Synthesis of classical and a nonclassical 2-amino-4-oxo-6-methyl-5- substituted pyrrolo[2,3-d]pyrimidine antifolate inhibitors of thymidylate synthase

Article abstract of DOI:10.1021/jm980586o

Compounds 2-5 were designed as potential antifolate nonpolyglutamatable inhibitors of thymidylate synthase (TS). These analogues are structurally related to 2-amino-4-oxo-5-substituted quinazolines and 2-amino-4-oxo-5- substituted pyrrolo[2,3-d]pyrimidine

Full text of DOI:10.1021/jm980586o

2272
J . Med. Chem. 1999, 42, 2272-2279
Syn th esis of Cla ssica l a n d a Non cla ssica l 2-Am in o-4-oxo-6-m eth yl-5-su bstitu ted
P yr r olo[2,3-d ]p yr im id in e An tifola te In h ibitor s of Th ym id yla te Syn th a se1
Aleem Gangjee,*,† Farahnaz Mavandadi,†, Roy L. Kisliuk,‡ 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, and Department of
Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202
Received October 14, 1998
Compounds 2-5 were designed as potential antifolate nonpolyglutamatable inhibitors of
thymidylate synthase (TS). These analogues are structurally related to 2-amino-4-oxo-5-
substituted quinazolines and 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines which have
shown excellent inhibition of TS and, for the quinazoline, significant promise as clinically useful
antitumor agents. Compounds 2-4 were synthesized by appropriate amine exchange reactions
on pivaloyl-protected 5-dimethylaminomethyl-substituted 6-methyl pyrrolo[2,3-d]pyrimidine
7 which in turn was obtained from the Mannich reaction of pivaloylated-6-methyl pyrrolo[2,3-
d]pyrimidine 6. In instances where the amine exchange reaction was sluggish, the Mannich
base was quaternized with methyl iodide which afforded much faster exchange reaction with
improved yields. For compound 5, 4-mercaptopyridine was used as the nucleophile and reacted
with 7. The analogues 2-4 inhibited Lactobacillus casei (lc) TS and recombinant human (h)
TS with IC50 in the 10-4 to 10-5 M range. Compound 5 inhibited lcTS and hTS 20% at 26 and
25 µM, respectively. In addition, compound 5 inhibited the growth of Pneumocystis carinii and
Toxoplasma gondii cells in culture by 76% at 32 × 10-6 M and 50% at 831 × 10-6 M, respectively.
In tr od u ction
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
hydrophobic interaction with Phe176 and the backbone
TS activity, in the absence of salvage, leads to “thym-
of Gly173. The propargyl moiety was thus determined
ineless death”. Thus inhibition of TS has long been an
to be essential for the potent activity of PDDF against
attractive goal for the development of antitumor agents.3,4
TS. Following the unsuccessful clinical trials of PDDF,
5-Fluorouracil (5-FU), a mechanism-based inhibitor
a series of 2-desamino modified analogues were reported
of TS, is metabolically transformed to 5-fluorodeox-
which improved the solubility characteristics.8 From
yuridinemonophosphate (FdUMP) and is the only TS
these studies emerged the 2-desamino-2-methyl-N10-
inhibitor antitumor agent FDA-approved for clinical use
methyl-5,8-dideaza analogue ZD1694 (Tomudex).9 This
compound is a weaker inhibitor of TS (Ki ) 670 nM), as
in the United States. Since several types of cancer either
do not respond to 5-FU or develop resistance to it, effort
compared to PDDF, however the 2-methyl moiety af-
continues to design folate analogues as potential TS
fords significantly improved solubility characteristics
inhibitors and as antitumor agents.5
compared to the 2-amino analogues. ZD1694 has been
The first antifolate TS inhibitor to enter clinical trials
approved in Europe as an anticancer agent and is
was 10-propargyl-5,8-dideazafolate (CB3717, PDDF)
currently undergoing clinical trials in the United States.
synthesized by J ones et al.6 PDDF inhibited human TS
Recent X-ray crystallographic studies of the ternary
with a Ki ≈ 10 nM and showed in vivo antitumor
activity. However, PDDF displayed nephrotoxicity as
well as hepatotoxicity, and clinical trials were discon-
tinued. The N10-propargyl moiety of PDDF provides a
10-fold increase in potency against TS as compared to
its N10-methyl analogue. The crystal structure of the
ternary complex PDDF with Escherichia coli (ec) TS and
dUMP7 shows that the N10-propargyl moiety is in
complex of ecTS-dUMP-ZD169410 show its structure to
be similar to that of ecTS-dUMP-PDDF.
† Duquesne University.
‡ Tufts University School of Medicine.
§Indiana University School of Medicine.
Current address: Centaur Pharmaceuticals, Inc., 484 Oakmead
Parkway, Sunnyvale, CA 94086.
Taylor et al.11 reported the 2-amino-4-oxo-5-substi-
tuted classical pyrrolo[2,3-d]pyrimidine LY231514 as a
multitarget antifolate which inhibits TS as well as
10.1021/jm980586o CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/27/1999

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2273
dihydrofolate reductase (DHFR).12 This compound is
currently in phase II clinical trials as an antitumor
agent and differs from PDDF and ZD1694 in that it
contains a 6-5-pyrrolo[2,3-d]pyrimidine ring system
rather than the 6-6-quinazoline ring system of PDDF
and ZD1694. LY231514 is a moderate TS inhibitor with
a Ki ) 340 nM (recombinant mouse); however, conver-
sion to the pentaglutamate increased its TS inhibitory
potency by 100-fold.12 Interestingly, LY231514 is also
a powerful DHFR inhibitor with a Ki ) 7.0 nM as its
monoglutamate.12
tumor cells and hence could be useful where resistance
is attributable to inefficient uptake systems.13
In an attempt to circumvent the drawbacks of poly-
glutamylation, Rosowsky et al.,26a-c Webber et al.,13
J ackman et al.,27 and Bavetsias et al.28a-c have designed
and synthesized a variety of nonpolyglutamatable TS
inhibitors as antitumor agents. The most successful of
these is ZD9331 which is a 2,7-dimethyl-3′-fluoro-γ-
tetrazole analogue of PDDF and was reported by Mar-
sham et al.29 This compound is a potent TS inhibitor
(Ki ) 0.44 nM) as well as a potent inhibitor of L1210
cell growth, utilizes the reduced folate carrier system,
and is currently in phase II clinical trials as an
antitumor agent. The presence of the 7-methyl moiety
as well as the tetrazole in ZD9331 prevents its poly-
glutamylation. Thus ZD9331 is currently the most
successful nonpolyglutamatable antifolate and attests
to the importance of nonpolygutamatable TS inhibitors
AG337 (Thymitaq) is a nonclassical, 5-thiopyridine-
substituted 2-amino-4-oxo-6-methylquinazoline anti-
folate which was designed as a TS inhibitor using the
X-ray crystal structure of E. coli TS with PDDF and
5FdUMP by Webber et al.13 This was the first nonclas-
sical antifolate TS inhibitor to reach clinical trials.14
AG337 is currently being developed by Zarix. AG337
had reached phase II/III clinical trials as an antitumor
agent for the treatment of solid tumors of the liver and
head and neck cancer by Agouron Pharmaceuticals.
as potential antitumor agents.
31
Gangjee et al.30,
reported both classical and non-
classical 5-substituted 2-amino-4-oxo-6-methylpyrrolo-
[2,3-d]pyrimidines which are hybrid analogues of AG337
and LY231514 and possess significant TS inhibitory
activity. For example, compounds 1a -e inhibited hu-
man TS with IC50 ) 340,30 42,30 1000,31 130,31 and 150
nM,31 respectively, compared to PDDF IC50 ) 180 nM31
and ZD1694 IC50 ) 880 nM.31 Interestingly compound
An important determinant of the antitumor activity
of classical antifolates is their ability to function as a
substrate for the enzyme folylpolyglutamate synthetase
(FPGS) which converts classical antifolates into long
chain noneffluxing poly-γ-glutamates within the cell.
The ineffectiveness of many classical antifolates against
resistant tumor cells has been attributed, in part, to
both reduced uptake15-18 and/or inefficient polyglut-
amylation.19-22 Several examples exist where inefficient
polyglutamylation contributes to poor antitumor activ-
ity, particularly with methotrexate (MTX), in solid
tumors such as soft-tissue sarcomas,23 cervical squa-
mous cell carcinomas,24 and head and neck squamous
cell carcinomas.25 In addition to solid tumors, certain
forms of leukemias also respond ineffectively to MTX
due to insufficient polyglutamylation.
Although polyglutamylation of certain classical anti-
folates appears necessary for cytotoxicity it has also
been implicated as a possible cause of toxicity to host
cells due to the prolonged retention of polyanionic forms
of polyglutamates. Potent inhibitors of TS which do not
require polyglutamylation would not only avoid drug
resistance due to inefficient polyglutamylation but could
also reduce the side effects associated with retention in
host cells via polyglutamylation. Further lipophilic,
nonclassical TS inhibitors such as AG337 do not require
the reduced folate transport systems to gain access to
1b is not a substrate for FPGS.30 Thus 1b was different
from PDDF, ZD1694, and LY231514 in that all three of
these antitumor agents are substrates for FPGS and
indeed require polyglutamylation for potent inhibition
of TS and antitumor activity. Compounds such as 1b
might therefore be utilized in instances where the tumor
is resistant to ZD1694 or LY231514 due to inefficient
FPGS activity in the tumor system. We were therefore
interested in providing additional analogues that were
potent TS inhibitors which would not require poly-
glutamylation for antitumor activity.
Molecular modeling using SYBYL 6.432 and superim-
posing the 6-5-pyrrolo[2,3-d]pyrimidine on to the 6-6-
quinazoline (PDDF and ZD1694) indicates that the
smaller 6-5 system has its 5- and 6-atoms closer to the
pyrimidine ring than the 6- and 7-atoms of the quinazo-
line. In compounds 1a -e not only was the bridge
attached to the 5-position of the pyrrolo[2,3-d]pyrimi-
dine, thus shortening the distance between the pyrimi-
dine ring and the side chain substituent as compared

2274 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Brief Articles
Sch em e 1a
to the quinazolines, but the bridge was also truncated
to a single atom as compared with PDDF, ZD1694, and
LY231514.
We reasoned that increasing the length of the bridge
of compound 1b by one atom would provide analogues
of LY231514. Further increasing the bridge length of
1b by an additional atom, providing a three-atom
bridge, would compensate for the smaller 6-5 ring
system as compared to the 6-6 ring system and would
place the p-aminobenzoyl-L-glutamate side chain at
approximately the same distance from the pyrimidine
ring as in the 6-6 ring system analogues, PDDF and
ZD1694. We thus designed compounds 2-5 as potential
antitumor agents. In the classical antifolate compounds
2-3, the two-atom bridge is a C8-N9 chain. Compound
3 contains the N9-propargyl moiety similar to PDDF.
Compound 4 is a three-atom bridged analogue. Com-
pound 5 is a bridge homologue of the significantly potent
compound 1a and was designed to compensate for the
contraction of the B-ring of 5 compared to the 6-6 ring
system of AG337.
a
Reagents and conditions: (a) dimethylamine/37% HCHO/
AcOH; (b) DMF/∆; (c) 1 N NaOH/sonicate/15 min; (d) 1 N NaOH/
sonicate/1 h/50 °C.
1). 2-Acylamino pyrrolo[2,3-d]pyrimidines have been
reported31 to afford the 5-substituted aminomethyl
compounds. The 2-acylamino group was proposed to
increase the nucleophilicity of the â-carbon of the pyrrole
ring compared to that of the unacylated analogue, thus
favoring the 5-substituted product. Hence, a Mannich
reaction was carried out on the pivaloylated pyrrolo-
[2,3-d]pyrimidine 6 utilizing the method described by
Benghiat and Crooks.34,35 The Mannich reagent was
prepared by the slow addition of 37% aqueous formal-
dehyde to a cold (0 °C) solution of N,N-dimethylamine
in glacial acetic acid. Compound 6 was added to the cold
reagent solution, and the mixture was heated to 70 °C
for 5 h until the disappearance of 6 on TLC. The product
7 was isolated as a white solid in 82% yield. Compound
7 was homogeneous on TLC and was characterized by
Webber et al.13 in their design of the nonclassical TS
inhibitor AG337 using the crystal structure of the
ternary complex of ecTS-5FdUMP-PDDF suggested that
the 6-methyl moiety of AG337 serves two important
functions. In AG337 it makes important hydrophobic
contacts with the Trp80 residue of ecTS and also
conformationally restricts the 5-position side chain of
AG337 into a favorable, low-energy conformation for
attachment to TS. Molecular modeling using Sybyl 6.4
and superimposition of compounds 2-5 on to the
ternary complex of ecTS-FdUMP-AG33733 indicates that
the pyrrolo[2,3-d]pyrimidine with a 6-methyl moiety
mimics the 6-methyl quinazoline of AG337 and, as
reported previously by Gangjee et al.30,31 for compounds
1a -e, provides for potent TS inhibition. In addition,
metabolite protection studies of 1b with FaDU squa-
mous cell carcinoma cells indicated that the primary
intracellular target of 1b is TS. In addition to the role
of the 6-methyl moiety to interact with Trp80 of ecTS
and to conformationally restrict the 5-substituted side
chain, Gangjee et al.30 have reported that the 6-methyl
group in classical 5-substituted pyrrolo[2,3-d]pyrim-
idines as in 1b also prevents the molecule from func-
tioning as a substrate for FPGS, and hence polyglutamy-
lation of 1b does not play a role in the cytotoxicity of
1b. Thus we elected to retain the 6-methyl group in the
design of compounds 2-5 as inhibitors of TS.
1
its H NMR and high-resolution mass spectrum.
The amine exchange reaction of 3-dimethylaminom-
ethylindole and its related compounds with an amine
(primary or secondary) was shown to proceed by an
elimination-addition mechanism to afford indoles with
a variety of aminomethyl side chains at the 3-position.
Akimoto et al.36 have used this technique to synthesize
the nucleoside, Queuosine, analogues. The 5-(substitut-
ed aminomethyl)pyrrolo[2,3-d]pyrimidine 7 was found
to undergo facile amine exchange reaction on warming
a solution of the compound with an excess of the
primary or secondary amine. The introduction of a new
amino function into the pyrrolo[2,3-d]pyrimidine ring
was presumed to have progressed via the formation of
a conjugated unsaturated ring system 7a by amine
elimination from 7 followed by the addition of the added
amine (Scheme 1).
Ch em istr y
Gangjee et al.31 recently reported the synthesis of the
pivaloyl-protected pyrrolo[2,3-d]pyrimidine 6 (Scheme
The analogues 2 and 3 were synthesized by a similar
amine exchange reaction between the intermediate 7

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2275
Sch em e 2a
Sch em e 3a
Ta ble 1. Inhibitory Concentrations (IC50, in µM) against TS
compd
L. caseia,39
human39
PDDF31
0.036
100
0.021
180
0.036
0.34
0.042
180
a
(a) Reagents and conditions: (a) DBDC; (b) isobutylchlorofor-
1a 30
mate/Et3N; (c) TFA/Me2Cl2/rt/10 min; (d) propargyl bromide/
Hunig’s base/toluene/90 °C/18-20 h.
1b30
2
3
4
5
90
ND
and the appropriate L-glutamate diethyl esters 8a ,37
8b,6 and 938 (Scheme 1). For compound 2 a mixture of
p-aminobenzoyl L-glutamate diethyl ester 8a and 7 was
heated at 100 °C in DMF for 53 h. Workup and
purification using silica gel column chromatography and
gradient elution (0-10% MeOH/CHCl3) afforded 10 in
47% yield. Compound 10 had a mp of 195 °C and was
homogeneous on TLC. Its mass spectrum showed an M+
peak at 582 and its 1H NMR spectrum showed the
8-CH2 at 4.31 ppm and the NH triplet at 6.45 ppm,
along with the appropriate protons from both the
glutamate and the pyrrolo[2,3-d]pyrimidine moieties,
corroborating the structure. The ester groups were
cleaved on sonicating a suspension of 10 in 1 N NaOH
until a clear brown solution was formed (15 min). The
product obtained upon acidification of this solution was
360
ND
>26 (20)a
>25 (20)a
chromatography using gradient elution with 0-5%
MeOH-CHCl3. Compounds 11 and 12 were character-
ized by their H NMR spectra and elemental analyses.
The diethyl ester moiety and the 2-pivaloyl groups were
cleaved by first dissolving the suspension of 11 or 12 in
1 N NaOH by sonication followed by heating at 50 °C
for 1 h. Compounds 3 and 4 were obtained, as solids,
upon acidification of this solution, to pH 2, with glacial
acetic acid. Target compounds 3 and 4 were character-
1
1
ized by their H NMR spectra and elemental analyses.
The nonclassical compound 5 was similarly synthe-
sized. In this case a sulfur nucleophile was required to
displace the dimethylamino moiety of 7 (Scheme 3).
Benghiat and Crooks34,35 have reported the preparation
of S-(3-indolylmethyl) derivatives of cysteine from
gramine, 2-methylgramine, and 3-(dimethylamino)-1H-
pyrrolo[2,3-b]pyridine. The dimethylamino group of
gramine was reported to be readily substituted by thiols
in aqueous sodium hydroxide. An analogous displace-
ment of the dimethylamino group of 7 with 4-mercap-
topyridine (16) was expected to yield the analogue 17.
Thus a solution of 16 in DMF was first activated with
NaH followed by the addition of 5-dimethylaminometh-
ylpyrrolo[2,3-d]pyrimidine 7. The reaction was allowed
to proceed for 12 h at 50 °C after which the product 17
was isolated in 38% yield following workup. The pivaloyl
group was cleaved by stirring a solution of 17 in a
mixture of MeOH-1 N NaOH at 50 °C for 1 h, which
afforded the target compound 5 in 51% yield. The
product was homogeneous on TLC and was character-
1
identified, by H NMR and elemental analysis, as the
pivaloyl protected acid, 13. The 2-pivaloyl group was
cleaved on stirring 13 in 1 N NaOH for 1 h at 50 °C.
Neutralization with glacial acetic acid afforded the
target compound 2, in 88% yield.
Compound 8b was synthesized using a slight modi-
fication of the literature method.6 Thus alkylation of
p-aminobenzoyl-L-glutamate with propargyl bromide in
the presence of Hunig’s base and heating in toluene for
18-20 h afforded the monoalkylated product (Scheme
2B). Purification of the crude product using silica gel
column chromatography with toluene/ethyl acetate (10:
2) as eluent afforded 8b which was identical to that
reported in the literature.6
For the analogue 4, p-aminomethylbenzoyl-L-glutamic
acid diethyl ester (9) was used for the amine exchange
reaction. The ester 9 was in turn obtained from 4-ami-
nomethyl benzoic acid as previously described by Gangjee
et al. (Scheme 2A).38
1
ized by its H NMR spectrum and elemental analysis.
Biologica l Eva lu a tion a n d Discu ssion
The amine exchange reaction between 7 and 8b and
7 and 9 was extremely slow and occurred only when the
dimethyl amino group of 7 was converted to a better
leaving group via quaternization using methyl iodide.
A solution of the compound 7 in DMF was stirred with
0.9 equiv of methyl iodide. The quaternization of the
dimethyl groups was assumed to have occurred when
the DMF solution turned turbid after stirring for 2 h at
room temperature. The amine 8b or 9 was added to this
turbid solution, and the mixture was sonicated until no
more starting material was detected on TLC (48 h).
Compounds 11 and 12 were isolated from the reaction
with 8b and 9, respectively, after silica gel column
The analogues 2-5 were evaluated as inhibitors of
Lactobacillus casei TS and recombinant human TS.39,40a,b
The inhibitory potencies (IC50) are listed in Table 1
along with that of PDDF, 1a , and 1b. The N-propargyl
analogue 3 was 2-fold and 4-fold more active against L.
casei TS than the corresponding N-unsubstituted ana-
logues 2 and 4 respectively. This result is in accord with
previous reports of increased TS inhibitory activity of
N-propargyl analogues as compared with the N-unsub-
stituted compounds.6 Compound 2 was also evaluated
and found to be poorly active against human TS. The
nonclassical analogue 5 inhibited both L. casei and

2276 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Brief Articles
human TS by 20% at 2.6 × 10-4 M and 2.5 × 10-4 M,
respectively. Higher concentrations of 5 could not be
tested due to interference with absorbance at 340 nm
used in the TS assay.
inhibition and will be the subject of a future com-
munication.
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 were accumulated by 16 K 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. Elemental
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 analytical samples
of antifolates were not 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.
In view of our interest in obtaining selective agents
for two prevalent opportunistic infections in AIDS
patients, we also evaluated the nonclassical analogue
5 as an inhibitor of the growth of Pneumocystis carinii
and Toxoplasma gondii cells in culture.41,42 Compound
5 inhibited the growth of P. carinii cells in culture by
76% at 32 µM and T. gondii cells in culture by 50% at
831 µM. The assay for the inhibition of the growth of P.
carinii cells in culture did not allow for a lower
concentration of 5 to be tested.
The lack of potent inhibitory activity of the classical
analogues 2-4 against TS was surprising, particularly
in light of the high potencies of the classical analogue
1b and LY231514 against TS. In addition, the low
potency of the nonclassical analogue 5 against human
TS compared to 1a was also surprising. On the basis of
the analogues reported in this study, along with the
previous reports of Gangjee et al.,30,31 it appears that
for 5-substituted pyrrolo[2,3-d]pyrimidines the 6-methyl
moiety is conducive for activity when the bridge at the
5-position is a single sulfur atom. This is true for the
classical as well as the nonclassical analogues. However,
if the bridge is homologated to a two-atom or three-atom
chain with a 6-methyl substituent, the analogue loses
activity as compared with a one-atom sulfur bridge
(compare 1a and 1b with 2-5). That homologation of
the bridge to two atoms is conducive to TS inhibitory
activity, at least in the classical pyrrolo[2,3-d]pyrimidine
analogues, is illustrated by LY231514. Thus it would
appear that the methyl moiety at the 6-position of
compounds 2-5 could be responsible, in part, for the
decrease in TS inhibitory activity. Taylor and Hu43 have
shown that placing a 6-methyl group on LY231514
substantially decreases the cytotoxicity of the molecule
against the growth of CEM cells in culture; however,
no TS inhibitory data was reported.
2-P iva loyla m in o-6-m eth yl-5-[(d im eth yla m in o)m eth yl]-
4-oxo-7H-p yr r olo[2,3-d ]p yr im id in e (7). Glacial AcOH (6
mL) was added dropwise over a period of 0.5 h to ice-cold 40%
w/v aqueous solution of dimethylamine (3 mL, 26.7 mmol).
Aqueous HCHO (37%) (2.20 mL, 29.3 mmol) was then added
followed by 2-pivaloylamino-6-methyl-4-oxo-7H-pyrrolo[2,3-d]-
pyrimidine (6) (1.93 g, 7.80 mmol). The suspension was
warmed to 70 °C, and the solution formed was stirred at 70
°C for 5 h, cooled to room temperature, and evaporated under
reduced pressure, azeotroping first with water (3 × 5 mL) and
then with MeOH (3 × 5 mL). Anhydrous ether (20 mL) was
then added to the oily residue which was then cooled at 0 °C
for 12 h. The white solid which separated was filtered, washed
with excess anhydrous ether, and dried under vacuum for 12
h to yield 1.90 g (82%) of 7 as an off-white powder: mp 115-
1
120 °C; TLC Rf ) 0.14 (MeOH/1 drop NH4OH, silica gel); H
Molecular modeling indicated that the 6-methyl moi-
ety of 2-5 occupies essentially the same position as the
6-methyl group of AG337 when bound to ecTS such that
it interacts with Trp80 of ecTS. Further, since this
interaction is conducive to potency against TS, it is
unlikely that this attribute of the 6-methyl moiety could
be responsible for the decreased inhibitory activity of
compounds 2-5. Webber et al.13 suggested that the
6-methyl group of AG337 also functions to conforma-
tionally orient the side chain of AG337 for optimal
interaction with TS. In compounds 2-5, which contain
a more flexible two- or three-atom bridge, the confor-
mational restricting influence of the 6-methyl moiety
is perhaps not as important as in 1a , 1b, and AG337
which contain a single sulfur atom bridge. It is conceiv-
able that the 6-methyl group of analogues 2-5 may
cause an unfavorable orientation of the side chain for
interaction with TS, and any gain in interaction with
Trp80 of the 6-methyl moiety is more than offset by the
necessity of the side chain to reorient itself into a less
stable conformation in order to interact with ecTS. The
synthesis of analogues of compounds 2-5, which lack
the 6-methyl group, are currently in progress to deter-
mine the contribution of the 6-methyl moiety to TS
NMR (Me2SO-d6) δ 1.23 (s, 9 H, C (CH3)3), 2.12 (s, 6 H,
N(CH3)2), 2.19 (s, 2 H, CH2), 3.52 (s, 3 H, 6 CH3), 11.28 (s, 1
H, NH); HRMS calculated for C15H23N5O2, m/z ) 305.1851;
found m/z ) 305.1861.
N-{2-P iva loyla m in o-4-oxo-6-m et h yl[(p yr r olo[2,3-d ]-
pyr im idin -5-yl)am in -m eth ylen e]ben zoyl}-L-glu tam ic Acid
Dieth yl Ester (10). A mixture of 6 (0.50 g, 1.64 mmol) and
p-aminobenzoylglutamic acid diethyl ester (8a ) (0.50 g, 1.55
mmol) in DMF (5 mL) was heated to 100 °C for 53 h. The
reaction mixture was then cooled to room temperature, and
the DMF evaporated under reduced pressure. The residue was
dissolved in MeOH, and 1.00 g of silica gel was added to this
solution which was evaporated to dryness to form a plug which
was loaded onto a dry silica gel column (2.4 cm × 20 cm) and
eluted with 0-10% MeOH:CHCl3 gradient. Fractions contain-
ing the product eluted in 10% MeOH:CHCl3 and were pooled
together and evaporated to yield 0.45 g (47%) of 10 as a light
brown powder: mp 195 °C; MS m/z 582 (M+); TLC Rf ) 0.74
1
(CHCl3/MeOH/NH4OH, 9:2:0.1, silica gel); H NMR (Me2SO-
d6) δ 1.15 (m, 6 H, CH2CH3), 1.23 (s, 9 H, C(CH3)3), 2.00 (m, 2
H, Glu â-CH2), 2.28 (s, 3 H, 6-CH3), 2.40 (t, 2 H, Glu γ-CH2),
4.05 (m, 4 H, OOC CH2 CH3), 4.31 (m, 3 H, N-CH2, Glu R-CH),
6.45 (t, 1 H, 9-NH), 6.68 (d, 2 H, 3′,5′-CH), 7.61 (d, 2 H, 2′,6′-
CH), 8.23 (d, 1 H, CONH), 10.75 (s, 1 H, NH), 11.45 (s, 1 H,
NH), 11.50 (s, 1 H, NH). Anal. (C29H38N6O7) C, H, N.
N-{2-P iva loyla m in o-4-oxo-6-m et h yl[(p yr r olo[2,3-d ]-
p yr im id in -5-yl)m eth yla m in om eth ylen e]ben zoyl}-L-glu t-

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2277
a m ic Acid Dieth yl Ester (12). To a solution of 7 (0.50 g, 1.64
mmol) in DMF (10 mL) was added CH3I (0.06 mL, 1.48 mmol),
and the mixture was stirred at room temperature for 2 h until
the solution turned turbid. The glutamate ester 9 (0.498 g,
1.48 mmol) was added to this mixture which was ultrasoni-
cated for 48 h until no more starting material could be detected
on TLC. The solution was evaporated to dryness under reduced
pressure. The residue was dissolved in MeOH, and 1.00 g of
silica gel was added to this solution and evaporated to dryness
to form a plug which was loaded onto a dry silica gel column
(2.4 cm × 20 cm) and eluted with a 0-5% MeOH:CHCl3
gradient. Fractions containing the product eluted in 5% MeOH:
CHCl3 and were pooled together and evaporated to yield 0.17
g (17%) of 12 as a light brown powder: mp >190 °C dec; TLC
Rf ) 0.58 (CHCl3/MeOH/NH4OH, 9:2:0.1, silica gel); 1H NMR
(Me2SO-d6) δ 1.17 (m, 6 H, CH2CH3), 1.21 (s, 9 H, C(CH3)3),
2.03 (m, 2 H, Glu â-CH2), 2.27 (s, 3 H, 6-CH3), 2.43 (t, 2 H,
Glu γ-CH2), 4.08 (m, 4 H, OOCCH2CH3), 4.24 (d, 4 H, CH2-
N-CH2), 4.44 (m, 1 H, Glu R-CH), 7.59 (d, 2 H, 3′,5′-CH), 7.94
(d, 2 H, 2′,6′-CH), 8.80 (d, 1 H, CONH), 9.36 (bm, 1 H, C-NH-
C), 10.97 (s, 1 H, NH), 11.80 (s, 1 H, NH), 12.22 (s, 1 H, NH).
2.33 (s, 3 H, CH3), 3.13 (t, 1 H, CtCH), 3.99 (s, 2 H, CH2),
4.42 (s, 2 H, CH2), 5.40 (m, 1 H, CH), 6.05 (bs, 2 H, NH2), 6.67
(d, 2 H, C6H5), 7.10 (d, 2 H, C6H5), 7.75 (d, 1 H, CONH), 10.38
(bs, 1 H, NH), 10.8 (bs, 1 H, NH). Anal. (C23H24N6O6‚0.5 H2O)
C, H, N.
N-{2-Am in o-4-oxo-6-m eth yl[(p yr r olo[2,3-d ]p yr im id in -
5-yl)m eth yla m in o m eth ylen e]ben zoyl}-L-glu ta m ic Acid
(4). A suspension of 12 (0.12 g, 0.20 mmol) in 1 N NaOH (0.5
mL) was ultrasonicated for 15 min until it formed a clear
brown solution. MeOH (5 mL) was added to this solution which
was stirred at 50 °C, for 1 h. The solution was then concen-
trated under reduced pressure, cooled to 0 °C and acidified
(pH 3.5) with glacial AcOH. The resulting suspension was
filtered, washed with water, and dried to yield 0.04 g (44%) of
4: mp > 200 °C; TLC Rf ) 0.15 (30% CHCl3/MeOH/NH4OH
9:2:0.1, silica gel); 1H NMR (Me2SO-d6) δ 2.03 (m, 2 H, Glu
â-CH2), 2.18 (s, 3 H, 6-CH3), 2.37 (t, 2 H, Glu γ-CH2), 4.07 (d,
2 H, N-CH2), 4.17 (d, 2 H, N-CH2), 4.37 (m, 1 H, Glu R-CH),
6.53 (s, 2 H, NH2), 7.57 (d, 2 H, 3′, 5′-CH), 7.93 (d, 3 H, 2′,
6′-CH, C-NH-C), 8.47 (d, 1 H, CONH), 11.16 (s, 1 H, NH).
Anal. (C21H24N6O6‚1.5 CH3COOH) C, H, N.
N-{2-P iva loyla m in o-4-oxo-6-m et h yl[(p yr r olo[2,3-d ]-
p yr im id in -5-yl) a m in om et h ylen e]b en zoyl}-L-glu t a m ic
Acid (13). A suspension of 10 (0.20 g, 0.34 mmol) in 1 N NaOH
(5 mL) was ultrasonicated for 15 min until a brown solution
had formed which was stirred for an additional 2 h at room
temperature and then neutralized with glacial AcOH. The
resulting suspension was refrigerated for 2 h and filtered to
yield 0.15 g (88%) of 13 as a bright yellow powder: TLC Rf )
0.14 (CHCl3/MeOH, 10:1, silica gel); 1H NMR (Me2SO-d6) δ 1.24
(s, 9 H, C(CH3)3), 1.91 (m, 2 H, Glu â-CH2), 2.08 (t, 2 H, Glu
γ-CH2), 2.28 (s, 3 H, 6-CH3), 4.31 (d, 2 H, N-CH2), 4.85 (m, 1
H, Glu R-CH), 6.45 (t, 1 H, 9-NH), 6.66 (d, 2 H, 3′,5′-CH), 7.64
(d, 2 H, 2′,6′-CH), 8.07 (d, 1 H, CONH), 10.78 (s, 1 H, NH),
11.34 (s, 1 H, NH), 11.86 (s, 1 H, NH). Anal. (C25H30N6O7‚1.0
H2O) C, H, N.
2-P iva loyla m in o-6-m et h yl-5-[(4′-p yr id in e)m er ca p t o-
m eth ylen e]-4-oxo-7H-p yr r olo-[2,3-d ]p yr im id in e (17). To
a solution of 4-mercaptopyridine 16 (1.00 g, 9.04 mmol) in DMF
(5 mL) was added NaH (0.10 g, 4.52 mmol), and the mixture
was stirred for 1 h at room temperature. The Mannich base 7
(1.15 g, 3.16 mmol) was then added, and the reaction mixture
was warmed to 50 °C for 48 h until no 7 was detected on TLC.
The reaction mixture was then cooled to room temperature,
and the solvent evaporated under reduced pressure. Water
(150 mL) was added to the residue which was then filtered
and dried to yield 0.50 g (42%) of 22: mp > 150 °C dec; TLC
1
Rf ) 0.65 (CHCl3/MeOH, 9:3, silica gel); H NMR (Me2SO-d6)
δ 1.24 (s, 9 H, C(CH3)3), 2.26 (s, 3 H, 6-CH3), 4.47 (d, 2 H,
CH2-S), 7.40 (d, 2 H, C5H4), 8.37 (d, 2 H, C5H4), 10.78 (s, 1 H,
NH), 11.46 (s, 1 H, NH), 11.83 (s, 1 H, NH). Anal. (C18H21N5-
SO2‚0.5 H2O) C, H, N, S.
2-Am in o-6-m eth yl-5-[(4′-pyr idin e)m er captom eth ylen e]-
4-oxo-7 H-p yr r olo-[2,3-d ]p yr im id in e (5). A solution of 17
(0.5 g, 1.3 mmol) in MeOH (45 mL) containing 1 N NaOH (4.5
mL) was stirred at 45-50 °C for 1 h until no more 17 was
detected on TLC. The solvents were evaporated under reduced
pressure and water (50 mL) was added to the residue which
was then neutralized with 50% HCl. The bright yellow
suspension was filtered, and the residue was recrystallized
N-{2-Am in o-4-oxo-6-m eth yl[(p yr r olo[2,3-d ]p yr im id in -
5-yl)a m in om eth ylen e]ben zoyl}-L-glu ta m ic Acid (2). A
suspension of 10 (0.20 g, 0.34 mmol) in 1 N NaOH (5 mL) was
ultrasonicated for 15 min until a brown solution had formed.
MeOH (10 mL) was added to this solution which was stirred
at 50 °C for 1 h. The solution was then concentrated under
reduced pressure, cooled to 0 °C, and neutralized with glacial
AcOH. The resulting bright yellow suspension was refrigerated
for 2 h and filtered to yield 0.13 g (87%) of 2: mp > 200 °C;
1
from
a mixture of ethyl acetate:glacial AcOH (9:1). The
TLC Rf ) 0.52 (30% NH4HCO3, cellulose); H NMR (Me2SO-
resulting light yellow solid was filtered and dried to yield 0.56
g (51%) of 5: mp > 175 °C dec; TLC Rf ) 0.56 (CHCl3/MeOH
9:3, silica gel); 1H NMR (Me2SO-d6) δ 2.14 (s, 3 H, 6-CH3), 4.38
(s, 2 H, CH2-S), 6.02 (s, 2 H, NH2), 7.31 (d, 2 H, C5H4), 8.33
(d, 2 H, C5H4), 10.28 (s, 1 H, NH), 10.91 (s, 1 H, NH). Anal.
(C13H13N5SO‚0.1 H2O‚0.3CH3COOC2H5) C, H, N, S.
d6) δ 1.86 (m, 2 H, Glu â-CH2), 2.08 (s, 3 H, 6-CH3), 2.23 (m,
2 H, Glu γ-CH2), 4.13 (m, 2 H, N-CH2), 4.24 (m, 1 H, Glu
R-CH), 5.94 (s, 2 H, NH2), 6.45 (t, 1 H, 9-NH), 6.54 (d, 2 H,
3′,5′-CH), 7.53 (d, 2 H, 2′,6′-CH), 8.02 (d, 1 H, CONH), 10.20
(s, 1 H, NH), 10.67 (s, 1 H, NH). Anal. (C20H22N6O6‚1.5 H2O)
C, H, N.
Th ym id yla te Syn th a se Assa y. Thymidylate synthase was
assayed spectrophotometrically at 30° and pH 7.4 in a mixture
containing 0.1 M 2-mercaptoethanol, 0.0003 M (6R,S)-tetrahy-
drofolate, 0.012 M formaldehyde, 0.02 M MgCl2, 0.001 M
dUMP, 0.04 M trisHCI, and 0.00075 M naEDTA.
This was the assay described by A. J . Wahba and M.
Friedkin,40a except that the dUMP concentration was increased
25-fold as per V. J . Davisson, W. Sirawaraporn, and D. V.
Santi.40b The reaction was initiated by the addition of an
amount of enzyme yielding a change in absorbance at 340 nm
of 0.016/min in the absence of inhibitor. The percent inhibition
was determined at a minimum of four inhibitor concentrations
within 20% of the 50% point. The standard deviations for
determination of the 50% points were within (10% of the
values given.
Ur a cil In cor p or a tion by Cu ltu r ed T. gon d ii. Uracil is
incorporated into nucleic acid by T. gondii grown in culture.44
T. gondii was originally isolated from an AIDS patient at
Indiana University School of Medicine. The organism was
isolated by growth in a BALB/c mouse and subsequently
amplifed by passage in mice. Organisms were taken from the
peritoneal fluid of infected mice and preserved by freezing.
N-{2-Am in o-4-oxo-6-m eth yl[(p yr r olo[2,3-d ]p yr im id in -
5-yl)pr opar gylam in om eth ylen e]ben zoyl}-L-glu tam ic Acid
(3). To a solution of 7 (0.50 g, 1.64 mmol) in DMF (15 mL)
was added CH3I (0.06 mL, 1.48 mmol), and the mixture stirred
at room temperature for 2 h until the solution turned turbid.
The propargyl glutamate ester (8b) (0.50 g, 1.48 mmol) was
added to this mixture which was heated at 100 °C for 48 h.
The solution was evaporated to dryness under reduced pres-
sure. The residue was dissolved in MeOH, and 1.00 g of silica
gel was added to this solution and evaporated to dryness to
form a plug which was loaded onto a dry silica gel column (2.4
cm × 20 cm) and eluted with a 0-5% MeOH:CHCl3 gradient.
Fractions containing the product eluted in 5% MeOH:CHCl3
and were pooled together and evaporated to afford the piv-
aloylated ester 11 which was dissolved in MeOH (5 mL)
containing 0.1 N NaOH. The mixture was heated at 50 °C for
1 h. The MeOH was evaporated, and the aqueous solution was
acidified with glacial AcOH to afford a brown precipitate. This
was filtered, washed with water, and dried over P2O5 to yield
0.030 g (4%) of 2 as a light brown powder: mp >200 °C dec;
1
TLC Rf ) 0.68 (CHCl3/MeOH/NH4OH, 9:2:0.1, silica gel); H
NMR (Me2SO-d6) δ 2.01 (m, 2 H, CH2), 2.22 (m, 2 H, CH2),

2278 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Brief Articles
Frozen stocks were used to inoculate cultures for drug testing.
The organism may be passed for several generations in culture
without loss of viability. Because mammalian cells do not
avidly incorporate uracil, the incorporation of uracil can be
used as an index of growth of T. gondii on host cells. For these
experiments T. gondii was grown on HEL (human embryonic
lung) cells with minimum essential medium (MEM) supple-
mented with glutamine (2 mM), penicillin/streptomycin (100
units/mL and 100 µg/mL, respectively), and fetal bovine serum
(10%). HEL cells were grown to confluency in 24-well tissue
culture plates using MEM as described above. To inoculate,
old medium was removed and replaced with 0.5 mL of fresh
medium containing 5 × 104 T. gondii tachyzoites obtained from
mouse peritoneal fluid or from tissue culture. Four hours after
addition of the inoculum, an additional 0.5 mL of medium
containing drug or control diluent was added to the wells.
Twenty-four hours after inoculation, 1 µCi of tritiated uracil
(New England Nuclear) was added to each well, and incubation
continued for an additional 24 h. At the end of this period,
the medium was sampled to determine remaining counts in
the supernatant. The cells were dislodged, suspended by
agitation, and filtered through glass fiber filters (Whatman)
using a well washing apparatus with a 24-place filter manifold.
Each well was washed with cold isotonic saline and added to
the filter. The filters were washed with approximately 20 mis
of cold isotonic saline, dried overnight, removed to scintillation
vials, and counted with Ultima Gold scintillant (Packard).
Cu ltu r e of P . ca r in ii for Dr u g Testin g. Compounds were
evaluated in short term culture using inocula from P. carinii-
infected rat lung and cell cultures of human embryonic lung
fibroblasts (HEL cells) as described.45-48 P. carinii was orgin-
ally obtained from a spontaneous infection in an immunosup-
pressed Sprague-Dawley rat. The lung of that rat was used
to make homogenates that contained viable P. carinii which
were then transtracheally inoculated into immunosuppressed
Sprague-Dawley rats that were initially free of any P. carinii.
The organism has been passaged in this way for over 10 years,
and frozen stocks are maintained to allow restart or expansion
of the colony for drug studies. The chromosome pattern
determined by PFGE has remained stable over time. This
strain of P. carinii is available through the AIDS Research
and Reference Reagent Program. Briefly, tissue cultures were
prepared using 24-well plates in which HEL cells had been
grown to confluency in MEM containing 10% fetal calf serum.
They were inoculated with 7 × 105 viable P. carinii tropho-
zoites per milliliter. Inoculum was prepared by grinding P.
carinii-infected rat lung in MEM, centrifuging at 250g to
remove fragments of tissue, and counting numbers of organ-
isms in 10 µL samples of supernate with Giemsa staining and
fluorescein diacetate/ethidium bromide viability stain. On the
basis of these counts, the number of organisms per milliliter
was adjusted by adding MEM to achieve the desired concen-
tration.
were done in the laboratory of Professors J ames W.
Smith and Marilyn S. Bartlett, Department of Pathol-
ogy, Indiana University School of Medicine.
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