Effect of C9-methyl substitution and C8-C9 conformational restriction on antifolate and antitumor activity of classical 5-substituted 2,4-diaminofuro[2,3-d]pyrimidines

Article abstract of DOI:10.1021/jm000130i

N-[4-[1-Methyl-2-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)ethyl]benz oyl]-L-glutamic acid (5) and its C8-C9 conformationally restricted E- and Z-isomers (6 and 7) were designed and synthesized in order to investigate the effect of incorporating a methyl grou

Full text of DOI:10.1021/jm000130i

J . Med. Chem. 2000, 43, 3125-3133
3125
Effect of C9-Meth yl Su bstitu tion a n d C8-C9 Con for m a tion a l Restr iction on
An tifola te a n d An titu m or Activity of Cla ssica l 5-Su bstitu ted
2,4-Dia m in ofu r o[2,3-d ]p yr im id in es1
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 April 1, 2000
N-[4-[1-Methyl-2-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid (5) and
its C8-C9 conformationally restricted E- and Z-isomers (6 and 7) were designed and synthesized
in order to investigate the effect of incorporating a methyl group at the C9 position and of
conformational restriction at the C8-C9 bridge of N-[4-[2-(2,4-diaminofuro[2,3-d]pyrimidin-5-
yl)ethyl]benzoyl]-L-glutamic acid (1) with respect to dihydrofolate reductase (DHFR) inhibitory
activity as well as antitumor activity. The compounds were synthesized by a Wittig reaction of
2,4-diamino-5-(chloromethyl)furo[2,3-d]pyrimidine with ethyl 4-acetylbenzoate followed by
catalytic reduction, hydrolysis, and standard peptide coupling with diethyl L-glutamate. The
biological results indicated that the addition of a 9-methyl group to the C8-C9 bridge, as in 5,
increased recombinant human (rh) DHFR inhibitory potency (IC50 ) 0.42 µM) as well as the
potency against the growth inhibition of tumor cells in culture (CCRF-CEM EC50 ) 29 nM,
A253 EC50 ) 28.5 nM, and FaDu EC50 ) 17.5 nM) compared with the 9-desmethyl analogue 1.
However, the conformationally restricted 4:1 Z/ E mixture of 7 and 6 was less potent than 5 in
both assays, and the pure E-isomer 6 was essentially inactive. These three classical analogues
were also evaluated as inhibitors of Lactobacillus casei, Escherichia coli, and rat and rh
thymidylate synthase (TS) and were found to be weak inhibitors. All three analogues 5-7
were good substrates for human folylpolyglutamate synthetase (FPGS). These data suggested
that FPGS is relatively tolerant to different conformations in the bridge region. Further
evaluation of the cytotoxicity of 5 and 7 in methotrexate (MTX)-resistant CCRF-CEM cell
sublines suggested that polyglutamylation was crucial for their mechanism of action. Metabolite
protection studies of 5 implicated DHFR as the primary intracelluar target. Compound 5 showed
GI50 values in 10-9-10-7 M range against more than 30 tumor cell lines in culture.
In tr od u ction
atom bridged compound 4 was poorly active against
DHFR (IC50 ) 1.0 × 10-5 M), whereas the three two-
atom bridged analogues 1-3 showed moderate to high
potency (IC50 ) 1.0 × 10-8 to 1.0 ×10-6 M). These
analogues were also potent inhibitors of tumor cell
growth in culture (EC50 ) 1.0 ×10-8 to 1.0 × 10-7 M).
The C9 analogue 1 (EC50 ) 1.0 × 10-7 M) was about
one-half as potent as its 9-aza analogue 2 against
cultured CCRF-CEM cells, whereas the N9-methyl
analogue 3 was about twice as potent.
Molecular modeling using Sybyl 6.28 and the crystal
structure of recombinant human DHFR (rhDHFR)9 with
methotrexate (MTX) in place of folate indicates that the
N9-methyl moiety of 3 (superimposed on MTX) is
positioned to interact with the hydrophobic Val115
residue of the enzyme active site. Thus substitution of
a methyl group at C9 of 1 would be predicted to enhance
binding and hence the inhibition of DHFR. In addition,
molecular modeling indicated that C9-methyl group
could introduce conformational restriction about the C8-
C9 bond thus decreasing the number of accessible low-
energy conformations compared with 1. Similar substi-
tutions of a methyl group in the C9-C10 bridge region
of 6-6 fused analogues have been reported by DeGraw
et al.10,11a,b in 2,4-diaminopteridine and 2,4-diaminopy-
Folate metabolism has been an attractive chemo-
therapeutic target due to its crucial role in the biosyn-
thesis of nucleic acid precursors.2 Tetrahydrofolate
(FH4), the key component of folate metabolisms, serves
as a cofactor precursor to carry one-carbon units for the
biosynthesis of purines and deoxythymidylate (dTMP).
During the synthesis of dTMP, FH4 is oxidized to 7,8-
dihydrofolate (FH2) and must be converted back to the
reduced form by dihydrofolate reductase (DHFR) to
continue DNA synthesis.3 Inhibition of DHFR depletes
the FH4 pool; thus DHFR inhibitors have found clinical
utility as antitumor, antibacterial, and antiprotozoan
agents.4,5
As part of a continuing effort to develop novel classical
antifolates as DHFR inhibitors and as antitumor agents,
Gangjee et al.6,7 previously reported the synthesis of
N-[4-[2-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)ethyl]ben-
zoyl]-L-glutamic acid (1) and its -CH2NH-, -CH2N-
(CH3)-, and -CH2NHCH2- analogues 2-4. The three-
* 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/jm000130i CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/07/2000

3126 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16
Gangjee et al.
addition, thymidylate synthases (TS), a crucial enzyme
in the biosynthesis of DNA which catalyzes the sole de
novo synthesis of dTMP from dUMP, is a folate-cofactor-
requiring enzyme.18 It was also of interest to evaluate
these new classical furo[2,3-d]pyrimidines 5-7 as in-
hibitors of TS.
Ch em istr y
The synthetic route to the target molecules 5-7 is
outlined in Scheme 1. A modification of the methodology
reported previously6 was utilized to assemble the skel-
eton of the two-carbon bridged target 5. The crucial
intermediate 8 was readily obtained by the reaction of
2,6-diaminopyrimidin-4-one and dichloroacetone in DMF
at room temperature as previously reported.19 Wittig
condensation of 8 and ethyl 4-acetylbenzoate in DMSO
solution, using excess n-Bu3P (3 equiv) and NaH (2.2
equiv) to form the ylide and methanol to quench the
reaction, furnished the olefin 9 in 42% yield as a mixture
of E- and Z-isomers. The mixture of the E- and Z-
isomers was established from its 1H NMR where the
characteristic C6 proton signal was observed at 7.52
ppm for the E-isomer, which is 0.85 ppm downfield from
the C6 proton of the Z-isomer (6.67 ppm). This compares
favorably with previously reported E- and Z-products.6
During the synthesis, some methyl ester exchange with
+ 20 % E
rido[2,3-d]pyrimidine analogues which enhanced inhibi-
tory activity against tumor cells in culture. As part of a
structure-activity relationship (SAR) study on the
relative conformational orientation of the furo[2,3-d]-
pyrimidine ring with respect to the benzoylglutamate
side chain of the two-atom bridged classical 2,4-diamino-
5-substituted-furo[2,3-d]pyrimidine antifolates, we de-
signed and synthesized N-[4-[1-methyl-2-(2,4-diamino-
furo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid
(5), the 9-methyl analogue of 1.
1
ethyl ester was detected in the H NMR spectrum. In
addition the removal of excess tributylphosphine and
phosphine oxide was also problematic. In an attempt
to improve the yield of 9, the amounts of tributylphos-
phine and NaH were reduced to 1.5 equiv each and
ethanol was used in the workup instead of methanol.
This afforded the olefin in 65% yield as a mixture (E:Z
A number of classical antifolate C9-C10 bridged
analogues have been reported in the 6-6 fused bicyclic
sytems, such as pteridines,10 pyrido[2,3-d]pyrimidines,11,12
and pyrido[3,2-d]pyrimidines,13 and C8-C9 bridged ana-
logues in the 6-5 fused bicyclic systems, such as pyrrolo-
[2,3-d]pyrimidines,14 cyclopenta[d]pyrimidines,15 and
furo[2,3-d]pyrimidines.6 There is however a dearth of
information regarding the E- and Z-isomers of these C-C
bridged classical analogues. The sole report of charac-
terized geometric isomers is by Harris et al.16 who
reported that in nonclassical 6-substituted 2,4-diami-
noquinazolines, the Z-isomer was more favorable for
bacterial DHFR inhibition than the E-isomer and was
similar in activity to the reduced bridge analogue. It is
surprising to note that despite the synthetic methodol-
ogy which affords the unsaturated precursors, none of
the geometrically restricted, unsaturated, classical ana-
logues, to our knowledge, were evaluated for DHFR
inhibitory activity or for their inhibition of the growth
of tumor cells in culture. Thus it was of considerable of
interest to synthesize and isolate the E/ Z mixture and
separate the E- and Z-isomers of 5 for biological evalu-
ation against DHFR and against the growth of tumor
cells in culture. Thus the C9-methylated C8-C9 confor-
mationally restricted E-6 and Z-7 N-[4-[1-methyl-2-(2,4-
diaminofuro[2,3-d]pyrimidin-5-yl)ethenyl]benzoyl]-L-
glutamic acids were designed as classical antifolates and
as antitumor agents.
1
ratio 2:1) as determined by H NMR. Catalytic hydro-
genation of 9 under optimized conditions followed by
column chromatography and recrystallization from
MeOH afforded the 8,9-dihydro derivative 10 in 75%
yield. Saponification of 10 with aqueous sodium hydrox-
ide afforded the free acid 11 in 90% yield. Subsequent
coupling of 11 with diethyl L-glutamate using isobutyl
chloroformate as the coupling agent afforded the product
12 in 85% yield. Final saponification with aqueous
sodium hydroxide at room temperature followed by
acidification to pH 4 in an ice bath afforded the diacid
5 in 97% yield. The structures of 5 and all intermediates
1
were confirmed by H NMR and elemental analysis.
It was more efficient to separate the E-isomer from
the mixture after coupling with the L-glutamate diester.
Thus hydrolysis of 9 with aqueous sodium hydroxide
afforded the free acid 13 of the E:Z mixture in 92% yield.
Subsequent coupling of 13 with diethyl L-glutamate
using isobutyl chloroformate afforded the desired coupled
products 14 and 15 as an E:Z mixture in 85% yield. All
attempts to separate the E-isomer 14 from the Z-isomer
15 using column chromatography were unsuccessful.
Partial separation was finally achieved by fractional
recrystallization from methanol/ethyl acetate (1:1). The
white crystals which separated contained the pure
E-isomer as confirmed by TLC and by its characteristic
C6 proton at 7.50 ppm. To further characterize the
structure of the E-isomer, a 500-MHz two-dimensional
proton-proton ROESY experiment was performed, and
the spectrum clearly indicated that the C6 proton
correlated to the C9-methyl group while the C8 proton
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 binding affinity for folate-
dependent enzyme.17a,b It was of interest to determine
the effect of adding a C9-methyl and C8-C9 bond
restriction with respect to FPGS activity as well. In

5-Substituted 2,4-Diaminofuro[2,3-d]pyrimidines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16 3127
Sch em e 1a
a
Reagents: (a) DMF, rt; (b) (i) Bu3P/DMSO, (ii) NaH, (iii) ethyl 4-acetylbenzoate; (c) 5% Pd-C/H2, 45 psi, MeOH/CHCl3/DMF; (d) (i)
1 N NaOH, DMSO/MeOH, (ii) 1 N HCl; (e) iBuOCOCl, Et3N, diethyl L-glutamate; (f) (i) 1 N NaOH, DMSO/MeOH, (ii) 1 N HCl.
Ta ble 1. Inhibitory Concentration (IC50, µM) against Isolated DHFR and TS
DHFR
TS
compd
rh
lc
ec
tg
rh
lc
ec
rat
5
0.42
42
2.2
1.0
0.45
0.22
0.022
2.1
84
4.4
0.1
0.56
0.22
0.011
1.1
11
1.1
nd
nd
2.1
110
4.4
nd
0.70
>360
>360
>360
220
>200
>200
>360
>360
>360
200
>200
>200
100
>360
>360
>360
>360
>360
6
7a
1b
nd
nd
2c
nd
nd
nd
nd
3c
nd
0.009
19.8
0.022
MTX
PDDF
0.15
0.055
0.1
0.36
a
b
Compound 7 is a 1:4 E:Z mixture; Data derived from ref 6. c Data derived from ref 7.
correlated with the 4-NH2 and the side chain phenyl
proton. This uneqivocally demonstrated the E-geometry
for compound 14. The Z-isomer remained in the filtrate
along with residual E-isomer. Separation of the pure
Z-isomer from this mixture was unsuccessful and it was
decided to carry this mixture to the next step. Final
saponification of pure 14 with aqueous sodium hydrox-
ide at room temperature followed by acidification to pH
4 in an ice bath afforded the E-diacid 6 in 97% yield.
The Z-diacid 7 obtained in 95% yield after saponification
of 15 was contaminated with about 20% of 6 as indicated
casei (lc), Escherichia coli (ec), Toxoplasma gondii (tg),
and recombinant human (rh) DHFR.20-22 The inhibitory
potency (IC50) values are listed in Table 1 and compared
with the value for methotrexate (MTX) and the previ-
ously reported values for 1-3.6,7 The reduced analogue
5 inhibited rhDHFR with an IC50 ) 0.42 µM and was
twice as potent as its 9-CH2 analogue 1. This inhibitory
activity of 5 is about one-twentieth that of MTX and is
similar to that of the 9-NH analogue 2 and about one-
half that of the N9-CH3 analogue 3. The 80/20% Z/ E
mixture 7 was 5-fold less potent than the reduced
analogue 5, while the E-isomer was only marginally
active against rhDHFR with an IC50 of 42 µM. In
contrast to analogue 1, which had a 10-fold increased
potency against lcDHFR compared with rhDHFR, all
three analogues 5-7 were more potent against rhDHFR
than lcDHFR or tgDHFR. Compound 5 was also more
potent against rhDHFR than ecDHFR. Thus compared
to the 9-desmethyl analogue 1, introduction of a 9-CH3
moiety not only increases potency but also reverses
selectivity for rhDHFR compared to lcDHFR. The bacte-
rial DHFR inhibitory activity of these classical ana-
logues 5-7 (Table 1) parallels that reported by Harris
1
by the H NMR on the basis of the C6 proton (Z at 6.64
ppm and E at 7.50 ppm) integrations of the mixture.
It should be noted that in our synthetic sequence, the
catalytic reduction of 9 using palladium on activated
carbon afforded 10 as a R,S racemic mixture at C9
which generated 5 as a mixture of diastereomers at C9.
Attempts to separate the diastereomers using column
chromatography were unsuccessful.
Biologica l Eva lu a tion a n d Discu ssion
The classical analogues 5 and 6 and the 80/20% Z/ E
mixture 7 were evaluated as inhibitors of Lactobacillus

3128 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16
Ta ble 2. Activity of 5-7 as Substrates for rhFPGSa
Gangjee et al.
lation may be important in the mechanism of action of
these novel furo[2,3-d]pyrimidines.
substrate
Km, µM
Vmax, rel
Vmax/Km
n
The target compounds 5-7 were also evaluated as
inhibitors of the growth of CCRF-CEM human leukemia
cells in culture during continuous exposure (Table
3).25-27 The reduced compound 5 was highly cytotoxic
against CCRF-CEM cells, had a potency similar to MTX,
and was about 10-fold more potent than 1, 5-fold more
potent than 2, and equipotent with 3. This indicates that
9-methylation (as in 3 and 5) is highly conducive to
tumor growth inhibition in culture. The pure confor-
mationally restricted E-isomer 6 was a very weak
inhibitor of CCRF-CEM cells growth, while analogue 7,
which is 80% Z-isomer, was moderately inhibitory.
These data suggest the Z-isomer is the only one in the
E/ Z pair which is biologically active. These three
classical analogues were further evaluated as inhibitors
of the growth of A253 and FaDu squamous cell carci-
noma of the head and neck cell lines in culture, during
continuous exposure (Table 3). The inhibitory activities
of 5-7 were consistent with the results obtained against
the CCRF-CEM cells. The most potent compound was
the reduced analogue 5 which was similar to MTX; the
80% Z-isomer 7 was much less potent and the E-isomer
6 was inactive. The activity of 5 further underscores the
importance of a 9-CH3 reduced bridge to tumor cell
inhibition in culture.
aminopterin
5
4.5 ( 0.9
1.2 ( 0.3
5.3 ( 0.8
10.3 ( 0.2
8.5 ( 2.1
1
0.23 ( 0.05
0.46 ( 0.09
0.15 ( 0.02
0.05 ( 0
6
2
2
2
3
0.52 ( 0.03
0.76 ( 0.01
0.56 ( 0
6
7b
1c
0.65 ( 0.01
0.07 ( 0.02
a
FPGS substrate activity was determined as described in the
Experimental Section. Values presented are average ( SD if n g
3 and average ( range for n ) 2. Vmax are calculated relative to
b
aminopterin within the same experiment. Compound 7 is a 1:4
E:Z mixture. c Data from ref 6.
et al.16 for nonclassical 2,4-diamino-6-substituted quinazo-
lines in that the C8-C9 reduced analogue is equipotent
with the Z-isomer and the E-isomer has significantly
reduced activity.
Analogues 5-7 were also evaluated as inhibitors of
lcTS, ecTS, rat (r) TS, and rhTS
or marginal inhibitors of these enzymes with IC50 values
> 360 µM in most cases (Table 1) compared to N10-
propargyl-5,8-dideazafolate (PDPF) as a control.
23,24
and were inactive
Since polyglutamylation plays a role in the mecha-
nism of action of some classical antifolates,17a,b it was
of interest to evaluate these analogues as substrates for
human FPGS (Table 2). The 9-methyl analogue 5 was
about twice as efficient (higher Vmax/Km) as aminopterin
(AMT) as an FPGS substrate; 5 also had a 7-fold lower
Km and 7-fold higher substrate efficiency relative to its
unmethylated parent 1.6 Since 5 is a mixture of dia-
stereomers at C9, it will be necessary to determine the
activity of each diastereomer to determine the effect of
chirality at C9 on FPGS activity. Of interest is the
finding that C9 methylation (1 vs 5) is highly beneficial
with regard to FPGS activity. This is in contrast to a
related series of C8-N9-containing analogues, N9 me-
thylation is slightly deterimental (2 vs 3;7) to FPGS
activity. The C8-C9 conformationally restricted E-iso-
mer 6 possessed substrate efficiency similar to that of
AMT but was 3-fold less efficient than that of the
unrestricted 5. Interestingly 6 was still more efficient
than unmethylated, unrestricted 1 (Km, 8.5; Vmax/Km,
0.076). The conformationally restricted Z:E (4:1) mixture
7 was a less efficient FPGS substrate than was the pure
E-isomer 6, primarily through an increase in Km;
however 7 was still about as efficient as unmethylated,
unrestricted 1. These data suggest that FPGS is rela-
tively tolerant to different conformations in the C8-C9
bridge region. These data further suggest polyglutamy-
Since compound 5 was 19-fold less potent than MTX
against isolated rhDHFR but only about 2-fold less
potent against the growth of tumor cells (CCRF-CEM,
FaDu, and A253) in culture, it was of interest to further
define its mechanism of action. Compounds 5 and 7 were
thus evaluated as inhibitors of the growth of three
CCRF-CEM sublines with defined mechanisms of re-
sistance to MTX.25-29 Against the increased DHFR
subline R1, compounds 5 and 7 were both about 20-fold
less potent than they were against parental cells. Thus
these compounds were qualitatively similar to MTX,
though for the latter drug this difference was 48-fold.
Despite their relatively low potency (compared to MTX)
as inhibitors of purified rhDHFR (Table 1), these data
are consistent with DHFR being the primary target of
these analogues. The MTX transport-deficient subline
R2 is cross-resistant to both 5 and 7 indicating that both
use the MTX/reduced folate carrier (RFC) for uptake.
However, the degree of cross-resistance to 5 (15-fold)
and 7 (8-fold) was much less than to MTX (111-fold)
which may suggest that the mutation in the MTX/RFC
Ta ble 3. Growth Inhibition of CCRF-CEM Human Leukemia Cells, Its MTX-Resistant Sublines, Human Squamous Cell Carcinoma
FaDu, and A253 Cells during Continuous Exposure (0-12 h)a
EC50, nM
resistance
mechanism
cell line
MTX
5
6
7b
1c
2d
3d
CCRF-CEM
sensitive
14.4 ( 1.0
(n ) 5)
29.2 ( 3.3
(n ) 5)
34000 ( 6000
(n ) 2)
1175 ( 83
(n ) 4)
290 ( 10
(n ) 3)
nde
142 ( 11
(n ) 4)
1523 ( 196
(n ) 3)
nde
47 ( 7
(n ) 4)
807 ( 68
(n ) 3)
nde
R1
increase
DHFR
deficient in
transport
decrease
FPGS
675 ( 35
685 ( 45
nde
32000 ( 1000
(n ) 2)
(n ) 2)
430 ( 60
(n ) 2)
220 ( 20
(n ) 2)
17.5 ( 0.5
(n ) 2)
(n ) 2)
17000 ( 0
(n ) 2)
6400 ( 900
(n ) 2)
1400 ( 100
(n ) 2)
R2(Bos)
R30dm
FaDu
A253
1600 ( 100
(n ) 2)
nde
nde
nde
14 ( 0
425 ( 50
(n ) 2)
180 ( 20
(n ) 2)
540 ( 90
(n ) 3)
663 ( 66
(n ) 3)
126 ( 34
(n ) 2)
nde
1030 ( 510
(n ) 3)
55 ( 15
(n ) 2)
nde
(n ) 2)
sensitive
11.3 ( 1.8
>10000
(n ) 2)
>10000
(n ) 2)
(n ) 2)
sensitive
14.5 ( 0.5
(n ) 2)
28.5 ( 2.5
(n ) 2)
700 ( 70
(n ) 2)
a
EC50 ) concentration of drug required to decrease cell viability as measured by MTA (MTT assay) by 50% after 5 days of treatment.
Compound 7 is a 1:4 E:Z mixture. c Data from ref 6. Data from ref 7. e nd ) not determined.
b
d

5-Substituted 2,4-Diaminofuro[2,3-d]pyrimidines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16 3129
Ta ble 4. Protection of FaDu Cells from the Growth Inhibitory
Effects of MTX and 5 by 50 µM Hx, 40 µM TdR, or the
Combination of 50 µM Hx + 40 µM TdRa
different tumor cell lines, with inhibitory values which
in some instances differed by 10 000-fold. This com-
pound is currently under further evaluation by the NCI
as an antitumor agent.
relative growth, % of control
drug
MTX 5.75 ( 1.25 11.25 ( 5.75 9.25 ( 1.75
5.75 ( 0.75 3.75 ( 0.75 8.0 ( 1.5
no addn
Hx
TdR
Hx + TdR
In summary, three classical 5-substituted, 2,4-diami-
nofuro[2,3-d]pyrimidine antifolates (5-7) modified in
the bridge region linking the furo[2,3-d]pyrimidine ring
and the side chain benzoyl-L-glutamic acid were de-
signed and synthesized, where the bridge flexibility is
partially restricted with a C9-methyl substitution (com-
pound 5) and completely restricted by a double bond
(compounds 6 and 7). The biological results indicated
that incorporating a C9-methyl group in the C8-C9
bridge region (analogue 5) increases DHFR inhibitory
activity and inhibition of the growth of CCRF-CEM
leukemic as well as FaDu and A253 cells in culture
compared to the C9 unsubstituted analogue 1, as well
as compared with the C8-N9 analogues 2 and 3.
Compared to 5, the conformationally restricted 80/20%
Z/ E mixture 7 was much less potent and the pure
E-isomer 6 was essentially inactive against rhDHFR
and against the growth of CCRF-CEM cells in culture.
This suggests that the conformation about the C8-C9
bridge of the classical 5-substiuted 2,4-diaminofuro[2,3-
d]pyrimidines is an important determinant for DHFR
and tumor inhibitory activity. The C8-C9 double bond
which provides complete restriction is detrimental to
DHFR inhibitory activity and to the inhibition of the
growth of tumor cells in culture compared to the C8-C9
reduced analogue 5 where the C8-C9 bridge maintains
some conformational flexibility.
102 ( 3.0
105.25 ( 4.75
5
a
Protection was determined as described in the Experimental
Section. Data are the average ( range of duplicate determinations
within a single experiment. The entire experiment was repeated
with similar results. Metabolites themselves had no effect on the
cell growth.
affects transport of 2,4-diaminofuro[2,3-d]pyrimidines
less than it does 2,4-diaminopteridines. Alternatively,
a second route for transport of 2,4-diaminofuro[2,3-d]-
pyrimidines may become available at higher concentra-
tions. It is also possible, however, that 5 and 7 are as
poorly transported as is MTX, but these analogues are
more readily converted to longer (Glug3), more potent
polyglutamate inhibitors and that this more efficient
metabolism compensates for the poor transport. Kinetic
constants for monoglutamyl 5 and 7 with human FPGS
(which primarily measures conversion to the diglutamate)
were determined (Table 2); the efficiency of conversion
to longer polyglutamates is under study. The cross-
resistance of the FPGS-deficient subline R30 to 5 (8-
fold) and 7 (5-fold) in continuous exposure suggests that
polyglutamylation plays a role in the mechanism of
action of these analogues, probably by increasing their
affinity for their target enzyme, DHFR. These data are
consistent with those (Table 2) showing that the ana-
logues are substrates for FPGS. Although DHFR is often
insensitive to the polyglutamylation status of substrates
and inhibitors,30 enhanced potency of polyglutamates
of a few novel antifolates against DHFR has been
reported.31
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. Melting points were determined on a MEL-
TEMP II melting point apparatus with FLUKE 51 K/J
electronic thermometer and are uncorrected. Nuclear magnetic
resonance spectra for proton (1H NMR) 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 ) mutiplet, br ) broad
singlet. The relative intergrals of peak areas agreed with those
expected for the assigned structures. Two-dimensional proton-
proton ROESY experiment was performed on a Bruker DRX500
(500 MHz) spectrometer. 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. Column chromatography was per-
formed on 230-400 mesh silica gel purchased from Aldrich,
Milwaukee, WI. Elemental analyses were performed by At-
lantic 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 Scien-
tific and were used as received.
Metabolite protection studies were carried out with
5 in order to further elucidate its mechanism of action.
Both 5 (at 100 or 200 nM) and MTX (at 40 and 50 nM),
drug levels that inhibited growth by 92-98%, were fully
protected by as little as 0.1 µM leucovorin indicating
that 5 acts as an antifolate (data not shown). Addition
of a combination of 40 µM thymidine (TdR) + 50 µM
hypoxanthine (Hx) was required to fully protect against
growth inhibition by 5 and by MTX (Table 4). With
either TdR or Hx alone, only marginal protection was
obtained. These results implicate DHFR as the primary
intracelluar target of 5 and are consistent with the
cross-resistance studies (above) and with previous data
on the classical furo[2,3-d]pyrimidine antifolates 1-3.6,7
Compound 5 was selected by the National Cancer
Institute (NCI)32 for evaluation in its in vitro preclinical
antitumor screening program. The ability of compound
5 to inhibit the growth of 54 tumor cell lines 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 5 showed GI50 values in the 10-9-10-7
M
Eth yl 4-[1-Meth yl-2-(2,4-d ia m in ofu r o[2,3-d ]p yr im id in -
5-yl)eth en yl]ben zoa te (9). To a solution of 2,4-diamino-5-
(chloromethyl)furo[2,3-d]pyrimidine (8)18 (1 g, 5 mmol) in
anhydrous DMSO (15 mL) was added tributylphosphine (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
range (Table 2 in Supporting Information). It is note-
worthy that in all leukemia as well as a variety of other
cell lines, compound 5 had GI50 values in the nanomolar
range or less. It was also interesting to note that 5 was
not a general cell poison but showed selectivity both
within a type of tumor cell line as well as across

3130 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16
Gangjee et al.
temperature. To this solution was added sodium hydride (92%
dispersion in mineral oil, 0.2 g, 7.5 mmol), followed by ethyl
4-acetylbenzoate (1.0 g, 5.5 mmol). The reaction mixture was
stirred at room temperature for 38 h. TLC showed that the
disappearance of the starting material (Rf 0.45) and appear-
ance of two spots at 0.58 and 0.60 (MeOH/CHCl3, 1:5, plus 1
drop of ammonia). The reaction was quenched with ethyl
acetate (25 mL), chloroform (25 mL) and 50 mL ethanol, the
resulting solution was evaporated and 100 mL methanol was
added to dissolve the residue, followed by 10 g 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), then sequentially with 300 mL 2-5% MeOH
in CHCl3. Fractions which showed the major spot at Rf 0.60
along with the spot at Rf 0.58 were pooled and evaporated to
dryness. Recrystallization from ethyl acetate afforded 1.35 g
(65%) of 9 (Z:E ratio 1:2) as yellow needles: mp 222-226 °C;
1H NMR (DMSO-d6) E-isomer δ 1.28-1.35 (t, 3 H, OCH2CH3),
2.25 (s, 1 H, 9-CH3), 4.28-4.35 (q, 2 H, OCH2CH3), 6.08 (s, 2
H, 4-NH2), 6.53 (s, 2 H, 2-NH2), 7.01 (s, 1 H, 8-CH), 7.52 (s,1
H, 6-CH), 7.78-7.81 (d, 2H, 3′-, 5′-CH), 7.94-7.97 (d, 2 H, 2′-,
6′-CH); Z-isomer δ 1. 28-1. 35 (t, 3 H, OCH2CH3), 2.20 (s, 3
H, 9-CH3), 4. 28-4.35 (q, 2 H, OCH2CH3), 6.02 (s, 2 H, 4-NH2),
6.32 (s, 1 H, 8-CH), 6.54 (s, 2 H, 2-NH2), 6.67 (s, 1 H, 6-CH),
7.33-7.36 (d, 2 H, 3′-, 5′-CH), 7.88-7.91 (d, 2 H, 2′-, 6′-CH).
Anal. (C18H19N4O3) C, H, N.
(s, 2 H, 4-NH2), 6.41 (s, 2 H, 2-NH2), 6.88 (s, 1 H, C6-CH),
7.12-7.15 (d, 2 H, 3′-, 5′-CH), 7.73-7.76 (d, 2 H, 2-′, 6′-CH).
Anal. (C16H16N4O3‚0.75H2O‚1.0HCl) C, H, N.
(R,S)-N-[4-[1-Meth yl-2-(2,4-d ia m in ofu r o[2,3-d ]p yr im i-
d in -5-yl)eth yl]ben zyl]-L-glu ta m ic Acid Dieth yl Ester (12).
To a solution of 11 (120 mg, 0.37 mmol) in anhydrous DMF (9
mL) was added triethylamine (130 µL) and the mixture 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 activated intermediate at Rf 0.53 and the disap-
pearance of the starting acid Rf 0.32. 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 subject to third round of activation and
coupling using the same the quantities as second round and
was stirred for additional 24 h. TLC showed the formation of
one major spot at Rf 0.54 (MeOH/CHCl3, 1:5). The reaction
mixture was evaporated to dryness under reduced pressure.
The residue was dissolved in 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
which showed the desired single spot at Rf 0.54 were pooled
and evaporated to dryness and crystallized from ethyl ether
and MeOH to afford 120 mg (75%) of 12 as a yellow solid: mp
118.8-120.9 °C; 1H NMR (DMSO-d6) δ 1.10-1.26 (m, 8 H, C9-
CH3, 2× OCH2CH3), 1.96-2.16 (m, 2 H, -CHCH2CH2CO-),
2.32-2.44 (m, 2 H, -CHCH2CH2CO-), 2.95-3.05 (br, 2 H,
C8-CH2), 3.06-3.15 (m, 1 H, C9-CH), 4.00-4.40 (q, 4 H, 2×
OCH2CH3), 4.41 (m, 1 H, -NHCH(CH2)-), 5.95 (s, 2 H, 4-NH2),
6.44 (s, 2 H, 2-NH2), 6.87 (s, 1 H, C6-CH), 7.30-7.33 (d, 2 H,
3′-, 5′-CH), 7.74-7.77 (d, 2 H, 2′-, 6′-CH), 8.61-8.63 (d, 1 H,
-CONH-). Anal. (C25H31N5O6) C, H, N.
Eth yl (R,S)-4-[1-Meth 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 (10). To a solution of 9 (200 mg,
0.6 mmol) in a mixture of DMF (2 mL), MeOH (30 mL) and
CHCl3 (70 mL) was added 5% palladium on activated carbon
(600 mg) and the suspension was hydrogenated in a Parr
apparatus at room temperature and 45 psi hydrogen pressure
for 5.5 h. TLC showed the disappearance of starting material
(Rf 0.60 and 0.58) and the formation of one major spot at 0.57
and one minor spot at 0.55 (MeOH/CHCl3, 1:5). The reaction
mixture was filtered through Celite and the residue was
washed with 20% DMF in methanol 100 mL. The filtrate was
evaporated to dryness under reduced pressure and the solid
was dissolved in 100 mL methanol. 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), then sequentially with 200 mL
2% MeOH in CHCl3, 200 mL 4% and 6% MeOH in CHCl3.
Fractions which showed the major spot at Rf 0.57 were pooled
and evaporated to dryness and recrystallized from ethyl ether
and MeOH to afford 150 mg (75%) of 10 as yellow needles:
mp 219.7-222 °C; 1H NMR (DMSO-d6) δ 1. 25-1.32 (m, 6 H,
C9-CH3, OCH2CH3), 2.95 (br, 2 H, C8-CH2), 3.03-3. 15 (m,
1 H, C9-CH), 4.27-4.29 (q, 2H, OCH2CH3), 5.99 (s, 2 H,
4-NH2), 6.47 (s, 2 H, 2-NH2), 6.88 (s, 1 H, C6-CH), 7.36-7.38
(d, 2 H, 3′-, 5′-CH), 7.84-7.86 (d, 2 H, 2′-, 6′-CH). Anal.
(C18H21N4O3) C, H, N.
(R,S)-N-[4-[1-Meth yl-2-(2,4-d ia m in ofu r o[2,3-d ]p yr im i-
d in -5-yl)eth yl]ben zyl]-L-glu ta m ic Acid (5). To a solution
of the diester 12 (110 mg, 0.2 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 0.54) 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 cooled in an ice bath and the pH 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 100 mg (95%) of 5 as
a white powder: mp 162.8-165 °C; TLC Rf 0.75 (MeOH-
(R,S)-4-[1-Meth 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 (11). To a solution of 10 (150 mg,
0.4 mmol) in MeOH/DMSO (2:1, 22 mL) was added 1 N NaOH
(6 mL) and the mixture was stirred under N2 at room
temperature for 16 h. TLC showed the disappearance of the
starting material (Rf 0.57) 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) and the solution was
filtered through Celite and the Celite pad was washed with 4
mL 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 125 mg (90%)
of 11 as a white powder: mp 266.6-268 °C; TLC Rf 0.32
(MeOH/CHCl3, 1:5); 1H NMR (DMSO-d6) δ 1.25-1. 32 (d, 6
H, C9-CH3), 2.90-3.10 (br, 3 H, C8-CH2 and C9-CH), 5.97
1
CHCl3-NH3OH, 3:7:2); H NMR (DMSO-d6) δ 1.25-1.27 (d,
3H, C9-CH3), 1.94-2.08 (2 sets of t, 2 H, Glu â-CH2), 2.31-
2.36 (t, 2 H, Glu γ-CH2), 2.96-2.98 (d, 2 H, C8-CH2), 3.03-
3.07 (m, 1 H, C9-CH), 4.37 (m, 1 H, Glu R-CH), 5.96 (s, 2 H,
4-NH2), 6.44 (s, 2 H, 2-NH2), 6.87 (s, 1 H, C6-CH), 7.30-7.33
(d, 2 H, 3′-, 5′-CH), 7.75-7.78 (d, 2 H, 2′-, 6′-CH), 8.49-8.52
(d, 1 H, -CONH-), 12.34 (br, 2 H, 2× COOH). Anal.
(C21H23N5O6‚1.5H2O) C, H, N.
Syn th esis of (E)- a n d (Z)-4-[1-Meth yl-2-(2,4-d ia m in o-
fu r o[2,3-d ]p yr im id in -5-yl) eth en yl]ben zoic Acid (13). To
a solution of 9 (150 mg, 0.4 mmol) in MeOH/DMSO (2:1, 22
mL) was added 1 N NaOH (6 mL) and the mixture stirred
under nitrogen at room temperature for 16 h. TLC showed the
disappearance of the starting material 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 (6 mL), the solution was

5-Substituted 2,4-Diaminofuro[2,3-d]pyrimidines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16 3131
filtered through Celite and the Celite pad was wash with 4
mL water. The filtrate was cooled in an ice bath and the pH
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 of 135 mg (97%) of 13
as a yellow solid: mp >300 °C dec; TLC Rf 0.34 (MeOH/CHCl3,
1:5); 1H NMR (DMSO-d6) E-isomer δ 2.24 (s, 3 H, 9-CH3), 6.10
(s, 2 H, 4-NH2), 6.55 (s, 2 H, 2-NH2), 6.99 (s, 1 H, 8-CH), 7.51
(s, 1 H, 6-CH), 7.75-7.78 (d, 2 H, 3′-, 5′-CH), 7.92-7.95 (d, 2
H, 2′-, 6′-CH),12.7 (br, 1 H, COOH); Z-isomer δ 2.19 (s, 3 H,
9-CH3), 6.04 (s, 2 H, 4-NH2), 6.32 (s, 1 H, 8-CH), 6.55 (s, 2 H,
2-NH2), 6.66(s, 1 H, 6-CH), 7.30-7.33 (d, 2 H, 3′-, 5′-CH), 7.86-
7.89 (d, 2 H, 2′-, 6′-CH), 12.7 (br, 1 H, COOH). Anal.
(C16H14N4O3‚0.4H2O) C, H, N.
The filtrate from the diester 14 was found to contain a
majority of the diester 15, Rf 0.58 (MeOH/CHCl3, 1:5), and was
directly used for the next step. Thus to 15 (100 mg, 0.2 mmol)
dissolved 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 0.58) and formation of one major spot at the origin
(MeOH/CHCl3, 1:5). The reaction mixture evaporated to dry-
ness under reduced pressure. The residue was dissolved in
distilled water (10 mL) and the resulting solution was cooled
in an ice bath and the pH adjusted to 2-3 by dropwise addition
of 1 N HCl. The resulting suspension was frozen in a dry ice-
acetone bath, 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 90
mg (95%) of 7 (E/ Z 1:4 mixture) as a yellow solid: mp 188.3-
198.3 °C; 1H NMR (DMSO-d6) E-isomer δ 1.97-2.13 (two sets
of triplets,2 H, Glu â-CH2), 2.25 (s, 3 H, 9-CH3), 2.26-2.39 (t,
2 H, Glu γ-CH2), 4.42 (m, 1 H, Glu R-CH), 6.10 (s, 2 H, 4-NH2),
6.92 (s, 1 H, 8-CH), 7.50 (s, 1H, 6-CH), 7.77-7.74 (d, 2 H, 3′-,
5′-CH), 7.91-7.94 (d, 2H, 2′-, 6′-CH), 8.24 (br, 2 H,2-NH2), 8.58
(d, 1 H, -NHCO-), 12.7 (br, 2 H, COOH); Z-isomer δ 2.00 (m,
2 H, Glu â-CH2), 2.19 (s, 3 H, 9-CH3),. 2.47-2.49 (t, 2 H, Glu
γ-CH2), 4.46 (m, 1 H, Glu R-CH), 6.02 (s, 2 H, 4-NH2), 6.34 (s,
1 H, 8-CH), 6.53 (s, 2 H, 2-NH2), 6.64 (s, 1 H, 6-CH), 7.28-
7.30 (d, 2 H, 3′-, 5′-CH), 7.80-7.82 (d, 2 H, 2′-, 6′-CH), 8.35 (d,
1 H, -NHCO-), 12.7 (br, 2H, COOH). Anal. (C21H21N5O6‚
1.5HCl‚1.5H2O) C, H, N, Cl.
Dih yd r ofola te Red u cta se (DHF R) Assa y.33 All enzymes
were assayed spetrophotometrically in a solution containing
50 µM dihydrofolate, 80 µM NADPH, 0.05 M Tris HCl, 0.001
M 2-mercaptoethanol, and 0.001 M EDTA at pH 7.4 and 30
°C. The reaction was initiated with an amount of enzyme
yielding a change in O.D. at 340 nm of 0.015/min.
Th ym id yla te Syn th a se (TS) Assa y. TS was assayed
spetrophotometrically at 30 °C and pH 7.4 in a mixture
containing 0.1 M 2-mercaptoethanol, 0.0003 M (6R,S)-terahy-
drofolate, 0.012 M formaldehyde, 0.02 M MgCl2, 0.001 M
dUMP, 0.04 M Tris HCl, and 0.00075 M NaEDTA. This was
the assay described by Wahba and Friedkin,34a except that the
dUMP concentration was increased 25-fold according to the
method of Davisson et al.34b The reaction was initiated by the
addition of an amount of enzyme yielding a change in absor-
bance at 340 nm of 0.016/min in the absence of inhibitor. 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.
Cell Lin es a n d Meth od s for Mea su r in g Gr ow th In -
h ibitor y P oten cy (Ta ble 3). Solutions used in cell culture
studies were standardized using extinction coefficients. Ex-
tinction coefficients were determined for 5 (pH 1, λmax-1 249
nm (22900), λmax-2 300 nm (8000); pH 7, λmax 249 nm (22400);
pH 13, λmax 249 nm (22500)), for 6 (pH 1, λmax 265 nm (28400);
pH 7, λmax 268 nm (30100); pH 13, λmax 268 nm (29700)), and
for 7 (pH 1, λmax 250 nm (22600); pH 7, λmax 261 nm (22500);
pH 13, λmax 261 nm (22600)). Extinction coefficients for
methotrexate (MTX), a generous gift of Immunex (Seattle,
WA), were from the literature.35
All cell lines were verified to be negative for mycoplasma
contamination using the GenProbe test kit. The human
T-lymphoblastic leukemia cell line CCRF-CEM25 and its meth-
otrexate-resistant sublines R1,26 R2 (Bos),27 and R30dm28 used
in these studies were cultured as described.28 R1 expresses
20-fold elevated levels of DHFR, the target enzyme of MTX.
R2 has dramatically reduced MTX uptake. R30dm expresses
only 1% of the FPGS activity of CCRF-CEM and is resistant
to short-term, but not continuous, MTX exposure; however,
R30dm is generally cross-resistant in continuous exposure to
antifolates requiring polyglutamylation to form potent potent
inhibitors. The A253 and FaDu human head and neck squa-
mous cell carcinoma monolayer cells were propagated in RPMI
1640/10% fetal calf serum (FCS); however, growth inhibition
was measured in medium containing 5% FCS36 to minimize
Syn th esis of (E)- a n d (Z)-N-[4-[1-Meth yl-2-(2,4-d ia m i-
n ofu r o[2,3-d ]p yr im id in -5-yl)eth en yl]ben zoyl]-L-glu ta m -
ic Acid (6 a n d 7). To a solution of 13 (180 mg, 0.37 mmol) in
a anhydrous DMF (9 mL) was added triethylamine (130 µL)
and the mixture 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 activated intermediate at Rf
0.61 and the disappearance of the starting acid Rf 0.34. 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 using half the quantities listed above. The
reaction mixture was stirred under nitrogen at room temper-
ature overnight. After 24 h, The reaction mixture was sub-
jected to third round of activation using the same the quan-
tities as the second round and was stirred for another 24 h.
TLC showed the formation of two major spots centered at Rf
0.59 (MeOH/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 chromato-
graphed on a silica gel column (2 × 15 cm) with 4% MeOH in
CHCl3 as eluent. Fractions which showed the spot at Rf 0.61-
0.58 were pooled and evaporated to dryness and crystallized
from ethyl acetate/methanol to afford 120 mg (55%) of 14 as
yellowish crystal as the single E-isomer: mp 180.8-181.5 °C;
1
Rf 0.61 (MeOH/CHCl3, 1:5); H NMR (DMSO-d6) δ 1.14-1.20
(m, 6 H, -COOCH2CH3 ×2), 2.13 (m, 2 H, CH-CH2-CH2-),
2.24 (s, 3 H, 9-CH3), 2.47-2.49 (t, 2 H, -CH2CH2CO-), 4.01-
4.15 (m, 4 H, -COOCH2CH3 ×2), 4.46 (m, 1 H, -CONH-CH-
), 6.09 (s, 2 H, 4-NH2), 6.52 (s, 2 H, 2-NH2), 6.97 (s, 1 H, 8-CH),
7.50 (s, 1 H, 6-CH), 7.73-7.76 (d, 2 H, 3′-, 5′-CH), 7.87-7.90
(d, 2 H, 2′-, 6′-CH), 8.76 (d, 1 H, -NHCO-). Anal. (C25H29N5O6)
C, H, N.
To a solution of the diester 14 (110 mg, 0.2 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 0.61)
and the 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) and the resulting solution was cooled in an ice bath
and the pH adjusted to 3-4 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 100 mg
(95%) of 6 as an off-white powder: mp 188.3-191.3 °C; 1H
NMR (DMSO-d6) δ 1.97-2.13 (two sets of triplets, 2 H, Glu
â-CH2), 2.25 (s, 3 H, 9-CH3), 2.26-2.39 (t, 2 H, Glu γ-CH2),
4.42 (m, 1 H, Glu R-CH), 6.10 (s, 2 H, 4-NH2), 6.92 (s, 1 H,
8-CH), 7.50 (s, 1H, 6-CH), 7.74-7.77 (d, 2 H, 3′-, 5′-CH), 7.91-
7.94 (d, 2 H, 2′-, 6′-CH), 8.24 (br, 2 H, 2-NH2), 8.58 (d, 1 H,
-NHCO-), 12.7 (br, 2 H, COOH). Anal. (C21H21N5O6‚1.0HCl‚
0.5H2O) C, H, N.

3132 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16
Gangjee et al.
(12) Piper, J . R.; J ohnson, C. A.; Otter, G. M.; Sirotnak, F. M.
Synthesis and Antifolate Evaluation of 10-Ethyl-5-methyl-5,10-
dideazaaminopterin and an Alternative Synthesis of 10-Ethyl-
10-deazaaminopterin (Edatrexate). J . Med. Chem. 1992, 35,
3002-3006.
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the levels of purine and pyrimidines introduced from FCS.
Growth inhibition of all cell lines by continuous drug exposure
was assayed as described. EC50 values were determined from
plots of percent control growth versus logarithm of drug
concentration.
Protection against growth inhibition of FaDu cells was
assayed by including metabolites simultaneously with
a
concentration of drug previously determined to inhibit growth
by 80-95%; the remainder of assay was as described. Metabo-
lites included were: leucovorin (LV, 0.1-10 µM); or thymidine
(TdR, 40 µM); or hypoxanthine (Hx, 50 µM); or a combination
of TdR and Hx at the same individual concentrations. Growth
inhibition was measured relative to the appropriate metabolite-
treated control; metabolites caused <9% growth inhibition in
the absence of drug, however.
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Activities of 6-Substituted 2,4-Diaminoquinazolines. Eur. J . Med.
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Ack n ow led gm en t. This work was supported in part
by Grant AI44661 (A.G.) from the National Institute of
Allergy and Infectious Diseases and Grants CA43500
(J .J .M.), Roswell Park Cancer Institute Core Grant CA
16065 (J .J .M.), and CA10914 (R.L.K.) from the National
Cancer Institute. The authors thank Dr. Fu-Tyan Lin,
Department of Chemistry, University of Pittsburgh, for
obtaining two-dimensional proton-proton ROESY spec-
tra. The authors also thank Mr. Gregory Nagel and Mr.
William Haile for performing biological and biochemical
studies.
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