Synthesis of classical, four-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/jm058213s

We report, for the first time, the biological activities of four-carbon-atom bridged classical antifolates on dihydrofolate reductase (DHFR), thymidylate synthase (TS), and folylpoly-glutamate synthetase (FPGS) as well as antitumor activity. Extension of

Full text of DOI:10.1021/jm058213s

J. Med. Chem. 2005, 48, 5329-5336
5329
Synthesis of Classical, Four-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,‡ 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 March 24, 2005
We report, for the first time, the biological activities of four-carbon-atom bridged classical
antifolates on dihydrofolate reductase (DHFR), thymidylate synthase (TS), and folylpoly-
glutamate synthetase (FPGS) as well as antitumor activity. Extension of the bridge homolo-
gation studies of classical two-carbon bridged antifolates, a 5-substituted 2,4-diaminofuro[2,3-
d]pyrimidine (1) and a 6-subsituted 2-amino-4-oxopyrrolo[2,3-d]pyrimidine (2), afforded two
four-carbon bridged antifolates, analogues 5 and 6, with enhanced FPGS substrate activity
and inhibitory activity against tumor cells in culture (EC50 e 10-7 M) compared with the two-
carbon bridged analogues. These results support our original hypothesis that the distance and
orientation of the side chain p-aminobenzoyl-L-glutamate moiety with respect to the pyrimidine
ring are a crucial determinant of biological activity. In addition, this study demonstrates that,
for classical antifolates that are substrates for FPGS, poor inhibitory activity against isolated
target enzymes is not necessarily a predictor of a lack of antitumor activity.
Introduction
bridge between the heterocycle and the p-aminobenzoyl-
L-glutamate portion. This lack of information is surpris-
ing given the vast literature on classical antifolates and
the remarkable activity of three-carbon-atom bridged
analogues.7 This study is the first report, to our knowl-
edge, of the biological activities of classical antifolates
with a four-carbon atom bridge.
Folate metabolism has long been recognized as an
effective target for chemotherapy because of its crucial
role in the biosynthesis of nucleic acid precursors.2
Inhibitors of folate-dependent enzymes in cancer, mi-
crobial, and protozoan cells provide compounds that
have found clinical utility as antitumor, antimicrobial,
and antiprotozoal agents.3,4
As part of a continuing effort to develop novel classical
antifolates as dihydrofolate reductase (DHFR) inhibitors
and as antitumor agents, Gangjee et al.8 previously
reported the synthesis of N-[4-[2-(2,4-diaminofuro[2,3-
d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid (1) as a
novel 6-5 bicyclic antifolate, which is reasonably potent
Variations in the atoms that link the heterocycle to
the p-aminobenzoyl-L-glutamate of classical antifolates
have been a particularly fruitful area of modification.2-4
Methotrexate (MTX) and the recently approved peme-
trexed (LY231514) (Figure 1) are both clinically used
antitumor agents and have variations in the “bridge
region”2-4 different from that of folic acid. MTX contains
a CH2-N(CH3) bridge, while pemetrexed contains a two-
carbon atom bridge. A vast number of linkers have been
used that involve replacing the -CH2-NH- bridge of
folic acid with carbon and heteroatoms as well as
homologation to three atom bridges.2-4 However, a
search of the literature indicates that there is a paucity
of information on extending the bridge to a four-atom
chain. The only two examples available are of the
synthesis of bishomofolic acid reported in 19675 with a
CH2CH2CH2NH bridge and a four-carbon-atom bridged
analogue.6 However, no biological activity of either
compound was or has been reported in the literature.
Thus, there are no reports in the literature on the
biological activities of antifolates with a four-atom
against the growth of tumor cells in culture (EC50
)
10-7-10-8 M). We have recently demonstrated that a
simple homologation of the C8-C9 bridge region to a
three-carbon bridge, analogue 3, enhances the antitu-
mor activity (EC50 ) 10-8-10-9 M).9 It was therefore
of interest to further explore the bridge homologation
strategy to study the optimal bridge length requirement
for classical 5-substituted furo[2,3-d]pyrimidines for
enzyme inhibitory activity and antitumor activity and
also to provide information on the biological and anti-
tumor effects of a four-carbon-atom bridge in classical
antifolates. Thus, compound 5, N-[4-[4-(2,4-diaminofuro-
[2,3-d]pyrimidin-5-yl)butyl]benzoyl]-L-glutamic acid, a
four-carbon bridged analogue, was designed and syn-
thesized.
Pemetrexed, a multitarget antifolate recently ap-
proved as an antitumor agent, is reported to be a dual
thymidylate synthase (TS)-DHFR inhibitor (hTS Ki )
340 nM; hDHFR Ki ) 7 nM).10 The 6-regio isomer of
pemetrexed, compound 2, reported by Taylor et al.11
was, however, inactive against the growth of tumor cells
in culture. A possible reason for its inactivity in culture
* To whom correspondence should be addressed. Phone: (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/jm058213s CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/09/2005

5330 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Gangjee et al.
Figure 1.
Scheme 1
could be that transposing the 5-ethylene bridge to the
6-position forces the benzoylglutamic acid side chain in
an orientation different from that required for optimal
enzyme interaction and antitumor activity. We specu-
lated that elongation of the ethylene bridge of 2 would
allow greater conformational flexibility and could per-
haps restore the inhibitory activity against the growth
of tumor cells in culture. Gangjee et al.9 recently
described a three-carbon bridged analogue of 2, com-
pound 4, which had enhanced antitumor activity in
culture. Thus, compound 6, N-[4-[4-(2-amino-3,4-dihy-
dro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-6-yl)butyl]benzoyl]-
L-glutamic acid was also synthesized to explore further
the bridge length potential for antitumor activity.
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 to folate-
dependent enzymes and antitumor activity.12,13 The
enhanced antitumor activities of analogues 3 and 4 were
attributed, in part, to their increased efficiency as
human FPGS substrates compared to their two-carbon
bridged parent analogues 1 and 2, respectively. Thus,
it was also of interest to determine the optimal bridge
length requirement of the classical 5-substituted 2,4-
diaminofuro[2,3-d]pyrimidine and 6-substituted 2-amino-
4-oxoprrolo[2,3-d]pyrimidine with respect to human
FPGS activity.
Scheme 2
Chemistry
Compounds 5 and 6 were obtained via a nine-step
synthesis from the commercially available methyl
4-formylbenzoate (7) using an R-chloromethyl ketone
condensation with pyrimidine as the key step as out-
lined in Schemes 1 and 2. Thus, a Wittig reaction14 of 7
with 3-(4-carbomethoxyphenyl)propyltriphenylphos-
phonium bromide 8 in 1:1 DMSO/THF with 2 equiv of
NaH in an ice bath and N2 atmosphere afforded the
penten-4-enoic acid 9 as a mixture of E- and Z-isomers
(Scheme 1). Hydrogenation of 9 afforded the pentanoic
acid 10, which was converted to the acid chloride 11 and
immediately reacted with diazomethane followed by
concentrated HCl to give the desired R-chloroketone
12.15
as solvent at room temperature for 24 h, compound 14
was obtained in 10% yield along with the recovery of
starting materials. Increasing the reaction temperature
and time afforded both the furo[2,3-d]pyrimidine 14 and
pyrrolo[2,3-d]pyrimidine 15. Optimal yields were ob-
tained at 40-45 °C for 3 days, and 14 and 15 were each
isolated, after chromatographic separation, in 38% yield
(76% total). Hydrolysis of 14 and 15 afforded the
corresponding free acids 16 and 17. Subsequent coupling
with L-glutamate diethyl ester using isobutyl chlorofor-
With the desired R-chloroketone 12 in hand, the next
step was the condensation of 2,4-diamino-6-hydroxypy-
rimidine 13 with 12 (Scheme 2). When DMF was used

Pyrimidine Analogues as Antifolates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5331
Table 1. Inhibitory Concentration (IC50 in µM) against
Table 2. 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, 5, or 6a
Isolated DHFR and TSa
DHFR
TS
rh
E. coli L. casei rh E. coli L. casei
EC50, nM
1b
1.0
9.0
NDd
7.0
0.1
220 NDd
NDd
R1b
(v DHFR)
R30dmd
(v Glun)
3c
10
>18 >180 >100
>17 >180 >100
>18 >100 >100
>17 >170 >100
drug CCRF-CEM
R2c (V uptake)
4c
5
6
>21 (16%) 22
>110
10
>110
21
21
100
MTX 13.0 ( 0
620 ( 40
4250 ( 50
g4600
860 ( 150
4850 ( 950
1500 ( 0
NDf
1050 ( 50
4350 ( 650
265 ( 25
8100 ( 400
14.5 ( 0.5
NDf
>20
0.022
NDd
1e
3g
4g
5
290 ( 10
60 ( 14
MTX
PDDF
pemetrexed 2.3
0.007 0.022
NDd NDd
0.18 0.09
NDd
0.009
38
84 ( 8
NDd
230
NDd
230
460 ( 10
120 ( 10
470 ( 60
3150 ( 150
18000h
19
38
6
1100 ( 100 3050 ( 150
a The percent inhibition was determined at a minimum of four
inhibitor concentrations within 20% of the 50% point. The standard
deviations for determination of 50% points were within (10% of
the value given. Recombinant human (rh) DHFR was kindly
provided by Dr. J. H. Freisheim, Medical College of Ohio, Toledo,
OH. E. coli DHFR was kindly provided by Dr. R. L. Blakley, St.
Jude Children’s Research Hospital, Memphis, TN. Recombinant
human (rh) TS and E. coli TS was kindly provided by Dr. Frank
Maley, New York State Department of Health, Albany, NY. PDDF
was kindly provided by Dr. M. G. Nair, University of South
Alabama, Mobile, AL. Pemetrexed was kindly provided by Dr.
Chuan Shih, Eli Lilly and Co., Indianapolis, IN. b Data derived
from ref 8. c Data derived from ref 9. d ND ) not determined.
a Values presented are the 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.14 c CCRF-CEM
subline resistant as a result of decreased uptake of MTX.15
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.16 e Data derived from ref 8. f ND ) not
determined. g Data from ref 9. h The first experiment with this line
indicated an EC50 greater than 10 000 nM. The value presented
is from a second experiment covering a higher range.
Table 3. Protection of CCRF-CEM Human Leukemia Cells
against the Growth Inhibitory Effects of MTX, 5, and 6 by 10
µM Hypoxanthine (Hx), 5 µM Thymidine (TdR), and Their
Combinationa
mate as the activating agent12 afforded the diesters 18
and 19. Final saponification of the diesters gave the
target compounds 5 and 6.
relative growth,b %
drug
no addition 5 µM TdR 10 µM Hx Hx + TdR
Biological Evaluation and Discussion
MTX (30 nM)
6 (5000 nM)
MTX (30 nM)
5 (5000 nM)
14 ( 1
21 ( 0
13 ( 0
20 ( 1
18 ( 1
18 ( 1
16 ( 0
20 ( 0
17 ( 0
85 ( 4
16 ( 1
21 ( 0
82 ( 2
85 ( 1
75 ( 7
74 ( 2
DHFR and TS Inhibition. Compounds 5 and 6 were
evaluated as inhibitors of Escherichia coli (ec), Lacto-
bacillus casei (lc), and recombinant human (rh) DHFR.15
The inhibitory potency (IC50) values are listed in Table
1 and compared with the values for MTX, 3, and 4 and
the previously reported values for 1.8 Both compounds
5 and 6 were poorly active against DHFR with IC50
values greater than 10-5 M.
Analogues 5 and 6 were also evaluated as inhibitors
of ecTS, lcTS, and rhTS12,13 and compared to pemetrexed
and PDDF, a standard TS inhibitor, as a control. Both
analogues 5 and 6 were inactive at the highest concen-
tration tested (Table 1).
In Vitro Human Tumor Cell Growth Inhibition.
Growth inhibitory potency of analogues 5 and 6 were
compared to that of MTX in continuous exposure against
the CCRF-CEM human lymphoblastic leukemia16 and
a series of MTX-resistant sublines17-19 (Table 2). Com-
pound 6 was about 85-fold less potent than MTX, while
5 was only 9-fold less potent than MTX. DHFR overex-
pressing line R1 was less than 3-fold cross-resistant to
6, suggesting that DHFR is probably not the primary
target of this analogue. In contrast, R1 was greater than
40-fold cross-resistant to 5, suggesting that it primarily
inhibits DHFR, as expected on the basis of its 2,4-
diaminofuropyrimidine structure. The MTX-resistant
transport-deficient subline R2, which does not express
functional reduced folate carrier (RFC),20 is 7-fold cross-
resistant to 6 and 2-fold cross-resistant to 5, while it is
115-fold resistant to MTX. The data suggest that 6
utilizes the RFC as its primary means of transport, but
at high extracellular levels it is able to diffuse through
the plasma membrane. The data also suggest that an
alternative carrier may transport 5 in CCRF-CEM cells.
A subline (R30dm) expressing low levels of FPGS is
highly cross-resistant to both analogues under continu-
a 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. b Deoxycytidine (dCyd, 10 µM) was
present in all the above cultures to prevent the inhibition of growth
caused in T-cell leukemias like CCRF-CEM by TdR (see Experi-
mental Section). In typical results, dCyd alone had no effect on
CCRF-CEM growth (96 ( 2% 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 (97 ( 1%
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 (94 ( 0% of control)
and was not protected against MTX-induced growth inhibition
(table above). Similarly, Hx + TdR + dCyd did not appreciably
inhibit growth of CCRF-CEM (93 ( 1% of control).
ous exposure conditions, suggesting that polyglutamate
forms of these analogues are essential to their mecha-
nisms of action. Both 5 and 6 had increased inhibitory
potency against CCRF-CEM cell growth in culture
compared to their two-carbon bridged parent analogues
but were less potent than the corresponding three-
carbon bridge analogues 3 and 4. These data suggest
that the three-carbon bridge may be optimal for the
classical 5-substituted 2,4-diaminofuro[2,3-d]pyrimidine
and 6-substituted 2-amino-4-oxopyrrolo[2,3-d]pyrimi-
dine with respect to antitumor activity.
Metabolite protection studies (Table 3) were per-
formed to further elucidate the mechanism of action of
5 and 6. At concentrations of drug that inhibited growth
of CCRF-CEM cells by g90%, leucovorin was able to
fully protect against the effects of MTX, 6, and 5 (data
not shown). This is consistent with an antifolate mech-
anism of action for both drugs. Further studies in CCRF-

5332 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Gangjee et al.
Table 4. Activity of 5 and 6 as Substrates for Recombinant
Table 5. Cytotoxicity Evaluation (GI50, M) of Compounds 5
and 6 against Selected Tumor Cell Lines23
Human FPGSa
substrate
Km, µM
Vmax,rel
1.00
0.65 ( 0.01
0.42 ( 0.02
0.57 ( 0.06
0.54 ( 0.02
0.54 ( 0.02
Vmax,rel/Km
n
cell lines
compound 5
compound 6
AMT
1c
5.4 ( 0.8
8.5 ( 17
0.3 ( 0
0.9 ( 0.01
2.0 ( 0.4
6.3 ( 0.4
0.19
0.07
1.4
0.63
0.27
0.086
7
2
2
3
3
3
leukemia
HL-60(TB)
SR
3.81 × 10-8
4.78 × 10-8
2.14 × 10-7
7.83 × 10-8
>1.0 × 10-4
6.56 × 10-6
6.09 × 10-7
3.98 × 10-7
3.81 × 10-6
8.98 × 10-7
3d
4d
5
K-562
colon cancer
SW-620
renal cancer
786-0
6
a FPGS substrate activity was determined as described in
Experimental Section at 2 mM L-[3H]glutamate. Values presented
b
are the average ( SD. Vmax,rel is calculated on the basis of the
apparent Vmax of a substrate relative to the apparent Vmax of AMT
within the same experiment. c Data derived from ref 8. d Data from
ref 9.
three- and four-carbon bridged), the data suggest that
they are both inhibitors of de novo purine synthesis. The
high degree of cross-resistance of the FPGS-deficient
subline to the homologue 6 suggests that polyglutamyl-
ation is required even in continuous exposure.
CEM cells examined the ability of thymidine (TdR) and/
or hypoxanthine (Hx) to protect against growth inhibition.
These metabolites can be salvaged to produce dTTP and
the purine dNTPs required for DNA synthesis and thus
bypass the MTX blockade.21 As described in the Experi-
mental Section, 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 protective effect on MTX either alone or in paired
combination with either Hx or TdR (Table 3; footnote).
The data (Table 3) show that for 6, Hx alone could
protect against growth inhibition, and addition of other
metabolites did not improve the protection. These data
suggest that 6 inhibits purine synthesis only; this is also
consistent with the low level of cross-resistance of the
DHFR overproducing the R1 subline (above). Since there
are two folate-dependent formyltransferases in purine
synthesis (i.e., GAR formyltransferase and AICAR
formyltransferase), it would be of interest to know which
is inhibited. Current anti-purine antifolates are prima-
rily targeted to GAR formyltransferase.22 For 5, neither
TdR nor Hx alone protected to any significant extent;
both metabolites were required to achieve significant
protection. This indicates that both purine and thymidy-
late synthesis are inhibited and is consistent with
DHFR being the primary target of 5.
FPGS Substrate Activity. The cross-resistance data
for FPGS-deficient subline R30dm (above) suggest that
5 and 6 must be substrates for human FPGS. Their
activity was evaluated in vitro with recombinant human
FPGS and compared to that of aminopterin (AMT), a
good substrate for FPGS. The data (Table 4) show that
both 5 and 6 are substrates for human FPGS. Com-
pound 6 was only half as efficient as AMT primarily
because of its decreased Vmax, while compound 5 was
slightly more efficient than AMT. These results suggest
that metabolism to polyglutamates must be considered
in the mechanism of action of both 5 and 6. Elongation
of the bridge region in the series of 2-amino-4-oxopyr-
rolo[2,3-d]pyrimidines, of which 6 is a member, showed
interesting effects on biological properties. This series
shows dramatic changes in Vmax as well as in Km for
FPGS activity. The three-carbon-bridge compound 4 has
a very low Km (0.9 µM) and a Vmax,rel of 0.57. Increasing
the length of the bridge to four carbons in 6 described
here caused an increase in Km of 7-fold with no effect
on Vmax. Growth inhibitory potency against CCRF-CEM
also shows an optimum at three carbons in this series.
Interestingly, for the compounds in this series that were
potent enough to perform metabolite protection (i.e.,
Elongation of the bridge region in the series of
substituted 2,4-diaminofuro[2,3-d]pyrimidines, of which
5 is a member, also has interesting effects on biological
activity. In contrast to the 2-amino-4-oxopyrrolo[2,3-d]-
pyrimidines, as the bridge is homologated from two to
four carbons, the Vmax,rel for human FPGS substrate
activity remains relatively constant; however, the Km
values vary widely without a consistent pattern. Com-
pound 3 has the lowest Km (0.3 µM) and is the most
efficient FPGS substrate in this group. The other chain
lengths each have similar growth inhibitory potencies,
but the three-carbon bridged compound 3 is the most
potent in this series. The two-carbon through four-
carbon bridged homologues all appear to be DHFR
inhibitors based on their metabolite protection profiles,
as expected from their 2,4-diamino substituents.
Cytotoxicity Assays. Compounds 5 and 6 were
selected by the National Cancer Institute23 for evalua-
tion of its in vitro preclinical antitumor screening
program. The ability of compounds 5 and 6 to inhibit
the growth of the sixty tumor cell lines of the NCI was
evaluated. The data for selected tumor cell lines mea-
sured as GI50 values, the concentration required to
inhibit the growth of tumor cells in culture by 50%
compared to a control, are reported in Table 5. Interest-
ingly, compound 5 was a potent inhibitor against the
growth of several tumor cell lines in culture, with GI50
values in the 10-8 M range (HL-60, SR, SW-620) (Table
5), and compound 6 also had moderate inhibitory
activity against several tumor cell lines with GI50 values
in the 10-6-10-7 M range (Table 5). The compounds
were relatively inactive (GI50 < 10-6 M) against other
tumor cell lines, indicating that these analogues are not
general cell poisons but afford selective inhibition of
some tumor cell lines. These data suggest that elonga-
tion of the bridge length from a two-carbon to a three-
or four-carbon bridge in the classical 5-substituted 2,4-
diaminofuro[2,3-d]pyrimidine and 6-substituted 2-amino-
4-oxopyrrolo[2,3-d]pyrimidine is conducive to the inhi-
bition of the growth of tumor cells in culture, with the
three-carbon bridge being the most potent.
In summary, compound 5, N-[4-[4-(2,4-diaminofuro-
[2,3-d]pyrimidin-5-yl)butyl]benzoyl]-L-glutamic acid, and
compound 6, N-[4-[4-(2-amino-3,4-dihydro-4-oxo-7H-
pyrrolo[2,3-d]pyrimidin-6-yl)butyl]benzoyl]-L-glutamic
acid, were designed and synthesized to explore, for the
first time, the biological effect of a four-carbon bridge
of classical antifolates on DHFR and TS inhibitory

Pyrimidine Analogues as Antifolates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5333
from ethyl ether to afford 4.4 g (94%) of 9 as white crystals:
mp 118-121 °C. 1H NMR (DMSO-d6) δ 2.21-2.30 (m, 4 H,
CH2-CH2-COOH), 6.1-6.6 (m, 2 H, -CHdCH-), 7.30-7.32
(d, 2 H, C6H4), 7.75-7.77 (d, 2 H, C6H4), 11.58 (s, 1H, COOH).
Anal. (C13H14O4) C, H.
activity, FPGS substrate efficiency, and antitumor
activity. Despite their poor inhibitory activity in isolated
enzyme assays, both analogues 5 and 6 had better
antitumor activity compared to the corresponding two-
carbon-bridge analogues 1 and 2, respectively, in tumor
cell culture. Further, both 5 and 6 are good substrates
for FPGS, which may account in part for their antitumor
activities. These data suggest that the elongation to a
four-carbon bridge of the two-carbon C8-C9 bridge of
classical 5-substituted 2,4-diaminofuro[2,3-d]pyrim-
idines and 6-substituted 2-amino-4-oxopyrrolo[2,3-d]-
pyrimidines is highly conducive to FPGS substrate
activity and tumor inhibitory activity. The differential
activities of the two-, three-, and four-carbon-atom
bridges support our original hypothesis that the dis-
tance and orientation of the side chain p-benzoyl-L-
glutamate moiety with respect to the pyrimidine ring
are crucial determinants of the biological activities. In
this series the three- and four-carbon bridges are better
than the two-carbon bridge and the three-carbon bridge
is optimal. These results also substantiate that for
classical antifolates that are substrates for FPGS, poor
activity against isolated target enzymes is not neces-
sarily a predictor of a lack of antitumor activity.
5-(4′-Carbomethoxyphenyl)pentanoic Acid (10). To a
solution of 9 (3 g, 15 mmol) in EtOAc/CHCl3 (2:1, 50 mL) was
added 10% Pd/C (500 mg). The resulting suspension was
hydrogenated in a Parr apparatus overnight at 45-50 psi
hydrogen pressure. TLC indicated the disappearance of the
starting material and the formation of one major spot at Rf )
0.29 (hexane/EtOAc, 3:1). The reaction mixture was filtered
through Celite and washed with methanol (30 mL). After
evaporation of the solvent, the residue was loaded on to a silica
gel column (4 × 20 cm) and flash-chromatographed with
hexane/EtOAc (3:1) and the desired fractions were pooled.
After evaporation of the solvent and recrystallization from
Et2O/EtOAc (2:1), 3 g (98%) of 10 was obtained as white
1
crystals: mp 86.9-88.5 °C; H NMR (DMSO-d6) δ 1.47-1.61
(m, 4 H, â-CH2 and γ-CH2), 2.20-2.51 (t, 2 H, R-CH2), 2.62-
2.67 (t, 2 H, benzylic-CH2), 3.82 (s, 3 H, OMe), 7.32-7.34 (d,
2 H, C6H4), 7.86-7.88 (d, 2 H, C6H4), 12.11 (s, 1H, COOH).
Anal. (C13H16O4) C, H.
Chloromethyl (4′-Carbomethoxyphenyl)butyl Ketone
(12). To a solution of 5-(4′-carbomethoxyphenyl)pentanoic acid
(10) (2.4 g, 10 mmol) in a 100 mL flask was 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 evaporation of solvent under reduced
pressure, the residue was dissolved in ethyl ether (20 mL).
The resulting solution was added dropwise to an ice-cooled
ether solution of diazomethane (generated in situ from 15 g
N-nitroso-N-methylurea) over 10 min. To this solution was
added concentrated HCl (20 mL). The resulting mixture was
refluxed for 1.5 h. After the mixture was cooled to room
temperature, the organic layer was separated and the aqueous
layer was extracted with ethyl ether (100 mL × 2). The
combined organic layers were washed with two portions of 10%
Na2CO3 solution and dried over Na2SO4. The solvent was
evaporated to afford 2 g (75%) of 12 as yellow needles: mp
74.6-75.6 °C; 1H NMR (DMSO-d6) δ 1.52-1.67 (m, 4 H, â-CH2
and γ-CH2), 2.51-2.56 (t, 2 H, R-CH2), 2.59-2.64 (t, 2 H,
benzylic CH2), 3.82 (s, 3 H, OMe), 4.50 (s, 2 H, CH2Cl), 7.33-
7.35 (d, 2 H, C6H4), 7.86-7.88 (d, 2 H, C6H4). Anal. (C14H17
O3Cl) C, H, Cl.
Methyl 4-[4-(2,4-Diaminofuro[2,3-d]pyrimidin-5-yl)bu-
tyl]benzoate (14) and Methyl 4-[4-(2-Amino-3,4-dihydro-
4-oxo-7H-pyrrolo[2,3-d]pyrimidin-6-yl)butyl]benzoate (15).
To a suspension of 2,4-diamino-6-hydroxypyrimidine (13) (0.8
g, 6 mmol) in anhydrous DMF (15 mL) was added 12 (1.6 g, 6
mmol). The resulting mixture was stirred under N2 at 40-50
°C for 3 days. TLC showed the disappearance of starting
materials and the formation of two major spots at Rf ) 0.32
and Rf ) 0.26 (CHCl3/MeOH, 6:1). After evaporation of solvent,
CH3OH (20 mL) was added followed by silica gel (5 g).
Evaporation of the solvent afforded a plug, which was loaded
onto a silica gel column (3.5 cm × 15 cm) and eluted initially
with CHCl3 followed by 3% MeOH in CHCl3 and then 4%
MeOH in CHCl3. Fractions showing Rf ) 0.32 were pooled and
evaporated, and the resulting solid was recrystallized from
MeOH to afford 770 mg (38%) of 14 as white crystals: mp
225.2-226.7 °C; 1H NMR (DMSO-d6) δ 1.55-1.65 (m, 4 H, C9-
CH2 and C10-CH2), 2.61-2.70 (m, 4 H, C11-CH2 and C8-
CH2), 3.82 (s, 3 H, OMe), 5.97 (s, 2 H, 4-NH2), 6.40 (s, 2 H,
2-NH2), 7.08 (s, 1 H, C6-CH), 7.32-7.35 (d, 2 H, C6H4), 7.85-
7.87 (d, 2 H, C6H4). Anal. (C18H20 N4O3) C, H, N.
Experimental Section
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 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 tetramethyl-
silane as internal standard: s ) singlet, d ) doublet, t )
triplet, q ) quartet, m ) mutiplet, 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, Milwau-
kee, 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 from Fisher Scientific
and were used as received.
(E/Z)-5-(4′-Carbomethoxyphenyl)-4-pentenoic Acid (9).
To a suspension of methyl 4-formylbenzoate (7) (3.2 g, 20
mmol) and (3-carboxypropyl)triphenylphosphonium bromide
8 (8.6 g, 20 mmol) in 80 mL DMSO/THF (1:1) was added 2
equiv of NaH (92%) (1.0 g, 40 mmol) in one portion in an ice
bath and N2 atmosphere. The resulting suspension was stirred
in an ice bath for an additional 30 min and slowly warmed to
room temperature for another 6 h. TLC indicated the disap-
pearance of the aldehyde spot and formation of two spots
centered at Rf ) 0.19 (hexane/EtOAc, 3:1). To the reaction
mixture cooled in an ice bath was added ice/water (100 g)
followed by concentrated HCl (10 mL). The resulting solution
was extracted with ether (100 mL × 3) and dried over Na2-
SO4. After evaporation of solvent, the residue was loaded on a
silica gel column (4 × 20 cm) and flash-chromatographed with
hexane/EtOAc (2:1) and the desired fractions were pooled.
After evaporation of solvent the residue was recrystallized
Fractions that showed an Rf ) 0.26 were pooled and
evaporated, and the resulting residue was recrystallized from
MeOH to afford 770 mg (38%) of 15 as light-yellow crystals:
mp 241.9-243.7 °C; 1H NMR (DMSO-d6) δ 1.58 (m, 4 H, C9-
CH2 and C10-CH2), 2.49-2.66 (m, 4 H, C11-CH2 and C8-
CH2), 3.82 (s, 3 H, OMe), 5.83 (s, 1 H, C5-CH), 5.96 (s, 2 H,
4-NH2), 7.32-7.34 (d, 2 H, C6H4), 7.85-7.87 (d, 2 H, C6H4),

5334 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Gangjee et al.
10.12 (s, 1 H, 3-NH), 10.80 (s, 1 H, 7-NH). Anal. (C18H20N4O3‚
γ-CH2), 2.50-2.66 (m, 4 H, C11-CH2, C8-CH2), 4.36-4.40 (br,
1 H, Glu R-CH), 5.80-5.95 (br, 2 H, 4-NH2), 6.40 (s, 2 H,
2-NH2), 7.20-7.50 (m, 3 H, C6-CH, C6H4), 7.79-7.82 (d, 2 H,
C6H4), 8.52-8.55 (d, 1 H, -CONH-),12.38-12.53 (br, 2 H, 2
× COOH). Anal. (C22H25N5O6‚1.4H2O) C, H, N.
0.1H2O) C, H, N.
4-[4-(2,4-Diaminofuro[2,3-d]pyrimidin-5-yl)butyl]ben-
zoic Acid (16). To a suspension of 15 (250 mg, 7 mmol) in
CH3OH/DMSO (1:1) was added 2 N NaOH (15 mL). 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 distilled water (15 mL). The solution was cooled in
an ice bath, and the pH was adjusted 3 to 4 using 2 N HCl.
The resulting suspension was chilled in a dry ice/acetone bath
and thawed to 4 °C overnight in a refrigerator. The precipitate
was filtered, washed with cold water, and dried in a desiccator
under reduced pressure using P2O5 to afford 230 mg (85%) of
16 as a light-yellow powder: mp >270 °C (dec); Rf ) 0.30
(CHCl3/MeOH, 5:1); 1H NMR (DMSO-d6) δ 1.54-1.66 (m, 4
H, C9-CH2 and C10-CH2), 2.67 (br, 4 H, C11-CH2 and C8-
CH2), 7.30-7.40 (m, 5 H, NH2, C6-CH, C6H4), 7.70 (s, 2 H,
NH2), 7.84-7.86 (d, 2 H, C6H4), 8.26 (s, 2 H, 2-NH2), 12.80 (s,
1 H, COOH). Anal. (C17H18N4O3‚1.0HCl‚0.60MeOH) C, H, N.
This acid was used directly for the next step without further
purification.
Diethyl N-{4-[4-(2,4-Diaminofuro[2,3-d]pyrimidin-5-yl-
)butyl]benzoyl}-L-glutamate (18). To a solution of 16 (175
mg, 0.45 mmol) in anhydrous DMF (9 mL) was added triethyl-
amine (130 µL), and the mixture was stirred under N2 at room
temperature for 5 min. The resulting solution was cooled to 0
°C. Isobutyl chloroformate (130 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 ) 0.57 and the disappearance of the starting
acid at Rf ) 0.30. Diethyl l-glutamate hydrochloride (240 mg,
1.0 mmol) was added to the reaction mixture followed im-
mediately by triethylamine (130 µL, 1.0 mmol). The reaction
mixture was allowed to slowly warm to room temperature and
stirred under N2 for 18 h. The reaction mixture was then
subjected to a second cycle of activation and coupling using
half the quantities listed above. The reaction mixture was
allowed to slowly warm to room temperature and stirred under
N2 for another 24 h. The reaction mixture was then evaporated
to dryness under reduced pressure. The residue was dissolved
in a minimum amount of CHCl3/MeOH, 4:1, chromatographed
on a silica gel column (2 cm × 15 cm), and eluted with 4%
MeOH in CHCl3. The desired fractions (TLC) were pooled,
evaporated to dryness, and recrystallized from ethyl ether to
afford 200 mg (85%) of 18 as yellow needles: mp 135.2-136.8
°C; Rf ) 0.57 (MeOH/CHCl3, 1:5); 1H NMR (DMSO-d6) δ 1.13-
1.20 (m, 6 H, OEt), 1.56-1.66 (m, 4 H, C9-CH2 and C10-
CH2), 1.99-2.11 (two sets of t, 2 H, Glu â-CH2), 2.40-2.45 (t,
2 H, Glu γ-CH2), 2.50-2.67 (m, 4 H, C11-CH2, C8-CH2),
4.00-4.13 (m, 4 H, OEt), 4.33-4.40 (m, 1 H, Glu R-CH), 5.94
(s, 2 H, 4-NH2), 6.38 (s, 2 H, 2-NH2), 7.08 (s, 1 H, C6-CH),
7.28-7.31 (d, 2 H, C6H4), 7.77-7.80 (d, 2 H, C6H4), 8.62-8.64
(d, 1 H, -CONH-). Anal. (C26H33N5O6) C, H, N.
4-[4-(2-Amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]py-
rimidin-6-yl)butyl]benzoic Acid (17). To a suspension of
15 (250 mg, 7 mmol) in CH3OH/DMSO (1:1, 40 mL) was added
3 N NaOH (15 mL). 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 MeOH. The combined
filtrate was evaporated to dryness under reduced pressure. To
this residue was added distilled water (15 mL). The solution
was cooled in an ice bath, and the pH was adjusted to 3-4
using 2 N HCl. The resulting suspension was chilled in a dry
ice/acetone bath and thawed to 4 °C overnight in a refrigerator.
The precipitate was filtered, washed with cold water, and dried
in a desiccator under reduced pressure using P2O5 to afford
260 mg (90%) of 17 as a brown powder: mp >266 °C (dec); Rf
1
) 0.18 (CHCl3/MeOH, 5:1); H NMR (DMSO-d6) δ 1.54-1.60
(br, 4 H, C9-CH2 and C10-CH2), 2.49-2.65 (m, 4 H, C11-
CH2, C8-CH2), 5.91 (s, 1 H, C5-CH), 6.64 (br, 2 H, 2-NH2),
7.29-7.31 (d, 2 H, C6H4), 7.83-7.85 (d, 2 H, C6H4), 10.71 (s, 1
H, 3-NH), 11.17 (s, 1 H, 7-NH), 12.75 (s, 1 H, COOH). Anal.
(C17H18N4O3‚1.0HCl) C, H, N. This acid was used directly for
the next step without further purification.
N-{4-[4-(2-Amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]-
pyrimidin-6-yl)butyl]benzoyl}-L-glutamic Acid (6). To a
solution of 17 (135 mg, 0.37 mmol) in anhydrous DMF (9 mL)
was added triethylamine (130 µL), and the mixture was stirred
under N2 at room temperature for 5 min. The resulting solution
was cooled to 0 °C. Isobutyl chloroformate (130 µL, 1.0 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 ) 0.55 and the disappearance
of the starting acid at Rf ) 0.18. Diethyl L-glutamate hydro-
chloride (240 mg, 1.0 mmol) was added to the reaction mixture
followed immediately by triethylamine (130 µL, 1.0 mmol). The
reaction mixture was slowly allowed to warm to room tem-
perature and stirred under N2 for 18 h. The reaction mixture
was then subjected to a second 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 ) 0.55 (MeOH/CHCl3, 1:5). The reaction mixture
was evaporated to dryness under reduced pressure. The
residue was dissolved in a minimum amount of CHCl3/MeOH,
4:1, and chromatographed on a silica gel column (2 cm × 15
cm) with 4% MeOH in CHCl3 as the eluent. Fractions that
showed the desired single spot at Rf ) 0.55 were pooled and
evaporated to dryness to afford 19 as a yellow syrup, which
was used directly for the next step.
To a solution of the diester 19 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 ) 0.55) and formation of one major
spot at the origin (MeOH/CHCl3, 1:5). The reaction mixture
was evaporated to dryness under reduced pressure. The
residue was dissolved in water (10 mL), the resulting solution
was cooled in an ice bath, and the pH was adjusted to 3-4
with dropwise addition of 1 N HCl. The resulting suspension
was frozen in a dry ice/acetone bath, thawed in a 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 (70%, two steps) of 6 as a yellow
powder: mp 171-173 °C; 1H NMR (DMSO-d6) δ 1.58 (br, 4 H,
C9-CH2 and C10-CH2), 1.94-2.06 (m, 2 H, Glu â-CH2), 2.34-
3.37 (t, 2 H, Glu γ-CH2), 2.49-2.69 (m, 4 H, C11-CH2, C8-
CH2), 4.39 (m, 1 H, Glu R-CH), 5.84 (s, 1 H, C5-CH), 5.99 (s,
2 H, 2-NH2), 7.26-7.29 (d, 2 H, C6H4), 7.77-7.80 (d, 2 H, C6H4),
8.50-8.53 (d, 1 H, -CONH), 10.16 (s, 1 H, 3-NH), 10.79 (s, 1
N-{4-[4-(2,4-Diaminofuro[2,3-d]pyrimidin-5-yl)butyl]-
benzoyl}-L-glutamic Acid (5). To a solution of the diester
18 (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) 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 and thawed in the refrigerator to 4-5 °C and
filtered. The residue was washed with a small amount of cold
water and ethyl acetate and dried in vacuo using P2O5 to afford
130 mg (92%) of 5 as a white powder: mp 163-165 °C; 1H
NMR (DMSO-d6) δ 1.56-1.66 (m, 4 H, C9-CH2 and C10-
CH2), 1.95-2.08 (m, 2 H, Glu â-CH2), 2.32-2.35 (t, 2 H, Glu

Pyrimidine Analogues as Antifolates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5335
H, 7-NH), 12.38-12.53 (br, 2 H, 2 × COOH). Anal. (C22H25N5O6‚
1.0HCl‚0.6H2O) C, H, N, Cl.
Folylpolyglutamate Synthetase (FPGS) Purification
and Assay. Recombinant human cytosolic FPGS was purified
and assayed as described previously.13 Both 5 (93% recovery)
and 6 (89% recovery) were themselves nearly quantitatively
recovered during the standard assay procedure, thus ensuring
that their polyglutamate products would also be quantitatively
recovered. A fourth milliliter of 0.1 N HCl (which recovers the
polyglutamate products) was required to obtain these recover-
ies of each analogue; however, kinetics constants were deter-
mined 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 concentra-
tions tested for 6; linearity could not be maintained as well
with 5, but the kinetics constants still represent good esti-
mates. Assays contained ∼400 units of FPGS activity; 1 unit
of FPGS catalyzes incorporation of 1 pmol of [3H]glutamate/
h. Because the Km value for 5 was low, assays to determine
its 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).
Dihydrofolate Reductase (DHFR) Assay.24 All DHFR
enzymes were assayed spectrophotometrically 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 at 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)-tetrahy-
drofolate, 0.012 M formaldehyde, 0.02 M MgCl2, 0.001 M
dUMP, 0.04 M Tris-HCl, and 0.000 75 M NaEDTA. This was
the assay described by Wahba and Friedkin25 except that the
dUMP concentration was increased 25-fold according to the
method of Davisson et al.26 The reaction was initiated by the
addition of an amount of enzyme, yielding a change in
absorbance at 340 nm of 0.016/min in the absence of inhibitor.
Cell Lines and Methods for Measuring Growth In-
hibitory Potency (Table 2). Drug solutions were standard-
ized using extinction coefficients. Extinction coefficients were
determined for 5 (pH 1, λmax ) 248 nm (23 300); pH 7, λmax
)
249 nm (22 500); pH 13, λmax ) 249 nm (22 200)) and for 6
(pH 1, λmax ) 229 nm (23 200); pH 7, λmax ) 250 nm (22 500);
pH 13, λmax ) 248 nm (21 900)). Extinction coefficients for
methotrexate (MTX), a gift of Immunex (Seattle, WA), were
from the literature.27 Aminopterin, hypoxanthine (Hx), thy-
midine (TdR), and deoxycytidine (dCyd) were purchased 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.
Acknowledgment. This work was supported in part
by National Institutes of Health Grants CA89300 (A.G.),
AI44661 (A.G.), CA43500 (J.J.M.), Roswell Park Cancer
Institute Core Grant CA16056 from the NCI, and Grant
CA10914 (R.L.K.). The authors thank Mr. William Haile
for performing growth inhibition studies and FPGS
activity assays.
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-CEM16 and its MTX-resistant sublines R1,17 R2,18 and
R30dm19 were cultured as described.19 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.19,28 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.
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
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
also 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, 5,
or 6 that would inhibit growth by ∼80% or more over a growth
period of ∼72 h. The growth period was limited because after
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.29 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).
Supporting Information Available: Analysis data for
5, 6, 9, 10, 12, 14-16, 21, and 22. This material is available
free of charge via the Internet at http://pubs.acs.org.
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