2-amino-4-oxo-5-substituted-pyrrolo[2,3-d]pyrimidines as nonclassical antifolate inhibitors of thymidylate synthase

Article abstract of DOI:10.1021/jm960097t

Six novel 2-amino-4-oxo-5-[(substituted phenyl)sulfanyl]pyrrolo[2,3- d]pyrimidines 7-12 were synthesized as potential inhibitors of thymidylate synthase (TS) and as antitumor and/or antibacterial agents. The analogues contain a 5-thio substituent with a p

Full text of DOI:10.1021/jm960097t

J . Med. Chem. 1996, 39, 4563-4568
4563
2-Am in o-4-oxo-5-su bstitu ted -p yr r olo[2,3-d ]p yr im id in es a s Non cla ssica l
An tifola te In h ibitor s of Th ym id yla te Syn th a se1a ,b
Aleem Gangjee,*,† Farahnaz Mavandadi,† Roy L. Kisliuk,‡ J ohn J . McGuire,§ and Sherry F. Queener
Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University,
Pittsburgh, Pennsylvania 15282, Department of Biochemistry, Tufts University School of Medicine,
Boston, Massachusetts 02111, Grace Cancer Drug Center, Roswell Park Cancer Institute, Elm and Carlton Streets,
Buffalo, New York 14263, and Department of Pharmacology and Toxicology, Indiana University School of Medicine,
Indianapolis, Indiana 46202
Received February 2, 1996X
Six novel 2-amino-4-oxo-5-[(substituted phenyl)sulfanyl]pyrrolo[2,3-d]pyrimidines 7-12 were
synthesized as potential inhibitors of thymidylate synthase (TS) and as antitumor and/or
antibacterial agents. The analogues contain a 5-thio substituent with a phenyl, 4′-chlorophenyl,
3′,4′-dichlorophenyl, 4′-nitrophenyl, 3′,4′-dimethoxyphenyl, and 2′-naphthyl on the sulfur, and
were synthesized from the key intermediate 2-(pivaloylamino)-4-oxo-6-methylpyrrolo[2,3-d]-
pyrimidine, 17. Appropriately substituted aryl thiols were appended to the 5-position of 17
via an oxidative addition reaction using iodine, ethanol, and water under conditions which
also resulted in the deprotection of the 2-amino group. The compounds were evaluated against
human, Lactobacillus casei, Escherichia coli, Streptococcus faecium, and Pneumocystis carinii
(pc) TSs and against human, rat liver (rl), pc, and Toxoplasma gondii (tg) DHFRs. The
nonclassical analogues with the 3′,4′-dichloro and the 4′-nitro substituents in the side chain (9
and 10) were more potent than N-[4-[N-[(2-amino-3,4-dihydro-4-oxo-6-quinazolinyl)methyl]-
N-prop-2-ynylamino]benzoyl]-L-glutamic acid (PDDF, 1) and N-[5-[N-[(3,4-dihydro-2-methyl-
4-oxo-6-quinazolinyl)methyl]-N-methylamino]-2-thenoyl]-L-glutamic acid (ZD1694, 2) against
human TS. Analogues with the 4′-chloro, 3′,4′-dimethoxy, and naphthyl side chains (8, 11
and 12) were more potent than the unsubstituted phenyl analogue (7) but less than 2, 9, and
10 by 1 order of magnitude. They were all poor inhibitors of human, rl, and pc DHFRs (IC50
) 10-5 M) but moderate inhibitors (IC50 ) 10-6 M) of tg DHFR. The 4-nitro analogue, 10
(EC50 1.5 µM), was comparable to PDDF in its potency as an inhibitor of the growth of the
FaDu human squamous cell carcinoma cell line.
In tr od u ction
against mouse recombinant TS) and as an effective
antitumor agent.
Thymidylate synthase (TS) is a crucial enzyme that
catalyzes the reductive methylation of 2′-deoxyuridine-
5′-monophosphate (dUMP) to 2′-deoxythymidine-5′-
monophosphate (dTMP) utilizing 5,10-methylenetet-
rahydrofolate, a cofactor which acts as the source of the
methyl group as well as the reductant.2 This is the
exclusive de novo source of dTMP; hence inhibition of
TS activity, in the absence of salvage, leads to “thym-
ineless death”. Thus inhibition of TS has long been an
attractive goal for the development of antitumor agents.3,4
The antifolates N-[4-[N-[(2-amino-3,4-dihydro-4-oxo-6-
quinazolinyl)methyl]-N-prop-2-ynylamino]benzoyl]-L-
glutamic acid (PDDF, 1)5 and its third generation
analogue N-[5-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quin-
azolinyl)methyl]-N-methylamino]-2-thenoyl]-L-glutam-
ic acid, tomudex, (ZD1694, 2)6 are classical inhibitors
of TS with antitumor activity, and 2 is currently in
phase III clinical trials. These analogues possess a 6-6
ring-fused system and a benzoyl- and thenoyl-L-glutamic
acid side chain at the 6-position, similar to that of
natural folates. Taylor et al.7 have reported the syn-
thesis of N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo-
[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid, a
B-ring-contracted 6-5 ring-fused classical antifolate,
(LY231514, 3), as an inhibitor of TS (Ki ) 340 nM
† Duquesne University.
A potential drawback of classical antifolates, such as
1, 2, and 3, is their requirement for the reduced folate
carrier system for transport into cells,8 the impairment
of which can lead to drug resistance.9-12 An additional
‡ Tufts University School of Medicine.
§ Roswell Park Center Institute.
Indiana University School of Medicine.
X Abstract published in Advance ACS Abstracts, October 1, 1996.
S0022-2623(96)00097-0 CCC: $12.00 © 1996 American Chemical Society

4564 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23
Gangjee et al.
cause of resistance to classical antifolates, particularly
of TS inhibitors, is that their antitumor activity is in
part determined by their ability to function as sub-
strates for the enzyme folylpoly-γ-glutamate synthetase
(FPGS).6,7,13,14 FPGS catalyzes the formation of poly-
γ-glutamates which are long-acting, noneffluxing forms
of the classical antifolates that lead to high intracellular
concentrations of these antitumor agents.6,7,15-18 Cells
that poorly polyglutamylate these antifolates are resis-
tant to these drugs.13,19 For both 2 and 3 it has been
shown that conversion of the monoglutamate forms to
their pentaglutamates produces a 60-fold (for 2)6 to a
130-fold (for 3)7 increase in TS inhibitory potency.
Although polyglutamylation is necessary for the cyto-
toxicity to tumor cells, it has also been implicated as a
possible cause of detrimental side effects, such as renal
and hepatic toxicities, in the host.20,21
The disadvantages of resistance to classical antifolate
TS inhibitors could be overcome by lipophilic nonclas-
sical antifolates. Such compounds would diffuse into
cells passively and circumvent drug resistance in cells
due to defective active transport. Further, the detri-
mental effects, of resistance and toxicity, associated with
polyglutamylation would also be eliminated. One such
lipophilic antifolate, which is incapable of being poly-
glutamylated and bears little structural resemblance to
the normal folate cofactor, is N6-[4-(N-morpholinosul-
fonyl)benzyl]-N6-methyl-2,6-diaminobenz[cd]indole glu-
curonate (AG331, 4).22 Compound 4 is currently in
phase I clinical trials as an antitumor agent.23 Webber
et al.24 recently reported a series of classical and
nonclassical 5-substituted quinazolines as inhibitors of
TS of which 2-amino-6-methyl-5-(pyridin-4-ylsulfanyl)-
3H-quinazolin-4-one (AG337, 5), was the most promis-
ing and is currently in phase II clinical trials as an
antitumor agent. On the basis of these reports and with
the goal of developing new cancer chemotherapeutic
agents, we designed, synthesized, evaluated, and re-
ported25 a nonclassical compound 6 which incorporates
the pyrrolo[2,3-d]pyrimidine ring of 3 and the side chain
of 5. Analogue 6 was active against human TS and
moderately cytotoxic against human CCRF-CEM leu-
kemic cells. Compound 6 also exhibited selectivity for
human vs bacterial TS, indicating a species difference
in TS from different sources.
were also of interest as selective antibacterial and
antifungal agents.
The potent nonclassical TS inhibitors in the literature
possess different electron-withdrawing groups in place
of the CO-glutamate of classical antifolates. Electron-
donating groups para to N10 have been postulated to
diminish the magnitude and alter the direction of the
dipole-dipole interaction between TS and the substi-
tuted phenyl ring of the inhibitor.24,26,27 The analogues
7-12 were synthesized as an extension of our previous
work25 to explore the effects on TS inhibition of electron-
withdrawing and -donating groups on the side chain
phenyl ring.
Ch em istr y
The key intermediate in the synthesis of analogues
7-12 was the 2-(pivaloylamino)-4-oxo-6-methylpyrrolo-
[2,3-d]pyrimidine, 17 (Scheme 1), to which various side
chains could be conveniently attached. Compound 17
was obtained via protection of the 2-amino group of 15
which we25 initially synthesized utilizing the method
reported by Secrist and Liu.28 This method involves
heating a mixture of 2,6-diamino-4-hydroxypyrimidine,
13, with chloroacetone, 14, in dimethylformamide at 60
°C for 2 days, which affords two products on TLC at Rf
0.36 and 0.56 corresponding to the pyrrolo[2,3-d]pyri-
midine 15 (55%) and the furo[2,3-d]pyrimidine 16,
respectively. In an attempt to improve the yield of the
desired pyrrolo[2,3-d]pyrimidine 15, in this study we
used a modification of the method reported by Ra-
masamy et al.29 which involved addition of chloroac-
etone, 14, to a solution of 2,6-diamino-4-hydroxypyri-
midine, 13, in sodium acetate and water. Although the
product begins to fall out of solution within 10 min, the
heating was continued at 100 °C for 4 h for completion
of the reaction. This method has the advantage of a
Species differences in bacterial vs human dihydro-
folate reductases (DHFR) have led to clinically useful
lipophilic inhibitors of DHFR, such as trimethoprim and
pyrimethamine, which are selective against bacteria and
fungi. Selectivity of DHFR inhibitors for different
species has been attributed to their differences in
inhibition and also to the fact that some microbes lack
cellular transport mechanism(s) for folate and reduced
folates and are, thus, unable to utilize preformed folates.
Two such organisms of concern that lack the transport
mechanism for classical folates and antifolates are
Pneumocystis carinii (pc) and Toxoplasma gondii (tg).
Infections caused by these organisms are the principal
cause of death in patients with the acquired immuno-
deficiency syndrome (AIDS). We reasoned that since a
difference in inhibitory potency exists between TS from
different sources, nonclassical, lipophilic inhibitors of
TS may also provide for selective inhibition of bacteria
and/or fungal TS. Thus the target nonclassical TS
inhibitors, 7-12, in addition to their antitumor effects,

Inhibitors of Thymidylate Synthase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23 4565
Ta ble 1. TS Inhibition IC50 (µM)31-33
Sch em e 1
compd
human L. casei
E. coli
0.037
5.3
S. faecium P. carinii
1 (PDDF)
0.18
0.095
8.8
0.037
8.8
0.09
0.53
ND
60
2 (ZD1694) 0.88
625
7
0.34
30
1.0
0.13
0.15
2.4
100
>29 (30)a 29
>30 (0) >30 (30)
>30 (20)
30
8
9
10
11
12
>2.6 (0) >26 (40)
5.3
45
5.1
45
13
>45 (31)
15
2.2
2.0
>24 (33) >24 (30)
>25 (32) >25 (36)
>24 (12)
>25 (0)
ND
ND
2.0
a
Number in parentheses indicates percent inhibition obtained
at that concentration.
complete within 4-5 h, indicating that the electron-
donating or -withdrawing effects of substituents in the
aryl thiol ring did not influence the oxidative addition
reaction for pyrrolo[2,3-d]pyrimidines, which is in agree-
ment with the observations of Beveridge and Harris30
for indoles and pyrroles. The deprotection step, how-
ever, varied from 16 to 20 h; analogues 7-10 required
16 h while analogues 11 and 12 required 20 h for
complete deprotection.
Biologica l Eva lu a tion a n d Discu ssion
Compounds 7-12 were evaluated31-33 as inhibitors
of human, Lactobacillus casei, Escherichia coli, Strep-
tococcus faecium, and Pneumocystis carinii TS (Table
1) along with PDDF and ZD1694. Replacing the thio-
pyridine ring of 6 with a phenyl ring, as in 7, diminished
inhibition for human TS, L. casei TS, and S. faecium
TS. For human TS, analogues with substitutions on the
phenyl ring were more inhibitory than the unsubsti-
tuted phenyl analogue. The electronic nature of the
substitutent on the side chain, however, was an impor-
tant factor in determining inhibitory potency. Electron-
withdrawing 4′-chloro, 3′,4′-dichloro, and 4′-nitro sub-
stituents in analogues 8, 9, and 10, respectively, showed
the most potent inhibition against isolated human TS,
and 9 and 10 were equipotent with PDDF and were
better than ZD1694 (2), which, as mentioned above, is
currently in phase III clinical trials as an antitumor
agent. A comparison of the inhibitory potencies of 6 and
7 against human TS indicates a significant loss of
potency in going from an electron-withdrawing sub-
stituent in 6 to an unsubstituted phenyl ring in 7. This
deficiency in 7 is more than overcome by the introduc-
tion of one or more electron-withdrawing groups as
illustrated by the greater than 30-fold increase in
potency of 8 and a greater than 200-fold increase in
potency of 9 and 10 compared to that of 7. Electron-
donating groups, as in 11, or the replacement of the
phenyl ring with a naphthalene ring, as in 12, increases
activity against human TS compared to the unsubsti-
tuted phenyl ring in 7, but are 1 order lower than that
of 9 and 10 which contain strong electron-withdrawing
groups. Thus for human TS, electron-withdrawing
groups are highly conducive to potency. For L. casei
TS a similar trend, as observed for human TS, was
apparent in that strong electron-withdrawing groups
present in 9 and 10 rendered these analogues more
potent than the unsubstituted phenyl analogue 7 and
electron-donating groups in 11 and a naphthyl ring
instead of a phenyl ring in 12 were more potent than 7.
Compound 10 was similar in potency to ZD1694 against
L. casei TS. With the exception of PDDF, all the
analogues tested were severalfold less potent against
shorter reaction time (4 vs 48 h28) and higher yields of
the pyrrolo[2,3-d]pyrimidine (70% vs 55%25,28). The
mixture of the pyrrolo[2,3-d]pyrimidine 15 and the furo-
[2,3-d]pyrimidine 16 was not separated but rather
converted to their more lipophilic pivaloyl-protected
derivatives 17 and 18 using pivaloyl anhydride and
potassium carbonate. The protected mixture was sepa-
rated by boiling in ethyl acetate. The highly lipophilic
2,4-bis(pivaloylamino)-5-methylfuro[2,3-d]pyrimidine (18)-
was soluble and was separated by filtration. The
desired compound 2-(pivaloylamino)-4-oxo-3,4-dihydro-
7H-pyrrolo[2,3-d]pyrimidine (17) was obtained as an
ethyl acetate insoluble solid in sufficiently pure form
for use in further reactions.
Analogues 7-12 were obtained in a one-step oxidative
addition of appropriately substituted aryl thiols to the
5-position of 17. Thus heating a mixture of 17 with the
substituted aryl thiols in a mixture of ethanol/water (2:
1) with 2 equiv of iodine at reflux for a period of 16-20
h afforded the desired target compounds 7-12. Within
1 h TLC of the reaction mixtures showed the formation
of a new spot at Rf 0.66-0.68 with the starting material
spot at Rf 0.64. On continued reflux an additional new
spot was detected at Rf 0.15-0.19 which after 16-20 h
was the major spot observed. Evaporation of the
solvents under reduced pressure, followed by cooling and
addition of cold distilled water to the residue, resulted
in the formation of a precipitate which was collected by
filtration. Washing with diethyl ether removed unre-
acted aryl thiol and afforded the target compounds 7-12
in 36-77% yields. The large upfield singlet correspond-
1
ing to the pivaloyl group protons was absent in the H
NMR spectra of these compounds which showed instead
a singlet exchangeable with deuterium oxide at 6.08-
6.21 ppm that integrated for the two protons of the
2-amino group. This along with the absence of the
5-aromatic proton and the presence of the appropriate
protons of the side chain for 7-12 confirmed that both
substitution and deprotection had occurred.
On the basis of TLC data it was apparent that the
initial substitution step was rapid in all cases and was

4566 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23
Gangjee et al.
Ta ble 2. Inhibitory Concentrations (IC50, µM) against DHFRs
L. casei TS than against human TS, clearly indicating
a difference in the two enzymes. Against E. coli TS,
similar inhibition profiles were observed as mentioned
above for L. casei TS. Here, as in human TS, electron-
withdrawing groups (9 and 10) were more conducive to
inhibition than the unsubstituted analogue 7. However,
similar to L. casei TS, the potencies of all the compounds
evaluated, except PDDF, were severalfold less than
against human TS. Analogues 7-10 along with PDDF
and ZD1694 were also evaluated as inhibitors of P.
carinii TS. Compounds 8, 9, and 10 with electron
withdrawing groups were from 12- to 30-fold more
potent than the unsubstituted analogue 7, indicating,
as with human TS, E. coli TS, and L. casei TS, that
compounds with electron-withdrawing groups were bet-
ter than the unsubstituted analogue 7.
and Selectivity Ratiosa
P. carinii rat liver selectivity T. gondii selectivity
compd
(pc)
(rl)
ratio rl/pc
(tg)
3.1
11.7
244
20
ratio rl/tg
8
9
10
11
40
>20
>15
>21
24.6
>20
7470
0.62
ND
ND
ND
7.9
>1.7
30.6
>1
>21
a
These assays were carried out at 37 °C under conditions of
substrate (90 µM dihydrofolic acid) and cofactor (119 µM NADPH)
in the presence of 150 mM KCl.35,36
Ta ble 3. Growth Inhibition of the FaDu Human Squamous
Cell Carcinoma Cell Line by Continuous (120 h) Exposure to
the Inhibitorsa
Compound
EC50, µM
n
7
8
10
11
>10
2
3
3
2
4
6
6.7 ( 1.5
1.5 ( 0.4
>10
Against S. faecium TS, the results were somewhat
different. Compounds 8 and 10 with electron-withdraw-
ing groups were more potent than 7. However, com-
pound 9 with 3′,4′-dichlorophenyl substitution was not
as inhibitory as 8 or 10 and was similar to 7. Since the
electronic nature of the phenyl substituent of 8, 9, and
10 are similar, the only difference of 9 is the additional
m-chloro substituent. Further, compound 11 with
electron-donating groups and compound 12 with a
naphthyl ring were less inhibitory than the unsubsti-
tuted phenyl analogue 7. These results of 9, 11, and
12 are in contrast to those observed for human TS, L.
casei TS, and E. coli TS, where 9, 11, and 12 were
consistently better inhibitors than 7. Comparison of 9,
11, and 12 at the same concentrations in S. faecium TS,
as in the other two bacterial enzymes L. casei TS and
E. coli TS, indicates a decrease in potency for S. faecium
TS. One plausible explanation for the lower potency of
9, 11, and 12 may be steric hindrance which is not well
tolerated by S. faecium TS. All three analogues (9, 11,
and 12) contain a meta substitution in addition to the
para substitution on the phenyl ring. This additional
meta substitution could provide for steric hindrance to
the attachment of 9, 11, and 12 to S. faecium TS,
resulting in the observed lower potency. Compound 10,
the most potent analogue of the series (6-12), was again
about half as potent as ZD1694.
PDDFb
AG331b
1.7 ( 0.2
1.0 ( 0.1
a
b
Compound 9 was poorly soluble and was not tested. McGuire
et al., manuscript in preparation.
compounds evaluated, and compound 9 and 10 along
with PDDF were the most potent inhibitors of human
TS.
Inspite of the homology between TS isolated thus
far,34 the selectivity of these analogues for human over
bacterial TS (Table 1) implies that a difference does
exist, which we are currently exploring with a variety
of different analogues in an attempt to provide agents
which are selective toward bacterial TS. In preliminary
experiments, analogue 7 gave 54% inhibition of P.
carinii growth in culture when tested at a concentration
of 36 µM. These results indicate that growth inhibition
by 7 occurs at concentrations comparable to the IC50 for
P. carinii TS (60 µM).
Analogues 8, 9, 10, and 11 were also evaluated35,36
as inhibitors of T. gondii, P. carinii, and rat liver DHFR
(Table 2). Although all the analogues were poor inhibi-
tors of P. carinii and rat liver DHFR, they were
moderate inhibitors of T. gondii DHFR. Compounds 8
and 10 were 8 and 31 times more selective, respectively,
for T. gondii DHFR compared to rat liver DHFR. This
parallels the trend we recently reported37 for nonclas-
sical 5-substituted-2,4-diaminopyrrolo[2,3-d]pyrimidine
inhibitors of DHFR.
In summary, for 5-substituted-2-amino-4-oxopyrrolo-
[2,3-d]pyrimidines, strong electron-withdrawing groups
and diCl) on the side chain phenyl ring afforded
(NO2
significantly better inhibitors of human TS, L. casei TS,
E. coli TS, and P. carinii TS compared to the unsubsti-
tuted phenyl ring. These results are similar to the
result observed for the quinazolines reported by Webber
et al.24 For human TS and L. casei TS, electron-
donating groups in the phenyl ring and the substitution
of a naphthyl ring for a phenyl also afforded better
inhibitors than the unsubstituted phenyl, but the dif-
ference in inhibitory potency was not as pronounced as
for strong electron-withdrawing groups. Against S.
faecium TS, though electron-withdrawing groups were
beneficial for inhibition, the presence of a meta sub-
stituent on the phenyl ring, irrespective of its electronic
nature, decreased activity. Further, none of the ana-
logues were significant inhibitors of bacterial TS and
shared this property with ZD1694. PDDF was the most
potent inhibitor of bacterial and fungal TS of the
The potent human TS inhibitory activity of com-
pounds 7, 8, 10, and 11 prompted the evaluation of these
analogues as inhibitors of the growth of the FaDu
human squamous cell carcinoma cell line in culture
(Table 3). Compound 9 could not be evaluated due to
its poor solubility. The growth inhibitory potency of 10
was comparable to that of PDDF and AG331 (Table 3).
Thus nonclassical analogues such as 10 could be useful
antitumor agents against tumors resistant to classical
antifolates24 such as PDDF, 2, and 3. These classical
analogues require active transport into tumors and need
polyglutamylation to exert their antitumor effects, both
of which are sources of tumor resistance8,11-14,17,20,21 and
neither of which is necessary for the cytotoxicity of 10.
We are currently exploring analogues of 10 as potential
antitumor agents, the results of which will be the topic
of future communications.

Inhibitors of Thymidylate Synthase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23 4567
was left at 4 °C for 6 h and filtered. The residue was washed
well with water and air-dried. The residue was then stirred
with diethyl ether (25 mL), filtered, washed with additional
amounts of ether to remove the unreacted thiophenol, and
dried to yield 0.09 g (77%) of 7: mp 275-280 °C dec; TLC Rf
0.40 (CHCl3/MeOH, 5:1, with 2 drops of NH4OH); 1H NMR
(Me2SO-d6) δ 2.18 (s, 3 H, 6-CH3), 6.11 (bs, 2 H, 2-NH2), 6.99
(m, 3 H, C6H5), 10.22 (s, 1 H, 7-NH), 11.41 (s, 1 H, 3-NH).
Anal. Calcd for (C13H11N4OS‚0.3H2O) C, H, N, S.
2-Am in o-6-m eth yl-5-[(4′-ch lor op h en yl)su lfa n yl]-3,4-d i-
h yd r o-4-oxo-7H-p yr r olo[2,3-d ]p yr im id in e (8). Compound
8 was synthesized as described for 7. 8: yield 40%; mp 255-
265 °C dec; TLC Rf 0.41 (CHCl3/MeOH, 5:1, with 2 drops of
NH4OH); 1H NMR (Me2SO-d6) δ 2.18 (s, 3 H, 6-CH3), 6.13 (bs,
2 H, 2-NH2), 6.99 (d, 3 H, C6H5), 10.22 (s, 1 H, 7-NH), 11.41
(s, 1 H, 3-NH). Anal. Calcd for (C13H11N4OS‚0.3H2O) C, H,
N, S.
Exp er im en ta l Section
All evaporations were carried out in vacuo with a rotary
evaporator. Analytical samples were dried in vacuo (0.2
mmHg) in an Abderhalden drying apparatus over P2O5 and
refluxing ethanol or toluene. Melting points were determined
on a Fisher-J ohns melting point apparatus and are uncor-
rected. Nuclear magnetic resonance spectra for proton (1H
NMR) were recorded on a Bruker WH-300 (300 MHz) spec-
trometer. Data was accumulated by 16K size with a 0.5 s
delay time and 70° tip angle. The chemical shift values are
expressed in ppm (parts per million) relative to tetramethyl-
silane as internal standard; s ) singlet, d ) doublet, dd )
doublet of doublet, t ) triplet, q ) quartet, m ) multiplet, bs
) broad singlet. The relative integrals of peak areas agreed
with those expected for the assigned structures. Thin layer
chromatography (TLC) was performed on POLYGRAM Sil
G/UV254 silica gel plates with fluorescent indicator, and the
spots were visualized under 254 and 366 nm illumination.
Proportions of solvents used for TLC are by volume. Elemen-
tal analyses were performed by Atlantic Microlabs Inc.,
Norcoss, GA. Analytical results indicated by element symbols
are within (0.4% of the calculated values. Fractional moles
of water or organic solvents frequently found in some analyti-
cal samples of antifolates were not removed in spite of 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.
2-Am in o-6-m eth yl-3,4-d ih yd r o-4-oxo-7H-p yr r olo[2,3-d ]-
p yr im id in e (15). Meth od A. To a suspension of 2,6-
diamino-4-hydroxypyrimidine (13) (5.04 g, 40.0 mmol) in N,N-
dimethylformamide (70 mL) was added chloroacetone (14)
(4.44 g, 48.0 mmol), and the mixture was stirred at 60 °C for
48 h under nitrogen. Two new spots at Rf 0.37 (corresponding
to 15) and Rf 0.56 (corresponding to 16) were seen on TLC
(CHCl3/MeOH, 4:1).28 The mixture was directly converted to
17 without separation of 15 and 16.
Meth od B. A suspension of 2,6-diamino-4-hydroxypyrimi-
dine (13) (1.26 g, 10 mmol) in 25 mL of water containing
sodium acetate (0.82 g, 10 mmol) was heated to 100 °C until
it formed a clear solution. Chloroacetone (14) (0.79 mL, 10
mmol) was added to this solution in one lot, following which a
precipitate began to form within 10 min. The reaction mixture
was heated with stirring at 100 °C for an additional 4 h, cooled
to 0 °C, and filtered to afford 1.15 g (70%) of 15: TLC Rf 0.37
(CHCl3/MeOH, 4:1); 1H NMR (Me2SO-d6) δ 2.15 (s, 3 H, 6-CH3),
5.84 (s, 1 H, 5-CH), 5.97 (bs, 2 H, 2-NH2), 10.14 (bs, 1 H, 7-NH),
10.79 (bs, 1 H, 3-NH).
2-(P iva loyla m in o)-6-m eth yl-3,4-d ih yd r o-4-oxo-7H-p yr -
r olo[2,3-d ]p yr im id in e (17). To a solution of 15 (2.00 g, 12.3
mmol) in N,N-dimethylformamide (50 mL) was added anhy-
drous K2CO3 (1.70 g, 12.3 mmol) followed by pivaloyl anhydride
(7.99 g, 42.9 mmol). The suspension was stirred under
nitrogen at 100 °C for 12 h. The reaction mixture was cooled
to room temperature and filtered, and the filtrate was evapo-
rated under reduced pressure. The residue was dissolved in
methylene chloride (200 mL) and washed with water (2 × 150
mL). The organic layer was dried (MgSO4) and filtered. After
evaporation of the filtrate, the residue was stirred in boiling
ethyl acetate (300 mL) and filtered. The residue was washed
with hot ethyl acetate (100 mL) and dried to afford 1.30 g (43%)
of 17 as an off-white solid: mp 290-294 °C; TLC Rf 0.54
(CHCl3/MeOH, 9:1, with 2 drops of concentrated NH4OH); 1H
NMR (Me2SO-d6) δ 1.23 (s, 9 H, C(CH3)3), 2.25 (s, 3 H, 6-CH3),
6.07 (s, 1 H, 5-CH), 10.75 (s, 1 H, 7-NH), 11.35 (s, 1 H, 3-NH),
11.78 (s, 1 H, CONH).
2-Am in o-6-m et h yl-5-(p h en ylsu lfa n yl)-3,4-d ih yd r o-4-
oxo-7H-p yr r olo[2,3-d ]p yr im id in e (7). To a solution of 17
(0.10 g, 0.43 mmol) in a mixture of ethanol/water (2:1, 30 mL)
was added iodine (0.22 g, 0.86 mmol) followed by thiophenol
(0.10 g, 0.86 mmol), and the reaction mixture was heated to
120-130 °C for 16 h. The mixture was cooled to room
temperature and concentrated under reduced pressure. Cold
water (100 mL) was added to the residue and the pH adjusted
to 8 with concentrated NH4OH with stirring. The suspension
2-Am in o-6-m et h yl-5-[(3′,4′-d ich lor op h en yl)su lfa n yl]-
3,4-d ih yd r o-4-oxo-7H-p yr r olo[2,3-d ]p yr im id in e (9). Com-
pound 9 was synthesized as described for 7. The residue was
recrystallized with ethyl acetate/acetic acid to afford 9 in 55%
yield: mp 235-245 °C dec; TLC Rf 0.41 (CHCl3/MeOH, 5:1,
1
with 2 drops of NH4OH); H NMR (Me2SO-d6) δ 2.19 (s, 3 H,
6-CH3), 6.16 (bs, 2 H, 2-NH2), 6.96 (m, 1 H, 5′-CH), 7.16 (s, 1
H, 2′-CH), 7.44 (m, 1 H, 6′-CH), 10.26 (bs, 1 H, 7-NH), 11.51
(bs, 1 H, 3-NH). Anal. Calcd for (C13H10N4OSCl2‚0.5H2O‚0.1HI)
C, H, N, S, Cl.
2-Am in o-6-m et h yl-5-[(4′-n it r op h en yl)su lfa n yl]-3,4-d i-
h yd r o-4-oxo-7H -p yr r olo[2,3-d ]p yr im id in e (10). Com-
pound 10 was synthesized as described for 7. 10: yield 60%;
mp 245-255 °C dec; TLC Rf 0.42 (CHCl3/MeOH, 5:1, with 2
drops of NH4OH); 1H NMR (Me2SO-d6) δ 2.19 (s, 3 H, 6-CH3),
6.21 (bs, 2 H, 2-NH2), 7.17 (d, 2 H, 2′,6′-CH), 8.06 (d, 2 H, 3′,5′-
CH), 10.35 (bs, 1 H, 7-NH), 11.58 (bs, 1 H, 3-NH). Anal. Calcd
for (C13H11N5O3S‚0.5H2O) C, H, N, S.
2-Am in o-6-m eth yl-5-[(3′,4′-d im eth oxyp h en yl)su lfa n yl]-
3,4-dih ydr o-4-oxo-7H-pyr r olo[2,3-d]pyr im idin e (11). Com-
pound 11 was synthesized as described for 7. 11: yield 75%;
mp 235-245 °C dec; TLC Rf 0.41 (CHCl3/MeOH, 5:1, with 2
drops of NH4OH); 1H NMR (Me2SO-d6) δ 2.21 (s, 3 H, 6-CH3),
3.68 (s, 3 H, OCH3), 3.75 (s, 3 H, OCH3), 6.08 (bs, 2 H, 2-NH2),
6.57 (d, 1 H, 5′-CH), 6.89 (s, 1 H, 2′-CH), 10.23 (s, 1 H, 7-NH),
11.32 (s, 1 H, 3-NH). Anal. Calcd for (C15H16N4O3S‚0.5H2O)
C, H, N, S.
2-Am in o-6-m eth yl-5-(2′-n a p h th ylsu lfa n yl)-3,4-d ih yd r o-
4-oxo-7H-p yr r olo[2,3-d ]p yr im id in e (12). Compound 12
was synthesized as described for 7. 12: yield 36%; mp 245-
255 °C dec; TLC Rf 0.39 (CHCl3/MeOH, 5:1, with 2 drops of
NH4OH); 1H NMR (Me2SO-d6) δ 2.22 (s, 3 H, 6-CH3), 6.13 (bs,
2 H, 2-NH2), 7.20 (d, 1 H, C10H7), 7.38 (m, 3 H, C10H7), 7.74
(m, 3 H, C10H7), 10.23 (bs, 1 H, 7-NH), 11.46 (bs, 1 H, 3-NH).
Anal. Calcd for (C17H14N4OS‚0.3H2O‚0.3HI) C, H, N, S.
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. The FaDu human squamous cell carci-
noma monolayer cell line was subcultured in RPMI 1640/10%
fetal calf serum in 100 mm cell culture dishes (Falcon) as
described.25 Inhibition of growth of these cell lines during
continuos drug exposure was measured as described.25 EC50
values were determined visually from plots of percent control
growth versus the logarithm of drug concentration. The cell
line was verified to be negative for mycoplasma contamination
using GenProbe test kit during the course of these studies.
Ack n ow led gm en t. This work was supported, in
part, by NIH Grants GM40998 (A.G.), AI30900 (A.G.),
CA16056 (J .J .M.), CA8550710A (J .J .M.), and CA10914
(R.L.K.), and NIH Contract N01-AI-87240 (S.F.Q.) Divi-
sion of AIDS.
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Doctor of Philosophy degree, J uly 1995. (b) Presented in part at
the 209th American Chemical Society National Meeting, Ana-
heim, CA, April 2-6, 1995; Abstr MEDI 128.

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