Design, synthesis, and biological activities of classical N-{4-[2-(2-amino-4-ethylpyrrolo[2,3-d]pyrimidin-5-yl)ethyl] benzoyl}-L-glutamic acid and its 6-methyl derivative as potential dual inhibitors of thymidylate synthase and dihydrofolate reductase and

Article abstract of DOI:10.1021/jm0203534

Two novel analogues, N-{2-amino-4-ethyl[(pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl}-L-glutamic acid (2) and N-{2-amino-4-ethyl-6-methyl[(pyrrolo[2,3-d]pyrimidin-5-yl) ethyl]benzoyl}-L-glutamic acid (4), were designed and synthesized as potent dual inhibi

Full text of DOI:10.1021/jm0203534

J . Med. Chem. 2003, 46, 591-600
591
Design , Syn th esis, a n d Biologica l Activities of Cla ssica l
N-{4-[2-(2-Am in o-4-eth ylp yr r olo[2,3-d ]p yr im id in -5-yl)eth yl]ben zoyl}-L-glu ta m ic
Acid a n d Its 6-Meth yl Der iva tive a s P oten tia l Du a l In h ibitor s of Th ym id yla te
Syn th a se a n d Dih yd r ofola te Red u cta se a n d a s P oten tia l An titu m or Agen ts1
Aleem Gangjee,*,† J ianming Yu,† Roy L. Kisliuk,‡ William H. Haile,§ Giulia Sobrero,§ and J ohn J . McGuire§
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 Grace Cancer Drug Center, Roswell Park Cancer Institute,
Elm and Carlton Streets, Buffalo, New York 14263
Received August 14, 2002
Two novel analogues, N-{2-amino-4-ethyl[(pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl}-L-
glutamic acid (2) and N-{2-amino-4-ethyl-6-methyl[(pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl}-
L-glutamic acid (4), were designed and synthesized as potent dual inhibitors of thymidylate
synthase (TS) and dihydrofolate reductase (DHFR) and as antitumor agents. Compound 2 had
inhibitory potency against human DHFR similar to N-{4-[2-(amino-3,4-dihydro-4-oxo-7H-
pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl}-L-glutamic acid (LY231514) and 1, whereas 4 was
inactive against human DHFR. Both 2 and 4 were more potent than LY231514 against E. coli
TS. Against human TS, 2 was 7-fold less potent than LY231514 and 4 showed similar inhibitory
activity as LY231514. In contrast to 2, which was an efficient substrate of human folypoly-
glutamate synthetase (FPGS), 4 was a poor substrate of FPGS. Compound 2 showed GI50 values
in the nanomolar range against more than 18 human tumor cell lines in the standard NCI
preclinical in vitro screen.
In tr od u ction
of a novel compound N-{2-amino-4-methyl[(pyrrolo[2,3-
d]pyrimidin-5-yl)ethyl]benzoyl}-L-glutamic acid 1 as a
potent, classical, dual inhibitor of DHFR and TS (Chart
1). Compound 1 was designed to bind in the “2,4-
diamino mode” to DHFR and in the “2-amino-4-methyl
mode” to TS, and hence function as a dual inhibitor
(Figure 1). The biological results showed that 1 was
more potent than a structurally related antifolate
LY231514 against both TS (Lactobacillus casei, Escheri-
chia coli, and rat) and DHFR (human, Toxoplasma
gondii, and Pneumocystis carinii). LY231514, a 2-amino-
4-oxo-5-substituted pyrrolo[2,3-d]pyrimidine, is cur-
rently in phases II and III clinical trials as an anticancer
agent. Compound 1 also demonstrated remarkable
inhibitory potency against the growth of tumor cells in
culture (GI50 < 1.00 × 10-8 M).9 The X-ray crystal
structure of 1 indicated that in P. carinii DHFR, the
pyrrolo[2,3-d]pyrimidine ring does indeed bind in the
“2,4-diamino mode”9 (Figure 1). In this binding mode,
the pyrrole nitrogen mimics the 4-amino moiety of 2,4-
diamino pyrimidines. TS binding of 1 was expected to
arise from the “2-amino-4-methyl mode” (Figure 1),
which is obtained by a 180° rotation about the 2-NH2-
C2 bond of the “2,4-diamino mode.” Molecular modeling
studies (SYBYL 6.510) with human DHFR and the
conformation of 1 from the X-ray crystal structure of 1
bound to P. carinii DHFR9 shows that the proximity of
the 4-methyl group of 1 to Phe31 and Leu22 of human
DHFR may be responsible for the increased inhibitory
activity of 1 compared with LY231514 (unfavorable
interaction of the 4-oxo moiety with Phe31 and Leu22).
In addition, molecular modeling of 1 in the crystal
structure of the quinazoline CB3717 (PDDF)11 or ZD1694
Folate metabolism represents an important and at-
tractive target for cancer chemotherapy because of its
crucial role in the biosynthesis of nucleic acid precur-
sors.2,3 Dihydrofolate reductase (DHFR)4 catalyzes the
reduction of folate or 7,8-dihydrofolate (FH2) to tetra-
hydrofolate (FH4) and couples with thymidylate syn-
thase (TS). TS is a crucial enzyme that catalyzes the
reductive methylation of 2′-deoxyuridine 5′-monophos-
phate (dUMP) to thymidine 5′-monophosphate (dTMP)
utilizing 5,10-methylenetetrahydrofolate as a cofactor
that functions as the source of the one-carbon group as
well as the reductant.5 This is the sole de novo source
of dTMP, and hence, inhibition of DHFR or TS activity
leads to “thymineless death”. Thus, TS and DHFR have
long been recognized as important targets for cancer
chemotherapy, and several DHFR and TS inhibitors
have found utility as antitumor agents.6 Usually a 2,4-
diamino-substituted pyrimidine ring is considered im-
portant for potent DHFR inhibitory activity, while a
2-amino-4-oxopyrimidine or 2-methyl-4-oxopyrimidine
ring is considered important for potent TS inhibitory
activity.6-8
As part of our efforts to design and synthesize dual
inhibitors of DHFR and TS that might be capable of
providing “combination therapy” in a single agent
without the pharmacokinetic disadvantages of two
separate agents, Gangjee et al.9 reported the synthesis
* To whom correspondence should be addressed. Phone: (412) 396-
6070. Fax: (412) 396-5593. E-mail: gangjee@duq.edu.
† Duquesne University.
‡ Tufts University School of Medicine.
§ Roswell Park Cancer Institute.
10.1021/jm0203534 CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/21/2003

592 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Gangjee et al.
Ch a r t 1
F igu r e 1.
accommodate groups larger than methyl at the 4-posi-
tion. Since the 4-methyl substitution provided potent
inhibition of both DHFR and TS, homologation of the
substitution to a 4-ethyl could perhaps further increase
potency and provide better antitumor agents. Thus, 2
was designed as a classical dual inhibitor of DHFR and
TS.
A major drawback of classical antifolates, such as
LY231514, is that they require transport into cells via
the reduced folate carrier (RFC) system, which when
impaired can lead to drug resistance.13-15 An additional
cause of resistance to classical antifolates is that their
antitumor activity is in part determined by their ability
to function as substrates for the enzyme folypoly-γ-
glutamate synthetase (FPGS).16,17 Thus, tumor cells
with decreased FPGS activity are resistant to agents
such as LY231514 that require FPGS for antitumor
activity. FPGS catalyzes the formation of folylpoly-γ-
glutamates, which are long-acting, noneffluxing forms
of the classical antifolates that lead to high intracellular
concentrations of these antitumor agents. Though poly-
glutamylation of these agents is necessary for cytotox-
icity to tumor cells, they have also been implicated as a
possible cause of host toxicity, such as renal and hepatic
toxicities in the host, since they are polyionic and are
retained within normal cells as well.18 Gangjee et al.19
reported that compound 3, bearing a 6-methyl group,
was a potent inhibitor of human and bacterial TS. This
analogue was neither a substrate nor an inhibitor of
human FPGS derived from human CCRF-CEM cells
and thus was devoid of the disadvantages mentioned
above with respect to FPGS. Therefore, compound 4,
which incorporates a 6-methyl group into compound 2,
was designed and synthesized. Compound 4 was also
anticipated to be a dual inhibitor of DHFR and TS and
was not expected to be a substrate for FPGS. Further,
the 6-methyl group was anticipated to sterically restrict
the number of low-energy conformations attainable by
the 5-substituted side chain. Such conformational re-
striction has been reported to enhance TS inhibition by
providing conformation(s) more conducive to TS bind-
ing.20 For DHFR inhibition, however, molecular model-
ing suggests that the 6-methyl group of 4 is expected to
lie in a region that contains the backbone carbonyl
oxygen of Val115 (hDHFR) and the phenolic hydroxy
group of Tyr121 (hDHFR). These unfavorable interac-
tions could result in a decrease in DHFR inhibitory
activity of 4 compared to 2. Thus, it was of interest to
determine the effect of the 6-methyl group on DHFR,
TS, and FPGS activity as well as its effect on the
inhibition of the growth of tumor cells in culture.
(Tomudex)12 with E. coli TS such that the pyrrole
nitrogen of 1 is positioned on the 4-oxo moiety of the
quinazoline predicts that the 4-methyl group mimics the
quinazoline C8 of CB3717 or ZD1694 and can interact
with Leu143 (3.42 Å) just as the C7 and C8 positions of
the quinazolines. The pyrrole nitrogen with its hydrogen
mimics the 4-oxo moiety and could hydrogen-bond with
Asp169 and be in contact with the 4CdO of dUMP and
Gly173. Further, the stacking interaction of the pyrrolo-
[2,3-d]pyrimidine of 1 is more appropriately aligned
with dUMP than is either CB3717 or ZD1694 and may
contribute to the additional binding of 1 compared to
LY231514. As a result, compound 1 exhibits greater TS
inhibitory activity than LY231514.9 Further, molecular
modeling studies indicated that there was sufficient
space in the lipophilic regions of both DHFR and TS to
Ch em istr y
Compound 5, which was previously reported, was
chosen as the starting material (Scheme 1).21-23 Com-
pound 5, refluxed with phosphorus oxychloride, afforded

Glutamic Acid and Its Derivative
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 593
Sch em e 1a
amine, which afforded 14 in 75% yield (Scheme 3).
Desilylation of 14 with fluoride ion gave 15 in 95% yield.
Compound 13 was finally obtained by coupling 15 with
diethyl N-(iodobenzoyl)-L-glutamate. The reaction yield
was low and the workup was tedious. Compound 13 was
reduced to afford 16 in 91% yield. Treatment of the
resulting ethano-bridged intermediate 16 with 1 N
NaOH resulted in simultaneous saponification of the
ester and removal of the pivaloyl protecting group to
afford 2 in 66% yield.
Compound 4 was synthesized from intermediate 20.
The scheme employed for the synthesis of 20 was a
modification of reports by Taylor et al.27 4-Hydroxy-1-
pentene 17 was reacted with methyl 4-bromobenzoate
via palladium acetate catalyzed coupling in the presence
of tetra-n-butylammonium chloride, lithium acetate, and
lithium chloride to afford 18 in 80% yield (Scheme 4).27a
Heating 18 under reflux in 2-methoxyethanol with 1
equiv of 2-amino-4-oxo-6-hydrazinopyrimidine27a af-
forded the requisite hydrazone 19 in 85% yield as a
brown solid. The subsequent cyclocondensation was first
attempted by thermolysis in refluxing tetralin (bp 207
°C); however, no reaction occurred. The reaction was
eventually accomplished by using diphenyl ether (bp 256
°C) as a solvent, under nitrogen, to afford 20 in 47%
yield. The purification of 20 was carried out by column
chromatography, which was different from that reported
by Taylor et al.27b To increase the solubility of 20 in
organic solvents and to improve the yield of the sub-
sequent 4-chlorination, the 2-amino group was piv-
aloylated using pivaloyl chloride and pyridine, which
afforded 21 in high yield (96%) as a pale-yellow solid.
Compound 21 was then refluxed for 2 h with phosphorus
oxychloride to afford 22 in 90% yield. Compound 22 was
coupled with tributylvinyltin in the presence of
Pd(PPh3)4 to afford 23. The reaction was refluxed in
toluene and went to completion after 5 h. Unfortunately,
the isolated yield, after several attempts, was always
very low (5-10%). Brathe et al.28 reported similar low
yields in the synthesis of 6-alkenylpurines. It was
mentioned in their work that 6-vinylpurine was prone
to decomposition on extensive drying under high vacuum
or storage. Thus, 23 was isolated by flash chromatog-
raphy and immediately subjected to catalytic hydro-
genation using palladium/charcoal to afford 24 in 62%
yield (over two steps). Saponification of 24 with aqueous
sodium hydroxide afforded the free acid 25 in 70% yield.
Peptide coupling of 25 with the diethyl ester of L-
glutamic acid afforded 26 in 76% yield. It was noted that
the number of equivalents of the diethyl ester of
L-glutamic acid required for the coupling was more than
that of 25 and the reaction was repeated by at least one
additional activation cycle (second cycle of addition of
isobutyl chloroformate, diethyl ester of L-glutamic acid,
and triethylamine). Final saponification with aqueous
sodium hydroxide at room temperature followed by
acidification to pH 4 in an ice bath afforded the diacid
4 in 52% yield.
a
Reagents and conditions: (a) POCl3, reflux; (b) NIS, DMF,
room temp, dark; (c) diethyl N-(4-ethynylbenzoyl)-L-glutamate,
Pd(PPh3)4, CuI, NEt3, DMF.
Sch em e 2a
a
Reagents and conditions: (a) tributylvinyltin, Pd(PPh3)4,
toluene, reflux; (b) 5% Pd/C, H2, 50 psi; (c) NIS, THF, room temp,
dark.
the 4-chloro derivative 6. The 5-position of 6 was
regiospecifically iodinated by treatment with N-iodo-
succinamide (NIS) in the dark for 1 h to afford 7.
Palladium-catalyzed cross-coupling of 7 with diethyl
N-(4-ethynylbenzoyl)-L-glutamate under Sonogashira
conditions gave 8. Compounds 6-8 have been previously
reported by Shih et al.24 However, the synthesis of 6 in
this report is different from that of Shih et al.24
Compounds 7 and 8 were obtained as described by Shih
et al.24 Since the palladium-catalyzed cross-coupling
between aryl halides with organostannanes (the Stille
reaction25,26) has been widely used for the construction
of carbon-carbon bonds, coupling of 8 with tributyl-
vinyltin in the presence of Pd(PPh3)4 was attempted for
the synthesis of diethyl N-{4-[2-(2-pivaloylamino-4-
vinylpyrrolo[2,3-d]pyrimidin-5-yl)ethynyl]benzoyl}-L-
glutamate (9). However, the reaction did not occur even
under reflux for 8 h. Changing the solvent from toluene
to DMF also did not afford the desired product. Piv-
aloylation of the 2-amino moiety allows for protection
as well as an increase of the organic solubility. An
alternative route started from 6 (Scheme 2), which was
subjected to Stille coupling with tributylvinyltin in the
presence of Pd(PPh3)4. The reaction was driven to
completion by vigorous reflux in toluene. Following
reduction of the double bond and regioselective iodina-
tion, compound 12 was obtained in a relatively high
yield. However, the coupling reaction of 12 with diethyl
N-(4-ethynylbenzoyl)-L-glutamate under Sonogashira
conditions was unsuccessful and did not afford 13.
An alternative route for the synthesis of 2 involved
the coupling of compound 12 with trimethylsilylacet-
ylene in the presence of Pd(PPh3)4, CuI, and triethyl-
Biologica l Eva lu a tion a n d Discu ssion
Compounds 2 and 4 were evaluated as inhibitors of
L. casei, E. coli, and human DHFR and compared to 1,
LY231514, methotrexate (MTX), and trimethoprim (TMP)
(Table 1). Compound 2 showed significant inhibitory

594 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Gangjee et al.
Sch em e 3a
a
Reagents and conditions: (a) trimethylsilylacetylene, Pd(PPh3)4, CuI, NEt3, THF; (b) n-Bu4NF, THF, room temp; (c) diethyl
4-iodobenzoyl-L-glutamate, Pd(PPh3)4, CuI, NEt3, THF; (d) 5% Pd/C, H2, 50 psi; (e) 1 N NaOH, then 1 N HCl.
Sch em e 4a
a
Reagents and conditions: (a) methyl 4-bromobenzoate, Pd(OAc)2, n-Bu4NCl, LiOAc, LiCl; (b) 2-amino-6-hydrazino-4-oxopyrimidine,
2-methoxyethanol, reflux; (c) diphenyl ether, reflux; (d) PivCl, pyridine, 120-130 °C; (e) POCl3, reflux; (f) tributylvinyltin, Pd(PPh3)4,
toluene, reflux; (g) 5% Pd/C, H2, 50 psi; (h) 1 N NaOH, then 1 N HCl; (i) diethyl L-glutamate hydrochloride, isobutyl chloroformate, NEt3;
(j) 1 N NaOH, then 1 N HCl.
activity against E. coli DHFR and was 250-fold and
2-fold more potent than LY231514 and 1, respectively,
but was much less potent than MTX and TMP. Com-
pound 2 had inhibitory activity against human DHFR
similar to that of LY231514 and 1, although it was less
potent than MTX. While 4 was 10-fold more potent than
LY231514 against E. coli DHFR, it was significantly less
potent than 1 against L. casei DHFR (3000-fold) and E.
coli DHFR (12-fold) (Table 1). Further, compound 4 was
inactive against human DHFR, indicating that the
6-methyl moiety in this system all but abolishes human
DHFR inhibitory activity. This decrease in activity
against human DHFR of 4 compared to 2 is probably
due to the unfavorable interaction of the 6-methyl
moiety of 4 with the carbonyl oxygen of Val115 and the
phenolic hydroxyl of Tyr121 of human DHFR.

Glutamic Acid and Its Derivative
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 595
Ta ble 1. Inhibitory Concentrations (IC50) against Isolated
DHFR
Ta ble 3. Growth Inhibition of CCRF-CEM Human Leukemia
Cells: Its MTX-Resistant Sublines with Defined Mechanism
during Continuous Exposure (0-120 h)
IC50 (M)
a
EC50
,
nM
compd
L. casei
E. colia
humanb
CCRF-
CEM
R1b
(vDHFR)
R2c
(Vuptake)
R30dmd
(VGlun)
1
2
4
2.4 × 10-8
1.8 × 10-6
9.2 × 10-7
2.2 × 10-5
2.3 × 10-4
7.0 × 10-9
1.5 × 10-8
1.2 × 10-6
9.2 × 10-7
2.2 × 10-5 (14c)
1.4 × 10-6
2.2 × 10-8
3.4 × 10-6
drug
MTX
e
7.0 × 10-5
2.3 × 10-4
2.2 × 10-8
6.2 × 10-7
13.7 ( 0.5
(n ) 7)
985 ( 215
(n ) 4)
2630 ( 95
(n ) 3)
12.8 ( 3.2
(n ) 3)
LY231514d
methotrexate
trimethoprim
1e
2
12.5 ( 2.1
(n ) 4)
158 ( 8
(n ) 2)
575 ( 25
(n ) 2)
18.5 ( 0.5
(n ) 2)
15.0 ( 1.0
(n ) 2)
695 ( 25
(n ) 2)
750 ( 160
(n ) 2)
18.5 ( 1.5
(n ) 2)
a
Kindly provided by Dr. R. L. Blakley, St. J ude Children’s
b
Research Hospital, Memphis, TN. Kindly provided by Dr. J . H.
4
>10000
(n ) 2)
>20000
(n ) 2)
>10000
(n ) 2)
>20000
(n ) 2)
Freisheim, Medical College of Ohio, Toledo, OH. c Percent inhibi-
d
tion. Kindly provided by Dr. Chuan Shih, Eli Lilly & Co.,
a
Indianapolis, IN. e Not determined.
EC50 ) concentration of drug required to decrease cell growth
b
by 50% after 5 days of treatment. CCRF-CEM subline resistant
Ta ble 2. Inhibitory Concentrations (IC50) against Isolated TS
to MTX solely as a result of a 20-fold increase in wild-type DHFR
protein and activity. c CCRF-CEM subline resistant as a result of
IC50 (M)
d
decreased uptake of MTX. CCRF-CEM subline resistant to MTX
compd
L. casei
E. colia
humanb
solely as a result of decreased polyglutamylation. This cell line
has 1% of the FPGS specific activity (measured with MTX as the
folate substrate) of parental CCRF-CEM. e Data from ref 9.
1
2
4
9.0 × 10-6
2.0 × 10-6
8.0 × 10-5
1.8 × 10-5
1.1 × 10-4
1.0 × 10-5
1.0 × 10-7
1.0 × 10-6
8.0 × 10-5
9.0 × 10-6
1.1 × 10-5
1.0 × 10-6
1.5 × 10-7
f
>1.0 × 10-4 (23b)
2.0 × 10-5
8.0 × 10-6
5.5 × 10-8
LY231514c
ZD1694d
PDDFe
induced growth inhibition. This indicates an antifolate
mechanism of action of 2. Since 4 did not inhibit growth
of CCRF-CEM cells even at 10 µM, the effect of leuco-
vorin was not tested.
a
Kindly provided by Dr. F. Maley, New York State Department
of Health, Albany, NY. Percent inhibition. c Kindly provided by
Dr. Chuan Shih, Eli Lilly & Co., Indianapolis, IN. Kindly
b
Since compound 2 was 42-fold less potent than MTX
against isolated hDHFR but equipotent against the
growth of tumor cells (CCRF-CEM) in culture, it was
of interest to further define its mechanism of action.
Compound 2 was thus evaluated in culture as an
inhibitor of the growth of three CCRF-CEM sublines
with defined MTX resistance mechanisms. A subline
with increased DHFR expression,30 R1, is 72-fold resist-
ant to MTX but only 13-fold cross-resistant to 2. A
subline with decreased MTX uptake,31 R2, is 179-fold
resistant to MTX but only 46-fold cross-resistant to 2.
The subline with diminished ability to polyglutamylate
MTX,17 R30dm, is not MTX-resistant in continuous
exposure (resistance is only seen in intermittent expo-
sure) because nonpolyglutamylated MTX is a tight-
binding DHFR inhibitor and transport is sufficient to
maintain the drug in excess of DHFR. R30dm displayed
only a 1.2-fold cross-resistance to 2 in continuous
exposure. The cross-resistance patterns of the CCRF-
CEM sublines are consistent with DHFR being the
primary target of 2. Cross-resistance of the MTX
transport deficient subline R2 to 2 indicates that it uses
the MTX/reduced folate carrier (RFC) for uptake. How-
ever, the degree of cross-resistance to 2 was much less
than to MTX. This could indicate that the drug is more
efficiently transported and/or polyglutamylated than is
MTX and the higher intracellular levels attained are
able to inhibit DHFR. The low level of cross-resistance
of the polyglutamylation-deficient cell line R30dm could
suggest that polyglutamylation of the analogue is not
required for potent target inhibition by 2 or that the
analogue is an extremely efficient FPGS substrate (see
below).
d
provided by Dr. Ann J ackman, Institute of Cancer Research,
Sutton, Surrey, U.K. e Kindly provided by Dr. M. G. Nair, Uni-
versity of South Alabama, Mobile, AL. f Not determined.
Compounds 2 and 4 were also evaluated as inhibitors
of L. casei, E. coli, and human TS (Table 2) along with
LY231514, ZD1694, and PDDF. Compounds 2 and 4
were slightly more potent than LY231514 against E. coli
TS. However compound 2 was 7-fold less potent than
LY231514 against human TS, while compound 4 showed
inhibitory activity similar to LY231514. Interestingly,
both 2 and 4 were less potent than 1 against E. coli TS
(9-fold and 40-fold, respectively) and human TS (9-fold
and 80-fold, respectively). In contrast to DHFR, com-
pound 4 was more potent than 2 against both E. coli
TS (5-fold) and human TS (8-fold), indicating that the
6-methyl moiety was conducive to TS inhibitory activity.
Thus, to some extent, analogues 2 and 4 do exhibit dual
inhibition of DHFR and TS, as anticipated from their
structural resemblance to 1, and the 6-methyl moiety
was conducive to TS inhibition but detrimental to DHFR
inhibitory activity. Both 2 and 4 were less potent than
ZD1694 and PDDF against TS.
The growth inhibitory potencies of 2 and 4 were
compared to the growth inhibitory potencies of 1 and
MTX in continuous exposure against CCRF-CEM29
human leukemia cells in culture (Table 3). Compound
2 was highly growth inhibitory with an EC50 of 15 nM,
which is similar to the potency of MTX and 1. Interest-
ingly, compound 4 was devoid of inhibitory activity
against the growth of CCRF-CEM cells in culture. This
indicates that 4-alkylation (as in 1 and 2) is highly
conducive to tumor growth inhibition in culture; how-
ever, minor changes (such as a 6-methyl group) in this
structure can profoundly affect the inhibitory activity.
CCRF-CEM cells treated with levels of 2 (50 and 70 nM)
inhibiting growth by 94-97% could be fully protected
by leucovorin at g0.1 µM (data not shown); this was
similar to the pattern of protection against MTX-
Since polyglutamylation can markedly enhance the
binding properties of substrate and inhibitors to most
folate-dependent enzymes,32 polyglutamates of 2 may
be responsible for the inhibition of DHFR and TS. Both
2 and 4 were also tested as substrates for human FPGS
to determine if polyglutamylation may be part of their
mechanism of action (Table 4). The data indicate that

596 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Gangjee et al.
Ta ble 4. Activity of Folate Analogues as Substrates for
compound 1, the 4-ethyl group in compound 2 is clearly
responsible for this increased spectrum of activity. In
contrast, compound 4 had an IC50 value below 1.00 ×
10-8 M in only one cell line (HL-60).
Recombinant Human FPGSa
substrate
MTXd
Km, µM
Vmax,rel
0.82
Vmax/Km
0.02
n
49
In summary, 2-amino-4-ethyl 5-substituted pyrrolo-
[2,3-d]pyrimidine 2 and its 6-methyl analogue 4 were
designed and synthesized as classical antifolates. The
biological results show that, as an analogue of compound
1, compound 2 retained similar activity against both
DHFR and TS and compound 4 exhibited relatively low
activity against DHFR but was a better inhibitor of TS
than 2. It is noteworthy that extending the 4-substituent
from methyl to ethyl in compound 2 dramatically
increased the inhibitory activity and the spectrum
against the growth of several tumor cells in culture.
Similar to compound 1, compound 2 was an excellent
substrate for FPGS. The poor FPGS activity of com-
pound 4 demonstrated that a 6-methyl group, as an-
ticipated from previous reports, is detrimental to FPGS
substrate activity.
aminopterin
4.9 ( 1.0
1
0.21
>0.17
>0.11
4
2
2
2
1b
2c
4
<1
0.17 ( 0.02
0.23
0.10 ( 0.01
<2
96 ( 19
0.001 ( 0.0001
a
FPGS substrate activity was determined as described in
Experimental Section. Values presented are the average ( SD if
b
n g 3 and are the average ( range for n ) 2. Data from ref 9.
c Substrate activity was maximal at 5 µM. Slight substrate
inhibition occurred at 5-50 µM. Vmax,rel is estimated on the basis
of maximum activity of substrate relative to the maximum for
d
aminopterin within the same experiment. Data from ref 38.
Ta ble 5. Cytotoxicity Evaluation against Selected Tumor Cell
Lines35
GI50, M
1
2
4
Leukemia
HL-60
MOLT-4
K-562
<1.00 × 10-8
<1.00 × 10-8
1.47 × 10-5
<1.00 × 10-8 <1.00 × 10-8
>1.00 × 10-4
1.00 × 10-7
1.43 × 10-7
Exp er im en ta l Section
<1.00 × 10-8
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
ethanol at reflux. Thin-layer chromatography (TLC) was
performed on silica gel plates with fluorescent indicator. Spots
were visualized by UV light (254 and 365 nm). All analytical
samples were homogeneous on TLC in at least two different
solvent systems. Purification by column and flash chromatog-
raphy was carried out using Merck silica gel 60 (200-400
mesh). The amount (weight) of silica gel for column chroma-
tography was in the range of 50-100 times the amount
(weight) of the crude compounds being separated. Columns
were dry-packed unless specified otherwise. Solvent systems
are reported as volume percent of mixture. Melting points were
determined on a Mel-Temp II melting point apparatus and are
uncorrected. Proton nuclear magnetic resonance (1H NMR)
Non-Small-Cell Lung Cancer
A549/ATCC
NCI-H226
NCI-H460
<1.00 × 10-8 >1.00 × 10-4
<1.00 × 10-8 >1.00 × 10-4
<1.00 × 10-8 >1.00 × 10-4
6.14 × 10-8
<1.00 × 10-8
Colon Cancer
HCT-15
HT29
SW-620
>1.00 × 10-4
>1.00 × 10-4
>1.00 × 10-4
<1.00 × 10-8 >1.00 × 10-4
<1.00 × 10-8 >1.00 × 10-4
<1.00 × 10-8 >1.00 × 10-4
CNS Cancer
SF-539
<1.00 × 10-8
<1.00 × 10-8 >1.00 × 10-4
Melanoma
LOX IMVI
UACC-257
UACC-62
1.15 × 10-5
>1.00 × 10-4
>1.00 × 10-4
<1.00 × 10-8
9.86 × 10-5
<1.00 × 10-8 >1.00 × 10-4
<1.00 × 10-8 >1.00 × 10-4
Ovarian Cancer
spectra were recorded on
a Bruker WH-300 (300 MHz)
OVCAR-4
>1.00 × 10-4
<1.00 × 10-8 >1.00 × 10-4
spectrometer. The chemical shift (δ) values are reported as
parts per million (ppm) relative to tetramethylsilane as
internal standard; s ) singlet, d ) doublet, t ) triplet, q )
quartet, m ) multiplet, br ) broad singlet, exch ) protons
exchangeable by addition of D2O. Elemental analyses were
performed by Atlantic Microlab, Inc., Norcross, GA. Elemental
compositions were within (0.4% of the calculated values.
Fractional moles of water or organic solvents frequently found
in some analytical samples of antifolates could not be removed
despite 24 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 except
anhydrous solvents, which were freshly dried in the laboratory.
2-P iva loyla m in o-4-ch lor op yr r olo[2,3-d ]p yr im id in e (6).
A mixture of 521 (0.45 g, 1.92 mmol) and phosphorus oxy-
chloride (4.5 mL) was heated under reflux for 2 h. Excess
phosphorus oxychloride was removed under vacuum. The
gummy residue was stirred vigorously with crushed ice (5 g),
and the pH was adjusted to 4 with concentrated ammonium
hydroxide. The resulting precipitate was filtered, washed with
cold water (10 mL × 2), and dried. The crude solid was
dissolved in methanol, silica gel (5 g) was added, and the
solvent was evaporated to form a plug, which was dried, loaded
on top of a silica gel column, and eluted with 5:2 hexanes/
ethyl acetate. Fractions containing the product (TLC) were
pooled, and the solvent evaporated to afford 0.20 g (41%) of 6
as a white solid: TLC Rf ) 0.58 (CHCl3/MeOH, 9:1); mp 212
°C, dec (lit.24 mp 217-220 °C, dec); 1H NMR (DMSO-d6) δ 1.22
(s, 9 H, C(CH3)3), 6.52 (d, 1 H, 5-H), 7.54 (t, 1 H, 6-H), 10.05
(br, 1 H, 2-NHPiv, exch), 12.19 (br, 1 H, 7-NH, exch).
Renal Cancer
ACHN
SN12C
<1.00 × 10-8
1.69 × 10-7 >1.00 × 10-4
>1.00 × 10-4
<1.00 × 10-8 >1.00 × 10-4
Breast Cancer
MCF7
MDA-MB-435
MAD-N
1.69 × 10-7
>1.00 × 10-4
>1.00 × 10-4
<1.00 × 10-8 >1.00 × 10-4
<1.00 × 10-8 >1.00 × 10-4
<1.00 × 10-8 >1.00 × 10-4
analogue 2 is nearly as efficient as aminopterin (AMT)
as an FPGS substrate; its low Km value and low Vmax
precluded determination of these kinetic constants.
Analogue 2 also has a 19-fold lower Km and greater than
220-fold higher substrate efficiency compared to ana-
logue 4. These results support the previous hypoth-
esis33 that the existence of a 6-methyl group in both 3
and 4 contributes to their poorer FPGS substrate
activity.
Compounds 2 and 4 were selected by the National
Cancer Institute for evaluation as antitumor agents in
the preclinical in vitro screening program.34 The ability
of compounds 2 and 4 to inhibit the growth of different
tumor cell lines was measured as GI50 values, the
concentration required to inhibit the growth of tumor
cells in culture by 50% compared to a control (Table 5).
Compound 2 showed potent antitumor activity and
displayed a much broader spectrum of activity (18 cell
lines) than 1 (6 cell lines) with IC50 values below 1.00
× 10-8 M. Compared with the 4-methyl group in
2-P iva loyla m in o-4-vin ylp yr r olo[2,3-d ]p yr im id in e (10).
A solution of 6 (0.10 g, 0.40 mmol) and tributylvinyltin (0.38

Glutamic Acid and Its Derivative
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 597
g, 1.20 mmol) in toluene (10 mL) was stirred under nitrogen
for 30 min, followed by the addition of tetrakis(triphenylphos-
phine)palladium(0) (0.05 g, 0.04 mmol). The reaction mixture
was then refluxed for 3 h, cooled to room temperature, and
filtered through a Celite pad. The solvent was removed in
vacuo to afford the crude product, which was further purified
by flash chromatography on silica gel and eluted with 1.5%
MeOH in CHCl3. Fractions containing the product (TLC) were
pooled, and the solvent was evaporated to afford 0.07 g (70%)
of 10 as a yellow solid: TLC Rf ) 0.37 (CHCl3/MeOH, 19:1);
mp 188-190 °C; 1H NMR (DMSO-d6) δ 1.23 (s, 9 H, C(CH3)3),
5.75 (dd, 1 H, J ) 10.8 Hz, J ) 1.0 Hz, CHdCH2), 6.53 (dd, 1
H, J ) 17.45 Hz, J ) 1.0 Hz, CHdCH2), 6.59 (d, 1 H, J ) 2.1
Hz, 5-H), 7.13 (dd, 1 H, J ) 10.8 Hz, J ) 17.45 Hz, CHdCH2),
7.48 (d, 1 H, J ) 2.9 Hz, 6-H), 9.67 (s, 1 H, 2-NHPiv, exch),
11.95 (s, 1 H, 7-NH, exch); HRMS (EI) calcd for C13H16N4O
244.1324, found 244.1334.
2-P iva loyla m in o-4-eth ylp yr r olo[2,3-d ]p yr im id in e (11).
To a Parr hydrogenation bottle was added 10 (0.72 g, 2.95
mmol) dissolved in a mixture of THF (15 mL) and methylene
chloride (15 mL), followed by the addition of 5% Pd/C (0.90 g).
This mixture was hydrogenated at 50 psi for 18 h. After the
catalyst was filtered and washed thoroughly with hot methanol
(40 mL), the filtrate was concentrated in vacuo. The crude
residue was then flash-chromatographed on silica gel and
eluted with a gradient of 0-1.5% MeOH in CHCl3. Fractions
containing the product (TLC) were pooled and the solvent was
evaporated to afford 0.65 g (91%) of 11 as a yellow foam: TLC
Rf ) 0.37 (CHCl3/MeOH, 19:1); 1H NMR (DMSO-d6) δ 1.23 (m,
12 H, CH2CH3 and C(CH3)3), 2.92 (q, 2 H, J ) 7.5 Hz, CH2CH3),
6.58 (d, 1 H, J ) 2.0 Hz, 5-H), 7.37 (d, 1 H, J ) 2.0 Hz, 6-H),
9.67 (s, 1 H, 2-NHPiv, exch), 11.85 (s, 1 H, 7-NH, exch).
14 (0.17 g, 0.50 mmol) and tetrabutylammonium fluoride (1
mL) dissolved in THF (10 mL). The reaction mixture was
heated at 50 °C under nitrogen for 30 min. The reaction was
then quenched by pouring the mixture into chloroform (80 mL)
and washing it with brine. The organic layer was separated,
dried over Na2SO4, and concentrated in vacuo. The residue was
then flash-chromatographed on silica gel and eluted with 3%
MeOH in CHCl3. Fractions containing the desired product
(TLC) were pooled and evaporated to afford 0.13 g (95%) of 15
as a pale-yellow solid: TLC Rf ) 0.40 (MeOH/CHCl3, 1:19);
1
mp 200 °C, dec; H NMR (DMSO-d6) δ 1.23 (s, 9 H, C(CH3)3),
1.32 (t, 3 H, J ) 7.5 Hz, CH2CH3), 3.13 (q, 2 H, J ) 7.5 Hz,
CH2CH3), 4.19 (s, 1 H, CH), 7.77 (s, 1 H, 6-H), 9.75 (s, 1 H,
2-NHPiv, exch), 12.26 (s, 1 H, 7-NH, exch); HRMS (EI) calcd
for C15H18N4O 270.1481, found 270.1484.
Dieth yl N-{4-[2-(2-P iva loyla m in o-4-eth ylp yr r olo[2,3-d ]-
p yr im id in -5-yl)eth yn yl]ben zoyl}-L-glu ta m a te (13). To a
25 mL round-bottomed flask, protected from light with alu-
minum foil, were added 15 (0.06 g, 0.22 mmol), copper(I) iodide
(0.05 g, 0.03 mmol), diethyl 4-iodobenzoyl-L-glutamate (0.29
g, 0.66 mmol), and tetrakis(triphenylphosphine)palladium(0)
(0.04 g, 0.035 mmol) dissolved in anhydrous THF (10 mL) and
triethylamine (0.5 mL). The solution was stirred at room
temperature for 4 h under nitrogen. The volatiles were
removed in vacuo, and the residue was dissolved in methylene
chloride (100 mL). The reaction mixture was washed with
brine, and the organic layer was separated, dried over Na2SO4,
and concentrated in vacuo. The crude residue was then flash-
chromatographed on silica gel and eluted with a gradient of
0-1% MeOH in CHCl3. Fractions containing the desired
product (TLC) were pooled and evaporated to afford 0.03 g
(24%) of 13 as a pale-yellow solid: TLC Rf ) 0.40 (MeOH/
CHCl3, 1:19); mp 223-225 °C; 1H NMR (DMSO-d6) δ 1.21 (m,
6 H, R- and γ-COOCH2CH3), 1.24 (s, 9 H, C(CH3)3), 1.38 (t, 3
H, J ) 7.4 Hz, CH2CH3), 2.10 (m, 2 H, â-CH2), 2.45 (t, 2 H, J
) 7.5 Hz, γ-CH2), 3.23 (q, 2 H, J ) 7.4 Hz, CH2CH3), 4.06 (m,
4 H, R- and γ-COOCH2CH3), 4.15 (m, 1 H, R-CH), 7.62 (d, 2
H, J ) 8.4 Hz, C6H4), 7.89 (d, 1 H, J ) 2.2 Hz, 6-H), 7.94 (d,
2 H, J ) 8.4 Hz, C6H4), 8.83 (d, 1 H, J ) 7.4 Hz, CONH, exch),
9.81 (s, 1 H, 2-NHPiv, exch), 12.43 (s, 1 H, 7-NH, exch); HRMS
(EI) calcd for C31H37N5O6 575.2744, found 575.2749.
Dieth yl N-{4-[2-(2-P iva loyla m in o-4-eth ylp yr r olo[2,3-d ]-
p yr im id in -5-yl)eth yl]ben zoyl}-L-glu ta m a te (16). To a Parr
hydrogenation bottle was added 13 (0.115 g, 0.20 mmol),
dissolved in methanol (15 mL) and methylene chloride (15 mL),
followed by the addition of 5% Pd/C (0.15 g). This mixture was
hydrogenated at 50 psi for 2 days. After the catalyst was
filtered and washed thoroughly with hot methanol (30 mL),
the filtrate was concentrated in vacuo. The crude residue was
then flash-chromatographed on silica gel and eluted with 1:19
MeOH/CHCl3. Fractions containing the desired product (TLC)
were pooled and evaporated to afford 0.109 g (91%) of 16 as a
white solid: TLC Rf ) 0.32 (MeOH/CHCl3, 1:19); mp 160.5-
162.5 °C; 1H NMR (DMSO-d6) δ 1.17 (m, 6 H, R- and
γ-COOCH2CH3), 1.23 (s, 9 H, C(CH3)3), 1.28 (t, 3 H, J ) 7.5
Hz, 4-CH2CH3), 2.09 (m, 2 H, â-CH2), 2.44 (t, 2 H, J ) 7.5 Hz,
γ-CH2), 2.95 (q, 2 H, J ) 7.5 Hz, 4-CH2CH3), 3.06 (m, 4 H,
CH2CH2), 4.09 (m, 4 H, R- and γ-COOCH2CH3), 4.43 (m, 1 H,
R-CH), 7.12 (s, 1 H, 6-H), 7.36 (d, 2 H, J ) 7.9 Hz, C6H4), 7.81
(d, 2 H, J ) 7.9 Hz, C6H4), 8.66 (d, 1 H, J ) 7.4 Hz, CONH,
exch), 9.62 (s, 1 H, 2-NHPiv, exch), 11.59 (s, 1 H, 7-NH, exch);
HRMS (EI) calcd for C31H41N5O6 579.3075, found 579.3057.
2-P iva loyla m in o-4-et h yl-5-iod op yr r olo[2,3-d ]p yr im i-
d in e (12). To a 25 mL round-bottomed flask, protected from
light with aluminum foil, were added 11 (0.41 g, 1.67 mmol)
and N-iodosuccinimide (0.41 g, 1.84 mmol) dissolved in anhy-
drous THF (10 mL). The reaction mixture was stirred under
nitrogen for 1 h. The solvent was removed in vacuo, and the
residue was dissolved in methylene chloride (100 mL). The
reaction mixture was washed with brine, and the organic layer
was separated, dried over Na2SO4, and concentrated in vacuo.
The crude residue was flash-chromatographed on silica gel and
eluted with 0.5% MeOH in CHCl3. Fractions containing the
desired product (TLC) were pooled and evaporated to afford
0.35 g (56%) of 12 as a pale-yellow solid: TLC Rf ) 0.33
1
(MeOH/CHCl3, 1:19); mp 206-208 °C; H NMR (DMSO-d6) δ
1.27 (s, 9 H, C(CH3)3), 1.32 (t, 3 H, J ) 7.5 Hz, CH2CH3), 3.13
(q, 2 H, J ) 7.5 Hz, CH2CH3), 7.62 (s, 1 H, 6-H), 9.75 (s, 1 H,
2-NHPiv, exch), 12.28 (s, 1 H, 7-NH, exch); HRMS (EI) calcd
for C13H17N4O 372.0447, found 372.0437.
2-P ivaloylam in o-4-eth yl-5-tr im eth ylsilyleth yn ylpyr r olo-
[2,3-d ]p yr im id in e (14). To a 50 mL round-bottomed flask,
protected from light with aluminum foil, were added 12 (0.29
g, 0.77 mmol), copper(I) iodide (0.057 g, 0.30 mmol), trimethyl-
silylacetylene (0.45 g, 4.6 mmol), and tetrakis(triphenylphos-
phine)palladium(0) (0.088 g, 0.077 mmol) dissolved in anhy-
drous THF (15 mL), anhydrous DMF (5 mL), and triethyl-
amine (0.1 mL). The solution was stirred at room temperature
for 4 h under nitrogen. The volatiles were removed in vacuo,
and the residue was dissolved in dichloromethane (100 mL).
The reaction mixture was washed with water, and the organic
layer was separated, dried over Na2SO4, and concentrated in
vacuo. The crude residue was then flash-chromatographed on
silica gel and eluted with 1% MeOH in CHCl3. Fractions
containing the desired product (TLC) were pooled and evapo-
rated to afford 0.20 g (75%) of 14 as a white solid: TLC Rf )
0.42 (MeOH/CHCl3, 1:19); mp 261-262.5 °C; 1H NMR (DMSO-
d6) δ 0.23 (s, 9 H, Si(CH3)3), 1.17 (s, 9 H, C(CH3)3), 1.29 (t, 3
H, J ) 7.5 Hz, CH2CH3), 3.14 (q, 2 H, J ) 7.5 Hz, CH2CH3),
7.76 (d, 1 H, J ) 2.0 Hz, 6-H), 9.75 (s, 1 H, 2-NHPiv, exch),
12.27 (s, 1 H, 7-NH, exch); HRMS (EI) calcd for C18H26N4OSi
342.1875, found 342.1885.
N-{4-[2-(2-Am in o-4-eth ylp yr r olo[2,3-d ]p yr im id in -5-yl)-
eth yl]ben zoyl}-L-glu ta m ic Acid (2). To a 15 mL round-
bottomed flask was added 16 (0.09 g, 0.16 mmol) suspended
in 1.0 N NaOH (2 mL). The reaction mixture was stirred at
50 °C for 3 days. The resulting solution was cooled in an ice
bath and acidified with 1.0 N HCl to pH 4, and the precipitate
was filtered, washed with water, and dried in vacuo to give
1
0.05 g (66%) of 2 as a white solid: mp 248-250 °C, dec; H
NMR (DMSO-d6) δ 1.25 (t, 3 H, J ) 7.2 Hz, CH2CH3), 1.95-
2.02 (m, 2 H, â-CH2), 2.37 (t, 2 H, J ) 7.0 Hz, γ-CH2), 2.85 (q,
2 H, J ) 7.2 Hz, CH2CH3), 2.97 (m, 4 H, CH2CH2), 4.27 (m, 1
H, R-CH), 5.91 (s, 2 H, 2-NH2, exch), 6.68 (d, 1 H, 6-H), 7.34
2-P iva loyla m in o-4-et h yl-5-et h yn ylp yr r olo[2,3-d ]p yr -
im id in e (15). To a 25-mL round-bottomed flask were added

598 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Gangjee et al.
(d, 2 H, J ) 8.0 Hz, C6H4), 7.80 (d, 2 H, J ) 8.0 Hz, C6H4),
8.52 (d, 1 H, J ) 7.4 Hz, CONH, exch), 10.75 (s, 1 H, 7-NH,
exch), 12.35 (br, 1 H, COOH, exch). Anal. (C22H25N5O5) C, H,
N.
Meth yl 4-[2-(2-Am in o-4-oxo-6-m eth ylp yr r olo[2,3-d ]p yr -
im id in -5-yl)eth yl]ben zoa te (20). A mixture of 19 (0.40 g,
1.16 mmol) in diphenyl ether (20 mL) was stirred under
nitrogen and heated under reflux for 5 h. After the mixture
was cooled to room temperature, hexanes (50 mL) were added
and the precipitated solid was collected by filtration. The
residue was suspended in methanol (50 mL), silica gel (10 g)
was added, and the solvent was evaporated to afford a plug.
The silica gel plug obtained was loaded onto a silica gel column
3.82 (s, 3 H, OCH3), 5.72 (dd, 1 H, J ) 10.8 Hz, J ) 1.0 Hz,
CHdCH2), 6.57 (dd, 1 H, J ) 17.4 Hz, J ) 1.0 Hz, CHdCH2),
7.29 (d, 2 H, J ) 7.7 Hz, 5-H), 7.32 (dd, 1 H, J ) 10.8 Hz, J )
17.4 Hz, CHdCH2), 7.84 (d, 2 H, J ) 7.7 Hz, C6H4), 9.56 (s, 1
H, 2-NHPiv, exch), 11.52 (s, 1 H, 7-NH, exch).
Meth yl 4-[2-(2-P iva loya m in o-4-eth yl-6-m eth ylp yr r olo-
[2,3-d ]p yr im id in -5-yl)eth yl]ben zoa te (24). To a Parr hy-
drogenation bottle was added 23 (0.12 g, 0.35 mmol) dissolved
in a mixture of THF (10 mL) and methylene chloride (10 mL),
followed by the addition of 5% Pd/C (0.10 g). This mixture was
hydrogenated at 50 psi for 18 h. After the catalyst was filtered
and washed thoroughly with methanol (30 mL), the filtrate
was concentrated in vacuo. The crude residue was then flash-
chromatographed on silica gel and eluted with a gradient of
0-1% MeOH in CHCl3. Fractions containing the product (TLC)
were pooled and the solvent was evaporated to afford 0.11 g
(92%) of 24 as a yellow sticky solid: TLC Rf ) 0.42 (CHCl3/
MeOH, 19:1); mp 72-74 °C; 1H NMR (DMSO-d6) δ 1.22-1.29
(m, 12 H, C(CH3)3 and 4-CH2CH3), 1.99 (s, 3 H, 6-CH3), 2.80-
3.20 (m, 6 H, CH2CH2 and 4-CH2CH3), 3.83 (s, 3 H, OCH3),
7.25 (d, 2 H, J ) 7.4 Hz, C6H4), 7.85 (d, 2H, J ) 7.4 Hz, C6H4),
9.55 (s, 1 H, 2-NHPiv, exch), 11.50 (s, 1 H, 7-NH, exch); HRMS
(EI) calcd for C24H30N4O3 422.2321, found 422.2318.
and eluted with
a gradient of 5-10% MeOH in CHCl3.
Fractions containing the product (TLC) were pooled and the
solvent was evaporated to afford 0.18 g (47%) of 20 as a pale-
yellow solid: TLC Rf ) 0.41 (CHCl3/MeOH, 5:1); mp 260 °C,
dec (lit.27 260 °C ,dec); 1H NMR (DMSO-d6) δ 1.82 (s, 3 H, CH3,
2.76 (t, 2 H, J ) 6.5 Hz, CH2), 2.90 (t, 2 H, J ) 6.5 Hz, CH2),
3.82 (s, 3 H, OCH3), 5.94 (br, 2 H, 2-NH2, exch), 7.26 (d, 2 H,
J ) 7.7 Hz, C6H4), 7.83 (d, 2 H, J ) 7.7 Hz, C6H4), 10.07 (br,
1
H, 3-NH, exch), 10.48 (br, 1 H, 7-NH, exch). Anal.
(C17H18N4O3‚0.5H2O) C, H, N.
Meth yl 4-[2-(2-P iva loya m in o-4-oxo-6-m eth ylp yr r olo-
[2,3-d ]p yr im id in -5-yl)eth yl]ben zoa te (21). To a 50 mL
round-bottomed flask was added 20 (0.15 g, 0.43 mmol) in
anhydrous pyridine (5 mL). To this solution was added pivaloyl
chloride (0.27 mL, 2.14 mmol). The reaction mixture was
heated to reflux for 3.5 h under nitrogen. The solvents were
removed in vacuo, and the residue was dissolved in MeOH (50
mL). Silica gel (10 g) was added to this solution and the solvent
was evaporated to afford a plug that was flash-chromato-
graphed on a silica gel column and that eluted with 0.5%
MeOH in CHCl3 to afford 0.18 g (96%) of 21 as a pale-yellow
solid: TLC Rf ) 0.31 (CHCl3/MeOH, 19:1); mp 246-248 °C;
1H NMR (DMSO-d6) δ 1.24 (s, 9 H, C(CH3)3), 1.91 (s, 3 H,
6-CH3), 2.87 (m, 4 H, CH2CH2), 3.82 (s, 3 H, OCH3), 7.26 (d, 2
H, J ) 7.8 Hz, C6H4), 7.83 (d, 2 H, J ) 7.8 Hz, C6H4), 10.74 (s,
1 H, 2-NHPiv or 3-NH, exch), 11.05 (s, 1 H, 2-NHPiv or 3-NH,
exch), 11.75 (s, 1 H, 7-NH, exch). Anal. (C22H27N4O3) C, H, N.
Meth yl 4-[2-(2-P ivaloyam in o-4-ch lor o-6-m eth ylpyr r olo-
[2,3-d ]p yr im id in -5-yl)eth yl]ben zoa te (22). A solution of 21
(0.18 g, 0.44 mmol) and phosphorus oxychloride (5 mL) was
refluxed at 120 °C for 2.5 h. The excess POCl3 was evaporated,
and the residue was neutralized with NH4OH to pH 4. The
solution was diluted by addition of methylene chloride (100
mL). The organic layer was washed with brine (50 mL × 2),
separated, dried over Na2SO4, and concentrated in vacuo. To
this solution were added methylene chloride (20 mL) and silica
gel (10 g), and the solvent was then evaporated. The silica gel
plug obtained was loaded onto a silica gel column and eluted
with 0.5% MeOH in CHCl3. Fractions containing the product
(TLC) were pooled and the solvent evaporated to afford 0.17 g
(90%) of 22 as a white solid: TLC Rf ) 0.38 (CHCl3/MeOH,
19:1); mp 202-204 °C; 1H NMR (DMSO-d6) δ 1.23 (s, 9 H,
C(CH3)3), 2.02 (s, 3 H, 6-CH3), 2.94 (t, 2 H, J ) 6.7 Hz,
CH2CH2), 3.01 (t, 2 H, J ) 6.7 Hz, CH2CH2), 3.83 (s, 3 H,
OCH3), 7.26 (d, 2 H, J ) 7.4 Hz, C6H4), 7.85 (d, 2 H, J ) 7.4
Hz, C6H4), 9.95 (s, 1 H, 2-NHPiv, exch), 11.95 (s, 1 H, 7-NH,
exch). Anal. (C22H25N4O3Cl) C, H, N, Cl.
4-[2-(2-Am in o-4-eth yl-6-m eth ylpyr r olo[2,3-d]pyr im idin -
5-yl)eth yl]ben zoic Acid (25). To a solution of 24 (0.12 g, 0.35
mmol) in MeOH (10 mL) was added 1 N NaOH (2 mL), and
the mixture was stirred under nitrogen at room temperature
for 2 days to complete the reaction. The reaction mixture was
concentrated in vacuo and cooled in an ice bath, and the
residual aqueous solution was acidified with 1 N HCl to pH 4.
The resulting suspension was filtered, washed with a small
amount of cold water and ethyl ether, and dried in vacuo to
afford 0.08 g (70%) of 25 as a buff-colored solid: mp 190 °C,
dec; 1H NMR (DMSO-d6) δ 1.24 (t, 3 H, J ) 7.3 Hz, 4-CH2CH3),
1.90 (s, 3 H, CH3), 2.82 (m, 6 H, CH2CH2 and 4-CH2CH3), 5.79
(s, 2 H, 2-NH2, exch), 7.22 (d, 2 H, J ) 7.4 Hz, C6H4), 7.82 (d,
2 H, J ) 7.4 Hz, C6H4), 10.66 (s, 1 H, 7-NH, exch), 12.70 (br,
1 H, COOH, exch); HRMS (EI) calcd for C18H20N4O2 324.1586,
found 324.1585.
Dieth yl N-{4-[2-(2-Am in o-4-eth yl-6-m eth ylp yr r olo[2,3-
d ]p yr im id in -5-yl)eth yl]ben zoyl}-L-glu ta m a te (26). To a
solution of 25 (0.10 g, 0.31 mmol) in anhydrous DMF (5 mL)
was added triethylamine (0.13 mL), and the mixture was
stirred under nitrogen at room temperature for 5 min. The
resulting solution was cooled to 0 °C, isobutyl chloroformate
(0.13 mL, 0.96 mmol) was added, and the mixture was stirred
at 0 °C for 30 min. At this time TLC (CHCl3/MeOH, 5:1)
indicated the formation of the activated intermediate at Rf )
0.55 and the disappearance of the starting acid (Rf ) 0.35).
Diethyl L-glutamate hydrochloride (0.24 g, 0.96 mmol) was
added to the reaction mixture followed immediately by tri-
ethylamine (0.13 mL, 0.96 mmol). The reaction mixture was
slowly warmed to room temperature and was stirred under
nitrogen for 6 h. The reaction mixture was then subjected to
another cycle of activation and coupling using half the quanti-
ties listed above. The reaction mixture was slowly warmed to
room temperature and was stirred for an additional 24 h. The
reaction mixture was then subjected to a third round of
activation and coupling using the same quantities as the
second round and was stirred for an additional 24 h. TLC
showed the formation of one major spot at Rf ) 0.52 (CHCl3/
MeOH, 5:1). The reaction mixture was evaporated to dryness
under reduced pressure. The residue was dissolved in MeOH
(5 mL), silica gel (10 g) was added, and the solvent was
removed to form a plug. The silica gel plug obtained was loaded
onto a silica gel column and eluted with a gradient of 0.5-5%
methanol in chloroform. Fractions containing the product
(TLC) were pooled and the solvent was evaporated to afford
0.12 g (76%) of 26 as a yellow solid: Rf ) 0.52 (CHCl3/MeOH,
5:1); mp 125.5-127.5 °C; 1H NMR (DMSO-d6) δ 1.20 (m, 9 H,
4-CH2CH3, R- and γ-COOCH2CH3), 1.64 (m, 2 H, â-CH2), 1.92
(s, 5 H, 6-CH3 and γ-CH2), 2.40 (m, 5H, 4-CH2CH3) 2.84 (m, 4
H, CH2CH2), 4.08 (m, 4 H, R- and γ-COOCH2CH3), 4.43 (m, 1
H, R-CH), 5.77 (br, 2 H, 2-NH2, exch), 7.28 (d, 2 H, J ) 7.4
Meth yl 4-[2-(2-P iva loya m in o-4-vin yl-6-m eth ylp yr r olo-
[2,3-d ]p yr im id in -5-yl)eth yl]ben zoa te (23). A solution of 22
(0.18 g, 0.42 mmol) and tributylvinyltin (0.40 g, 1.26 mmol)
in toluene (15 mL) was stirred under nitrogen for 30 min,
followed by the addition of Pd(PPh3)2Cl2 (0.06 g, 0.084 mmol).
The reaction mixture was then refluxed for 4 h, cooled to room
temperature, and filtered through a Celite pad. The filtrate
was evaporated in vacuo to afford the crude product that was
further purified by flash chromatography on silica gel and
eluted with 1% MeOH in CHCl3. Fractions containing the
product (TLC) were pooled and the solvent was evaporated to
afford 0.12 g (67%) of 23 as a yellow sticky foam: TLC Rf )
0.37 (CHCl3/MeOH, 19:1); 1H NMR (DMSO-d6) δ 1.22 (s, 9 H,
C(CH3)3), 1.96 (s, 3 H, 6-CH3), 2.70-3.30 (m, 4 H, CH2CH2),

Glutamic Acid and Its Derivative
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Hz, C6H4), 7.79 (d, 2H, J ) 7.4 Hz, C6H4), 8.65 (d, 1 H, J ) 7.1
Hz, CONH, exch), 10.66 (s, 1 H, 7-NH, exch); HRMS (EI) calcd
for C27H35N5O5 509.2638, found 509.2627.
N-{4-[2-(2-Am in o-4-et h yl-6-m et h ylp yr r olo[2,3-d ]p yr -
im id in -5-yl)eth yl]ben zoyl}-L-glu ta m ic Acid (4). To a solu-
tion of 26 (0.10 g, 0.20 mmol) in methanol (10 mL) was added
1 N NaOH (2 mL). After 12 h of being stirred at room
temperature, the reaction mixture was concentrated under
reduced pressure, and the residual aqueous solution, cooled
in an ice bath, was acidified to pH 4 with 1 N HCl. The
precipitated solid was collected by filtration, washed with
brine, and dried in vacuo to afford 0.05 g (51%) of 4 as an off-
white solid: mp 238-240 °C; 1H NMR (DMSO-d6) δ 1.26 (t, 3
H, J ) 6.9 Hz, 4-CH2CH3), 1.92 (m, 5 H, 6-CH3 and â-CH2),
2.09 (m, 2 H, γ-CH2), 2.32 (t, 2 H, J ) 6.9 Hz, 4-CH2CH3),
2.85 (m, 4 H, CH2CH2), 4.38 (m, 1 H, R-CH), 6.19 (bs, 2 H,
2-NH2, exch), 7.23 (d, 2 H, J ) 7.2 Hz, C6H4), 7.84 (d, 2 H, J
) 7.2 Hz, C6H4), 8.54 (d, 1 H, J ) 7.2 Hz, CONH, exch), 10.97
(s, 1 H, 7-NH, exch), 12.48 (br, 1 H, COOH, exch). Anal.
(C23H27N5O5) C, H, N.
F olylp olyglu ta m a te Syn th eta se (F P GS) P u r ifica tion
a n d Assa y. A BaculoGold transfection kit (Pharmingen, San
Diego, CA) was used to cotransfect Sf9 insect cells with
pVL1392/cFPGS shuttle vector (a generous gift from Dr. B.
Shane, UC Berkeley), and BaculoGold DNA was used accord-
ing to manufacturer’s instructions to yield recombinant human
cytosolic FPGS-encoding baculovirus. After titering, recombi-
nant virus was used to infect (MOI ) 4) Sf9 monolayers and
cells were harvested at 72 h. FPGS activity was extracted36
and purified by (NH4)2SO4 fractionation and BioGel A-0.5M
chromatography (specific activity 6.4 × 106 pmol [3H]Glu h-1
mg-1 protein). Km values for AMT and MTX as substrates are
the same for this recombinant FPGS as those reported for
human CCRF-CEM FPGS.37 FPGS was assayed as previously
described.37 Drug solutions used in FPGS assays and cell
culture studies (below) were standardized using extinction
coefficients. For 2: pH 1, 241 nm (33 200 cms-1 m-1); pH 7,
237 nm (31 500 cms-1 m-1); pH 13, 236 nm (32 000 cms-1 m-1).
For 4: pH 1, 243 nm (35 600 cms-1 m-1); pH 7, 239 nm (35 500
cms-1 m-1); pH 13, 239 nm (35 700 cms-1 m-1). Extinction
coefficients for methotrexate (MTX), a generous gift of Immu-
nex (Seattle, WA), and aminopterin were from the literature.35
Cell Cu ltu r e. Human T-lymphoblastic leukemia cell line
CCRF-CEM and its MTX transport deficient subline R2, their
culture, and inhibition of their growth in continuous (120 h)
drug exposure are as described in ref 17. Cell lines were
negative for mycoplasma (Mycoplasma Plus PCR primers,
Stratagene, La J olla, CA). EC50 values were interpolated from
plots of percent growth relative to solvent-treated control
versus logarithm of the drug concentration. Protection against
growth inhibition of CCRF-CEM cells was assayed by including
leucovorin (0.1-10 µM) with a concentration of drug previously
determined to inhibit growth by 90-95%; the remainder of
assay was as described. Growth inhibition was measured
relative to the appropriate leucovorin-treated control; leuco-
vorin caused no growth inhibition in the absence of drug,
however.
Ack n ow led gm en t. This work was supported in part
by the National Institutes of Health, Grants AI41743
(A.G.), AI44661 (A.G.), and CA43500 (J .J .M.), and by
Roswell Park Cancer Institute Core Grants CA16056
(J .J .M.) and CA10914 (R.L.K.). Giulia Sobrero was a
participant of the 49th Roswell Park Summer Research
Program and was supported by R25 Grant CA84473.
The authors thank Dr. J ianxia Guo for her expertise in
expressing the rhcFPGS in Sf 9 insect cells.
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J M0203534