured into an appropriately sized
column and washed sequentially with 100 mg/mL ascorbate,
5 mg/mL sodium metabisulfite, and 10 mM phosphate buffer
pH 7 with 20 mM 2-mercaptoethanol (ME). This treatment
reduces the folate on the column to dihydrofolate, the substrate
for DHFR. After washing to remove all traces of sodium
metabisulfite, the sample containing DHFR is slowly loaded
onto the column. When the entire sample has been loaded,
the column is washed with ME until protein in the eluate
becomes undetectable. Elution continues with stepwise wash-
ings of about 2 column volumes of ME containing 0.1 M NaCl,
ME with 0.5 M NaCl, and ME with 1 M NaCl and 3 mM folate.
The activity of DHFR from affinity columns can be maintained
with folate and with glycerol. These materials are used during
the concentration steps that follow the affinity column. Glyc-
erol is added to all samples and they are frozen at -70 °C.
Aliquots are analyzed for protein content, DHFR activity, and
purity by SDS-polyacrylamide gel electrophoresis (SDS-
PAGE).
DHF R fr om Cu ltu r ed T. gon d ii Tested with Kn own
In h ibitor s of th e En zym e. IC50 values calculated for metho-
trexate and pyrimethamine were 0.014 and 0.24 µM, respec-
tively; the IC50 values agree closely with the reported values
of 0.021 and 0.76 µM. The IC50 value for pyrimethamine with
DHFR prepared from T. gondii harvested from mice was 0.39
µM, a value closely in agreement with the IC50 for the enzyme
from cultured T. gondii. The IC50 value for trimethoprim was
1.8, 2.9, and 3.5 µM in three independent trials with enzyme
from cultured T. gondii but was reported by others to be 14.5
µM for the enzyme prepared from organisms harvested from
mice.51
Kinetics for cofactor and substrate were determined for
DHFR from rat liver and T. gondii. Both forms of DHFR
displayed kinetics for dihydrofolic acid under our conditions
of assay that differed from the Michaelis-Menten model.52
Curves suggested substrate inhibition at concentrations above
that used in the standard assay. The kinetic parameters of
rat liver DHFR and T. gondii DHFR were similar, with half-
maximal rates at 11-13 µM dihydrofolic acid. Others have also
suggested that substrate kinetics of the two forms of DHFR
are similar.53 Kinetics for NADPH followed the Michaelis-
Menten model and yielded linear double reciprocal plots; Km
values of 11 and 23 µM were determined for DHFR from T.
gondii and rat liver, respectively. Others reported a Km of 6.7
µM for NADPH with DHFR from T. gondii.53 These kinetic
studies and studies using known inhibitors of T. gondii DHFR
confirmed that the enzyme from cultured T. gondii was similar
to preparations previously reported. On the basis of these
studies with DHFR, we have designated cultured T. gondii as
our standard source for enzyme preparations.
Isola t ion of P . ca r in ii DHFR. Cytosolic enzymes of P.
carinii were isolated as previously described for DHFR.49
Isolated organisms were resuspended in 50 mM phosphate
buffer (pH 7.0) containing leupeptin (20 µg/mL), phenylmeth-
ylsulfonyl fluoride (9 µg/mL), soybean trypsin inhibitor (50 µg/
mL), aprotinin (50 µg/mL), and 20 mM 2-mercaptoethanol,
sonicated, and centrifuged at 100000g. The supernate, which
contained both DHFR and DHPS activity, was stored in liquid
nitrogen.
Recombinant P. carinii DHFR was also produced for enzyme
assays. The gene sequence was identical to that previously
reported. The expression system used pET8C, which employs
the T7 RNA polymerase.55 Host E. coli containing the ap-
propriate plasmid construction was grown in Luria broth
culture with 75 µg/mL kanamycin at 37 °C on a rotary shaker.
The culture was transferred to fresh medium and OD590 was
monitored. When the OD590 reaches 0.4, the culture was shifted
to 42 °C for 30 min to induce the gene for T7 RNA polymerase.
Rifampin (200 µg/mL) was added to suppress E. coli RNA
polymerase. After 30 min, the culture was shifted back to 37
°C for 90 min. Cells were harvested by centrifugation, washed,
and suspended in appropriate buffer containing protease
inhibitors and 2-mercaptoethanol as described above. Bacterial
cells were ruptured by sonication. The 100000g supernate
containing recombinant P. carinii DHFR has been the stan-
dard enzyme used in the screen under contract NO1-AI-35171.
Studies with [35S]methionine incorporation have shown this
preparation to contain predominantly one heavy band on
Ack n ow led gm en t. This work was supported in part
by grants from the National Institute of Allergy and
Infectious Diseases (AI44661, A.G.; AI41743, A.G.), the
National Cancer Institute (CA43500, J .J .M.; CA10914,
R.L.K.) and Roswell Park Cancer Institute Core Grant
(CA16065, J .J .M.). The authors thank Ms. Cynthia
Russell for performing the cell culture studies and Mr.
William Haile for the other enzyme inhibition studies.
Su p p or tin g In for m a tion Ava ila ble: Analysis data for
6-17, 21, 23, and 31. This material is available free of charge
via the Internet at http/::pubs.acs.org.
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