Benzoyl ring halogenated classical 2-amino-6-methyl-3,4-dihydro-4-oxo-5- substituted thiobenzoyl-7H-pyrrolo[2,3-d]pyrimidine antifolates as inhibitors of thymidylate synthase and as antitumor agents

Article abstract of DOI:10.1021/jm040144e

In an attempt to circumvent resistance to and toxicity of clinically used folate-based thymidylate synthase (TS) inhibitors that require folylpoly-γ-glutamate synthetase (FPGS) for their antitumor activity, we designed and synthesized two classical 6-5 ring-fused analogues, N-[4-[(2-amino-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)thio] -2′-fluorobenzoyl]-L-glutamic acid (4) and N-[4-[(2-amino-6-methyl-3,4- dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)thio]-2′-chlorobenzoyl] -L-glutamic acid (5), as TS inhibitors and antitumor agents. The key intermediates in the synthesis of these classical analogues were the mercaptans 10 and 11, which were obtained from the corresponding nitro compounds 6 and 7 respectively, by reduction of the nitro groups followed by diazotization of the amines. The syntheses of analogues 4 and 5 were achieved via the oxidative addition of the sodium salt of ethyl 2-halo-substituted-4-mercaptobenzoate (16 or 17) to 2-amino-6-methyl-3,4-dihydro-4-oxo-7H-pyirolo[2,3-d]pyrimidine (18) in the presence of iodine. The esters obtained from the reaction were deprotected and coupled with diethyl-L-glutamate followed by saponification. Compounds 4 and 5 were both more potent inhibitors of human TS (IC₅₀ values of 54 and 51 nM, respectively) than were PDDF and the clinically used ZD1694 and LY231514. Compounds 4 and 5 were not substrates for human FPGS up to 250 μM. In addition, 4 and 5 were growth inhibitory against CCRF-CEM cells as well as a number of other tumor cell lines in culture, and protection studies established TS as the principal target of these analogues.

Full text of DOI:10.1021/jm040144e

6730
J. Med. Chem. 2004, 47, 6730-6739
Benzoyl Ring Halogenated Classical
2-Amino-6-methyl-3,4-dihydro-4-oxo-5-substituted
Thiobenzoyl-7H-pyrrolo[2,3-d]pyrimidine Antifolates as Inhibitors of
Thymidylate Synthase and as Antitumor Agents1
Aleem Gangjee,*,† Hiteshkumar D. Jain,† 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 July 23, 2004
In an attempt to circumvent resistance to and toxicity of clinically used folate-based thymidylate
synthase (TS) inhibitors that require folylpoly-γ-glutamate synthetase (FPGS) for their
antitumor activity, we designed and synthesized two classical 6-5 ring-fused analogues, N-[4-
[(2-amino-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)thio]-2′-fluorobenzoyl]-
L-glutamic acid (4) and N-[4-[(2-amino-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-
5-yl)thio]-2′-chlorobenzoyl]-L-glutamic acid (5), as TS inhibitors and antitumor agents. The key
intermediates in the synthesis of these classical analogues were the mercaptans 10 and 11,
which were obtained from the corresponding nitro compounds 6 and 7 respectively, by reduction
of the nitro groups followed by diazotization of the amines. The syntheses of analogues 4 and
5 were achieved via the oxidative addition of the sodium salt of ethyl 2-halo-substituted-4-
mercaptobenzoate (16 or 17) to 2-amino-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidine
(18) in the presence of iodine. The esters obtained from the reaction were deprotected and
coupled with diethyl-L-glutamate followed by saponification. Compounds 4 and 5 were both
more potent inhibitors of human TS (IC50 values of 54 and 51 nM, respectively) than were
PDDF and the clinically used ZD1694 and LY231514. Compounds 4 and 5 were not substrates
for human FPGS up to 250 µM. In addition, 4 and 5 were growth inhibitory against CCRF-
CEM cells as well as a number of other tumor cell lines in culture, and protection studies
established TS as the principal target of these analogues.
Introduction
(MTA). ZD1694 has been approved in several countries
for colorectal cancer. LY231514, in combination with
cisplatin, has been recently approved for the treatment
of malignant pleural mesothelioma.8b Some classical
antifolates such as ZD1694 and LY231514 have an
N-benzoyl-L-glutamic acid side chain, which makes
them substrates for the enzyme folylpoly-γ-glutamate
synthetase (FPGS).7-12 FPGS catalyzes the formation
of poly-γ-glutamates, which leads to high intracellular
concentrations of these antitumor agents and in some
cases increases TS inhibitory activity for certain anti-
folates such as ZD16947 (60-fold) and LY2315148a (130-
fold) compared to their monoglutamate forms.10-13
Although polyglutamylation of certain antifolates is
necessary for cytotoxicity, it has also been implicated
in toxicity to normal host cells due to cellular retention
of the polyanionic poly-γ-glutamate metabolites, which
do not efflux host cells.14 In addition, the problem of
resistance in tumors, due to low or defective FPGS, has
placed limitations on the use of classical antifolates,
which depend on polyglutamylation for their antitumor
effects.15-18 In an attempt to circumvent the potential
problems associated with FPGS including resistance,
Gangjee et al.19 and others20 have designed compounds
that are potent inhibitors of isolated human TS in their
monoglutamate forms and do not need FPGS for potent
TS inhibitory activity. A compound not subject to
Due to the involvement of folates in the biosynthesis
of nucleic acid precursors, inhibitors of folate metabo-
lism have provided important therapeutic agents useful
in cancer chemotherapy.2,3 Thymidylate synthase (TS)
catalyzes the reductive methylation of 2′-deoxyuridine-
5′-monophosphate (dUMP) to 2′-deoxythymidine-5′-
monophosphate (dTMP) utilizing 5,10-methylenetet-
rahydrofolate as the source of the methyl group as well
as the reductant.4 This represents the sole de novo
source of dTMP; hence, inhibition of TS, in the absence
of salvage, leads to “thymineless death”. Thus inhibition
of TS is an attractive goal for the development of
antitumor agents.3,5 PDDF,6 ZD16947 (Tomudex), and
LY2315148a (Alimta) are important examples of TS
inhibitors (Figure 1). PDDF and ZD1694 are quinazoline
analogues and are characterized by the presence of a
6-6 ring-fused system similar to natural folates and
have a polar N-benzoyl-L-glutamic acid side chain
appended to the 6-position. On the other hand, LY231514
is characterized by the presence of a 6-5 ring-fused
system and is designated a multitargeted antifolate
* To whom correspondence should be addressed. Phone: 412-396-
6070. Fax: 412-396-5593. E-mail: gangjee@duq.edu.
† Duquesne University.
‡ Roswell Park Cancer Institute.
§ Tufts University School of Medicine.
10.1021/jm040144e CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/04/2004

Pyrrolo[2,3-d]pyrimidines as Inhibitors of TS
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6731
Figure 1.
activation by polyglutamylation needs to have high
intrinsic potency as a TS inhibitor. Gangjee et al.19
previously reported a classical pyrrolo[2,3-d]pyrimidine,
compound 1, as a potent inhibitor of isolated human TS
with an IC50 value of 42 nM and a reasonable inhibitor
of human recombinant DHFR with an IC50 value of 2.2
µM in its monoglutamate form, thus potentially provid-
ing dual inhibitory activity of TS and DHFR. Compound
1 was equipotent with PDDF and was more potent than
the clinically used ZD1694 and LY231514 against
isolated human TS in their monoglutamate forms.19 In
addition, compound 1 was a reasonably potent inhibitor
of the growth of CCRF-CEM human leukemic cells in
culture with an EC50 value of 0.45 µM as compared with
PDDF (EC50 ) 0.72 µM). A potential advantage of
compound 1 over ZD1694 and LY231514 is that com-
pound 1 was not a substrate for human FPGS from
CCRF-CEM cells at concentrations up to 1045 µM.19 The
lack of FPGS substrate activity of 1 was attributed, in
part, to the presence of the 6-methyl group on the
pyrrolo[2,3-d]pyrimidine of 1.19 In addition, molecular
modeling (SYBYL 6.8)21 suggested that the 6-methyl
group in compound 1 makes important hydrophobic
contacts with Trp109 in human TS (Figure 2) and also
serves to lock the 5-position side chain into favorable,
low-energy conformations. This probably contributes to
the high inhibitory activity against TS of compound 1
in its monoglutamate form. The fact that LY231514, a
pyrrolo[2,3-d]pyrimidine, much like 1, lacks a 6-methyl
group and is a substrate for FPGS lends further
credence to the involvement of the 6-methyl moiety in
preventing FPGS substrate activity in 1. In the 6-6
quinazoline analogues of ICI 198583, incorporation of
a 7-methyl group was shown to afford compounds with
both potent TS (both in vivo and in vitro) inhibitory
activity and a lack of FPGS substrate activity.22 Fur-
ther, in these series of compounds a 2′-fluoro substituent
in the phenyl ring gave a 2-3-fold enhancement in TS
inhibitory activity as well as in the growth inhibitory
potency against L1210 cells, as exemplified by ZM214888,
the 2,7-dimethyl-2′-F benzoyl analogue of PDDF.23
Further modification of the L-glutamic acid side chain
led to ZD9331, which is currently undergoing phase I/II
clinical trials as an antitumor agent.24-26 Similarly,
halogen substituents in the benzoyl portion of metho-
trexate27 have afforded potent DHFR inhibitors. Re-
cently, Gangjee et al.28 showed that nonclassical ana-
logues of 1 with electron-withdrawing groups in the
phenyl ring of the side chain enhanced TS inhibitory
activity. The SAR indicated that analogues with an
electron-withdrawing group at both the 3′- and 4′-
positions of the phenyl side chain provided optimum
inhibitory potency against TS. Some of the nonclassical
analogues reported28 were much more potent than the
clinically used classical antitumor agents ZD1694 and
LY231514. Electron-donating groups on the phenyl ring
of the side chain were found to diminish TS inhibitory
activity. Similarly, substitution in the benzoyl ring with
electron-donating groups in the classical analogues of
ZM214888 were found to diminish TS inhibitory activ-
ity.23 In an attempt to increase the TS inhibitory activity
of the classical analogue 1 and to provide potent tumor
cell inhibitory activity, electron-withdrawing fluorine
and chlorine atoms were incorporated separately at the
2′-position of the benzoyl ring of compound 1 to give
compounds 4 and 5, respectively. The synthesis and
biological activities of analogues 4 and 5 are presented
in this report.
Chemistry
The method used for the synthesis of the classical
compounds 4 and 5 essentially followed a modified

6732 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Gangjee et al.
Figure 2. Stereoview compound 1 superimposed on LY231514 (not shown) in human TS (PDB code 1JU6).50
Scheme 1a
a Conditions: (a) 10% Pd-C, H2, 1 atm, rt; (b) concentrated HCl, 0 °C, NaNO2; (c) potassium xanthogenate, 80 °C; (d) 6 N KOH, reflux;
(e) concentrated HCl, 0 °C; (f) 30% H2O2, rt; (g) EtI, NaHCO3, rt, 5 days; (h) NaBH4, rt, 30 min.
sequence previously reported19 for compound 1. The key
intermediates in the synthesis of 4 and 5 were the
appropriately substituted aryl thiols, 10 and 11 (Scheme
1). There was no reported synthesis for 10 and 11 in
the literature. However, methods29,30 were reported for
the synthesis of aryl thiols from the corresponding
amines. The anilino precursors 8 and 9 for the synthesis
of 10 and 11, respectively, were not commercially
available. The corresponding nitro compounds 6 and 7
were used as the logical starting materials for the
synthesis of the aryl thiols 10 and 11. Thus, reduction23
of the nitro group in the commercially available 2-halo-
substituted 4-nitrobenzoic acids 6 and 7 with 10% Pd-C
and H2 at 1 atm pressure afforded the corresponding
2-halo-substituted 4-aminobenzoic acids 8 and 9 in 100%
and 98% yields, respectively. The amines were then
converted to the corresponding 2-halo-substituted-4-
mercaptobenzoic acids 10 and 11 using the standard
diazotization method.29,30 This involved diazotization of
8 and 9 with sodium nitrite in aqueous acid, followed
by nucleophilic displacement of the diazo moiety with
ethyl xanthic acid potassium salt and subsequent hy-
drolysis of the resulting xanthate esters with potassium
hydroxide (Scheme 1). Acidification of the potassium salt
of xanthic acid so obtained resulted in the evolution of
carbon oxysulfide and the formation of an oil, which was
extracted into ether. The crude oil obtained on evapora-
tion of the solvent afforded the desired 2-halo-substi-
tuted 4-mercaptobenzoic acids, 10 and 11. Oxidation31
of 10 and 11 with 30% H2O2 in ethanol gave the
corresponding substituted aryl disulfides 12 and 13 in
35% and 25% yield, respectively. Oxidation with 30%
H2O2 at room temperature was found to give better
yields than oxidation with iodine in ethanol. The

Pyrrolo[2,3-d]pyrimidines as Inhibitors of TS
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6733
Scheme 2a
a Conditions: (a) I2, 16 or 17, EtOH/H2O (2:1), 100-110 °C; (b) 1 N NaOH, 80 °C; (c) iBuOCOCl, NEt3, diethyl-L-glutamate hydrochloride,
0 °C to rt; (d) 1 N NaOH, 0 °C.
Table 1. Inhibitory Concentrations (IC50) against Isolated TS
substituted aryl disulfides 12 and 13 were then alkyl-
and DHFR
ated19 with ethyl iodide to afford the corresponding
TS IC50 (nM)
humana E. colia
DHFR IC50 (µM)
diethyl 4,4′-dithio-2,2′-dihalo-substituted-bis(benzoate)
14 and 15, respectively, as oils, which were used without
further purification. Reduction19 of 14 and 15 to the
corresponding sodium salt of ethyl 2-halo-substituted-
4-thiobenzoates 16 and 17 was achieved using sodium
borohydride in ethanol as reported previously.19 Reac-
tion of the sodium salts of the mercaptans 16 and 17
with the pyrrolo[2,3-d]pyrimidine 1828 gave the corre-
sponding esters 19 and 20 (Scheme 2) in reasonably
good yields without the necessity of protecting the
2-amino group of 18, as reported previously.19 Com-
pound 19 was obtained in 41% yield, while 20 was
obtained in 52% yield. This method had the advantage
over our previously reported method19 of shorter reac-
tion times (4 h vs 16 h) and that it can be carried out
without protecting and deprotecting the 2-amino group,
which occurs in poor yields (20-30%) over two steps.
The structures of 19 and 20 were established by 1H
NMR, where the 3′-aromatic proton of 19 coupled with
the adjacent 2′-fluorine to afford the expected triplet at
7.68-7.74 ppm. This, along with the disappearance of
the 5-aryl proton at 5.83 ppm, present in 18, and the
presence of the requisite protons of the side chain
confirmed the structure of 19. Similarly, the splitting
pattern of the aromatic protons in 20 along with the
disappearance of the 5-aryl proton, present in 18,
confirmed the structure of 20. Ester hydrolysis of 19 and
20 with 1 N NaOH at 80 °C afforded the corresponding
acids 21 and 22 in 85% and 92% yields, respectively.19
Peptide coupling of the acids 21 and 22 with diethyl-L-
glutamate using the mixed anhydride method19 with
isobutyl chloroformate and triethylamine afforded 23
compd
humanb
E. colic
1
4
5
54
54
270
720
5100
72
5700
2.1
2.1
21
21
51
11
40
PDDFd
72
380
ND
ND
ND
ND
ZD1694e
(Tomudex)
LY231514f
(Alimta)
MTX
9500
ND
76000
ND
6.6
0.009
230
0.032
a Kindly provided by Dr. Frank Maley, New York State Depart-
ment of Health. b Kindly provided by Dr. J. H. Freisheim, Medical
College of Ohio, Toledo, OH. c Kindly provided by Dr. R. L. Blakley,
St. Jude Children’s hospital, Memphis, TN. d Kindly provided by
Dr. M. G. Nair, University of South Alabama. e Kindly provided
by Dr. Ann Jackman, Institute of Cancer Research, Sutton, Surrey,
UK. f Kindly provided by Dr. Chuan Shih, Eli Lilly and Co.
Biological Evaluation and Discussion
The classical analogues 4 and 5 were evaluated as
inhibitors of TS32 and DHFR33 from human and Es-
cherichia coli. The inhibitory potencies (IC50) are listed
in Table 1 along with those of PDDF, ZD1694, LY231514,
and methotrexate (MTX). The classical analogues 4 and
5 were both excellent inhibitors of human TS. Both 4
and 5 were equipotent with the previously reported
compound 1 and PDDF. Interestingly, 4 and 5 were
remarkably more potent than ZD1694 (7-fold) and
LY231514 (176-fold). Similar results were obtained with
E. coli TS. Both 4 (13-fold) and 5 (100-fold) were more
potent in inhibiting human TS than E. coli TS, indicat-
ing a significant species difference. Compound 4 was 10-
fold less while 5 was 71-fold less potent in inhibiting E.
coli TS than PDDF. The inhibitory data for compound
4 against human TS was unexpected, in that it was
essentially the same as that for the unhalogenated
compound 1 and did not result in an increase in TS
inhibitory activity. Jackman et al.23 have reported that
benzoyl ring substitution with an electron-withdrawing
fluorine atom at the 2′-position results in a 2-5-fold
increase in TS inhibitory activity in their series of
compounds. A plausible explanation for the observed
increase in TS inhibition was that the 2′-fluorine atom
1
and 24 in yields of 20% and 30%, respectively. The H
NMR spectrum of 23 and 24 in deuterated dimethyl
sulfoxide revealed the expected peptide NH doublet at
8.49 and 8.74-8.76 ppm, respectively, which exchange
on addition of D2O. Hydrolysis19 of the diesters 23 and
24 with aqueous sodium hydroxide at room tempera-
ture, followed by acidification with 3 N HCl in the cold,
afforded the desired target compounds 4 and 5 in 60%
and 68% yields, respectively.

6734 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Gangjee et al.
Table 3. 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
Table 2. Growth Inhibition of Parental CCRF-CEM Human
Leukemia Cells and Sublines with Single, Defined
Meachanisms of MTX Resistance during Continous Exposure
(0-120 h) to MTX, 4, or 5e
EC50 (nM)
relative growth (%)a,b
R1a
(v DHFR)
R2b
(V uptake)
R30dmc
(V Glun)
no
drug
CCRF-CEM
drug
addition 5 µM TdR 10 µM Hx Hx + TdR
MTX
4
5
17.5 ( 0.5
215 ( 25
690 ( 110
680 ( 30
505 ( 15
1900 ( 100
2100 ( 290
2450 ( 150
12000d
16.5 ( 0.5
305 ( 5
1200 ( 0
MTX (40 nM)
4 (5000 nM)
5 (2400 nM)
10 ( 1
10 ( 1
11 ( 0
10 ( 1
96 ( 4
92 ( 3
11 ( 1
11 ( 0
12 ( 1
100 ( 2
103 ( 1
96 ( 5
a CCRF-CEM subline resistant to MTX solely as a result of a
20-fold increase in wild-type DHFR protein and activity.44 b C-
CRF-CEM subline resistant as a result of decrease uptake of
MTX.45 c 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.17 d The first experiment with this line
indicated an EC50 > 10 000 nM. The value presented is from a
second experiment covering a higher concentration range. e Values
presented are average ( range for n ) 2.
a Deoxycytidine (dCyd; 10 µM) was present in all the above
cultures to prevent the inhibition of growth caused by TdR in T-cell
leukemia cell lines such as CCRF-CEM (see the methods). In
control results from the experiment above, dCyd alone had no
effect on CCRF-CEM growth (107 ( 1% of control) and did not
protect against growth inhibition by any of these drugs (data not
shown). Hx alone (101 ( 0% of control) or in the presence of dCyd
(110 ( 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 (40 ( 1% of control),
but TdR+dCyd was not growth inhibitory (106 ( 2% of control)
and neither protected against MTX-induced growth inhibition
(table above). Similarly, Hx + TdR + dCyd did not inhibit growth
of CCRF-CEM (110 ( 3% of control). b Growth is expressed relative
to quadruplicate cultures not treated with either drug or metabo-
lite and is the average ( range for duplicate treated samples. The
experiment was repeated with similar results.
hydrogen bonds with the amide hydrogen atom, which-
restricts this part of the molecule in a more favorable
conformation for binding to TS than in the analogue
without the 2′-fluorine. A spin-spin coupling constant
of 6.4 Hz between these two atoms was observed in the
1H NMR of an analogue of ICI198583 (Figure 1), where
Y ) F instead of H.23 However, in the 1H NMR of
compound 4, no such coupling was observed between
the 2′-fluoro and the amide hydrogen atom and may
account at least in part for the lack of increase in TS
inhibitory activity. Compounds 4 and 5 were inhibitors
of both human DHFR and E. coli DHFR (Table 1) and
were 10- and 2-fold more potent, respectively, in inhibit-
ing human DHFR than E. coli DHFR. It is well
documented that both the interaction of the 4-amino
protons and the protonated N1 of 2,4-diaminopyrimi-
dine-containing antifolates such as MTX are essential
for tight binding to DHFR.34 In compounds 4 and 5, this
binding orientation can be achieved by rotating the bond
between the C2 and 2-NH2 by 180° such that the pyrrole
NH mimics the 4-amino group while the 3-NH proton
mimics the protonated N1 of MTX, thereby allowing for
DHFR inhibition. Such dual binding orientations of
pyrrolo[2,3-d]pyrimidine antifolates has been proposed
for the 2,4-diaminopyrrolo[2,3-d]pyrimidines by Miwa
et al.35 and is further supported by the potent DHFR
inhibitory activity reported for the 4-desaminopyrrolo-
[2,3-d]pyrimidine antifolates.36,37
Growth inhibitory potency of 4 and 5 were compared
to that of MTX in continuous exposure against CCRF-
CEM human lymphoblatic leukemia and a series of
MTX-resistant sublines (Table 2). Compound 4 was only
12-fold less potent than MTX, while 5 was 39-fold less
potent than MTX. The DHFR overexpressing cell line
R1 was <3-fold cross-resistant to both 4 and 5, suggest-
ing that DHFR is probably not the primary target of
these analogues. The MTX-resistant transport-deficient
subline R2, that does not express functional reduced
folate carrier38 (RFC), is >17-fold cross-resistant to 5
and 11-fold cross-resistant to 4, while it is 120-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, they are able to diffuse
through the plasma membrane. A subline (R30dm)
expressing low levels of folylpolyglutamate synthetase
(FPGS) is only marginally cross-resistant to both ana-
logues under continuous exposure conditions, suggesting
that polyglutamate forms of 4 and 5 are not essential
to their mechanisms of action.
Metabolite protection studies were performed to
further elucidate the mechanism of action of 4 and 5.
At a concentration of MTX that inhibited growth of
CCRF-CEM cells by 95%, leucovorin at 0.1 mM was able
to fully protect against the effects of MTX (6 ( 4%
growth inhibition). In contrast, at concentrations of
either 4 or 5 that inhibited growth by 95%, even 10 mM
leucovorin only afforded marginal protection (90 ( 3%
growth inhibition). Although this might suggest that
these analogues are not antifolates, some validated
antifolates (e.g., BW1843U8939) are poorly protected by
leucovorin. These data do suggest, however, that leu-
covorin “rescue”, as used clinically with high-dose
MTX,40 would not be successful with these analogues.
Further studies in CCRF-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.41 As described in the Experimental Section,
in T-lymphoblast cell lines such as CCRF-CEM, TdR can
only be tested in the presence of deoxycytidine (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
a). The data (Table 3) show that for 4 and 5, TdR alone
protects against growth inhibition, while Hx alone does
not; addition of Hx to TdR does not affect the protection.
These data suggest that 4 and 5 inhibit only thymidy-
late synthesis, indicating that TS is the target of these
drugs. Thus, these halogenated analogues have the
same mechanism of action as their unhalogenated
parent 1.
The substrate activity of 4 and 5 was evaluated in
vitro with recombinant human FPGS and compared to
that of AMT, a good substrate for FPGS. The data (Table
4) showed that neither 4 nor 5 is a substrate for human

Pyrrolo[2,3-d]pyrimidines as Inhibitors of TS
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6735
Table 4. Activity of Folate Analog as Substrates for
alone is able to protect against the growth inhibitory
effects of compounds 1, 4, and 5, all three target TS
directly. Interestingly, leucovorin cannot provide com-
plete protection for 1, 4, or 5. None of the three
analogues is a substrate for human FPGS and thus
polyglutamates are not critical to their mechanism of
action. This lack of substrate activity of compounds 4
and 5 could be an important attribute to overcome
potential resistance to classical TS inhibitors such as
ZD1694 and LY231514, which depend on intracellular
polyglutamylation to exert their cytotoxic effects.
Recombinant Human FPGSa
b
substrate
Km, µM
4.3 ( 0.6
inactive e 250 µM
inactive e 250 µM
Vmax rel
,
Vmax rel
,
/Km
n
AMT
4
5
1.00
0
0
0.23
0
0
4
2
2
a FPGS substrate activity was determined as described in
Experimental section. Values presented are the average ( SD.
b Vmax,rel is calculated based on the apparent Vmax of a substrate
relative to the apparent Vmax of AMT within the same experiment.
Table 5. Cytotoxicity Evaluation against Selected Tumor Cell
Lines40
Experimental Section
GI50 (M)
All evaporations were carried out in vacuo with a rotary
evaporator. Analytical samples were dried in vacuo (0.2
mmHg) in an Abderhalden drying apparatus over P2O5.
Melting points were determined on a Fisher-Johns melting
point apparatus and are uncorrected. Nuclear magnetic reso-
nance spectra for proton (1H NMR) were recorded on a Bruker
WH-300 (300 MHz) spectrometer. Data were accumulated by
16K size with a 0.5 s delay time and 70° tip angle. The
chemical shift values are expressed in ppm (parts per million)
relative to tetramethylsilane as internal standard; s ) singlet,
d ) doublet, dd ) doublet of doublet, t ) triplet, q ) quartet,
m ) multiplet, bs ) broad singlet. The relative integrals of
peak areas agreed with those expected for the assigned
structures. Mass spectra were recorded on a VG-7070 double-
focusing mass spectrometer or in a LKB-9000 instrument in
the electron ionization (EI) mode. 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. Elemental analyses were per-
formed by Atlantic Microlabs Inc., Norcoss, GA. Analytical
results indicated by element symbols 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 removed, despite 24-48 h of drying in vacuo, and
were confirmed where possible by their presence in the 1H
NMR spectrum. All solvents and chemicals were purchased
from Aldrich Chemical Co. and Fisher Scientific and were used
as received.
cell line
4
5
leukemia
CCRF-CEM
non-small-cell lung cancer
EKVX
colon cancer
HCC-2998
CNS cancer
SF-268
7.12 × 10-7
6.83 × 10-6
<1.00 × 10-8
3.47 × 10-7
ND
7.58 × 10-7
1.48 × 10-6
7.44 × 10-6
breast cancer
NCI/ADR-RES
BT-549
3.83 × 10-7
8.54 × 10-6
2.21 × 10-6
<1.00 × 10-8
FPGS at up to 250 mM. The lack of FPGS substrate
activity is consistent with the minimal cross-resistance
of the FPGS-deficient subline R30dm (above) to both 4
and 5 and underscores the absence of polygluatamyla-
tion for the cytotoxicity of 4 and 5. Marsham et al.20
have reported a 7-CH3 analogue of 2-desamino-2-methyl
PDDF that was not a substrate for human FPGS. This
further substantiates the idea that methyl substituents
in the 6-position of classical pyrrolo[2,3-d]pyrimidines
and in the 7-position of quinazolines prevent FPGS
substrate activity as a consequence of bulk and/or
hydrophobicity, which is not tolerated by human FPGS.
Compounds 4 and 5 were selected by the National
Cancer Institute42 for evaluation as antitumor agents
in the preclinical in vitro screening program. The ability
of 4 and 5 to inhibit the growth of different tumor cell
lines was measured as GI50 values, the concentration
required to inhibit the growth of tumor cells in culture
by 50%. Compounds 4 and 5 displayed potent antitumor
activity with GI50 values in the 8.54 × 10-6 to <1.00 ×
10-8 M range in six cell lines. GI50 values for compounds
4 and 5 are shown in Table 5.
In summary, benzoyl ring modification of the 2-amino-
4-oxo-5-thiobenzoyl-6-methylpyrrolo[2,3-d]pyrimidine
with electron-withdrawing halogens such as chlorine
and fluorine has modest effects on biological activity as
compared to the parent unhalogenated compound. Ana-
logues 4 and 5 were comparable to the unhalogenated
analogue 1 in inhibiting human TS. This result indicates
that benzoyl ring modification of classical pyrrolo[2,3-
d]pyrimidine with halogen substitution in the 2′-position
has little or no effect on human TS inhibitory activity.
The unhalogenated derivative, compound 1, is slightly
more potent than the 2′-chloro analogue 5 and slightly
less potent than the 2′-fluoro analogue 4 as an inhibitor
of CCRF-CEM cell growth. Cross-resistance data cor-
roborate a similar mechanism of action for 1, 4, and 5.
On the basis of metabolite protection studies that TdR
4-Amino-2-fluorobenzoic Acid (8). To a solution of 2-fluoro-
4-nitrobenzoic acid, 6 (5 g, 27 mmol), in ethyl acetate in a Parr
hydrogenation bottle was added 1 g of 10% Pd-C. The
resulting mixture was hydrogenated at 1 atm of pressure until
complete reduction was indicated by TLC (ca. 4 h), following
which the mixture was filtered through Celite and the Celite
was washed with excess ethyl acetate until the washings did
not give a spot on TLC. The combined washings were then
evaporated under reduced pressure to afford 4.17 g (ca 100%)
of 8 as a light cream powder: mp 211.3-214 °C; TLC Rf ) 0.54
(CHCl3/MeOH 5:1); 1H NMR (DMSO-d6) δ 6.16 (s, 2 H, 4-NH2),
6.24-6.29 (d, 1 H, 5-CH), 6.35-6.37 (d, 1 H, 6-CH), 7.51-7.57
(t, 1 H, 3-CH), 12.15 (br s, 1 H, COOH); HRMS calcd for C7H6-
NO2F m/z ) 155.0382, found m/z ) 155.0375.
4-Amino-2-chlorobenzoic Acid (9). To a solution of
2-chloro-4-nitrobenzoic acid, 7 (5 g, 24.8 mmol), in ethyl acetate
in a Parr hydrogenation bottle was added 1 g of 10% Pd-C.
The resulting solution was hydrogenated at 1 atm of pressure
until complete reduction was indicated by TLC (ca. 4.5 h),
following which the mixture was filtered through Celite and
the Celite was washed with excess ethyl acetate until the
washings did not give a spot on TLC. The combined washings
were then evaporated under reduced pressure to afford 4.25 g
(ca 98%) of 9 as a light green powder: mp 208-210 °C (lit.43
mp 205-207 °C); TLC Rf ) 0.52 (CHCl3/MeOH 5:1); 1H NMR
(DMSO-d6) δ 6.07 (s, 2 H, 4-NH2), 6.47-6.51 (d, 1 H, 5-CH),
6.61 (s, 1 H, 3-CH), 7.62-7.65 (d, 1 H, 6-CH).
2,2′-Difluoro-4,4′-dithiobis(benzoic acid) (12). To a stirred
solution of 12.5 mL of concentrated HCl and the same amount
of water was added the substituted aniline 8 (5 g, 32 mmol)

6736 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Gangjee et al.
and the suspension was warmed to 70 °C. After this, the
solution was cooled to 0 °C and diazotized with NaNO2 (2.2 g,
32 mmol) over 20 min. This solution was then added dropwise
over 1 h to potassium xanthogenate (19 g, 117 mmol) solution
kept at 80 °C, after which the oil that separated was extracted
with diethyl ether, dried over MgSO4 for 2 h, and then
evaporated under low pressure to give a brownish oil. The oil
that was obtained was dissolved in 50 mL of ethanol to which
10 g of KOH in ethanol was added slowly over 30 min and the
solution was heated at 80 °C for 8 h. The ethanol was removed
under reduced pressure and the residue that was obtained was
dissolved in water and slowly neutralized with concentrated
HCl at 0 °C to pH 5 and then extracted with ethyl acetate.
The ethyl acetate was then dried over MgSO4 and removed
under low pressure to give the aryl thiol 10 as a solid with
strong thiol odor. The thiol 10 that was obtained was sus-
pended in ethanol and to this suspension was added 30% H2O2
(3.8 mL) and the mixture stirred at room temperature under
N2 for 24 h. The solvent was then removed and the residue
that was obtained was chromatographed on silica gel with
1-3% MeOH in CHCl3 to give 1.95 g (35%) of the disulfide 12
as a light cream solid: mp >245 °C (dec); TLC Rf ) 0.76
100 °C. At this point, iodine (1.0 g, 4 mmol) was added and
the resulting solution was refluxed for 4 h, when TLC indicated
the disappearance of starting material at Rf 0.33 and the
formation of a major spot at Rf 0.41 (CHCl3/MeOH 5:1). To
the resulting solution was added excess Na2S2O3 and the
reaction mixture was evaporated to dryness. To the resulting
residue were added silica gel (5 g) and methanol, and the
solvent was evaporated under low pressure to give a plug
which was chromatograhed on silica gel using a gradient of
1-5% MeOH in CHCl3. Fractions containing the desired spot
(TLC) were pooled and evaporated to dryness to afford 300
mg (41%) of 19 as a light cream solid: mp 297-303 °C; TLC
Rf ) 0.49 (CHCl3/MeOH 5:1); 1H NMR (DMSO-d6) δ 1.24-1.29
(t, 3 H, CH2CH3), 2.17 (s, 3 H, 6-CH3), 4.22-4.29 (q, 2 H, CH2-
CH3), 6.16 (s, 2 H, 2-NH2), 6.77-6.81 (d, 1 H, C6H3), 6.99-
6.93 (d, 1 H, C6H3), 7.68-7.71 (t, 1 H, C6H3), 10.27 (s, 1 H,
7NH), 11.55 (s, 1 H, 3NH). Anal. Calcd for (C16H15N4SO3F‚
0.1H2O) C, H, N, S, F.
Ethyl 4-[(2-Amino-6-methyl-3,4-dihydro-4-oxo-7H-pyr-
rolo[2,3-d]pyrimidine-5-yl)sulfanyl]-2′-chlorobenzoate
(20). To a solution of the disulfide 13 (1 g, 2.66 mmol) in
anhydrous N,N-dimethylacetamide (10 mL) was added pow-
dered NaHCO3 (0.9 g, 11 mmol) followed by ethyl iodide (1.72
g, 11 mmol), and the reaction mixture was stirred under N2
for 5 days. The mixture was then diluted with water (25 mL)
and extracted with ethyl acetate (3 × 25 mL). The combined
organic layer was washed with water (50 mL) and brine (50
mL) and then dried (MgSO4) and filtered. The filtrate was
evaporated to a dark brown oil under reduced pressure. This
oil was chromatographed on silica gel and eluted with CH2-
Cl2. Fractions containing the desired spot (TLC) were pooled
and evaporated to dryness to afford a tan oil which slowly
crystallized to afford an solid that was used immediately in
the next step. To a solution of crude 15 (1.00 g, 2.3 mmol) in
absolute ethanol (10 mL) was added NaBH4 (0.087 g, 2.3 mmol)
all at once and the mixture stirred under N2 for 30 min. To
this ethanolic solution of the sodium salt of ethyl 2-chloro-4-
mercaptobenzoate was added pyrrolopyrimidine 18 (0.38 g, 2.3
mmol), the ratio of ethanol/water was adjusted to 2/1 (40 mL),
and the resulting suspension was heated to 100 °C. At this
point, iodine (1.16 g, 4.6 mmol) was added and the resulting
solution was refluxed for 4 h, when TLC indicated the
disappearance of starting material at Rf 0.33 and the formation
of a major spot at Rf 0.44 (CHCl3/MeOH 5:1). To the resulting
solution was added excess Na2S2O3 and the reaction mixture
was evaporated to dryness. To the resulting residue were
added silica gel (5 g) and methanol, and the solvent was
evaporated under low pressure to give a plug which was
chromatograhed on silica gel using a gradient of 1-5% MeOH
in CHCl3. Fractions containing the desired spot (TLC) were
pooled and evaporated to dryness to afford 460 mg (52%) of
20 as a light cream solid: mp 295-300 °C; TLC Rf ) 0.47
1
(CHCl3/MeOH 5:1); H NMR (DMSO-d6) δ 7.39-7.46 (t, 2 H,
5-CH and 6-CH), 7.78-7.83 (t, 1 H, 3-CH); HRMS calcd for
C14H8O4S2F2 m/z ) 341.9832, found m/z ) 341.9835.
2,2′-Dichloro-4,4′-dithiobis(benzoic acid) (13). To a
stirred solution of 12.5 mL of concentrated HCl and the same
amount of water was added the substituted aniline 9 (5 g, 29
mmol) and the suspension was warmed to 70 °C. After this,
the solution was cooled to 0 °C and diazotized with NaNO2 (2
g, 29 mmol) over 20 min. This solution was then added
dropwise over 1 h to potassium xanthogenate (19 g, 117 mmol)
solution (10 mL) kept at 80 °C, after which the oil that
separated was extracted with diethyl ether, dried over MgSO4
for 2 h, and then evaporated under low pressure to give a
brownish oil. The oil that was obtained was dissolved in 50
mL of ethanol to which 10 g of KOH in ethanol was added
slowly over 30 min and the solution was heated at 80 °C for 8
h. The ethanol was removed under reduced pressure and the
residue that was obtained was dissolved in water and slowly
neutralized with concentrated HCl at 0 °C to pH 5 and then
extracted with ethyl acetate. The ethyl acetate was then dried
over MgSO4 and removed under low pressure to give the
substituted aryl thiol 11 as a solid with a strong thiol odor.
The thiol 11 that was obtained was suspended in ethanol, to
this suspension was added 30% H2O2 (3.8 mL), and the mixture
was stirred at room temperature under N2 for 24 h. The solvent
was then removed and the residue that was obtained was
chromatographed on silica gel with 1-3% MeOH to give 1.34
g (25%) of the disulfide 13 as a light yellow solid: mp >185
°C (dec); TLC Rf ) 0.74 (CHCl3/MeOH 5:1); 1H NMR (DMSO-
d6) δ 7.60 (d, 1 H, 5-CH), 7.69 (s, 1 H, 3-CH), 7.79 (d, 1 H,
6-CH).
1
(CHCl3/MeOH 5:1); H NMR (DMSO-d6) δ 1.25-1.30 (t, 3 H,
Ethyl 4-[(2-Amino-6-methyl-3,4-dihydro-4-oxo-7H-pyr-
rolo[2,3-d]pyrimidine-5-yl)sulfanyl]-2′-fluorobenzoate (19).
To a solution of the disulfide 12 (1.05 g, 3.0 mmol) in
anhydrous N,N-dimethylacetamide (10 mL) was added pow-
dered NaHCO3 (0.9 g, 11 mmol) followed by ethyl iodide (1.72
g, 11 mmol), and the reaction mixture was stirred under N2
for 5 days. The mixture was then diluted with water (25 mL)
and extracted with ethyl acetate (3 × 25 mL). The combined
organic layer was washed with water (50 mL) and brine (50
mL) and then dried (MgSO4) and filtered. The filtrate was
evaporated to a dark brown oil under reduced pressure. This
oil was chromatographed on silica gel and eluted with CH2-
Cl2. Fractions containing the desired spot (TLC) were pooled
and evaporated to dryness to afford 14 as a tan oil that was
used immediately in the next step. To a solution of 14 (0.8 g,
2 mmol) in absolute ethanol (10 mL) was added NaBH4 (0.075
g, 2 mmol) all at once and the mixture stirred under N2 for 30
min. To this ethanolic solution of the sodium salt of ethyl
2-fluoro-4-mercaptobenzoate, 16, was added pyrrolopyrimidine
18 (0.33 g, 2 mmol), the ratio of ethanol/water was adjusted
to 2/1 (40 mL), and the resulting suspension was heated to
CH2CH3), 2.18 (s, 3 H, 6-CH3), 4.25-4.30 (q, 2 H, CH2CH3),
6.16 (s, 2 H, 2-NH2), 7.02-7.04 (m, 2 H, C6H3), 7.65-7.68 (d,
1 H, C6H3), 10.27 (s, 1 H, 7NH), 11.54 (s, 1 H, 3NH). Anal.
Calcd for (C16H15N4SO3Cl‚0.6H2O) C, H, N, S, Cl.
4-[(2-Amino-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo-
[2,3-d]pyrimidin-5-yl)thio]-2′-fluorobenzoic Acid (21). To
a solution of 19 (800 mg, 2.33 mmol) in ethanol (80 mL) was
added aqueous 1 N NaOH and the reaction mixture stirred at
room temperature for 24 h. The solution was evaporated to
dryness, and the sodium salt was dissolved in water (40 mL)
and carefully acidified to pH 4 by dropwise addition of 3 N
HCl. The resulting suspension was left at 0 °C for 24 h and
filtered. The residue was washed with water, acetone, and
diethyl ether and dried over P2O5 at 78 °C to afford 662 mg
(85%) of the free acid 21 as a light brown solid: mp >320 °C
1
(dec); TLC Rf 0.32 (CHCl3/MeOH 5:1); H NMR (DMSO-d6) δ
2.17 (s, 3 H, 6-CH3), 6.23 (s, 2 H, 2-NH2), 6.74-6.78 (d, 1 H,
C6H3), 6.87-6.89 (d, 1 H, C6H3), 7.67-7.72 (t, 1 H, C6H3), 10.34
(s, 1 H, 7NH), 11.56 (s,1 H, 3NH); HRMS calcd for C14H11N4O3-
SF m/z ) 334.05359, found m/z ) 334.05326.

Pyrrolo[2,3-d]pyrimidines as Inhibitors of TS
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6737
4-[(2-Amino-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo-
[2,3-d]pyrimidin-5-yl)thio]-2′-chlorobenzoic Acid (22). To
a solution of 20 (110 mg, 0.29 mmol) in ethanol (10 mL) was
added aqueous 1 N NaOH and the reaction mixture stirred at
room temperature for 24 h. The solution was evaporated to
dryness, and the sodium salt was dissolved in water (8 mL)
and carefully acidified to pH 4.0 by dropwise addition of 3 N
HCl. The resulting suspension was left at 5 °C for 24 h and
filtered. The residue was washed with water, acetone, and
diethyl ether and dried over P2O5 at 78 °C to afford 94 mg
(92%) of the free acid 22 as a light brown solid: mp 290-295
(5 mL), and the solution was stirred for a further 24 h. The
solution was then cooled in an ice bath and acidified carefully
to pH 4.0 with dropwise addition of 3 N HCl. This suspension
was left at 5 °C for 24 h and filtered. The residue was washed
well with water and ether and dried over P2O5/vacuum to
afford 110 mg (60%) of 4 as a light cream solid: mp 192-200
°C; TLC Rf ) 0.58 (CHCl3/MeOH/NH4OH 3:9:1); 1H NMR
(DMSO-d6) δ 1.88-2.04 (m, 2 H, Glu â-CH2), 2.19 (s, 3 H,
6-CH3), 2.30-2.32 (t, 2 H, Glu γ-CH2), 4.35 (m, 1 H, Glu R-CH),
6.17 (s, 2 H, 2-NH2), 6.75-6.79 (d, 1 H, 5′-CH), 6.88-6.91 (d,
1 H, 6′-CH), 7.44-7.49(t, 1 H, 3′-CH), 8.36 (d, 1 H, CONH),
10.28 (s, 1 H, 7NH), 11.53 (s, 1 H, 3NH), 12.50 (br s, COOH).
Anal. Calcd for (C19H18N5SO6F‚0.6H2O) C, H, N, S, F.
N-[4-[(2-Amino-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo-
[2,3-d]pyrimidin-5-yl)thio]-2′-chlorobenzoyl]-L-glutam-
ic Acid (5). To a solution of 24 (120 mg, 0.22 mmol) in ethanol
(5 mL) was added 1 N NaOH (1 mL) and the solution stirred
at room temperature for 24 h. The ethanol was evaporated
under reduced pressure, the residue was dissolved in water
(5 mL), and the solution was stirred for a further 24 h. The
solution was then cooled in an ice bath and acidified carefully
to pH 4.0 with dropwise addition of 3 N HCl. This suspension
was left at 5 °C for 24 h and filtered. The residue was washed
well with water and ether and dried over P2O5/vacuum to
afford 77 mg (68%) of 5 as a light brown solid: mp 221-225
°C; TLC Rf ) 0.56 (CHCl3/MeOH/NH4OH 3:9:1); 1H NMR
(DMSO-d6) δ 1.90-1.94 (m, 2 H, Glu â-CH2), 2.19 (s, 3 H,
6-CH3), 2.30 (t, 2 H, Glu γ-CH2), 4.28 (m, 1 H, Glu R-CH), 6.23
(s, 2 H, 2-NH2), 6.97-6.98 (m, 2 H, C6H3), 7.28-7.30 (d, 1 H,
C6H3), 8.31 (d, 1 H, CONH), 10.43 (s, 1 H, 7NH), 11.50 (s, 1 H,
3NH). Anal. Calcd for (C19H18N5SO6Cl‚1.6H2O‚0.1HCl) C, H,
N, S, Cl.
Drugs and Chemicals. Drug solutions were standardized
using extinction coefficients. Extinction coefficients were
determined for 5 [pH 1, λmax-1 264 nm (22 600); pH 7, λmax 264
nm (22 700); pH 13, λmax 268 (24 300)] and for 4 [pH 1, λmax
272 nm (22 300); pH 7, λmax 271 nm (23 000); pH 13, λmax 277
nm (24 900)]. Extinction coefficients for methotrexate (MTX),
a gift of Immunex (Seattle, WA), were from the literature.44
Aminopterin was purchased from Sigma Chemical Co. (St.
Louis, MO). Calcium leucovorin (LV) was purchased from
Schircks Laboratoties (Jona, Switzerland). Hypoxanthine (Hx),
thymidine (TdR), and deoxycytidine (dCyd) were purchased
from Sigma Chemical Co, (St. Louis, MO). Other chemicals
and reagents were reagent grade or higher.
Cell Lines and Methods for Measuring Growth In-
hibitory Potency. Cell lines were verified to be negative for
mycoplasma contamination (Mycoplasma Plus PCR primers,
Stratagene, La Jolla, CA). The human T-lymphoblastic leu-
kemia cell line CCRF-CEM45 and its MTX-resistant sublines
R1,46 R2,47 and R30dm17 were cultured as described.17 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 DH-
FR. 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 continuous exposure to antifolates that require
polyglutamylation to form potent inhibitors. Growth inhibition
of all cell lines by continuous drug exposure was assayed as
described.17,39 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 logarithm of drug concentration.
1
°C; TLC Rf ) 0.34 (CHCl3/MeOH 5:1); H NMR (DMSO-d6) δ
2.18 (s, 3 H, 6-CH3), 6.18 (s, 2 H, 2-NH2), 6.98-7.01 (d, 2 H,
C6H3), 7.65-7.68 (d, 1 H, C6H3), 10.29 (s, 1 H, 7NH), 11.54 (s,
1 H, 3NH) 13.08 (bs, 1 H, COOH); HRMS calcd for C14H11N4O3-
-
SCl m/z ) 305.0263, found m/z ) 305.0257 (M COOH)+.
Diethyl N-[4-[(2-Amino-6-methyl-3,4-dihydro-4-oxo-7H-
pyrrolo[2,3-d]pyrimidin-5-yl)thio]-2′-fluorobenzoyl]-L-
glutamate (23). To a suspension of the acid 21 (535 mg, 1.6
mmol) in anhydrous DMF (21 mL) under N2 was added
triethylamine (675 µL, 4.8 mmol) and the suspension was
heated to 80 °C to form a solution. This solution was cooled to
0 °C and isobutyl chloroformate (632 µL, 4.8 mmol) was added,
followed 15 min later by diethyl-L-glutamate hydrochloride
(1.15 g, 4.8 mmol) and immediately followed by triethylamine
(675 µL, 4.8 mmol). The reaction mixture was slowly allowed
to warm to room temperature and stirred for 24 h. The DMF
was evaporated using an oil pump at room temperature. To
the resulting residue were added 25 mL of methanol and silica
gel (4 g), and the suspension was evaporated to dryness. The
silica gel plug was loaded on a wet (CHCl3) silica gel column
and eluted with a gradient of 1-5% MeOH in CHCl3. Fractions
containing the desired spot (TLC) were pooled and evaporated
to dryness under vaccum to give 170 mg (20%) of 23 as a light
cream solid: mp 132-134 °C; TLC Rf ) 0.62 (CHCl3/MeOH
5:1); 1H NMR (DMSO-d6) δ 1.14-1.21 (m, 6 H, CH2CH3), 1.87-
2.03 (m, 2 H, Glu â-CH2), 2.25 (s, 3 H, 6-CH3), 2.40 (t, 2 H,
Glu γ-CH2), 4.00-4.10 (q, 4 H, CH2CH3), 4.39 (m, 1 H, Glu
R-CH), 6.13 (s, 2 H, 2-NH2), 6.75-6.90 (m, 2 H, C6H3), 7.43-
7.46 (t, 1 H, C6H3), 8.50 (d, 1 H, CONH), 10.25 (s, 1 H, 7NH),
11.51 (s, 1 H, 3NH). Anal. Calcd for (C23H26N5SO6F‚0.3H2O)
C, H, N, S, F.
Diethyl N-[4-[(2-Amino-6-methyl-3,4-dihydro-4-oxo-7H-
pyrrolo[2,3-d]pyrimidin-5-yl)thio]-2′-chlorobenzoyl]-L-
glutamate (24). To a suspension of the acid 22 (400 mg, 1.14
mmol) in anhydrous DMF (21 mL) under N2 was added
triethylamine (481 µL, 3.42 mmol) and the suspension was
heated to 80 °C to form a solution. This solution was cooled to
0 °C and isobutyl chloroformate (450 µL, 3.42 mmoL) was
added, followed 15 min later by diethyl-L-glutamate hydro-
chloride (820 mg, 3.42 mmoL) and immediately followed by
triethylamine (481 µL, 3.42 mmol). The reaction mixture was
slowly allowed to warm to room temperature and stirred for
24 h. The DMF was evaporated using an oil pump at room
temperature. To the resulting residue were added 15 mL of
methanol and silica gel (2 g), and the suspension evaporated
to dryness. The silica gel plug was loaded on a wet silica gel
column and eluted with a gradient of 1-5% MeOH in CHCl3.
Fractions containing the desired spot (TLC) were pooled and
evaporated to dryness under vaccum to give 185 mg (30%) of
24 as a light cream solid: mp 145-155 °C; TLC Rf ) 0.61
(CHCl3/MeOH 5:1); 1H NMR (DMSO-d6) δ 1.14-1.21 (m, 6 H,
CH2CH3), 1.87-2.03 (m, 2 H, Glu â-CH2), 2.18 (s, 3 H, 6-CH3),
2.42 (t, 2 H, Glu γ-CH2), 4.03-4.11 (q, 4 H, CH2CH3), 4.35 (m,
1 H, Glu R-CH), 6.15 (s, 2 H, 2-NH2), 6.98-7.02 (m, 2 H, C6H3),
7.24-7.26 (d, 1 H, C6H3), 8.73-8.76 (d, 1 H, CONH), 10.26 (s,
1 H, 7NH), 11.51 (s, 1 H, 3NH). Anal. Calcd for (C23H26N5SO6-
Cl‚0.2H2O) C, H, N, S, Cl.
Protection against growth inhibition of CCRF-CEM cells was
assayed by including leucovorin ((6R,S)-5-formyltetrahydro-
folate) at 0.1-10 mM 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 mM, 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,
N-[4-[(2-Amino-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo-
[2,3-d]pyrimidin-5-yl)thio]-2′-fluorobenzoyl]-L-glutam-
ic Acid (4). To a solution of 23 (200 mg, 0.38 mmol) in ethanol
(5 mL) was added 1 N NaOH (1 mL) and the solution stirred
at room temperature for 24 h. The ethanol was evaporated
under reduced pressure, the residue was dissolved in water

6738 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Gangjee et al.
(11) Jackman, A. L.; Newell, D. R.; Gibson, W.; Jodrell, D. I.; Taylor,
G. A.; Bishop, J. A.; Hughes, L. R.; Calvert, A. H. The Biochemi-
cal Pharmacology of the Thymidylate Synthase Inhibitor 2-De-
samino-2-methyl-N10-propargyl-5,8-dideazafolic Acid (IC1 198583).
Biochem. Pharmacol. 1991, 41, 1885-1895.
(12) Nair, M. G.; Abraham, A.; McGuire, J. J.; Kisliuk, R. L.; Galivan,
J. Polyglutamylation as a Determinant of Cytotoxicity of Clas-
sical Folate Analogue Inhibitors of Thymidylate Synthase and
Glycinamide Ribonucleotide Formyltransferase. Cell. Pharmacol.
1994, 1, 245-249.
(13) Bisset, G. M. F.; Pawelczak, K.; Jackman, A. L.; Calvert, A. H.;
Hughes, L. R. Syntheses and Thymidylate Synthase Inhibitory
Activity of the Poly-γ-glutamyl Conjugates of N-[5-[N-(3,4-
Dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-
2-thenoyl]-L-glutamic Acid (IC1 D1694) and Other Quinazoline
Antifolates. J. Med. Chem. 1992, 35, 859-866.
TdR + dCyd), or all together (Hx + TdR + dCyd) with
concentrations of MTX, 4, or 5 that would inhibit growth by
>80% over a growth period of ≈72 h; horse serum was
decreased to 5% (normally 10%) in these studies to reduce its
contribution of metabolites. 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.48 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 inhibi-
tion was measured 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.49 Both 4 (93% recovery)
and 5 (86% recovery) were themselves nearly quantitatively
recovered during the assay procedure, thus ensuring that their
polyglutamate products would also be quantitatively recovered.
Kinetic constants for AMT 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 AMT concentrations tested. Assays con-
tained ≈400 units of FPGS activity; one unit of FPGS catalyzes
incorporation of 1 pmol of [3H]glutamate/h.
(14) Bisset, G. M. F.; Bavetsias, V.; Thornton, T. J.; Pawelczak, K.;
Calvert, A. H.; Hughes, L. R.; Jackman, A. L. The Synthesis and
Thymidylate Synthase Inhibitory Activity of L-γ-L-Linked Dipep-
tide and L-γ-Amide Analogues of 2-Desamino-2-methy1-N10
-
propargyl-5,8-dideazafolic acid (IC1 198583). J. Med. Chem.
1994, 37, 3294-3302.
(15) Jackman, A. L.; Kelland, L. R.; Kimbell, R.; Brown, M.; Gibson,
W.; Aherne, W.; Hardcastle, A.; Boyle, F. T. Mechanisms of
Acquired Resistance to the Quinazoline Thymidylate Synthase
Inhibitor ZD1694 (Tomudex) in One Mouse and Three Human
Cell Lines. Br. J. Cancer. 1995, 71, 914-924.
(16) Barakat, R. R.; Li, W. W.; Lovelace, C.; Bertino, J. R. Intrinsic
Resistance of Squamous Cell Carcinoma Cell Lines as a Result
of Decreased Accumulation of Intracellular Methotrexate Poly-
glutamates. Gynecol. Oncol. 1993, 51, 54-60.
(17) McCloskey, D. E.; McGuire, J. J.; Russell, C. A.; Rowan, B. G.;
Bertino, J. R.; Pizzorno, G.; Mini, E. Decreased Folylpoly-
glutamate Synthetase Activity as a Mechanism of Methotrexate
Resistance in CCRF-CEM Human Leukemia Sublines. J. Biol.
Chem. 1991, 266, 6181-6187.
(18) Braakhuis, B. J.; Jansen, G.; Noordhius, P.; Kegel, A.; Peters,
G. J. Importance of Pharmacodynamics in the In Vitro Antipro-
liferative Activity of the Antifolates Methotrexate and 10-EDAM
Against Head and Neck Squamous Cell Carcinoma. Biochem.
Pharmacol. 1993, 46, 2155-2161.
(19) Gangjee, A.; Devraj, R.; McGuire, J. J.; Kisliuk, R. L. 5-Arylthio
Substituted 2-Amino-4-oxo-6-methylpyrrolo[2,3-d]pyrimidine An-
tifolates as Thymidylate Synthase Inhibitors and Antitumor
Agents. J. Med. Chem. 1995, 38, 4495-4502.
(20) Marsham, P. R.; Jackman, A. L.; Barker, A. J.; Boyle, F. T.; Pegg,
S. J.; Wardleworth, J. M.; Kimbell, R.; O’Connor, B. M.; Calvert,
A. H.; Hughes, L. R. Quinazoline Antifolate Thymidylate Syn-
thase Inhibitors: Replacement of Glutamic Acid in the C2-
Methyl Series. J. Med. Chem. 1995, 38, 994-1004.
(21) Tripos Inc., 1699 South Hanley Road, St. Louis, MO 63144.
(22) Sanghani, P. C.; Jackman, A.; Evans, V. R.; Thornton, T.;
Hughes, L.; Calvert, A. H.; Moran, R. G. A strategy for the
Design of Membrane-Permeable Folypoly-gamma-glutamate
Synthetase Inhibitors: “Bay-Region”-Substituted 2-Desamino-
2-methyl-5,8-dideazafolate Analogues. Mol. Pharmacol. 1994, 45,
341-351.
(23) Marsham, P. R.; Jackman, A. L.; Oldfield, J.; Hughes, L. R.;
Thornton, T. J.; Bisset, G. M. F.; O’ Connor, B. M.; Bishop, J. A.
M.; Calvert, A. H. Quinazoline-Antifolate Thymidylate Synthase
Inhibitors: Benzoyl Ring Modifications in the C2-Methyl Series.
J. Med. Chem. 1990, 33, 3072-3078.
(24) Jackman, A. L.; Kimbell, R.; Aherne, G. W.; Brunton, L.; Jansen,
G.; Stephens, T. C.; Smith, M. N.; Wardleworth, J. M.; Boyle, F.
T. Cellular Pharmacology and In Vivo Activity of a New
Anticancer Agent, ZD9331: A Water-Soluble, Nonpolyglutamat-
able, Quinazoline-Based Inhibitor of Thymidylate Synthase.
Clin. Cancer Res. 1997, 3, 9911-921.
Acknowledgment. This work was supported, in
part, by grants from the National Institutes of Health,
NCI, CA89300 (A.G.), CA43500 (J.J.M.), and CA10914
(R.L.K.), and Roswell Park Cancer Institute Core Grant
CA16065. The authors thank Mr. William Haile for per-
forming growth inhibition studies and FPGS activity
assays.
References
(1) Presented, in part, at the 13th International Conference on
Pterins, Folates and Biogenic Amines, Maui, HI, March 30-
April 4, 2003, and, in part, at the American Association for
Cancer Research, Washington, DC, July 11-14, 2003, Abstr
3465.
(2) Jackson, R. C. Antifolate Drugs: Past and Future Perspectives.
In Antifolate Drugs in Cancer Therapy; Jackman, A. L., Ed.;
Humana Press: Totowa, NJ, 1999; pp 1-12.
(3) Berman, E. M.; Werbel, L. M. The Renewed Potential for Folate
Antagonists in Contemporary Cancer Chemotherapy. J. Med.
Chem. 1991, 34, 479-485.
(4) Carreras, C. W.; Santi, D. V. Catalytic Mechanism and Structure
of Thymidylate Synthase. Annu. Rev. Biochem. 1995, 64, 721-
762.
(5) Douglas, K. T. The Thymidylate Synthesis Cycle and Anticancer
Drugs. Med. Res. Rev. 1987, 7, 441-445.
(6) Jones, T. R.; Calvert, A. H.; Jackman, A. L.; Brown, S. J.; Jones,
M.; Harrap, K. R. A Potent Antitumor Quinazoline Inhibitor of
Thymidylate Synthase: Synthesis, Biological Properties and
Therapeutic Results in Mice. Eur. J. Cancer 1981, 17, 11-19.
(7) Jackman, A. L.; Taylor, G. A.; Gibson, W.; Kimbell, R.; Brown,
M.; Calvert, A. H.; Judson, I. R.; Hughes, L. R. ICI D1694, A
Quinazoline Antifolate Thymidylate Synthase Inhibitor That is
a Potent Inhibitor of L1210 Tumour Cell Growth In Vitro and
In Vivo: A New Agent for Clinical Study. Cancer Res. 1991, 51,
5579-5586.
(25) Boyle, F. T.; Wardleworth, J. M.; Hennequin, L. F.; Kimbell, R.;
Marsham, P. R.; Stephens, T. C.; Jackman, A. L. ZD9331-Design
of a Novel Nonpolyglutamatable Quinazoline-Based Inhibitor of
Thymidylate Synthase. Proc. Am. Assoc. Cancer. Res. 1994, 35,
302.
(26) Wardleworth, J. M.; Boyle, F. T.; Barker, R. J.; Hennequin, L.
F.; Pegg, S. J.; Stephens, T. C.; Kimbell, R.; Brown, M.; Jackman,
A. L. ZD9331, The Design and Synthesis of a Novel Nonpoly-
glutamatable TS Inhibitor. Ann. Oncol. 1994, 5, 247.
(27) Misra, D. K.; Humphreys, S. R.; Friedkin, M.; Goldin, A.;
Crawford, E. J. Increased Dihydrofolic Reductase Activity as a
Possible Basis of Drug Resistance in Leukemia. Nature 1961,
189, 39-42.
(8) (a)Taylor, E. C.; Kuhnt, D.; Shih, C.; Rinzel, S. M.; Grindey, G.
B.; Barredo, J.; Jannatipour, M.; Moran, R. A Dideazatetrahy-
drofolate Analogue Lacking a Chiral Center at C-6, N-[4-[2-(2-
Amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-y1)eth-
ylbenzoyl]-L-glutamic Acid, Is an Inhibitor of Thymidylate
Synthase. J. Med. Chem. 1992, 35, 4450-4454. (b) (http://
www.alimta.com/) Mesothelioma and Safety Information from
Eli Lilly.
(9) Gibson, W.; Bisset, G. M. F.; Marsham, P. R.; Kelland, L. R.;
Judson, I. R.; Jackman, A. L. The Measurement of Poly-
glutamate Metabolites of the Thymidylate Synthase inhibitor,
ICI D1694, in Mouse and Human Cultured Cells. Biochem.
Pharmacol. 1993, 45, 863-869.
(28) Gangjee A.; Mavandadi F.; Kisliuk R. L.; McGuire J. J.; Queener
S. F. 2-Amino-4-oxo-5-substituted-pyrrolo[2, 3-d]pyrimidines as
Nonclassical Antifolate Inhibitors of Thymidylate Synthase. J.
Med. Chem. 1996, 39, 4563-4568.
(10) Sikora, E.; Jackman, A. L.; Newell, D. F.; Calvert, A. H.
Formation and Retention and Biological Activity of N10-Prop-
argyl-5,8-dideazafolic Acid (CB 3717) Polyglutamates in L1210
Cells In Vitro. Biochem. Pharmacol. 1988, 37, 4047-4054.

Pyrrolo[2,3-d]pyrimidines as Inhibitors of TS
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6739
(29) Hung, J.; McNamara, D. J.; Werbel, L. M. Antimalarial Drugs.
Communication 52. Synthesis of 3,4-Dihydrothioxanthene-1,9-
dione Analogues as Potential Antimalarial Agents. J. Heterocycl.
Chem. 1983, 20, 1575-1580.
(30) Gangjee, A.; Dubash, N. P.; Queener, S. F. The Synthesis of New
2,4-Diaminofuro[2,3-d]pyrimidines with 5-Biphenyl, Phenoxy-
phenyl and Tricyclic Substitutions as Dihydrofolate Reductase
Inhibitors. J. Heterocycl. Chem. 2000, 37, 935-942.
(31) Kesavan, V.; Bonnet-Delpon, D.; Begue, J.-P. Oxidation in Fluoro
Alcohols: Mild and Efficient Preparation of Disulfides from
Thiols. Synthesis 2000, 2, 223-225.
(40) Bertino, J. R. Rescue Techniques in Cancer Chemotherapy: Use
of Leucovorin and Other Rescue Agents after Methotrexate
Treatment. Sem. Oncol. 1977, 4, 203-216.
(41) Hakala, M. T.; Taylor, E. The Ability of Purine and Thymine
Derivatives and of Glycine to Support the Growth of Mammalian
Cells in Culture. J. Biol. Chem. 1959, 234, 126-128.
(42) We thank the Developmental Therapeutics Program of the
National Cancer Institute for performing the in vitro anticancer
evaluation.
(43) Kamogawa, H.; Hirose, T.; Nanasawa, M. Mesomorphic Phenyl
Benzoate and 1,3-Dioxane Derivatives Bearing a Terminal Vinyl
Group. Bull. Chem. Soc. Jpn. 1983, 56, 3517-3518.
(44) Blakley, R. L. The Biochemistry of Folic acid and Related
Pteridines; Amsterdam: Elsevier, 1969; p 569.
(45) 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.
(46) Mini, E.; Srimatkandada, S.; Medina, W. D.; Moroson, B. A.;
Carman, M. D.; Bertino, J. R. Molecular and Karyological
Analysis of Methotrxate-Resistant and -Sensitive Human Leu-
kemic CCRF-CEM Cells. Cancer Res. 1985, 45, 317-325.
(47) Rosowsky, A.; Lazarus, H.; Yuan, G. C.; Beltz, W. R.; Mangini,
L.; Abelson, H. T.; Modest, E. J.; Frei, E.; III Effects of
Methotrexate Esters and other Lipophilic Antifolates on Meth-
otrexate-Resistant Human Leukemic Lymphoblasts. Biochem.
Pharmacol. 1980, 29, 648-652.
(48) Grindey, G. B.; Wang, M. C.; Kinahan, J. J. Thymidine Induced
Perturbations in Ribonucleoside Triphosphate Pools in Human
Leukemic CCRF-CEM Cells. Mol. Pharmacol. 1979, 16, 601-
606
(49) Gangjee, A.; Yu, J.; Kisliuk, R. L.; Haile, W. H.; Sobrero, G.;
McGuire, J. J. Design, Synthesis and Biological Activities of
Classical N-[4-[2-(2-Amino-4-ethylpyrrolo[2,3-d]pyrimidin-5-yl)-
ethyl]benzoyl]-L-glutamic acid and its 6-Methyl Derivative as
Potential Dual Inhibitors of Thymidylate Synthase and Dihy-
drofolate Reductase and as Potential Antitumor Agents J. Med.
Chem. 2003, 46, 591-600.
(50) Sayre, P. H.; Finer-Moore, J. S.; Fritz, T. A.; Biermann, D.; Gates,
S. B.; MacKellar, W. C.; Patel, V. F.; Stroud, R. M. Multi-targeted
Antifolates Aimed at Avoiding Drug Resistance Form Covalent
Closed Inhibitory Complexes with Human and Escherichia coli
Thymidylate Synthases. J. Mol. Biol. 2001, 313, 813-829.
(32) Wahba, A. J.; Friedkin, M. The Enzymatic Synthesis of Thymidy-
late. Early Steps in the Purification of Thymidylate Synthetase
of Escherichia coli. J. Biol. Chem. 1962, 237, 3794-3801.
(33) Kisliuk, R. L.; Strumpf, D.; Gaumont, Y.; Leary, R. P.; Plante,
L. Diastereoisomers of 5,10-Methylene-5,6,7,8-tetrahydropteroyl-
D-glutamic Acid. J. Med. Chem. 1977, 20, 1531-1533.
(34) Blakley, R. L. Dihydrofolate Reductase. In Folate and Pterins
Chemistry and Biochemistry of Folates; Blakley., R. L., Benkovic,
S. J., Eds.; Wiley: New York, 1984; Vol. I, pp 191-253.
(35) Miwa, T.; Hitaka, T.; Akimoto, H.; Nomura, H. Novel Pyrrolo-
[2,3-d]pyrimidine Antifolates: Synthesis and Antitumor Activi-
ties. J. Med. Chem. 1991, 34, 555-560.
(36) Shih, C.; Gosset, L. S. The Synthesis of N-{2-Amino-4-substituted-
[(pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl}-L-glutamic Acids as
Antineoplastic Agents. Heterocycles 1993, 35, 825-841.
(37) Shih, C.; Gosset, L. S.; Hu, Y.; Mendelsohn, L. G.; Habeck, L.
L.; Grindey, G. B.; Schultz, R. M.; Andis, S. L.; Moran, R. G.;
Freisheim, J. H. The Discovery of 4-Deoxy-5,10-Dideazatetrahy-
drofolic Acid (4-H DDATHF) and N-{2-Amino-[5,6-dihydro-
(pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl}-L-glutamic Acid (4-
H-5,6-dihydro-LY231514): A New and Novel Class of Dihydrofo-
late Reductase Inhibitors. Presented at the Tenth International
Symposium, Chemistry and Biology of Pteridines and Folates,
Orange Beach, AL, March 21-26, 1993; Abstr F14.
(38) Wong, S. C.; Zhang, L.; Witt, T. L.; Proefke, S. A.; Bhushan, A.;
Matherly, L. H. Impaired Membrane Transport in Methotrexate-
Resistant CCRF-CEM Cells Involves Early Translation Termi-
nation and Increased Turnover of a Mutant Reduced Folate
Carrier. J. Biol. Chem. 1999, 274, 10388-10394.
(39) McGuire, J. J.; Magee, K. J.; Rusell, C. A.; Canestrari, J. M.
Thymidylate Synthase as a Target for Growth Inhibition in
Methotrexate Sensitive and -Resistant Human Head and Neck
Cancer and Human Leukemia Cell Lines. Oncology Res. 1997,
9, 139-147.
JM040144E