Synthesis of classical, three-carbon-bridged 5-substituted furo[2,3-d]pyrimidine and 6-substituted pyrrolo[2,3-d]pyrimidine analogues as antifolates

Article abstract of DOI:10.1021/jm040123k

Bridge homologation of the previously reported classical two-carbon-bridged antifolates, a 5-substituted 2,4-diaminofuro[2,3-d]pyrimidine (1) [which is a 6-regioisomer of LY231514 (Alimta)] and a 6-subsituted 2-amino-4-oxopyrrolo[2,3- d]pyrimidine, afforded the three-carbon-bridged antifolates analogues 4 and 5, with enhanced inhibitory activity against tumor cells in culture (EC ₅₀ values in the 10^-8-10^-7 M range or less). These two analogues were synthesized via a 10-step synthetic sequence starting from methyl 4-bromobenzoate (14), which was elaborated to the α-chloromethyl ketone (8) followed by condensation with 2,6-diamino-pyrimidin-4-one (7) to afford the substituted furo[2,3-d]pyrimidine 9 and the pyrrolo[2,3-d]-pyrimidine 10. Subsequent coupling of each regioisomer with diethyl-L-glutamate followed by saponification afforded 4 and 5. The biological results indicate that elongation of the C8-C9 bridge of the classical 5-substituted 2,4-diaminofuro[2,3-d]pyrimidine and 6-substituted 2-amino-4-oxopyrrolo[2,3-d]pyrimidine are highly conducive to antitumor activity in vitro, despite a lack of increase in inhibitory activity against the target enzymes. This supports our original hypothesis that truncation of the B-ring of a highly potent 6-6 ring system to a 6-5 ring system can be compensated by bridge homologation to restore the overall length of the molecule.

Full text of DOI:10.1021/jm040123k

J. Med. Chem. 2004, 47, 6893-6901
6893
Synthesis of Classical, Three-Carbon-Bridged 5-Substituted
Furo[2,3-d]pyrimidine and 6-Substituted Pyrrolo[2,3-d]pyrimidine Analogues as
Antifolates1
Aleem Gangjee,*,† Yibin Zeng,†,‡ John J. McGuire,§ Farideh Mehraein,§ 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 June 30, 2004
Bridge homologation of the previously reported classical two-carbon-bridged antifolates, a
5-substituted 2,4-diaminofuro[2,3-d]pyrimidine (1) [which is a 6-regioisomer of LY231514
(Alimta)] and a 6-subsituted 2-amino-4-oxopyrrolo[2,3-d]pyrimidine, afforded the three-carbon-
bridged antifolates analogues 4 and 5, with enhanced inhibitory activity against tumor cells
in culture (EC50 values in the 10-8-10-7 M range or less). These two analogues were synthesized
via a 10-step synthetic sequence starting from methyl 4-bromobenzoate (14), which was
elaborated to the R-chloromethyl ketone (8) followed by condensation with 2,6-diamino-
pyrimidin-4-one (7) to afford the substituted furo[2,3-d]pyrimidine 9 and the pyrrolo[2,3-d]-
pyrimidine 10. Subsequent coupling of each regioisomer with diethyl-L-glutamate followed by
saponification afforded 4 and 5. The biological results indicate that elongation of the C8-C9
bridge of the classical 5-substituted 2,4-diaminofuro[2,3-d]pyrimidine and 6-substituted
2-amino-4-oxopyrrolo[2,3-d]pyrimidine are highly conducive to antitumor activity in vitro,
despite a lack of increase in inhibitory activity against the target enzymes. This supports our
original hypothesis that truncation of the B-ring of a highly potent 6-6 ring system to a 6-5
ring system can be compensated by bridge homologation to restore the overall length of the
molecule.
Introduction
against tumor cells in culture with EC50 values in the
nanomolar range as compared to 1.6,7 The increased
potency against DHFR as well as against the growth of
tumor cells in culture of 2 and 3 were, in part, attributed
to increased hydrophobic interaction of the C9-alkyl
moiety with DHFR and by their increased lipophilicity.
In this report we describe an alternate approach to
improve the antitumor activity of 1 via bridge homolo-
gation.
Folate metabolism has long been recognized as an
effective target for chemotherapy, due to its crucial role
in the biosynthesis of nucleic acid precursors.2 Inhibitors
of folate-dependent enzymes have found clinical utility
as antitumor, antimicrobial, and antiprotozoal agents.3,4
As part of a continuing effort to develop novel classical
antifolates as DHFR inhibitors and as antitumor agents,
Gangjee et al.5 reported the synthesis of N-[4-[2-(2,4-
diaminofuro[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutam-
ic acid (1) as a novel 6-5 bicyclic antifolate. Despite its
reasonably potent inhibitory activity against the growth
of tumor cells in culture (EC50‚10-7-10-8 M), its dihy-
drofolate reductase (DHFR) inhibitory potency was low
(IC50 ) 10-6 M) compared to 2,4-diamino 6-6 bicyclic
analogues such as methotrexate (MTX) (IC50 ) 22 ×
10-9 M). We have recently demonstrated that a simple
alkylation of the C9 position of the C8-C9 bridge region
of 1 enhances the inhibition of DHFR such as in
compounds 2 and 3.6,7 Compared to 1, the C9-methyl
analogue 2 (IC50 ) 0.42 × 10-6 M) was twice as
inhibitory against recombinant human (rh) DHFR,
while the C9-ethyl analogue 3 (IC50 ) 0.22 × 10-6 M)
was twice as potent as analogue 2.6,7 Both 2 and 3 were
about 10-fold greater in their growth inhibitory potency
A possible reason for the low DHFR inhibitory activity
of 1 and its relatively low activity against tumor cell
growth in culture compared to MTX could be that
truncation of the 6-6 bicyclic system (of MTX-like
compounds) to a 6-5 ring system shortens the overall
length of the molecule, which results in less than
optimal enzyme interaction and antitumor activity.
8
Molecular modeling studies using SYBYL 6.6 and its
energy minimization options suggested that the distance
and orientation of the R-carboxyl group of the glutamate
of 1 with respect to the pyrimidine ring fell short by
approximately one carbon, compared to that of the
bound conformation of MTX in rhDHFR. The R-car-
boxylate group has important charge-mediated interac-
tions with DHFR.9 Thus, homologation of the two-
carbon bridge of 1 to a three-carbon bridge might restore
the appropriate position for the R-carboxyl group and
thus increase the potency against DHFR and perhaps
improve the antitumor activity against tumor cells in
culture as well. Support for this idea comes from TNP-
351,10 a three-carbon-bridged pyrrolo[2,3-d]pyrimidine
analogue of 1 that is a highly potent DHFR inhibitor
* To whom correspondence should be addressed. Tel: (412) 396-
6070. Fax: (412) 396-5593. E-mail: gangjee@duq.edu.
† Duquesne University.
‡ Current address: Department of Medicinal Chemistry, The Uni-
versity of Kansas, Lawrence, KS 66045.
§ Roswell Park Cancer Institute.
| Tufts University School of Medicine.
10.1021/jm040123k CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/08/2004

6894 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Gangjee et al.
pyrrolo[2,3-d]pyrimidine 5. Thus it was of interest to
synthesize compound 5 as a homologue of the 6-regio-
isomer of LY231514 (Figure 1) with increased confor-
mational flexibility in the side chain.
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 enzymes and antitumor activity.6,11 Thus, it
was also of interest to determine the effect of homolo-
gation of the bridge length of the classical, two-carbon-
bridged, 5-substituted 2,4-diaminofuro[2,3-d]pyrimidine
and 6-substituted 2-amino-4-oxopyrrolo[2,3-d]pyrimi-
dine to three carbons with respect to FPGS activity.
Chemistry
It was anticipated that a Wittig condensation of
5-chloromethyl-2,4-diaminofuro[2,3-d]pyrimidine (6) with
a suitably substituted phenyl acetylaldehyde followed
by catalytic reduction would afford the skeleton of target
compound 4 (route A, Figure 2). However, in a model
reaction using phenylacetaldehyde, the olefinic inter-
mediate was isolated in 35% yield as an E/Z mixture.
Reduction of the double bond afforded the desired
product in only 20% isolated yield. Furthermore, syn-
thesis of the commercially unavailable, substituted
phenylacetaldehyde afforded low yields.
An alternate route was devised from the retrosyn-
thetic analysis of the target skeletons (9 and 10) (route
B, Figure 3), where a single step cyclocondensation of
2,6-diaminopyrimidin-4-one 7 with a suitable R-halo
ketone 815 would afford both the furo[2,3-d]pyrimidine
and the pyrrolo[2,3-d]pyrimidine.
Figure 1.
and which is currently in clinical trials in Japan as an
antitumor agent. Thus, compound 4, the three-carbon-
bridged analogue of 1 and a furo isostere of TNP-351,
was designed and synthesized to explore its antitumor
activity.
Gangjee et al.11 recently reported that in 6-5 ring-
fused pyrrolo[2,3-d]pyrimidine systems such as the
4-methyl analogue of LY231514, AGJY161 (Figure 1),
the 7-NH mimics the 4-amino group of MTX by rotation
of 180° around the C2-NH2 bond when bound to DHFR.
LY231514 is reported to be a dual TS-DHFR inhibitor
(hTS Ki ) 340 nM; hDHFR Ki ) 7 nM).12 Since the
2-amino-4-oxo portion of the pyrimidine ring is the
preferred binding mode for classical antifolates to
TS [e.g. 2-amino-4-oxo-N10-propargyl-5,8-dideazafolate
(PDDF)],13 and the 2,4-diamino portion of the pyrimi-
dine ring is the preferred binding mode for DHFR (e.g.
MTX),9 a possible reason for the dual TS and DHFR
inhibitory activity of LY131514 could be the ability of
the 2-amino-4-oxopyrrolo[2,3-d]pyrimidine to adopt both
the “2-amino-4-oxo” mode as well as “2,4-diamino” mode
(Figure 1) via rotation of the C2-NH2 bond by 180°,
using the pyrrole NH to mimic the 4-amino group of
MTX.
In our recent investigations16 of the cyclocondensation
of 7 with various R-chloro ketones, we found that both
12 and 13 could be obtained as a mixture of regioisomers
in a single reaction by careful monitoring of the reaction
conditions. Separation of the isomers afforded pure 12
and 13 in good yield (50-70%). The desired R-chloro-
methyl ketone 8 could, in turn, be obtained from
commercially available substituted bromobenzene and
3-butyn-1-ol.
Thus, palladium-catalyzed coupling of methyl 4-
bromobenzoate (14) with 3-butyn-1-ol (15)17 (Scheme 1)
afforded phenylbutynyl alcohol 16 (75%), which was
catalytically hydrogenated to give the saturated alcohol
17 in quantitative yield. Subsequent oxidation of 17
using Jones’ reagent18 afforded the carboxylic acid 18
(65%), which was converted to the acid chloride 19 and
immediately reacted with diazomethane followed by
The 6-regioisomer of LY231514 (Figure 1) was previ-
ously synthesized by Taylor et al.14 and was reported
to be inactive in cell culture. The first step in the
synthetic sequence for 4 (Scheme 2) also afforded the
precursor to the 6-substituted three-carbon-bridged
Scheme 1

Furo- and Pyrrolo[2,3-d]pyrimidines as Antifolates
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6895
Scheme 2
Table 1. Inhibitory Concentration (IC50 in µM) against
concentrated HCl to give the desired R-chloro ketone 8
in 75% yield over three steps.19
Isolated DHFR and TSa
DHFR
ecd
NDb
7.0
>21 (16%) 22
TS
ece
With the desired R-chloro ketone 8 at hand, the next
step was the condensation of 2,6-diaminopyrimidin-4-
one 7 with 8 (Scheme 2). Using DMF as the solvent at
room temperature for 24 h, compound 9 was obtained
in 10% yield along with the recovery of starting materi-
als. Increasing the reaction temperature and time
afforded both 9 and 10. Optimal yields were obtained
at 40-45 °C for 3 days and 9 and 10 were each isolated,
after chromatographic separation, in 40% yield (80%
total).
rhc
lc
0.1
11
rhe
lc
1b
1.0
9.0
220
ND
200
4
>18 >180 >100
5
>110 >17 >180 >100
MTX
0.022
ND
0.007 0.022 ND
ND
230
ND
ND
0.009
38
PDDFf
ND
230
0.18 0.09
LY231514g 2.3
19
38
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 IC50 values were within (10% of
the value given. b Data derived from ref 5; ND ) not determined.
c rhDHFR kindly provided by J. H. Freisheim, Medical College of
Ohio, Toledo, OH. d E. coli DHFR kindly provided by Dr. R. L.
Blakley, St. Jude’s Children’s Research Hospital, Memphis, TN.
e rh TS and E. coli TS kindly provided by Dr. Frank Maley, New
York State Department of Health, Albany, NY. f PDDF kindly
provided by Dr. M. G. Nair, University of South Alabama, Mobile,
AL. g LY231514 (Alimta) kindly provided by Dr. Chuan Shih, Eli
Lilly and Co., Indianapolis, IN.
Hydrolysis of 9 and 10 afforded the corresponding free
acids 20 and 21 (90-92%). Subsequent coupling with
L-glutamate diethyl ester using isobutyl chloroformate
as the activating agent17 afforded the diesters 22 and
23. Final saponification of the diesters gave the desired
diacids 4 and 5 respectively.
Figure 2.
Biological Evaluation and Discussion
DHFR and TS Inhibition. Compounds 4 and 5 were
evaluated as inhibitors of Escherichia coli (ec), Lacto-
bacillus casei (lc), and recombinant human (rh) DHFR.17
The inhibitory potency (IC50) values are listed in Table
1 and compared with MTX as well as the previously
reported values for 1.5 For rhDHFR, the 5-substituted,
three-carbon-bridged furo[2,3-d]pyrimidine analogue 4
was about 400-fold less potent than MTX, 4-fold less
potent than LY231514, and 9-fold less potent than the
two-carbon-bridged analogue 1. The 6-substituted, three-
carbon-bridged analogue 5 was poorly active with an
IC50 > 21 µM.
Figure 3.

6896 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Gangjee et al.
Table 2. Activity of 4 and 5 as Substrates for Recombinant
Table 3. Growth Inhibition of Parental CCRF-CEM Human
Leukemia Cells and Sublines with Single, Defined Mechanisms
of MTX Resistance during Continuous (0-120 h) Exposure to
MTX, 4, or 5a
Human FPGSa
substrate
Km, µM
Vmax,rel
1.00
Vmax/Km
n
aminopterin
4.5 ( 1.4
8.5 ( 2.1
0.3 ( 0
0.22
0.07
1.4
7
3
2
2
EC50, nM
1b
4
5
0.65 ( 0.01
0.42 ( 0.02
0.57 ( 0.06
R1b
(v DHFR)
R2c
(V uptake)
R30dmd
(V Glun)
0.9 ( 0.1
0.63
drug
CCRF-CEM
a FPGS substrate activity was determined as described in the
ref 6 at 2 mM L-[3H]glutamate. Values presented are average (
SD if n g 3 and average ( range for n ) 2. Vmax values are
calculated relative to aminopterin within the same experiment.
b Data from ref 5.
MTX
8.2 ( 0.2
290 ( 10
60 ( 14
660 ( 50
ndf
g4600
860 ( 150
1500 ( 0
ndf
1050 ( 50
4350 ( 650
7.9 ( 0.4
4250 ( 50
84 ( 8
1e
4
5
460 ( 10
470 ( 60
a All values presented are average ( range for n ) 2. b CCRF-
CEM subline resistant to MTX solely as a result of a 20-fold
increase in wild-type DHFR protein and activity.20 c CCRF-CEM
subline resistant as a result of decreased uptake of MTX.21
d CCRF-CEM subline resistant to MTX 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.22 e Data from ref 5. f nd ) not determined.
Against ecDHFR, compound 4 had similar potency as
against rhDHFR and compound 5 was 3-fold less potent.
Compound 4 also inhibited lcDHFR at a level similar
to its inhibition of rhDHFR and compound 5 was
essentially inactive, while the parent two-carbon-
bridged analogue 1 had a 10-fold increase in potency
compared to its rhDHFR inhibition.
DHFR overexpressing line R1 was >76-fold cross-
resistant to 4, similar to the resistance to MTX, indicat-
ing that DHFR is likely the primary target of this 2,4-
diaminofuropyrimidine. In contrast, R1 was <2-fold
cross-resistant to 5, suggesting that it primarily inhibits
a site other than DHFR. The MTX-transport-resistant
subline R2, which does not express functional reduced
folate carrier (RFC),25 is 17-fold cross-resistant to 4 and
10-fold resistant to 5, while it is 180-fold resistant to
MTX. The data suggest that both 4 and 5 utilize the
RFC as their primary means of transport, but at high
extracellular levels, both drugs are able to diffuse
through the plasma membrane. A cell line expressing
low levels of folylpolyglutamate synthetase (FPGS) is
not cross-resistant to either analogue under continuous
exposure conditions. This means either that each mono-
glutamate is a potent inhibitor of its respective target
(as is MTX), which is inconsistent with the enzyme
inhibition data (Table 1), or that each analogue is
efficiently polyglutamylated even at low FPGS levels,
indicating that they are efficient substrates, which is
consistent with their high efficiency as FPGS substrates
(Table 2).
Metabolite protection studies were performed to
further elucidate the mechanism of action of 4 and 5.
At concentrations of drug that inhibited the growth of
CCRF-CEM cells by g90%, leucovorin was able to fully
protect against the effects of MTX, 4, and 5 (data not
shown). This is consistent with an antifolate mechanism
of action for 4 and 5. Further studies in CCRF-CEM
cells examined the ability of thymidine (TdR) and
hypoxanthine (Hx) to protect against growth inhibition.
These metabolites can be salvaged to produce dTTP and
the purine NTPs required for DNA synthesis and thus
bypass the MTX blockade.26 As described in the meth-
ods, in T-lymphoblast cell lines such as CCRF-CEM,
TdR can only be tested in the presence of dCyd, which
reverses its toxic effects; however, dCyd has no protec-
tive effect on MTX either alone or in paired combination
with either Hx or TdR (Table 4, footnote a). The data
(Table 4) show that for 4, neither TdR nor Hx alone
protected to any significant extent; both metabolites
were required to achieve significant protection. This
indicates that both purine and thymidylate synthesis
are inhibited, and this is consistent with DHFR being
the primary target of 4. In contrast, Hx alone could
protect against growth inhibition by 5 and addition of
Analogues 4 and 5 were also evaluated as inhibitors
of ecTS, lcTS, and rhTS6,7 and compared to PDDF as a
control. Both analogues were inactive at the concentra-
tions tested (Table 1).
FPGS Substrate Activity. Since polyglutamylation
by the enzyme FPGS can significantly enhance the
antitumor activity of some classical antifolates, such as
LY231514 and TNP-351,14,20 it was of interest to evalu-
ate these analogues as substrates of FPGS. Their
activity was evaluated in vitro with recombinant human
FPGS and compared to that of AMT, a good substrate
for FPGS. The data (Table 2) show that both 4 and 5
are very efficient substrates for human FPGS, despite
their 2-fold lower Vmax values, because of substantial
decreases in Km. Compound 4 was 6-fold more efficient
than AMT, while 5 was 3-fold more efficient than AMT.
These results suggest that metabolism to polyglutamates
must be considered in the mechanism of action of both
4 and 5. It is interesting to note that modification of
the bridge region of the classical 2,4-diaminofuropyri-
midine 1 has significant effects on FPGS substrate
activity. Compared to the two-carbon-bridged compound
1, the three-carbon-bridged compound 4 is 20-fold more
efficient as a FPGS substrate.
In Vitro Human Tumor Cell Growth Inhibition.
The growth inhibitory potency of 4 and 5 were compared
to that of MTX in continuous exposure against the
CCRF-CEM human lymphoblastic leukemia21 and a
series of MTX-resistant sublines22-24 (Table 3). The
three-carbon-bridged 2,4-diaminofuro[2,3-d]pyrimidine
analogue 4 was highly cytotoxic against the growth of
CCRF-CEM cells in culture with an EC50 of 60 nM,
which was about 5-fold more potent than its parent
compound 1. Interestingly, compound 5, the three-
carbon-bridged 2-amino-4-oxopyrrolo[2,3-d]pyrimidine
analogue, also had reasonable inhibitory potency against
the growth of CCRF-CEM cells in culture with an EC50
of 460 nM, which was unexpected since the 6-regioiso-
mer of LY231514 was reported to be inactive in culture
with an EC50 > 60 µM.11 This indicates that elongation
of the bridge length from two carbons to three carbons
for the classical 5-substituted 2,4-diaminofuro[2,3-d]-
pyrimindine and the 6-substituted 2-amino-4-oxo-
pyrrolo[2,3-d]pyrimidine is highly conducive to tumor
growth inhibition in culture and could be the result of
the polyglutamate metabolites of 4 and 5.

Furo- and Pyrrolo[2,3-d]pyrimidines as Antifolates
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6897
Table 4. Protection of CCRF-CEM Human Leukemia Cells
against the Growth Inhibitory Effects of MTX, 4, and 5 by 10
µM Hypoxanthine (Hx), 5 µM Thymidine (TdR), and Their
Combination
elongation of the C8-C9 bridge of the classical, 5-sub-
stiuted 2,4-diaminofuro[2,3-d]pyrimidine and 6-substi-
tuted 2-amino-4-oxopyrrolo[2,3-d]pyrimidine are highly
conducive to tumor inhibitory activity.
relative growth (%)a,b
drug
no addition 5 µM TdR 10 µM Hx Hx + TdR
Experimental Section
MTX (30 nM)
13 ( 0
21 ( 0
11 ( 0
12 ( 0
16 ( 0
25.5 ( 0.5 20 ( 0
12.5 ( 0.5 13.5 ( 0.5 73.5 ( 0.5
11 ( 0 80.5 ( 0.5 76 ( 1
15.5 ( 0.5 74.5 ( 6.5
All evaporations were carried out in vacuo with a rotary
evaporator. Analytical samples were dried in vacuo (0.2
mmHg) in a CHEM-DRY drying apparatus over P2O5 at 80
°C. 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 protons (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 tetramethyl-
silane 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 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 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 could not be 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. or Fisher Scientific and
were used as received.
Methyl 4-(4-Hydroxybutynyl)benzoate (16). To a 250-
mL round-bottomed flask, equipped with a magnetic stirrer
and gas inlet, was added a mixture of palladium chloride (45
mg, 0.26 mmol), triphenylphosphine (125 mg, 0.48 mmol),
methyl 4-bromobenzoate (14) (10 g, 46 mmol), and diethyl-
amine (300 mL). To the stirred mixture, under N2, were added
copper(I) iodide (90 mg) and 3-butyn-1-ol (15) (3.3 g, 47 mmol),
and the reaction mixture was stirred at room temperature
overnight (17-18 h). Diethylamine was removed under re-
duced pressure, brine (80 mL) was added, and the mixture
was extracted with EtOAc. The extracts were pooled and dried
over Na2SO4 and passed over a short silica gel column (3 × 6
cm) to remove the catalyst. The fractions collected were pooled
and concentrated under reduced pressure, and the residue was
recrystallized from EtOAc/hexane (1:5) to give 8 g (80%) of 16
as white flakes: mp 95.3-96.3 °C (lit. mp 95.5-96 °C); 1H
NMR (DMSO-d6) δ 2.56-2.60 (t, 2 H, propargyl CH2), 3.56-
3.60 (t, 2 H, CH2-OH), 3.84 (s, 3 H, OMe), 4.92-4.95 (t, 1 H,
OH), 7.50-7.53 (d, 2 H, C6H4), 7.89-7.92 (d, 2 H, C6H4).
Methyl 4-(4-Hydroxybutyl)benzoate (17). To a Parr flask
were added 16 (5 g, 25 mmol), 5% palladium on activated
carbon (500 mg), and MeOH (100 mL). Hydrogenation was
carried out at 45-50 psi of H2 for 12 h. The reaction mixture
was filtered through Celite, washed with MeOH/CHCl3 (1:1)
(100 mL), passed through a short silica gel column (3 × 5 cm),
and concentrated under reduced pressure to give 5.1 g
(quantitative) of 17 as a clear oil: 1H NMR (DMSO-d6) δ 1.42-
1.65 (m, 4 H, 2′-CH2, 3′-CH2), 2.60-2.65 (t, 2 H, benzylic CH2),
3.38-3.42 (t, CH2-OH), 3.81 (s, 3 H, OMe), 4.39 (br, 1 H, OH),
7.30-7.33 (d, 2 H, C6H4), 7.85-7.88 (d, 2 H, C6H4). This alcohol
was directly used for the next step.
4-(4′-Carbomethoxyphenyl)butyric Acid (18). A solution
of methyl 4-(4-hydroxybutyl)benzoate (17) (3 g, 15 mmol) in
acetone (30 mL) was added dropwise to a cold solution (ice
bath) of CrO3 (8 g) in sulfuric acid (60 mL) and water (180
mL). After the addition, the resulting solution was stirred in
an ice bath for an additional 2 h and the solution was allowed
to warm to room temperature overnight. TLC indicated the
disappearance of the starting alcohol and the formation of one
major spot at Rf ) 0.35 (hexane/EtOAc 2:1). The solution was
4
73.5 ( 2.5
MTX
5
a Deoxycytidine (dCyd; 10 µM) was present in all the above
cultures to prevent the inhibition of growth caused in T-cell
leukemias such as CCRF-CEM by TdR (see the methods). In
typical results, dCyd alone had no effect on CCRF-CEM growth
(97 ( 3% of control) and did not protect against growth inhibition
by any of these drugs (data not shown). Hx alone (98 ( 1% of
control) or in the presence of dCyd (94 ( 0% of control) did not
affect CCRF-CEM growth and did not protect against MTX-
induced growth inhibition (Table above). TdR alone inhibited
growth of CCRF-CEM (35 ( 0% of control), but TdR+dCyd was
essentially not growth inhibitory (95 ( 2% of control) and neither
protected against MTX-induced growth inhibition (table above).
Similarly, Hx + TdR + dCyd did not appreciably inhibit growth
of CCRF-CEM (95.5 ( 2.5% of control). b Growth is expressed
relative to quadruplicate cultures not treated with either drug or
metabolite and is the average ( range for duplicate treated
samples. Each experiment was repeated for each drug with similar
results.
other metabolites did not improve the protection. These
data suggest that 5 inhibits purine synthesis only; this
is also consistent with the lack of cross-resistance of the
DHFR overproducing R1 (Table 3). Since there are two
folate-dependent formyltransferases in purine synthesis,
it would be of interest to know which is inhibited.
Current anti-purine antifolates are primarily targeted
to GAR formyltransferase.27
Compounds 4 and 5 were selected by the National
Cancer Institute28 for evaluation in its in vitro preclini-
cal antitumor screening program. The ability of com-
pounds 4 and 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 the control. In more than 10 cell
lines, compound 4 showed GI50 values in the 10-9-10-7
M range (Table 5) and compound 5 showed GI50 values
in the 10-8-10-7 M in eight cell lines. It was interesting
to note that compounds 4 and 5 were not general cell
poisons but showed selectivity both within a type of
tumor cell line as well as across different tumor cell lines
with inhibitory values that in some instances differed
by 10 000-fold. Compound 4 is currently under further
evaluation by the NCI as an antitumor agent.
In summary, classical, three-carbon-bridged, 5-sub-
stituted 2,4-diaminofuro[2,3-d]pyrimidine (4) and 6-sub-
situted 2-amino-4-oxopyrrolo[2,3-d]pyrimidine (5) were
designed and synthesized. The biological results indicate
that elongation of the C8-C9 bridge of the classical,
5-substituted furo[2,3-d]pyrimidine to a three-carbon
bridge (analogue 4) increases (5-fold) the inhibitory
activity against the growth of tumor cells in culture
compared to the two-carbon-bridged parent, compound
1. Homologation of the C8-C9 bridge of the classical,
6-substituted pyrrolo[2,3-d]pyrimidine to a three-carbon-
bridge (analogue 5) also resulted in an increase of
inhibitory activity against the growth of tumor cells in
culture compared to the previously reported 6-regio-
isomer of LY231514.14 These data suggest that the

6898 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Gangjee et al.
Table 5. Cytotoxicity Evaluation (GI50, M) of Compounds 4 and 5 against Selected NCI Tumor Cell Lines26
cell lines
leukemia
compd 4
compd 5
cell lines
compd 4
compd 5
CNS cancer
SF-268
melanoma
SK-MEL-28
renal cancer
786-0
prostate cancer
PC-3
breast cancer
MCF7
CCRF-CEM
HL-60(TB)
K-562
4.29 × 10-8
<1.0 × 10-8
<1.0 × 10-8
<1.0 × 10-8
4.76 × 10-6
1.11 × 10-6
6.5 × 10-8
2.27 × 10-7
1.70 × 10-7
9.99 × 10-7
4.71 × 10-7
6.48 × 10-7
>1.0 × 10-4
1.23 × 10-7
1.25 × 10-8
1.74 × 10-7
2.14 × 10-8
2.23 × 10-8
5.58 × 10-6
2.95 × 10-7
6.28 × 10-7
3.80 × 10-5
2.21 × 10-6
1.30 × 10-6
1.95 × 10-6
SR
non-small cell lung cancer
A549/ATCC
1.66 × 10-8
NCI-H460
HOP-92
colon cancer
HCT-116
SW-620
<1.0 × 10-8
9.59 × 10-5
<1.0 × 10-8
<1.0 × 10-8
nd
MDA-MB-435
MDA-N
9.56 × 10-7
extracted with 3 × 200 mL of ethyl ether and dried over Na2-
SO4. After evaporation of the solvent under reduced pressure,
the resulting residue was flash chromatographed through
silica gel column (3.5 × 15 cm) using hexane/EtOAc (2:1) as
eluent. The desired fraction (TLC) was collected and the
solvent evaporated under reduced pressure. Recrystallization
of the resulting residue from Et2O afforded 2 g (63%) of 18 as
Fractions with Rf ) 0.38 (TLC) were pooled and evaporated,
and the resulting residue was recrystallized from CH3OH (10
mL) to afford 800 mg (40%) of 10 as light yellow crystals: mp
240.2-242 °C; 1H NMR (DMSO-d6) δ 1.83-1.95 (m, 2 H, C9-
CH2), 2.64-2.69 (t, 2 H, C10-CH2), 2.73-2.97 (two sets of t,
2 H, C8-CH2), 3.82 (s, 3 H, OMe), 5.89 (s, 1 H, C5-CH), 5.96
(s, 2 H, 4-NH2), 7.34-7.37 (d, 2 H, Ph-CH), 7.86-7.89 (d, 2 H,
Ph-CH), 10.15 (s, 1 H, 3-NH), 10.83 (s, 1 H, 7-NH). Anal. C17H18
N4O3·0.2H2O (C, H, N).
1
white crystals: mp 76.5-78 °C; H NMR (DMSO-d6) δ 1.76-
1.86 (m, 2 H, â-CH2), 2.20-2.25 (t, 2 H, R-CH2), 2.64-2.69 (t,
2 H, benzylic CH2), 3.83 (s, 3 H, OMe), 7.32-7.35 (d, 2 H,
C6H4), 7.86-7.89 (d, 2 H, C6H4), 12.29 (s, 1 H, COOH). Anal.
C12H14 O4 (C, H).
4-[3-(2,4-Diaminofuro[2,3-d]pyrimidin-5-yl)propyl]-
benzoic Acid (20). To a suspension of the ester 9 (300 mg, 9
mmol) in MeOH/DMSO (1:1) (40 mL) was added 3 N NaOH
(15 mL). The resulting mixture was stirred at 40-50 °C under
N2 for 5 h. TLC indicated the disappearance of starting
material and the formation of one major spot at the origin.
The resulting solution was passed through Celite and washed
with a minimum amount of CH3OH. The combined filtrate was
evaporated under reduced pressure to dryness. To this residue
was added distilled water (15 mL), the resulting solution was
cooled in an ice bath, and the pH was adjusted to 3 to 4 using
3 N HCl. The resulting suspension was frozen using a dry ice-
acetone bath and thawed to 4 °C overnight in a refrigerator.
The suspension was filtered, washed with cold water, and dried
in a desiccator using P2O5 under reduced pressure to afford
280 mg (92%) of 20 as a white powder: mp >370 °C (dec); Rf
Chloromethyl 3-(4′-Carbomethoxyphenyl)propyl Ke-
tone (8). To 4-(4′-carbomethoxyphenyl)butyric acid (18) (2.3
g, 10 mmol) in a 100 mL flask were added oxalyl chloride (5
mL) and anhydrous CH2Cl2 (10 mL). The resulting solution
was refluxed for 1 h and then cooled to room temperature.
After evaporating the solvent under reduced pressure, the
residue (19) was dissolved in 20 mL of Et2O. The resulting
solution was added dropwise to an ice-cooled diazomethane
(generated in situ from 15 g of N-nitroso-N-methylurea) and
ether solution in an ice bath over 10 min. The resulting
mixture was allowed to stand for 30 min and then stirred for
an additional 30 min. To this solution was added concentrated
HCl (20 mL). The resulting mixture was refluxed for 1.5 h.
After cooling to room temperature, the organic layer was
separated and the aqueous layer extracted with Et2O (100 mL
× 2). The combined organic layer and Et2O extract was washed
with two portions of 10% Na2CO3 solution and dried over Na2-
SO4. Evaporation of the solvent afforded 2 g (80%) of 8 as
1
) 0.38 (CHCl3/MeOH 5:1); H NMR (DMSO-d6) δ 1.80-1.85
(m, 2 H, C9-CH2), 2.65-2.95 (m, 4 H, C10-CH2, C8-CH2),
6.85 (s, 2 H, 2- or 4-NH2), 7.20-7.39 (m, 5 H, 4- or 2-NH2,
C6-CH, C6H4), 7.84-7.86 (d, 2 H, C6H4), 12.80 (s, 1 H, COOH).
Anal. C16H16N4O3·1.0HCl·0.30H2O (C, H, N). This acid was used
directly for the next step without further purification.
1
yellow needles: mp 30-33 °C; H NMR (DMSO-d6) δ 1.77-
Diethyl N-{4-[3-(2,4-Diaminofuro[2,3-d]pyrimidin-5-
yl)propyl]benzoyl}-L-glutamate (22). To a solution of 20
(200 mg, 0.57 mmol) in anhydrous DMF (9 mL) was added
triethylamine (200 µL) and the mixture stirred under N2 at
room temperature for 5 min. The resulting solution was cooled
to 0° C, isobutyl chloroformate (200 µL, 1.5 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 acti-
vated intermediate (Rf ) 0.62) and the disappearance of the
starting acid (Rf ) 0.38). Diethyl-L-glutamate hydrochloride
(390 mg, 1.5 mmol) was added to the reaction mixture followed
immediately by triethylamine (200 µL, 1.5 mmol). The reaction
mixture was slowly allowed to warm to room temperature and
stirred under N2 for 18 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 N2
for an additional 24 h. TLC showed the formation of one major
spot at Rf ) 0.62 (MeOH/CHCl3 1:5). The reaction mixture was
evaporated to dryness under reduced pressure. The residue
was dissolved in a minimum amount of MeOH/CH3Cl (1:4) and
chromatographed on a silica gel column (2 × 15 cm) with 2%
MeOH in CHCl3 as eluent. The desired fractions (TLC) were
pooled and evaporated to dryness, and the residue was
crystallized from Et2O to afford 240 mg (60%) of 22 as yellow
1.91 (m, 2 H, â-CH2), 2.50-2.54 (t, 2 H, R-CH2), 2.61-2.66 (t,
2 H, benzylic CH2), 3.82 (s, 3 H, OMe), 4.50 (s, 2 H, CH2Cl),
7.40-7.43 (d, 2 H, C6H4), 7.86-7.89 (d, 2 H, C6H4). Anal. C13H15
O3Cl (C, H, Cl).
Methyl 4-[3-(2,4-Diaminofuro[2,3-d]pyrimidin-5-yl)-
propyl]benzoate (9) and Methyl 4-[3-(2-Amino-3,4-dihydro-4-
oxo-7H-pyrrolo[2,3-d]pyrimidin-6-yl)propyl]benzoate (10).
To a suspension of 2,6-diaminopyrimidin-4-one (7) (0.8 g, 6
mmol) in anhydrous DMF (15 mL) was added chloromethyl
3-(4′-carbomethoxyphenyl)propyl ketone (8) (1.5 g, 6 mmol).
The resulting mixture was stirred under N2 at 40-50 °C for 3
days. TLC showed the disappearance of the starting materials
and the formation of two major spots at Rf ) 0.58 and 0.39
(CHCl3:MeOH 5:1). After evaporation of solvent under reduced
pressure, MeOH (20 mL) was added followed by silica gel (5
g) and the solvent evaporated. The resulting plug was loaded
on to a silica gel column (3.5 × 12 cm) and eluted with CHCl3
followed by 3% MeOH in CHCl3 and then 4% MeOH in CHCl3.
Fractions with an Rf ) 0.58 (TLC) were pooled and evaporated
and the resulting residue was recrystallized from MeOH to
afford 800 mg (40%) of 9 as white crystals: mp 221.2-222.8
°C; 1H NMR (DMSO-d6) δ 1.80-1.88 (m, 2 H, C9-CH2), 2.57-
2.65 (t, 2 H, C10-CH2), 2.69-2.75 (t, 2 H, C8-CH2), 3.82 (s,
3 H, OMe), 5.96 (s, 2 H, 2-NH2), 6.47 (s, 2 H, 4-NH2), 7.13 (s,
1 H, C6-CH), 7.35-7.37 (d, 2 H, C6H4), 7.86-7.88 (d, 2 H,
C6H4). Anal. C17H18 N4O3 (C, H, N).
1
needles: mp 130.6-132.2 °C; H NMR (DMSO-d6) 1.08-1.25
(m, 6 H, OEt), δ 1.78-1.80 (m, 2 H, C9-CH2), 1.97-2.11 (2

Furo- and Pyrrolo[2,3-d]pyrimidines as Antifolates
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6899
sets of t, 2 H, Glu â-CH2), 2.40-2.45 (t, 2 H, Glu γ-CH2), 2.57-
2.80 (m, 4 H, C10-CH2, C8-CH2), 4.00-4.13 (m, 4 H, OEt),
4.33-4.40 (m, 1 H, Glu R-CH), 5.97 (s, 2 H, 4-NH2), 6.41 (s, 2
H, 2-NH2), 7.13 (s, 1 H, C6-CH), 7.33-7.35 (d, 2 H, C6H4),
7.79-7.81 (d, 2 H, C6H4), 8.64-8.66 (d, 1 H, -CONH-). Anal.
C25H31N5O6 (C, H, N).
afford 240 mg (60%) of 23 as a yellow syrup which was used
directly for the next step.
To a solution of the diester 23 (150 mg, 0.3 mmol) in MeOH
(10 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 ) 0.45) and 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, thawed to
4-5 °C in the refrigerator, and filtered. The residue was
washed with a small amount of cold water and ethyl acetate
and dried in vacuo using P2O5 to afford 135 mg (95%) of 5 as
a yellow powder: mp 187.1-190 °C; 1H NMR (DMSO-d6) δ
1.87-2.11 (m, 4 H, C9-CH2, Glu â-CH2), 2.33-2.38 (t, 2 H,
Glu γ-CH2), 2.47-2.68 (m, 4 H, C10-CH2, C8-CH2), 4.38-
4.43 (m, 1 H, Glu R-CH), 5.89 (s, 1 H, C5-CH), 6.02 (s, 2 H,
2-NH2), 7.30-7.32 (d, 2 H, C6H4), 7.78-7.80 (d, 2 H, C6H4),
8.52-8.54 (d, 1 H, -CONH), 10.19 (s, 1 H, 3-NH), 10.82 (s, 1
H, 7-NH). Anal. (C21H23N5O6·1.0HCl) (C, H, N, Cl)
N-{4-[3-(2,4-Diaminofuro[2,3-d]pyrimidin-5-yl)propyl]-
benzoyl}-L-glutamic Acid (4). To a solution of the diester
22 (150 mg, 0.3 mmol) in MeOH (10 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 ) 0.62) (MeOH/CHCl3 1:5) and formation
of one major spot at the origin. The reaction mixture was
evaporated to dryness under reduced pressure and the residue
dissolved in water (10 mL). The resulting solution was 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, 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 (92%) of 4 as a white powder: mp 159-161 °C;
1H NMR (DMSO-d6) 1.83-2.08 (m, 4 H, C9-CH2, Glu â-CH2),
2.32-2.36 (t, 2 H, Glu γ-CH2), 2.57-2.93 (m, 4 H, C10-CH2,
C8-CH2), 4.36-4.38 (m, 1 H, Glu R-CH), 5.97 (s, 2 H, 4-NH2),
6.40 (s, 2 H, 2-NH2), 7.13 (s, 1 H, C6-CH), 7.30-7.32 (d, 2 H,
C6H4), 7.80-7.82 (d, 2 H, C6H4), 8.50-8.52 (d, 1 H, -CONH-
), 12.38-12.53 (br, 2 H, 2 × COOH). Anal. C21H23N5O6·1.2H2O
(C, H, N).
Dihydrofolate Reductase (DHFR) Assay.29 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 OD at 340 nm of 0.015/min.
Thymidylate Synthase (TS) Assay. TS was assayed
spectrophotometrically at 30 °C and pH 7.4 in a mixture
containing 0.1 M 2-mercaptoethanol, 0.0003 M (6R,S)-tetra-
hydrofolate, 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,30 except that the
dUMP concentration was increased 25-fold according to the
method of Davisson et al.31 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.
Cell Lines and Methods for Measuring Growth Inhibi-
tory Potency (Table 2). Drug solutions were standardized
using extinction coefficients. Extinction coefficients were
determined for 4 [pH 1, λmax-1 249 nm (21 500), λmax-2 302 nm
(7100); pH 7, λmax 250 nm (21 100); pH 13, λmax 251 nm
(21 300)] and for 5 [pH 1, λmax 229 nm (21 900); pH 7, λmax 251
nm (20 900); pH 13, λmax 251 nm (20 600)]. Extinction coef-
ficients for methotrexate (MTX), a gift of Immunex (Seattle,
WA), were from the literature.32 Aminopterin, hypoxanthine
(Hx), thymidine (TdR), and deoxycytidine (dCyd) were pur-
chased from Sigma Chemical Co. (St. Louis, MO). Calcium
leucovorin (LV) was purchased from Schircks Laboratories
(Jona, Switzerland). Other chemicals and reagents were
reagent grade or higher.
Cell lines were verified to be negative for mycoplasma
contamination (Mycoplasma Plus PCR primers, Stratagene,
La Jolla, CA). The human T-lymphoblastic leukemia cell line
CCRF-CEM21 and its MTX-resistant sublines R1,22 R2,23 and
R30dm24 were cultured as described.24 R1 expresses 20-fold
elevated levels of dihydrofolate reductase (DHFR), the target
enzyme of MTX. R2 has dramatically reduced MTX uptake
but normal levels of MTX-sensitive DHFR. R30dm expresses
1% of the folylpolyglutamate synthetase (FPGS) activity of
CCRF-CEM and is resistant to short-term, but not continuous,
MTX exposure; however, R30dm is cross-resistant in continu-
ous exposure to antifolates that require polyglutamylation to
form potent inhibitors. Growth inhibition of all cell lines by
continuous drug exposure was assayed as described.20,24 EC50
values (drug concentration effective at inhibiting cell growth
by 50%) were determined visually from plots of percent growth
relative to a solvent-treated control culture versus the loga-
rithm of drug concentration.
4-[3-(2-Amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]-
pyrimidin-6-yl)propyl]benzoic Acid (21). To a suspension
of 10 (250 mg, 7 mmol) in CH3OH/DMSO (1:1) was added 15
mL of 3 N NaOH. The resulting mixture was stirred under
N2 at 40-50 °C for 5 h. TLC indicated the disappearance of
starting material and the formation of one major spot at the
origin. The resulting solution was passed through Celite and
washed with a minimum amount of CH3OH. The combined
filtrate was evaporated under reduced pressure to dryness. To
this residue was added 15 mL of distilled water, and the
solution, in an ice bath, was adjusted to pH 3-4 using 2 N
HCl. The resulting suspension was frozen in an acetone-dry
ice bath, thawed to 4 °C overnight in a refrigerator, filtered,
washed with cold water, and dried in a desiccator under
reduced pressure using P2O5 to afford 260 mg (90%) of 21: mp
>280 °C (dec); Rf ) 0.32 (CHCl3: MeOH 5:1); 1H NMR (DMSO-
d6) δ 1.88-1.97 (m, 2 H, C9-CH2), 2.66-2.97 (m, 4 H, C10-
CH2, C8-CH2), 5.96 (s, 1 H, C5-CH), 6.60 (br, 2 H, 2-NH2),
7.32-7.34 (d, 2 H, C6H4), 7.85-7.87 (d, 2 H, C6H4), 10.68 (s, 1
H, 3-NH), 11.17 (s, 1 H, 7-NH), 13.0 (s, 1 H, COOH). Anal.
C16H16 N4O3·1.0HCl·0.30H2O (C, H, N). This acid was used
directly for the next step without further purification.
N-{4-[3-(2-Amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]-
pyrimidin-6-yl)propyl]benzoyl}-L-glutamic Acid (5). To
a solution of 21 (200 mg, 0.57 mmol) in anhydrous DMF (9
mL) was added triethylamine (200 µL) and the mixture stirred
under N2 at room temperature for 5 min. The resulting solution
was cooled to 0 °C, isobutyl chloroformate (200 µL 1.5 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 0.44 and the disappearance
of the starting acid (Rf 0.32). Diethyl-L-glutamate hydrochloride
(390 mg, 1.5 mmol) was added to the reaction mixture followed
immediately by triethylamine (200 µL, 1.5 mmol). The reaction
mixture was slowly allowed to warm to room temperature and
stirred under N2 for 18 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 N2
for 24 h. TLC showed the formation of one major spot at Rf )
0.45 (MeOH/CHCl3 1:5). The reaction mixture was evaporated
to dryness under reduced pressure. The residue was dissolved
in a minimum amount of CH3Cl/MeOH (4:1) and chromato-
graphed on a silica gel column (2 × 15 cm) with 4% MeOH in
CHCl3 as the eluent. Fractions that showed the desired single
spot at Rf ) 0.45 were pooled and evaporated to dryness to
Protection against growth inhibition of CCRF-CEM cells was
assayed by including leucovorin ((6R,S)-5-formyltetrahydro-
folate) at 0.1-10 µM with a concentration of drug previously
determined to inhibit growth by 90-95%; the remainder of

6900 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Gangjee et al.
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the assay was as described. Growth inhibition was measured
relative to the appropriate leucovorin-treated control; leuco-
vorin, even at 10 µM, caused no growth inhibition in the
absence of drug, however. Protection against growth inhibition
of CCRF-CEM cells was assayed by including Hx (10 µM), TdR
(5 µM), or dCyd (10 µM) individually, in pairs (Hx + dCyd,
TdR + dCyd), or all together (Hx + TdR + dCyd) with
concentrations of MTX, 4, or 5 that would inhibit growth by
90-95% over a growth period of ≈72 h. Compound 4 could
only be tested up to 1500 nM, a concentration that inhibited
growth by only 80%. The growth period was limited, because
beyond 72 h CCRF-CEM cells deplete TdR in the growth media
and drug effects are no longer protected. dCyd is added only
to alleviate the growth inhibitory effects of 5 µM TdR against
CCRF-CEM cells.33 Controls with metabolites alone (no drug)
in the combinations described above (in duplicate), controls
with drug alone with no metabolites (in duplicate), and
untreated controls with neither drugs nor metabolites (in
quadruplicate) were performed. Growth inhibition was mea-
sured as percent growth relative to untreated control cells
(absence of drugs and metabolites).
Folylpolyglutamate Synthetase (FPGS) Purification
and Assay. Recombinant human cytosolic FPGS was purified
and assayed as described previously.11 Both 4 and 5 were
themselves quantitatively recovered during the standard assay
procedure, thus ensuring that their polyglutamate products
would also be quantitatively recovered. Kinetic constants were
determined by the hyperbolic curve fitting subroutine of
SigmaPlot (Jandel) or Kaleidagraph (Synergy Software) using
a g10-fold range of substrate concentration. Activity was linear
with respect to time at the highest and lowest substrate
concentrations tested. Assays contained ≈400 units of FPGS
activity; one unit of FPGS catalyzes incorporation of 1 pmol
of [3H]glutamate/h. Because Km values for 4 and 5 were low,
the assays to determine kinetic constants were modified to
include 2 mM L-[3H]glutamate, instead of the standard 4 mM.
The resulting lower background allowed quantitation at lower
levels of product synthesis. The Km value for AMT was the
same whether 2 or 4 mM glutamate was used (data not
shown).
(16) Gangjee, A.; Yang, J.; McGuire, J. J.; Kisliuk, R. L. Classical
2,4-Diamino-5-substituted Furo[2,3-d]pyrimidine and 2-Amino-
4-oxo-6-substituted Pyrrolo[2,3-d]pyrimidine on Antifolate Activ-
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10-Dideaza-5,6,7,8-tetrahydrofolic Acid and 5,10-Dideaza-5,6,7,8-
tetrahydrohomofolic Acid. An Effective Principle for Carbonyl
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(18) Wipf, P.; Coish, P. D. G. Total Synthesis of (()-Nisamycin. J.
Org. Chem. 1999, 64, 5053-5061.
Acknowledgment. This work was supported in part
by grants from the National Institute of Health CA89300
(A.G.), AI44661 (A.G.), CA43500 (J.J.M.) and Roswell
Park Cancer Institute Core Grant CA16065 from the
NCI, and CA10914 (R.L.K.). The authors thank Mr.
William Haile for performing growth inhibition studies
and FPGS activity assays.
Supporting Information Available: Elemental analysis
data for 4, 5, 8-10, 18, 20-22. This material is available free
of charge via the Internet at http://pubs.acs.org.
(19) DeGraw, J. L.; Kisliuk, R. L.; Gaumont, Y.; Baugh, C. M.; Nair,
M. G. Synthesis and Antifolate Activity of 10-Deazaaminopterin.
J. Med. Chem. 1974, 17, 552-553.
(20) McGuire, J. J.; Magee, K. J.; Russell, C. A.; Canestrari, J. M.
Thymidylate Synthase as a Target for Growth Inhibition in
Methotrexate-Sensitive and -Resistant Human Head and Neck
Cancer and Leukemia Cell Lines. Oncology Res. 1997, 9, 139-
147.
(21) Foley, G. F.; Lazarus, H.; Farber, S.; Uzman, B. G.; Boone, B.
A.; McCarthy, R. E. Continuous Culture of Lymphoblasts from
Peripheral Blood of a Child with Acute Leukemia. Cancer 1965,
18, 522-529.
(22) Mini, E.; Srimatkandada, S.; Medina, W. D.; Moroson, B. A.;
Carman, M. D.; Bertino, J. R. Molecular and Karyological
Analysis of Methotrexate-Resistant and -Sensitive Human
Leukemic CCRF-CEM Cells. Cancer Res. 1985, 45, 317-325.
(23) Rosowsky, A.; Lazarus, H.; Yuan, G. C.; Beltz, W. R.; Mangini,
L.; Abelson, H. T.; Modest, E. J.; Frei, E., III. Effects of
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(24) McCloskey, D. E.; McGuire, J. J.; Russell, C. A.; Rowan, B. G.;
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