Synthesis of N-[4-[1-ethyl-2-(2,4-diaminofuro[2,3-d] pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid as an antifolate1

Article abstract of DOI:10.1021/jm010575m

N-[4-[1-Ethyl-2-(2,4-diaminofuro[2,3-d] pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid 3 was designed and synthesized to investigate the effect of homologation of a C9-methyl to an ethyl on dihydrofolate reductase (DHFR) inhibition and on antitumor activit

Full text of DOI:10.1021/jm010575m

1942
J . Med. Chem. 2002, 45, 1942-1948
Syn th esis of
N-[4-[1-Eth yl-2-(2,4-d ia m in ofu r o[2,3-d ]p yr im id in -5-yl)eth yl]ben zoyl]-L-glu ta m ic
Acid a s a n An tifola te1
Aleem Gangjee,*,† Yibin Zeng,† J ohn J . McGuire,‡ and Roy L. Kisliuk§
Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University,
Pittsburgh, Pennsylvania 15282, Grace Cancer Drug Center, Roswell Park Cancer Institute, Elm and Carlton Streets,
Buffalo, New York 14263, and Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111
Received December 19, 2001
N-[4-[1-Ethyl-2-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid 3 was
designed and synthesized to investigate the effect of homologation of a C9-methyl to an ethyl
on dihydrofolate reductase (DHFR) inhibition and on antitumor activity. Compound 3 was
obtained via a concise seven step synthesis starting from palladium-catalyzed carbonylation
of 4-propionylphenol, followed by a Wittig reaction with 2,4-diamino-5-(chloromethyl)furo[2,3-
d]pyrimidine (6), catalytic hydrogenation, hydrolysis, and standard peptide coupling with diethyl
L-glutamate. The biological results indicated that extending the C9-methyl group to an ethyl
on the C8-C9 bridge region (analogue 3) doubled the inhibitory potency against recombinant
human (rh) DHFR (IC50 ) 0.21 µM) as compared to the C9-methyl analogue 1 and was 4-fold
more potent than the C9-H analogue 2. As compared to 1, compound 3 demonstrated increased
growth inhibitory potency against several human tumor cell lines in culture with GI50 values
< 1.0 × 10-8 M. Compound 3 was also a weak inhibitor of rh thymidylate synthase. Compounds
1 and 3 were efficient substrates of human folylpolyglutamate synthetase (FPGS). Further
evaluation of the cytotoxicity of 3 in methotrexate-resistant CCRF-CEM cell sublines and
metabolite protection studies implicated DHFR as the primary intracelluar target. Thus,
alkylation of the C9 position in the C8-C9 bridge of the classical 5-substituted 2,4-diaminofuro-
[2,3-d]pyrimidine is highly conducive to DHFR and tumor inhibitory activity as well as FPGS
substrate efficiency.
In tr od u ction
Folate metabolism is an attractive target for cancer
chemotherapy, due to its crucial role in the biosynthesis
of nucleic acid precursors.2 Inhibition of folate-depend-
ent enzymes in cancer cells, microbial cells, and in
protozoan cells provides compounds that have found
clinical utility as antitumor, antimicrobial, and anti-
protozoal agents.3,4
As part of a continuing effort in our laboratory to
develop novel classical antifolates as dihydrofolate re-
ductase (DHFR) inhibitors and as antitumor agents,
Gangjee et al.5,6 reported the synthesis of N-[4-[1-
methyl-2-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)ethyl]-
benzoyl-L-glutamic acid (1) and its C9-H analogue 2
(Figure 1). The C9-methyl analogue 1 was twice as
inhibitory against recombinant human (rh) DHFR and
about 10 times greater in its growth inhibitory potency
against tumor cells in culture (CCRF-CEM EC50 29.2
nM, FaDu EC50 17.5 nM, and A253 EC50 28.3 nM) as
compared to 2. The increased potency against DHFR
and against the growth of tumor cells in culture of 1
was, in part, attributed to increased hydrophobic inter-
action of the C9-methyl moiety with DHFR and by its
increased lipophilicity. Molecular modeling studies us-
F igu r e 1.
ing Sybyl 6.57 suggested that extending the C9-methyl
to an ethyl moiety could further enhance both the
hydrophobic interaction with the enzyme and increase
the lipophilicity, which might provide more potent
inhibition of the growth of tumor cells. Similar homo-
logation of a methyl moiety to an ethyl in the C9-C10
bridge region of 6-6 fused bicylic analogues derived
from deaza aminopterin (AMT) has been extensively
studied8-11 and led to the discovery of edatrexate,10 the
10-ethyl-10-deaza AMT analogue, which was advanced
to phase III clinical trials. DeGraw et al.11a,b reported
that in the 10-alkyl-5,10-dideaza AMT analogues, ho-
mologation of the 10-methyl moiety to an ethyl in-
creased its growth inhibitory potency against tumor
cells in culture and, more importantly, alkylation of the
* To whom correspondence should be addressed. Tel.: (412)396-
6070. Fax: (412)396-5593. E-mail: gangjee@duq.edu.
† Duquesne University.
‡ Roswell Park Cancer Institute.
§ Tufts University School of Medicine.
10.1021/jm010575m CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/21/2002

L-Glutamic Acid as an Antifolate
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1943
F igu r e 2.
Sch em e 1
Sch em e 2
bridge C10 carbon enhanced transport of the antifolate
into tumor cells. As part of a structure-activity rela-
tionship study on the C8-C9 bridge region of classical
2,4-diamino-5-substituted-furo[2,3-d]pyrimidine anti-
folates, we designed and synthesized N-[4-[1-ethyl-2-
(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-
glutamic acid 3, the 9-ethyl homologue of 1, to investigate
the effect of a C9-ethyl on the inhibitory activity against
DHFR and against the growth of tumor cells in culture.
employed enolate chemistry starting from commercially
available 4-acetylbenzoic acid ester 7. The enolate was
generated in situ using LDA at -78 °C and subsequent
reaction with MeI. The desired product was difficult to
separate from the starting methyl ketone. Repeated
recrytallization afforded the pure ethyl ketone 6 in only
40% isolated yield.
Polyglutamylation via folylpolyglutamate synthetase
(FPGS) is an important mechanism for trapping clas-
sical folates and antifolates within the cell thus main-
taining high intracellular concentrations and in some
instances for increasing the binding affinity for folate-
dependent enzymes.12a,b Alkylation on the bridge carbon
C10 of the 10-deaza AMT analogue was reported to
enhance the substrate efficiency of the analogue for
FPGS.13 It was thus of interest to also determine the
effect of a C9-ethyl modification with respect to FPGS
activity.
The second method was an alkylation reaction, using
the appropriate Grignard reagent, of methyl 4-formyl-
benzoate (8) followed by a PCC oxidation. Though the
secondary alcohol was isolated in 90% yield, the PCC
oxidation resulted in degradation of the methyl ester
and the pure ethyl ketone 6 was isolated in only 45%
yield. The final strategy employed a modified Gerlach’s
palladium-catalyzed carbonylation15 from the 4-propio-
nylphenol triflate. Thus, 4-propionylphenol triflate was
obtained in quantitative yield from the commercially
available 4-hydroxypropiophenone 9 (Scheme 1) and
was directly subjected to palladium-catalyzed carbony-
lation using dppp/Pd(OAc)2 (1/1.3 ratio) as catalyst (5-
6% equivalent to that of triflate) and dimethyl forma-
mide (DMF)/MeOH (4/3) as solvent and afforded the
desired ethyl ketone 6 in 92% yield over two steps.
In addition, thymidylate synthase (TS), which cata-
lyzes the sole de novo synthesis of deoxythymidylate
(dTMP) from deoxyuridine monophosphate (dUMP), is
a folate cofactor-requiring enzyme.14 It was also of
interest to evaluate 3 as an inhibitor of TS.
Ch em istr y
From a retrosynthetic point of view (Figure 2), it was
anticipated that a Wittig type condensation of the
5-chloromethyl group of 2,4-diamino-5-(chloromethyl)-
furo[2,3-d]pyrimidine (5) with an appropriately substi-
tuted propiophenone 6 would afford the desired 9-ethyl
furo[2,3-d]pyrimidine nucleus 4 of the target compound.
Sequential reduction, hydrolysis, peptide coupling, and
saponification would afford the desired target 3.
With the desired ethyl ketone 6 in hand, the next step
was the condensation with intermediate 5 (Scheme 2),
which in turn was obtained from 2,6-diaminopyrimidin-
4-one 10 and 1,3-dichloroacetone 11.6,16 Wittig conden-
sation of 5 and 6 in dimethyl sulfoxide (DMSO), using
excess n-Bu3P and NaH to form the ylide and methanol
to quench the reaction,5 furnished the olefin 12 in 67%
yield as a mixture of E and Z isomers. Catalytic
hydrogenation of 12 under optimized conditions followed
Thus, the first step was the synthesis of 4-propionyl-
benzoic acid ester (6). Three possible synthetic ap-
proaches are shown in Scheme 1. The first attempt

1944 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Gangjee et al.
Ta ble 1. Inhibitory Concentration (IC50 in µM) against
stituted furo[2,3-d]pyrimidines is highly beneficial with
regard to FPGS substrate activity. These data further
suggest that polyglutamylation may be important in the
mechanism of action of these novel furo[2,3-d]pyrim-
idines. Because 3 is a mixture of diastereomers at C9,
it will be necessary to determine the activity of each
diastereomer to determine the effect of chirality at C9
on FPGS activity.
Isolated DHFR and TSa
DHFR
E. coli
TS
rh
T. gondii
rh
E. coli
1b
0.42
1.0
0.22
0.022
ND
1.1
ND
0.44
0.009
ND
2.1
ND
1.1
0.011
ND
>360
220
220
ND
0.15
100
ND
220
ND
0.1
2c
3
MTX
PDDF
The target compound 3 was also evaluated as an
inhibitor of the growth of CCRF-CEM human leukemia
cells in culture during continuous exposure (Table
3).22-24 Compound 3 was highly cytotoxic with an
EC50 of 35.5 nM, which is similar to that of MTX and
is 8-fold more potent than 2 and equipotent with 1.
This indicates that C9-alkylation (as in 1 and 3) is
also highly conducive to tumor growth inhibition in
culture.
a
The percent inhibition was determined at a minimum of four
inhibitor concentrations with 20% of the 50% point. The standard
deviations for determination of 50% points were within (10% of
the value given. Data derived from ref 5. c Data derived from ref
6; ND ) not determined.
b
Ta ble 2. Activity of 3 as a Substrate for rh FPGSa
substrate
Km (µM)
Vmax (rel)
Vmax/Km
n
AMT
3
3.2 ( 0.3
1.1 ( 0.1
1.2 ( 0.3
8.5 ( 2.1
1
0.31 ( 0.03
0.34 ( 0.05
0.46 ( 0.09
0.07 ( 0.02
3
2
2
2
0.37 ( 0.01
0.52 ( 0.03
0.65 ( 0.01
Because compound 3 was 10-fold less potent than
MTX against isolated rhDHFR but only about 2-fold less
potent against the growth of tumor cells (CCRF-CEM)
in culture, it was of interest to further define its
mechanism of action. Compound 3 was thus evaluated
as an inhibitor of the growth of three CCRF-CEM
sublines with defined mechanisms of resistance to
MTX.22-26 Against the increased DHFR subline R1,
compound 3 was about 80-fold less potent than against
parent CCRF-CEM. Thus, it was quantitatively similar
to MTX (72-fold). These data are consistent with DHFR
being the primary target of analogue 3. Cross-resistance
of the MTX transport deficient subline R2 to 3 indicated
that it uses the MTX/reduced folate carrier (RFC) for
uptake. However, the degree of cross-resistance to 3 (25-
fold) was much less than to MTX (180-fold), which may
suggest that the mutation in the MTX/RFC affects
transport of 2,4-diaminofuro[2,3-d]pyrimidines to a
lesser extent than it does 2,4-diaminopteridines. Alter-
natively, a second route for transport of 2,4-diaminofuro-
[2,3-d]pyrimidines may become available at higher
concentrations. It is also possible, however, that 3 is as
poorly transported as is MTX but it is more readily
converted to longer (Glug3), more potent polyglutamate
inhibitors and that this more efficient metabolism
compensates for the poor transport. The kinetic constant
for monoglutamyl 3 with human FPGS (which primarily
measures conversion to the diglutamate) has been
determined (Table 2). The data show that 3 is a much
more efficient FPGS substrate than is MTX.
1b
2c
a
FPGS substrate activity was determined as described in ref
6. Values presented are average ( SD if n g 3 and average (
range for n ) 2. Vmax is calculated relative to AMT within the same
experiment. Data from ref 5. c Data from ref 6.
b
by column chromatography and recrystallization from
MeOH afforded 4 in 80% yield. Saponification of 4 with
aqueous sodium hydroxide gave the free acid 13 in 95%
yield. Subsequent coupling of 13 with diethyl L-
glutamate using isobutyl chloroformate as the coupling
reagent afforded the product 14 in 90% yield. Final
saponification with aqueous sodium hydroxide at room
temperature followed by acidification to pH 4 in an ice
bath afforded the diacid 3 in 95% yield. The structures
of 3 and all intermediates were characterized by 1H
nuclear magnetic resonance (NMR) and elemental
analysis.
Biologica l Eva lu a tion a n d Discu ssion
Compound 3 was evaluated as an inhibitor of Es-
cherichia coli (ec), Toxoplasma gondii (tg), and rh
DHFR.17-19 The inhibitory potency (IC50) values are
compared with methotrexate (MTX) in Table 1 and the
previously reported values for 1 and 2.5,6 The inhibitory
potency of 3 is twice that of its C9-methyl analogue 1
and four times that of analogue 2. Against ecDHFR and
tgDHFR, compound 3 was also twice as potent as the
C9-methyl, 1.
Interestingly, compound 3 showed only a slight
decrease in potency against the FPGS deficient cell line
R30dm, which suggests that either the polyglutamates
are not required for the antitumor activity of 3 in
continuous exposure or the polyglutamates are formed
very efficiently. These two possibilities are currently
being explored. However, on the basis of the data in
Table 2 showing that analogue 3 is a good substrate for
FPGS, it is more likely that the later possibility oper-
ates. Although DHFR is often insensitive to the poly-
glutamylation status of substrates and inhibitors,27
enhanced potency of polyglutamates of antifolates against
DHFR has been reported.28a,b
Analogue 3 was also evaluated as an inhibitor of ecTS
and rhTS20,21 and was a marginal inhibitor of these
enzymes with IC50 values g 200 µM in most cases (Table
1).
Because polyglutamylation plays a role in the mech-
anism of action of some classical antifolates,12a,b it was
of interest to evaluate analogue 3 as a substrate for
human FPGS (Table 2). The data indicate that the
9-ethyl analogue 3 is as efficient as AMT as a FPGS
substrate; both 3 and 1 also have a 7-fold lower Km and
greater than 5-fold higher substrate efficiency relative
to the unalkylated parent 2.5 This result was consistent
with those obtained for 10-deaza AMT analogues, in
which alkylation of the C10 by an ethyl increased its
FPGS substrate efficiency as compared to the C10
unalkylated analogue.13 The results obtained with 1 and
3 suggest that alkylation of the C9 of classical 5-sub-
Metabolite protection studies were carried out with
3 in order to further elucidate its mechanism of action.
Both 3 (at 100 or 200 nM) and MTX (at 40 and 50 nM)
drug levels that inhibited growth by 95% were fully

L-Glutamic Acid as an Antifolate
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1945
Ta ble 3. Growth Inhibition of CCRF-CEM Human Leukemia Cells, Its MTX-Resistant Sublines with Defined Mechanism during
Continuous Exposure (0-120 h) EC50 in nMa
cell lines
MTX
1b
2c
3
CCRF-CEM
R30dm
R1
14.5 ( 0.5 (n ) 4)
15.0 ( 0 (n ) 2)
1050 ( 50 (n ) 2)
2600 ( 100 (n ) 2)
29.2 ( 0.5 (n ) 5)
220 ( 20 (n ) 2)
685 ( 45 (n ) 2)
430 ( 60 (n ) 2)
290 ( 10 (n ) 3)
35.5 ( 1.5 (n ) 2)
50 ( 8 (n ) 2)
2100 ( 100 (n ) 2)
890 ( 110 (n ) 2)
4250 ( 50 (n ) 2)
ND
ND
R2
a
b
EC50 ) concentration of drug required to decrease cell growth by 50% after 5 days of treatment. Data from ref 5. c Data from ref 6;
ND ) not determined.
Ta ble 4. Cytotoxicity Evaluation (GI50, M) of Compound 3a Against Selected Tumor Cell Lines as Compared to Compound 129
cell lines
compd 3
compd 15
cell lines
compd 3
compd 15
Ovarian Cancer
<1.0 × 10-8
Melanoma
2.37 × 10-8
8.18 × 10-8
Colon Cancer
<1.0 × 10-8
2.39 × 10-8
<1.0 × 10-8
Renal Cancer
<1.0 × 10-8
<1.0 × 10-8
3.40 × 10-7
<1.0 × 10-8
Breast Cancer
<1.0 × 10-8
<1.0 × 10-8
<1.0 × 10-8
IGROV1
OVCAR-5
7.14 × 10-8
6.68 × 10-8
LOX IMVI
SK-MEL-5
<1.0 × 10-8
7.22 × 10-7
7.39 × 10-8
<1.0 × 10-8
<1.0 × 10-8
<1.0 × 10-8
Nonsmall Cell Lung Cancer
1.38 × 10-8
A549/ATCC
NCI-H460
NCI-H522
5.81 × 10-8
HCT-15
HT29
SW-620
<1.0 × 10-8
1.73 × 10-8
<1.0 × 10-8
ND
CNS Cancer
<1.0 × 10-8
SF-268
SF-539
SF-295
U251
6.13 × 10-8
8.78 × 10-8
8.94 × 10-7
2.32 × 10-8
<1.0 × 10-8
<1.0 × 10-8
<1.0 × 10-8
<1.0 × 10-8
<1.0 × 10-8
ACHN
CAKI-1
TK-10
UO-31
1.43 × 10-8
<1.0 × 10-8
ND
<1.0 × 10-8
>1.0 × 10-4
<1.0 × 10-8
2.32 × 10-8
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
RPMI-8226
SR
<1.0 × 10-8
MCF7
MDA-MB-435
MDA-N
2.70 × 10-8
<1.0 × 10-8
<1.0 × 10-8
<1.0 × 10-8
<1.0 × 10-8
<1.0 × 10-8
<1.0 × 10-8
Sensitivity of the other tumor cell lines in the NCI panel to 3: GI50 values > 1.0 × 10-4 M are SK-MEL-2, EKVX, NCI-H226, RXF
a
393, MDA-MB-231/ATCC, HS 578T, BT-549, T-47D, and SNB-75; GI50 values in 10-5 to 10-6 M is H-23; GI50 values in 10-6 to 10-7 M are
HOP-62 and HOP-92; GI50 values in 10-7 to 10-8 M are NCI/ADR-RES, SN12C, and HCT-116; GI50 values in 10-8 M are UACC-257,
UACC-62, and SK-OV-3; GI50 values < 1.0 × 10-8 M are OVCAR-8, UO-31, and 786-0.
protected by as little as 0.1 µM leucovorin indicating
that 3 acts as an antifolate (data not shown). These
results are consistent with DHFR being the primary
intracellular target of 3 (although other targets are not
excluded) and are also consistent with the cross-
resistance studies (Table 3) and with previous data on
the classical furo[2,3-d]pyrimidine antifolates 1 and 2.5,6
Compound 3 was selected by the National Cancer
Institute (NCI)29 for evaluation in its in vitro preclinical
antitumor screening program. The ability of compound
3 to inhibit the growth of tumor cells was measured as
GI50 values, the concentration required to inhibit the
growth of tumor cells in culture by 50% as compared to
a control. In more than 30 cell lines, compound 3 showed
GI50 values in the 10-8 M range or less. It is noteworthy
that in a variety of tumor cell lines (Table 4), compound
3 had more than 5-fold increased inhibitory potency as
compared to the C9-methyl analogue 1. This increased
potency in continuous exposure may reflect the greater
inhibitory potency of the C9-ethyl analogue against
human DHFR (Table 1), although differences in DHFR
sensitivity, drug transport, and intracellular poly-
glutamate accumulation may also contribute in various
cell lines. It was also interesting to note that (data not
shown), like compound 1, compound 3 was not a general
cell poison but showed selectivity both within a type of
tumor cell line as well as across different tumor cell
lines, with inhibitory values which in some instances
differed by 10 000-fold. These data also support the
previous hypothesis that alkylation of the bridge carbon
of the classical deaza folate analogues could enhance
the transport into certain tumor cells.11,30 This com-
pound is currently under further evaluation by the NCI
as an antitumor agent.
In summary, C9-ethyl classical 5-substituted, 2,4-
diaminofuro[2,3-d]pyrimidine antifolate 3 was designed
and synthesized, where the C9-methyl was homologated
to an ethyl. The biological results show that extending
the C9-methyl group to an ethyl in the C8-C9 bridge
(analogue 3) increases DHFR inhibitory activity as
compared to the C9-methyl analogue 1 and the C9-
unalkylated analogue 2. As compared to compound 1,
extending the C9-methyl to an ethyl also results in
increased inhibitory potency against the growth of
several tumor cell lines in culture. Both 1 and 3 are
more efficient as FPGS substrates than 2. Compounds
1 and 3 are also more potent than 2 (>8-fold) as
inhibitors against the growth of CCRF-CEM leukemia
cells in culture. These data show that the alkylation of
the C9 position in the C8-C9 bridge of classical
5-substituted 2,4-diaminofuro[2,3-d]pyrimidine is highly
conducive to DHFR and tumor inhibitory activity as well
as FPGS substrate efficiency.
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 mm
Hg) in an Abderhalden drying apparatus over P2O5 and
refluxing ethanol. Melting points were determined on a MEL-
TEMP II melting point apparatus with FLUKE 51 K/J
electronic thermometer and are uncorrected. NMR spectra for
proton (1H) were recorded on a Bruker WH-300 (300 MHz)
spectrometer. The chemical shift values are expressed in ppm
(parts per million) relative to tetramethylsilane as internal
standard; s ) singlet, d ) doublet, t ) triplet, q ) quartet, m
) multiplet, br ) broad singlet. The relative integrals of peak
areas agreed with those expected for the assigned structures.
Thin-layer chromatography (TLC) was performed on POLY-
GRAM Sil G/UV254 silica gel plates with fluorescent indicator,
and the spots were visualized under 254 and 366 nm illumina-

1946 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Gangjee et al.
tion. Proportions of solvents used for TLC are by volume.
Column chromatography was performed on 230-400 mesh
silica gel purchased from Aldrich, Milwaukee, WI. Elemental
analyses were performed by Atlantic Microlab, Inc., Norcross,
GA. Element compositions 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
prevented despite 24-48 h of drying in vacuo and were
confirmed where possible by their presence in the 1H NMR
spectra. All solvents and chemicals were purchased from
Aldrich Chemical Co. and Fisher Scientific and were used as
received.
7.28-7.31(d, 2 H, Ph-CH), 7.90-7.93 (d, 2 H, Ph-CH). Anal.
(C18H18 N4O3‚0.3CH3COOEt) C, H, N.
Meth yl (R,S)-4-[1-Eth yl-2-(2,4-d ia m in ofu r o[2,3-d ]p yr i-
m id in -5-yl)eth yl]ben zoa te (4). To a solution of 12 (175 mg,
0.5 mmol) in a mixture of MeOH (20 mL) and CHCl3 (50 mL)
was added 10% palladium on activated carbon (350 mg), and
the suspension was hydrogenated in a Parr apparatus at room
temperature and 45 psi hydrogen pressure for 8 h. TLC showed
the disappearance of starting material (Rf 0.78 and 0.73) and
the formation of one major spot at Rf 0.76 (MeOH/CHCl3, 1:5).
The reaction mixture was filtered through Celite, and the
residue was washed with MeOH/CHCl3 (1:1) 100 mL. The
filtrate was evaporated to dryness under reduced pressure, and
100 mL of methanol was added to the solid. To this solution
was added 3 g of silica gel, and the solvent was evaporated to
dryness under reduced pressure. This silica gel plug was
loaded on a dry silica gel column (2 × 15 cm) and flash
chromatographed initially with CHCl3 (200 mL) and then
sequentially with 200 mL of 2% MeOH in CHCl3 and 200 mL
of 4 and 6% MeOH in CHCl3. Fractions that showed the major
spot at Rf 0.57 were pooled and evaporated to dryness and
recrystallized from ethyl ether and MeOH to afford 145 mg
(80%) of 4 as white needles; mp 199-201.5 °C. TLC Rf 0.76
(MeOH/CHCl3, 1:5). 1H NMR (DMSO-d6): δ 0.66-0.71 (t, 3
H, C9-CH2CH3), 1.58-1.80 (m, 2 H, C9-CH2CH3), 2.87-3.17
(m, 3 H, C9-CH, C8-CH2), 3.83 (s, 3 H, OMe), 5.93 (s, 2 H, 4-
or 2-NH2), 6.40 (s, 2 H, 2- or 4-NH2), 6.83 (s, 1 H, C6-CH),
7.31-7.34 (d, 2 H, Ph-CH), 7.81-7.86 (d, 2 H, Ph-CH). Anal.
(C18H20 N4O3‚0.4 H2O) C, H, N.
(R,S)-4-[1-Eth yl-2-(2,4-d ia m in ofu r o[2,3-d ]p yr im id in -5-
yl)eth yl]ben zoic Acid (13). To a solution of 4 (130 mg, 0.38
mmol) in MeOH/DMSO (2:1, 22 mL) was added 1 N NaOH (6
mL), and the mixture was stirred under N2 at room temper-
ature for 16 h. TLC showed the disappearance of the starting
material (Rf 0.76) and formation of one major spot at the origin
(MeOH/CHCl3, 1:5). The reaction mixture was evaporated to
dryness under reduced pressure. The residue was dissolved
in distilled water (6 mL), the solution was filtered through
Celite, and the Celite pad was washed with 4 mL of water.
The filtrate was cooled in an ice bath, and the pH was adjusted
to 4 by dropwise addition of 1 N HCl. The resulting suspension
was frozen in a dry ice-acetone bath and thawed in the
refrigerator to 4-5 °C and filtered. The residue was washed
with a small amount of cold water and ethyl acetate and dried
in vacuo using P2O5 to afford 120 mg (95%) of 13 as a yellow
powder. TLC Rf 0.40 (MeOH/CHCl3, 1:5); mp 256.5-258.3 °C.
1H NMR (DMSO-d6): δ 0.66-0.71 (t, 3 H, C9-CH2CH3), 1.58-
1.80 (m, 2 H, C9-CH2CH3), 2.87-3.17 (m, 3 H, C9-CH, C8-
CH2), 5.93 (s, 2 H, 4- or 2-NH2), 6.40 (s, 2 H, 2- or 4-NH2),
7.09 (s, 1 H, C6-CH), 7.30-7.32 (d, 2 H, Ph-CH), 7.81-7.83
(d, 2 H, Ph-CH), 12.83 (br, 1 H, COOH). Anal. (C17H18 N4O3‚
0.2 H2O) C, H, N.
(R,S)-N-[4-[1-Eth yl-2-(2,4-diam in ofu r o[2,3-d]pyr im idin -
5-yl)eth yl]ben zoyl]-L-glu ta m ic Acid Dieth yl Ester (14).
To a solution of 13 (120 mg, 0.37 mmol) in anhydrous DMF (9
mL) was added triethylamine (134 µL), and the mixture was
stirred under nitrogen at room temperature for 5 min. The
resulting solution was cooled to 0 °C, isobutyl chloroformate
(130 µL 0.96 mmol) was added, and the mixture was stirred
at 0 °C for 30 min. At this time, TLC (MeOH/CHCl3, 1:5)
indicated the formation of the activated intermediate at Rf 0.75
and the disappearance of the starting acid Rf 0.40. Diethyl-L-
glutamate hydrochloride (240 mg, 0.96 mmol) was added to
the reaction mixture followed immediately by triethylamine
(130 µL, 0.96 mmol). The reaction mixture was slowly allowed
to warm to room temperature and stirred under nitrogen for
6 h. The reaction mixture was then subjected to another cycle
of activation and coupling using half the quantities listed
above. The reaction mixture was slowly allowed to warm to
room temperature and stirred under nitrogen for 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 0.76 (MeOH/
Meth yl 4-P r op ion ylben zoa te (6). A 7.5 g (50 mmol)
amount of 4-hydroxy propiophenone (9) was suspended in 300
mL of anhydrous CH2Cl2 and cooled to -30 °C. A 6.42 g (60
mmol) amount of 2,6-lutidine, 1.22 g (10 mmol) of 4-N,N-
dimethylaminopyridine, and 17 g (60 mmol) of trifluoromethane-
sulfonic acid anhydride were added. The resulting mixture was
stirred under N2 at ambient temperature for 2 h and washed
with 2 × 50 mL saturated NH4Cl solution and 2 × 100 mL of
1 N HCl. The organic layer obtained was dried under anhy-
drous Na2SO4 and after evaporation of the solvent under
reduced pressure afforded 14 g of 4-propionylphenol trifluo-
romethanesulfonate as a yellowish oil, which was used directly
for the next step.
A mixture of 4-propionylphenol trifluoromethanesulfonate
(14 g, 50 mmol) and triethylamine (25 mL, 180 mmol) in 150
mL of DMF was charged with palladium acetate (0.9 g, 4
mmol), 1,3-bis(diphenylphosphino)propane (1.4 g, 3 mmol), and
110 mL of anhydrous methanol. The resulting suspension was
purged with carbon monoxide for 5 min and then stirred under
carbon monoxide for 4 h at 70 °C. A 300 mL amount of water
was added to the reaction mixture, and the resulting mixture
was extracted with 3 × 200 mL ethyl acetate and dried under
Na2SO4. After evaporation and column chromatography (4 ×
20 cm) on silica gel (Hexane/EtOAc, 2:1), the mixture afforded
8.9 g (92%, lit.12 80%) of methyl 4-propinoylbenzote as white
crystals; mp 80-81.5 °C (lit.31 80-81 °C). 1H NMR (CDCl3): δ
1.21-1.25 (t, 3 H, -COCH2CH3), 2.99-3.07 (q, 2 H, -COCH2-
CH3), 3.94 (s, 3 H, OMe), 7.99-8.02 (d, 2 H, Ph-CH), 8.09-
8.12 (d, 2 H, Ph-CH); Anal. (C11H12O3) C, H.
Meth yl (E/Z)-4-[1-Eth yl-2-(2,4-d ia m in ofu r o[2,3-d ]p yr i-
m id in -5-yl)eth en yl]ben zoa te (12). To a solution of 2,4-
diamino-5-(chloromethyl)furo[2,3-d]pyrimidine 66,16 (1 g, 5
mmol) in anhydrous DMSO (15 mL) was added tributylphos-
phine (92%, 1.6 g, 7.5 mmol), and the resulting mixture was
stirred at 60 °C in an oil bath for 3 h under N2 to form the
phosphonium salt. The deep orange solution was then cooled
to room temperature. To this solution was added sodium
hydride (92% dispersion in mineral oil, 0.2 g, 7.5 mmol),
followed by methyl 4-propionylbenzoate (1.2 g, 5.5 mmol). The
reaction mixture was stirred at room temperature for 38 h.
TLC showed the disappearance of the starting material (Rf
0.45) and appearance of two spots at 0.73 and 0.78 (MeOH/
CHCl3, 1:5). The reaction was quenched with MeOH (80 mL).
The resulting solution was evaporated, and 100 mL of metha-
nol was added to dissolve the residue, followed by 10 g of silica
gel. This mixture was evaporated under reduced pressure to
dryness to afford a silica gel plug, which was loaded on a dry
silica gel column (4 × 20 cm) and flash chromatographed
initially with CHCl3 (300 mL) and then sequentially with 300
mL of 2-5% MeOH in CHCl3. Fractions that showed the major
spot at Rf 0.73 along with the spot at Rf 0.78 were pooled and
evaporated to dryness. Recrystallization from ethyl acetate
afforded 1.15 g (67%) of 12 (Z:E ratio 1:1) as yellow needles;
1
mp 189.9-193.4 °C. H NMR (DMSO-d6): E-isomer: δ 0.95-
1.00 (t, 3 H, C9-CH2CH3), 2.50-2.55 (q, 2 H, C9-CH2CH3),
3.86 (s, 3 H, OMe), 6.09 (s, 2 H, 4- or 2-NH2), 6.47 (s, 2 H, 2-
or 4-NH2), 6.86 (s, 1 H, C8-CH), 7.44 (s, 1 H, C6-CH), 7.71-
7.74 (d, 2 H, Ph-CH), 7.94-7.97 (d, 2 H, Ph-CH); Z-isomer: δ
0.95-1.00 (t, 3 H, C9-CH2CH3), 2.69-2.74 (q, 2 H, C9-CH2-
CH3), 3.84 (s, 3 H, OMe), 6.02 (s, 2 H, 4- or 2-NH2), 6.21 (s, 1
H, C8-CH), 6.50 (s, 2 H, 2- or 4-NH2), 6.62 (s, 1 H, C6-CH),

L-Glutamic Acid as an Antifolate
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1947
CHCl3, 1:5). The reaction mixture was evaporated to dryness
under reduced pressure. The residue was dissolved in the
minimum amount of CH3Cl/MeOH (4:1) and chromatographed
on a silica gel column (2 × 15 cm) and with 4% MeOH in CHCl3
as the eluent. Fractions that showed the desired spot were
pooled and evaporated to dryness and crystallized from ethyl
ether and MeOH to afford 170 mg (90%) of 14 as yellow
crystals; mp 99.7-102.1 °C. 1H NMR (DMSO-d6): δ 0.66-0.71
(t, 3 H, C9-CH2CH3), 0.93-1.20 (m, 6 H, 2 × OCH2CH3),
1.58-1.80 (two sets of m, 2 H, C9-CH2CH3), 1.98-2.10 (m, 2
H, -CHCH2CH2CO-), 2.39-2.45 (m, 2 H, -CHCH2CH2CO-),
2.87-3.08 (m, 3 H, C9-CH, C8-CH2), 4.03-4.13 (m, 4 H, 2 ×
OCH2CH3), 4.42 (m, 1 H, -NHCH(CH2)-), 5.94 (s, 2 H, 4- or
2-NH2), 6.41 (s, 2 H, 2- or 4-NH2), 6.83 (s, 1 H, C6-CH), 7.27-
7.29 (d, 2 H, 3′-, 5′-CH), 7.74-7.76 (d, 2 H, 2′-, 6′-CH), 8.61-
8.63 (d, 1 H, -CONH-). Anal. (C26H33N5O6) C, H, N.
(R,S)-N-[4-[1-Eth yl-2-(2,4-diam in ofu r o[2,3-d]pyr im idin -
5-yl)eth yl]ben zoyl]-L-glu ta m ic Acid (3). To a solution of the
diester 14 (150 mg, 0.29 mmol) in MeOH (10 mL) was added
1 N NaOH (6 mL), and the mixture was stirred under nitrogen
at room temperature for 16 h. TLC showed the disappearance
of the starting material (Rf 0.76) and formation of one major
spot at the origin (MeOH/CHCl3, 1:5). The reaction mixture
was evaporated to dryness under reduced pressure. The
residue was dissolved in water (10 mL), the resulting solution
was cooled in an ice bath, and the pH was adjusted to 3-4
with dropwise addition of 1 N HCl. The resulting suspension
was frozen in a dry ice-acetone bath and thawed in the
refrigerator to 4-5 °C and filtered. The residue was washed
with a small amount of cold water and ethyl acetate and dried
in vacuo using P2O5 to afford 130 mg (95%) of 3 as a white
powder; mp 171.3-173.5 °C. TLC Rf 0.80 (MeOH/CHCl3/NH3-
OH, 3:7:2). 1H NMR (DMSO-d6): δ 0.66-0.71 (t, 3 H, C9-
CH2CH3), 1.58-1.80 (two sets of m, 2 H, C9-CH2CH3), 1.94-
2.08 (2 sets of t, 2 H, Glu â-CH2), 2.31-2.36 (t, 2 H, Glu γ-CH2),
2.87-3.16 (m, 3 H, C8-CH2, C9-CH), 4.41 (m, 1 H, Glu R-CH),
6.17 (s, 2 H, 4- or 2-NH2), 6.66 (s, 2 H, 2- or 4-NH2), 6.88 (s, 1
H, C6-CH), 7.27-7.29 (d, 2 H, 3′-, 5′-CH), 7.75-7.77 (d, 2 H,
2′-, 6′-CH), 8.52-8.54 (d, 1 H, -CONH-), 12.38-12.53 (br, 2
H, 2 × COOH). Anal. (C22H25N5O6‚0.5H2O) C, H, N.
simultaneously with a concentration of drug previously de-
termined to inhibit growth by about 95%; the remainder of
the assay was as described.5,6 Growth inhibition was measured
relative to the appropriate LV-treated control; LV caused no
significant growth inhibition in the absence of drug.
Ack n ow led gm en t. This work was supported, in
part, by grants from the National Institute of Health
AI44661 (A.G.), AI41743 (A.G.), CA89300 (A.G.), CA43500
(J .J .M.) Core Grant CA16056 and CA10914 (R.L.K.). We
thank Mr. Gregory Nagel and Mr. William Haile for
performing biological and biochemical studies.
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1948 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Gangjee et al.
(17) Recombinant human DHFR was kindly supplied by Dr. J . H.
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