Synthesis and biological activities of tricyclic conformationally restricted tetrahydropyrido annulated furo[2,3-d]pyrimidines as inhibitors of dihydrofolate reductases

Article abstract of DOI:10.1021/jm9705420

The synthesis of seven 2,4-diamino-5,6,7,8-tetrahydro-7-substituted pyrido[4',3':4,5]furo[2,3-d]pyrimidines 1-6 are reported as nonclassical antifolate inhibitors of dihydrofolate reductase (DHFR) and compound 7 as a classical antifolate inhibitor of tumor cells in culture. The compounds were designed as conformationally restricted analogues of trimetrexate. The synthesis was accomplished from the cyclocondensation of 3-bromo-4-piperidone with 2,4-diamino-6-hydroxypyrimidine to afford regiospecifically 2,4-diamino- 5,6,7,8-tetrahydropyrido-[4',3':4,5]furo[2,3-d]pyrimidine-7-hydrobromide (16). This in turn was alkylated with the appropriate benzyl halide to afford the target compounds 1-6. The classical antifolate 7 utilized 4- (chloromethyl)benzoyl-L-glutamic acid diethyl ester (17) instead of the benzyl halide for alkylation, followed by saponification to afford 7. Compounds 1-6 showed moderate inhibitory potency against DHFR from Pneumocystis carinii, Toxoplasma gondii, Mycobacterium avium, and rat liver. The classical analogue 7 was 88-fold more potent against M. avium DHFR than against rat liver DHFR. The classical analogue was also inhibitory against the growth of tumor cells, CCRF-CEM, and FaDu, in culture.

Full text of DOI:10.1021/jm9705420

J . Med. Chem. 1998, 41, 1409-1416
1409
Syn th esis a n d Biologica l Activities of Tr icyclic Con for m a tion a lly Restr icted
Tetr a h yd r op yr id o An n u la ted F u r o[2,3-d ]p yr im id in es a s In h ibitor s of
Dih yd r ofola te Red u cta ses1
Aleem Gangjee,*,† Elfatih Elzein,† Sherry F. Queener,‡ and J ohn J . McGuire§
Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University,
Pittsburgh, Pennsylvania 15282, Department of Pharmacology and Toxicology, Indiana University School of Medicine,
Indianapolis, Indiana 46202, and Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York 14263
Received November 14, 1997
The synthesis of seven 2,4-diamino-5,6,7,8-tetrahydro-7-substituted pyrido[4’,3’:4,5]furo[2,3-
d]pyrimidines 1-6 are reported as nonclassical antifolate inhibitors of dihydrofolate reductase
(DHFR) and compound 7 as a classical antifolate inhibitor of tumor cells in culture. The
compounds were designed as conformationally restricted analogues of trimetrexate. The
synthesis was accomplished from the cyclocondensation of 3-bromo-4-piperidone with 2,4-
diamino-6-hydroxypyrimidine to afford regiospecifically 2,4-diamino-5,6,7,8-tetrahydropyrido-
[4’,3’:4,5]furo[2,3-d]pyrimidine-7-hydrobromide (16). This in turn was alkylated with the
appropriate benzyl halide to afford the target compounds 1-6. The classical antifolate 7 utilized
4-(chloromethyl)benzoyl-L-glutamic acid diethyl ester (17) instead of the benzyl halide for
alkylation, followed by saponification to afford 7. Compounds 1-6 showed moderate inhibitory
potency against DHFR from Pneumocystis carinii, Toxoplasma gondii, Mycobacterium avium,
and rat liver. The classical analogue 7 was 88-fold more potent against M. avium DHFR than
against rat liver DHFR. The classical analogue was also inhibitory against the growth of tumor
cells, CCRF-CEM, and FaDu, in culture.
Infections with Pneumocystis carinii and Toxoplasma
gondii remain the leading cause of death in patients
with acquired immunodeficiency syndrome (AIDS).2
Standard treatment of P. carinii pneumonia utilizes
pentamidine or sulfonamides with diaminopyrimidine
antifolates.3 Therapy for T. gondii infection relies
mainly on the antifolate pyrimethamine in combination
with sulfonamides.4 However, all agents currently used
to treat P. carinii and T. gondii infections in AIDS
patients cause side effects that are sometimes severe
enough to require cessation of therapy.5,6 The current
approach to the treatment of P. carinii and T. gondii
infections with dihydrofolate reductase (DHFR) inhibi-
tors takes advantage of the fact that these organisms
are permeable to lipophilic nonclassical antifolates but,
unlike mammalian cells, lack a carrier-mediated active
transport mechanism for uptake of classical folates with
polar glutamate side chains.7 Thus, in principle, one
can selectively protect sensitive host tissues from the
toxic effects of nonselective lipophilic antifolates by
coadministration (protection) or subsequent administra-
tion (rescue) of a reduced folate, leucovorin. Two
lipophilic antifolates that have been utilized in the
treatment of P. carinii and T. gondii infections are the
potent DHFR inhibitors trimetrexate (TMQ) and piri-
trexim (PTX).8-13 A major disadvantage of the therapy
of P. carinii and T. gondii infections with TMQ or PTX
is that TMQ and PTX inhibit mammalian DHFR with
equal to or greater potency than that displayed toward
nonmammalian forms of DHFR.14 As a result, the use
of TMQ is associated with host toxicity including bone
marrow depression. Recently approved by the FDA,
TMQ along with host protection or rescue by leucovorin
has proven to be a viable alternate therapy for P. carinii
infections in patients with AIDS. However, the high
cost of leucovorin, as well as its failure to protect host
cells in certain instances, has prompted the search for
potent analogues with greater selectivity for P. carinii
and T. gondii DHFR than that of TMQ or PTX.
† Duquesne University.
Efforts in our laboratory have resulted in the synthe-
sis of a variety of bicyclic, lipophilic 6-5 fused furo[2,3-
‡ Indiana University School of Medicine.
§ Roswell Park Cancer Institute.
S0022-2623(97)00542-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 03/26/1998

1410 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9
Gangjee et al.
MIN options of SYBYL with 10° increments of the
torsion angles of the flexible side chain. Molecular
modeling indicated that the 7-position of attachment of
the side chain on the 2,4-diaminotetrahydropyridofuro-
[2,3-d]pyrimidine, for compounds 1-6, was optimal.
Substitutions on the phenyl ring of compounds 1-6
were selected on the basis of previous reports from our
laboratory15,16 which showed that methoxy and chloro
substituents on the side chain phenyl ring of 2,4-
diamino-5-substituted-furo[2,3-d]pyrimidines and 5-sub-
stituted-pyrrolo[2,3-d]pyrimidines are conducive to po-
tent and/or selective inhibition of P. carinii and T. gondii
DHFR. In addition, the clinically used agents TMP and
pyrimethamine contain similar substituents. Thus, the
design of the conformationally restricted 2,4-diamino-
5,6,7,8-tetrahydro-7-(substituted benzyl)pyrido[4’,3’:4,5]-
furo[2,3-d]pyrimidines 1-6 was based on molecular
modeling with the idea that conformational restriction
of the bridge would afford orientations of the side chain
that are conducive to binding to P. carinii and/or T.
gondii DHFR, which in turn should enhance selectivity.
Gangjee et al.15 recently synthesized classical anti-
folate analogues containing a novel furo[2,3-d]pyrimi-
dine ring system (8, 9) as inhibitors of DHFR and as
antitumor agents. These analogues showed significant
inhibitory activities against DHFR from rat liver,
(recombinant) human, P. carinii, T. gondii, and L. casei.
The most potent inhibition of DHFR was observed with
analogue 9 against P. carinii DHFR (IC50 ) 3.5 × 10-8
M). The compounds were also significantly cytotoxic
against the growth of a variety of human tumor cell
lines in culture. Compound 9 had an IC50 ) 4.3 × 10-8
M against leukemia CCRF-CEM cells in culture and
was about an order of magnitude better than 8.
F igu r e 1. Compound 2 superimposed on hDHFR bound TMQ.
d]pyrimidines,15 and most recently, the 5-substituted-
pyrrolo[2,3-d]pyrimidines.16 These studies strongly
suggest that B-ring contracted (6-5 ring systems)
analogues are conducive to selectivity for P. carinii and
T. gondii DHFR compared to human DHFR.
Trimethoprim (TMP) shows selectivity for bacterial
DHFR compared to vertebrate DHFR. This selectivity
is based, in part, on conformational differences in the
DHFR bound orientations of the trimethoxyphenyl side
chain of TMP, as elucidated by X-ray crystallographic
studies.17,18,19 The 2,4-diaminopyrimidine ring of TMP
binds to Escherichia coli DHFR in the same manner as
it does to chicken liver DHFR. The trimethoxyphenyl
group of TMP, however, occupies a “lower” binding
pocket, pointing “downwards” in E. coli DHFR, while
in chicken liver DHFR, the trimethoxyphenyl group
occupies an “upper" binding pocket and is oriented
opposite to that in E. coli DHFR. The lack of selectivity
of TMQ and PTX could arise in part from the confor-
mational flexibility of the side chains which are capable
of adopting a variety of different conformations and are
thus able to appropriately bind to a variety of DHFR,
resulting in potent inhibition without selectivity.
We reasoned that conformational restriction of the
C9-N10 bridge of TMQ could result in analogues with
a favorable orientation of the lipophilic side chain for
selective inhibition of P. carinii DHFR and/or T. gondii
DHFR. Molecular modeling with SYBYL20 and its
SEARCH and MAXIMIN options suggested that ana-
logues of TMQ with the C9-N10 bridge conformation-
ally restricted, by incorporation into a tetrahydropyri-
dine ring fused to a 2,4-diaminofuro[2,3-d]pyrimidine,
would provide suitably restricted analogues. Superim-
position of the energy-minimized conformations of the
tetrahydropyrido annulated 2,4-diaminofuro[2,3-d]py-
rimidines 1-6 on the E. coli DHFR bound conformation
of TMQ21 with the pyrimidine ring of the target com-
pounds 1-6 superimposed on the pyrimidine ring of
TMQ showed that the side chains of compounds 1-6
overlap reasonably well but are not identical with the
side chain of E. coli DHFR bound TMQ. This is
illustrated for compound 2 in Figure 1. The energy-
minimized conformations of 1-6, for the superimposi-
tion, were obtained by using the SEARCH and MAXI-
Compounds 8 and 9 were both substrates for human
folylpolyglutamate synthase (FPGS) from CCRF-CEM
cells, and their first-order rate constants were as good
as aminopterin (AMT), which is a good substrate for the
ligation of the first glutamic acid. The N-CH3 analogue
9 was a more potent inhibitor of DHFR as well as of
tumor cells in culture than its N-desmethyl analogue
8. A possible explanation for this difference in activities
is that, for the desmethyl analogue 8, there is greater
flexibility in the side chain about τ1,τ2, and τ3, and the
benzoyl glutamate moiety could be oriented in a con-
formation that may not be conducive to optimum
binding. In contrast, in the N-methyl analogue 9, the
methyl group induces some constraint on the flexibility
of τ2 and τ3 which may result in a more favorable
conformation of the benzoyl glutamate side chain. In
addition, the N-9 methyl moiety is known to increase
potency against DHFR depending on the nature of the
heterocycle as well as the substituents on the side chain
phenyl ring.15 These studies provided the impetus for
the design and synthesis of the classical tricyclic com-
pound 7, as a conformationally restricted analogue of 8
and 9 and as a potential antitumor agent. Molecular
modeling studies using SYBYL20 showed that in tricyclic
rigid analogues such as compound 10 (which we have
previously synthesized)22 the orientation of the phenyl
side chain is dictated entirely by the conformation of
the C ring, and this orientation was not conducive to
DHFR binding. Thus we incorporated an additional
methylene bridge between the nitrogen in the C ring

Inhibitors of Dihydrofolate Reductases
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1411
Sch em e 1a
pyrimidines 15 and 16 without any of the 2-amino-4-
oxopyrrolo[2,3-d]pyrimidines as has been observed for
other bicyclic systems.23 The fact that the furo[2,3-d]-
pyrimidine had formed rather than the pyrrolo[2,3-d]-
1
pyrimidine was supported by H NMR data indicated
above as well as the observation of Secrist and Liu23
who established that cyclic R-halo ketones such as
R-chlorocyclohexanone regiospecifically afford the 2,4-
diaminofuro[2,3-d]pyrimidine when cyclocondensed with
2,4-diamino-6-hydroxypyrimidine. In addition, the ab-
sence of the characteristic downfield (9-12 ppm) pyrrole
1
NH proton in the H NMR along with the presence of
the 2- and 4-amino protons confirms that the product
was exclusively the 2,4-diamino-5,6,7,8-tetrahydropy-
ridofuro[2,3-d]pyrimidine rather than the pyrrolo[2,3-
d]pyrimidine.
Two positional isomers of the tricyclic pyridofuro[2,3-
d]pyrimidine are possible in the cyclocondensation of
14 (or 13) with 2,4-diamino-6-hydroxypyrimidine to
afford the desired N-Boc-2,4-diamino-5,6,7,8-tetrahy-
dropyrido[4′,3′:4,5]furo[2,3-d]pyrimidine regioisomer 15
or the alternate N-Boc-2,4-diamino-5,6,7,8-tetrahydro-
pyrido[3′,4′:4,5]furo[2,3-d]pyrimidine regioisomer 15a .
Using the 13C chemical shift prediction software,24 two
distinct 13C NMR were predicted for the two isomers
15 and 15a . The critical differences were that for
compound 15 the chemical shift of the 4b carbon was
predicted at 109.16 ppm while for 15a it was predicted
at 100.07 ppm, and the 13C shift for the 8a carbon was
predicted at 139.03 for 15 and 148.84 for 15a . In the
13C NMR of the product obtained, the 13C chemical shift
for the 4b carbon was at 110.08 ppm, and there was no
signal around 100.07 ppm; the next high field signal was
at 91.90 ppm. Further, the 8a carbon was observed at
140.31 ppm in accord with the predicted value of 139.03
ppm for 15, and there was no signal in the vicinity of
148.84 ppm predicted for 15a . This supports the
structure of the product as 15 rather than 15a .
a
(a) Di-tert-butyl dicarbonate, DMF, triethylamine; (b) chloroform/
bromine/room temperature; (c) DMF/room temperature; (d) DMSO/
potassium carbonate/room temperature.
and the side chain to allow for an appropriate orienta-
tion of the p-aminobenzoyl-L-glutamic acid in compound
7 as a semirigid analogue of compounds 8 and 9, in
which part of the side chain was incorporated in a third
ring thus restricting τ1 and τ2 of 8 and 9.
Ch em istr y
The synthesis of the tricyclic ring system was envi-
sioned via a one-step cyclocondensation of a biselectro-
phile derived from an appropriate piperidone and 2,4-
diamino-6-hydroxypyrimidine, similar to that employed
for the synthesis of the bicyclic furo[2,3-d]pyrimidines.15
The synthesis of compounds 1-6 is shown in Scheme
1. Protection of the commercially available 4-piperidone
hydrochloride 11 using di-tert-butyl dicarbonate in
dimethylformamide (DMF) at room temperature af-
forded the N-Boc-4-piperidone 12 in 86% yield. Bromi-
nation of 12 with 1 equiv of bromine in chloroform at
room temperature resulted in a mixture of the 3-bromo-
4-piperidone hydrobromide (13) as a white precipitate
(82% yield) and about 10% of the N-Boc brominated
product 14. Condensation of 13 or 14 with 2,6-diamino-
4-hydroxypyrimidine in anhydrous DMF at room tem-
perature for 48 h afforded 16 in 58% yield and 15 in
33% yield, respectively. Since this cyclocondensation
reaction could occur to afford the tetrahydropyrido
annulated 2-amino-4-oxopyrrolo[2,3-d]pyrimidine or the
tetrahydropyrido annulated 2,4-diaminofuro[2,3-d]py-
rimidine, it was essential to establish the structure of
the product. The reaction in each case afforded a single
product. The 1H NMR spectrum in deuterated dimethyl
sulfoxide of 15 and 16 indicated the presence of both
the 2- and 4-amino protons as singlets, exchangeable
with deuterium oxide. This along with the absence of
the H5 proton of the pyrimidine and the presence of the
methylene protons of the C ring confirmed that the
condensation of 13 and 14 with 2,6-diamino-4-hydroxy-
pyrimidine was regiospecific and afforded exclusively
the tetrahydropyrido annulated 2,4-diaminofuro[2,3-d]-
To further establish the structure of 15 as distinct
from 15a , a NOESY was performed between the 4-NH2
and the 5-CH2. In structure 15a this NOESY would be
expected to occur with the most downfield CH2 moiety
which would be the 5-CH2. However, in 15 this 5-CH2
would not be the most downfield CH2. The NOESY for
the product obtained shows a cross peak between the
4-NH2 and the CH2 (3.58 ppm) which is not the most
downfield CH2 thus establishing 15 rather than 15a as
the structure of the regiospecific product. In addition,
a CH correlation via long-range coupling (or COLOC)
experiment was performed which indicates CH relation-
3
ships through both two and three bonds (2J CH and J CH
)
applicable to quaternary carbon atoms. Such a COLOC
was observed with the 4b carbon at 110.08 and all three
CH2 moieties of the tetrahydropyrido ring, indicating a
two or three bond relationship. Such a relationship for
all three CH2s would be absent for the structure 15a
since the 7-CH2 protons would be four bonds away and
would not afford a COLOC with C4b, thus confirming
the structure of the product as 15.
Having established the structure of 15 and hence the
regiospecific nature of the cyclocondensation of 14 (and
hence 13) with 2,4-diamino-6-hydroxypyrimidine, we
turned our attention to the synthesis of the target
compounds. Stirring 16 in anhydrous DMSO with 2

1412 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9
Gangjee et al.
Sch em e 2a
infection in HIV-infected patients. Compound 7 showed
significant inhibitory activity against M. avium DHFR
and has a selectivity ratio of 88 versus rlDHFR. This
encouraging result suggested that such tricyclic ana-
logues may be selective agents against M. avium.
However, the nonclassical analogues 1-6 were poor
inhibitors of M. avium DHFR. Nevertheless, the sig-
nificant selectivity of 7 for M. avium DHFR allows it to
function as a lead compound for structural modifica-
tions, which are currently underway.
The ability of 7 to inhibit human CCRF-CEM DHFR
was also determined. Compound 7 (IC50 ) 11.8 ( 3.3
µM) was more than 104-fold less potent than methotr-
exate (MTX) (IC50 ) 0.7 ( 1 nM) against CCRF-CEM
DHFR.
a
(a) DMF, isobutyl chloroformate, triethylamine, L-glutamic
The activity of the classical analogue 7 was compared
to MTX as inhibitors of the growth of CCRF-CEM
human leukemia and FaDu squamous cell carcinoma
of the head and neck cell lines during continuous
exposure in vitro (Table 2). Both parent cell lines are
similarly sensitive to MTX under these conditions.
Compound 7 was 200-fold less potent than MTX as a
growth inhibitor of CCRF-CEM cells and >5000-fold less
potent than MTX against FaDu.
acid diethyl ester; (b) DMSO/potassium carbonate/room temper-
ature; (c) methoxyethanol, 1 N NaOH.
equiv of anhydrous potassium carbonate and the ap-
propriate benzyl halide for 48-72 h at room tempera-
ture afforded the desired products 1-6. The classical
analogue was synthesized in a manner similar to that
employed for the synthesis of 1-6 using 4-(chlorometh-
yl)benzoyl-L-glutamic acid diethyl ester 17 instead of the
benzyl halide (Scheme 2). The isolation of the products
was greatly simplified by adding an excess of water to
the reaction mixture and stirring at room temperature
for 1-3 h, which allows the products to separate.
Chromatographic purification afforded analytically pure
target compounds 1-7 in yields ranging from 38 to 52%.
The diester 17 was in turn prepared by coupling
4-(chloromethyl)benzoic acid with glutamic acid diethyl
ester using the mixed anhydride method and triethy-
lamine as the base.15
Compound 7 was also tested as a growth inhibitor of
two CCRF-CEM sublines with defined mechanisms of
MTX resistance (Table 2). R30dm27 has 1% of the
parental FPGS level; it is not MTX resistant in continu-
ous exposure (resistance is only seen in intermittent
exposure) because the nonpolyglutamylated parent drug
is a tight-binding DHFR inhibitor and transport is
sufficient to maintain drug in excess of DHFR. How-
ever, on continuous exposure to 7, R30dm is highly cross
resistant, indicating that polyglutamylation must be
essential to its mechanism of action. It is well docu-
mented that some classical antifolates that inhibit
thymidylate synthase (TS) have significant cross resis-
tance to R30dm.28 This result is attributed to the
necessity of these classical antifolates to be poly-
glutamylated in order to exert their antitumor activity.
R1,29 with amplified DHFR expression, displayed 40-
fold resistance to MTX under continuous exposure. R1
is only about 3-fold cross resistant to 7. Thus DHFR
does not appear to be the primary target of 7 despite
its 2,4-diamino structure, since it is a weak DHFR
inhibitor and a DHFR-amplified cell line is only mini-
mally cross resistant to this agent.
Growth inhibition of CCRF-CEM cells in culture by
MTX (EC96) and 7 (EC91) are completely protected by
the simultaneous presence of 10 µM leucovorin (data
not shown). This indicates that the inhibitory effects
of 7 appear to be antifolate in nature.
Most classical antifolates (which contain a p-amino-
benzoyl-L-glutamate) are substrates for human folyl-
polyglutamate synthetase (FPGS), although their rela-
tive substrate activity is a function of their structure.
Gangjee et al.15,30 have shown that furo[2,3-d]pyrimi-
dine analogues containing CH2NH, CH2N(CH3), CH2-
CH2, and CH2NHCH2 bridges from the 5-position of
the furo[2,3-d]pyrimidine to the p-benzoylglutamate
moiety serve as efficient FPGS substrates. Since com-
pound 7 showed significant cross resistance to R30dm
cell line (Table 3), its activity as a substrate for human
FPGS was evaluated and compared to that of aminop-
Biologica l Eva lu a tion a n d Discu ssion
Nonclassical analogues 1-6 were evaluated as inhibi-
tors of DHFR from P. carinii (pcDHFR), T. gondii
(tgDHFR), and rat liver (rlDHFR).25,26 The inhibitory
potencies (IC50) are listed in Table 1. Surprisingly,
analogues 1-6 were weak inhibitors of these DHFR
with IC50 values ranging from 1.4 to >35 µM. There
are three possible explanations for the lack of DHFR
inhibitory activity of theses analogues: first, that pro-
tonation of the basic alicyclic nitrogen in the C ring, at
physiological pH, may result in a positive charge on the
nitrogen which is not conducive to DHFR binding;
second, the tricyclic ring system may be too rigid and/
or bulky to allow for optimum orientation of the phenyl
side chain upon binding to DHFR; third, the distance
between the lipophilic side chain and the 5-position of
the bicyclic furo[2,3-d]pyrimidine ring system may be
one carbon atom too long, which does not permit proper
interaction of the substituted side chain with appropri-
ate portions of DHFR. A study involving the synthesis
of additional analogues is currently underway to cir-
cumvent the above possible reasons for the inactivity
of compounds 1-6. The classical analogue 7 also
showed weak inhibitory activity against pcDHFR and
tgDHFR. Compound 7 did however display selectivity
ratios of 7.9 and 4 against pcDHFR and tgDHFR,
respectively, versus rlDHFR.
Compounds 1-7 were also evaluated against DHFR
from Mycobacterium avium, an agent of opportunistic

Inhibitors of Dihydrofolate Reductases
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1413
Ta ble 1. Enzyme Inhibitory Concentrations (IC50, µM) against DHFRs and Selectivity Ratios25,26
compd
P. carinii
rat liver
rl/pc
ND
ND
ND
2.3
0.9
2.0
7.9
1.4
T. gondii
rl/tg
M. avium
rl/ma
1
2
3
4
>9.0
>35.0
>22.8
22.6
24.1
40.5
10.9
0.90
0.035
12.0c
0.042c
0.001c
15.0
>35.0
22.8
50.9
20.6
1.4
>35.0
>20.4
13.1
22.3
31.7
>10.0
ND
ND
3.9
ND
2.5
4.0
1.9
0.021
49.0
0.30
0.21
>15.0
12.1
ND
>2.9
ND
ND
ND
ND
88.0
>20.0
>50.0
>20.0
>81.2
0.97
5
6
81.2
85.8
7a
21.5
0.70
8b
1.3
0.43
NT
NT
9b
12.2
11.1
19.8
TMP
TMQ
MTX
133.0c
0.003d
0.003d
2.7d
0.01
0.01d
0.014
0.014d
a
b
d
IC50 against CCRF-CEM DHFR ) 11.8 µM. Data from ref 15. c Data from ref 25. Data from ref 26. ND ) not done, NT ) not
tested.
Ta ble 2. Growth Inhibition (EC50, nM) of Parental
CCRF-CEM, Sublines with Single, Defined Mechanism of MTX
Resistance, and FaDu Human Squamous Cell Carcinoma of the
Head and Neck during Continuous (0-120 h) Exposure to MTX
and 7a
Ta ble 3. Activity of 7 as Substrate for CCRF-CEM Human
Leukemia Cell Folylpolyglutamate Synthetasea
substrate
Km, µM
Vmax(rel)
Vmax(rel)/Km
n
aminopterin
7
4.8 ( 0.7
6.2 ( 1.4
1
0.21
0.05
6
2
0.29 ( 0.05
CEM/R30dmb
CEM/R1cc
a
drug CCRF-CEM
V(Glun)
v(DHFR)
FaDu
FPGS substrate activity was determined as described in the
Experimental Section. Values presented are average ( SD if n g
3 and average ( range for n ) 2. Vmax are calculated relative to
aminopterin within the same experiment.
MTX 14.3 ( 1.11
(n ) 12)
8.0 ( 2.7
(n ) 5)
>50000
(n ) 2)
567 ( 29
(n ) 3)
17.2 ( 1.5
(n ) 5)
7
3600 ( 1200
(n ) 7)
10300 ( 5400 >100000
(n ) 3)
(n ) 2)
tal analyses were performed by Atlantic Microlabs Inc.,
Norcross, GA. Analytical results indicated by an element
symbol are within (0.4% of the calculated values. Fractional
moles of water frequently found in some analytical samples
of antifolates were not removed despite 24-48 h of drying in
vacuum and were confirmed, where possible, by their presence
in the 1H NMR spectrum.
a
Average values are presented ( range for n ) 2 and ( SD for
b
n g 3. CCRF-CEM sublines 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.27 c CCRF-CEM subline resistant to MTX
solely as a result of a 20-fold increase in wild-type DHFR protein
and activity.28
N-(Bu t oxyca r b on yl)-4-p ip er id on e (12). 4-Piperidone
hydrochloride monohydrate 11 (2.0 g, 13.0 mmol) was dissolved
in 30 mL of N,N-dimethylformamide (DMF) at 110-115 °C.
The solution was cooled to room temperature, and to it was
added triethylamine (2.60 g, 26.0 mmol) and a solution of di-
tert-butyl dicarbonate (3.06 g, 14.0 mmol) in 10 mL of DMF.
The mixture was stirred for 24 h at room temperature. The
DMF was removed under reduced pressure. Water (100 mL)
was added to the residue, the mixture was extracted with 2 ×
100 mL of ethyl ether, the organic layer was dried (MgSO4)
and filtered, and ether was removed under reduced pressure
to afford 2.33 g (90%) of 12 as a white powder: mp 113-115
terin (AMT), which is a good substrate for ligation of
the first γ-glutamic acid (Table 3). Although compound
7 is a less efficient FPGS substrate than AMT, poly-
glutamylation must still be considered as a potential
component of its mechanism of action. The current
findings show that replacement of simple bridges (CH2-
NH, CH2N(CH3), CH2CH2, and CH2NHCH2) from the
5-position of furo[2,3-d]pyrimidine with a constrained
annular bridge involving the 5- and 6-positions allows
the analogue to retain FPGS substrate activity with a
Km similar to those of the open chain bicyclic analogues
and only a slight decrease in Vmax. These results show
that the substrate binding site of FPGS tolerates
significant bulk in the so-called C-region,31 as has been
suggested based on the excellent substrate activity of
BW1843U89.32 The importance of polyglutamate me-
tabolism of compound 7 to its mechanism is underscored
by the cross resistance of a cell line with decreased
FPGS activity (Table 2); in this regard it is similar to
our earlier reported furo[2,3-d]pyrimidine analogues.15
°C;
1H NMR (CDCl3) δ 1.48 (s, 9 H, Boc), 2.41 (t, 4 H, 2-CH2,
6-CH2), 3.67 (t, 4 H, 3-CH2, 5-CH2).
3-Br om o-4-p ip er id on e Hyd r obr om id e (13). N-(Butoxy-
carbonyl)-4-piperidone 12 (2.60 g, 13.0 mmol) was dissolved
in 70 mL of chloroform. To it was added Br2 (2.08 g, 13.0
mmol) over a period of 30 min. The reaction was continued
for 2 h at room temperature during which time a white
precipitate of 13 was formed. The reaction mixture was
filtered and the solid washed with chloroform and ether to
1
afford 2.76 g (82%) of 13: mp 200-201 °C; H NMR (Me2SO-
d6) spectrum was complex due to interference from DMSO-d6
and water (due to the hygroscopic nature of the product).
However, the proton R to the carbonyl and the bromine was
observed at δ 5.09 ppm. In addition, the spectrum showed
the absence of the Boc group. This compound was unstable
for long durations at room temperature and was used in the
next step without further purification.
Exp er im en ta l Section
Melting points were determined on a Mel-Temp II melting
point apparatus and are uncorrected. Nuclear magnetic
1
resonance spectra for H NMR were recorded on a Bruker WH-
2,4-Dia m in o-5,6,7,8-tetr a h yd r op yr id o[4′,3′:4,5]fu r o[2,3-
d ]p yr im id in e-7-Hyd r obr om id e (16). To a suspension of
2,4-diamino-6-hydroxypyrimidine (0.50 g, 4.0 mmol) in 3 mL
of anhydrous DMF under nitrogen was added dropwise a
solution of 13 (1.83 g, 4.0 mmol) in 10 mL of anhydrous DMF.
The reaction became a clear solution after 1 h and was left at
room temperature for 48 h. The white precipitate which
formed was collected by filtration and air-dried to afford 0.66
g (58%) of 16: mp 302-304 °C; 1H NMR (Me2SO-d6) spectrum
indicated a mixture of two hydrobromide salts, δ 2.73-2.97
300 (300 MHz) spectrometer, and the chemical shifts are
presented relative to TMS as the internal standard. Chemical
shifts listed for multiplets were measured from the ap-
proximate centers, and 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. Elemen-

1414 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9
Gangjee et al.
(overlapped m, 4 H, 6-CH2), 3.40, (overlapped m, 4 H, 5-CH2),
4.18, 4.29 (overlapped m, 4 H, 8-CH2), 6.24 (br s, 2 H, 4-NH2),
8.11 (br s, 2 H, 4-NH2), 8.90 (br s, 2 H, 2-NH2), 9.34 (br s, 2 H,
2-NH2), 10.42 (s, 1 H, N-H), 11.09 (s, H, N-H). Anal. Calcd
for (C9H11N5O‚1.5HBr‚0.3H2O) C, H, N, Br.
5.93 (s, 2 H, 4-NH2), 6.40 (m, 3 H, 2-NH2, 4′-H), 6.52 (d, 2 H,
2′,6′-H). Anal. Calcd for (C18H21N5O3‚0.06H2O) C, H, N.
2,4-Diam in o-5,6,7,8-tetr ah ydr o-7-(2′,4′-dich lor o-ben zyl)-
p yr id o-[4′,3′:4,5]fu r o[2,3-d ]p yr im id in e (4). This compound
was prepared and purified in a manner similar to that
described for 1 using 2′,4′-dichlorobenzyl chloride, instead of
benzyl bromide, to afford 0.15 g (48%) of 4: mp 265-267 °C;
2,4-Dia m in o-5,6,7,8-tetr a h yd r o-7-(bu toxyca r bon yl)p y-
r id o[4′,3′:4,5]fu r o[2,3-d ]p yr im id in e (15). The filtrate ob-
tained in the synthesis of 13 was diluted with 150 mL of
chloroform and washed with water, saturated sodium bicar-
bonate, and then brine. The organic layer was dried over
anhydrous MgSO4 and filtered and the chloroform removed
under reduced pressure to afford a viscous brown oil. The
residue was immediately dissolved in 5 mL of anhydrous DMF
and added to a suspension of 2,4-diamino-6-hydroxypyrimidine
(0.25 g, 2.0 mmol) in anhydrous DMF. The mixture was
stirred for 48 h at room temperature. The DMF was removed
under reduced pressure, the residue was dissolved in 50 mL
of methanol, 1.70 g of silica gel was added, and the mixture
was evaporated to dryness under reduced pressure. Ethyl
ether (50 mL) was added to the silica gel plug and the plug
collected after filtration was air-dried and placed on top of a
dry silica gel column (1.5 × 10 cm) and eluted with CHCl3/
MeOH (15:1). Fractions containing the desired product were
pooled and evaporated to dryness under reduced pressure to
afford 0.03 g (33%) of 15: mp 246-248 °C; TLC Rf 0.63 (CHCl3/
MeOH, 9:1, silica gel); 1H NMR (Me2SO-d6) δ 1.43 (s, 9 H, Boc),
2.67, (t, 2 H, 6-CH2), 3.58 (t, 2 H, 5-CH2), 4.37 (s, 2 H, 8-CH2),
5.99 (s, 2 H, 4-NH2), 6.43 (s, 2 H, 2-NH2); 13C NMR (125 MHz,
Me2SO-d6) δ 21.5 (C5), 28.0 (Boc CH3), 41.7 (C8 or C6), 42.9
(C6 or C8), 79.5 (Boc C), 91.9 (C4a), 110.1 (C4b), 140.3 (C8a),
155.0 (C4), 158.4 (C2 or Boc-CO), 160 (Boc-CO or C2), 168
(C9a). Anal. Calcd for (C14H19N5O3) C, H, N.
1
TLC Rf 0.66 (CHCl3/MeOH, 2:1, silica gel); H NMR (Me2SO-
d6) δ 2.74 (overlapped m, 4 H, 5-CH2, 6-CH2), 3.52 (s, 2 H,
8-CH2), 3.78 (s, 2 H, benzylic-CH2), 5.93 (s, 2 H, 4-NH2), 6.38
(s, 2 H, 2-NH2), 7.44 (m, 1 H, 6′-H), 7.57 (m, 2 H, 3′-H, 5′-H).
Anal. Calcd for (C16H15N5O Cl2) C, H, N, Cl.
2,4-Dia m in o-5,6,7,8-tetr a h yd r o-7-(3′,4′-d ich lor oben zyl)-
p yr id o[4′,3′:4,5]fu r o[2,3-d ]p yr im id in e (5). This compound
was prepared and purified in a manner similar to that
described for 1 using 3′,4′-dichlorobenzyl chloride, instead of
benzyl bromide, to afford 0.13 g (45%) of 5: mp 275-278 °C;
1
TLC Rf 0.64 (CHCl3/MeOH, 2:1, silica gel); H NMR (Me2SO-
d6) δ 2.71 (overlapped m, 4 H, 5-CH2, 6-CH2), 3.32 (s, 2 H,
8-CH2), 3.71 (s, 2 H, benzylic CH2), 5.93 (s, 2 H, 4-NH2), 6.37
(s, 2 H, 2-NH2), 7.36 (d, 1 H, 6′-H), 7.59 (m, 2 H, 2′-H, 5′-H).
Anal. Calcd for (C16H15N5O Cl2) C, H, N, Cl.
2,4-Dia m in o-5,6,7,8-tetr a h yd r o-7-(2′,6′-d ich lor oben zyl)-
p yr id o[4′,3′:4,5]fu r o[2,3-d ]p yr im id in e (6). This compound
was prepared and purified in a manner similar to that
described for 1 using 2′,6′-dichlorobenzyl chloride, instead of
benzyl bromide, to afford 0.10 g (39%) of 6: mp 280-282 °C;
1
TLC Rf (CHCl3/MeOH, 2:1, silica gel); H NMR (Me2SO-d6) δ
2.71 (overlapped m, 4 H, 5-CH2, 6-CH2), 3.43 (s, 2 H, 8-CH2),
3.71 (s, 2 H, benzylic CH2), 5.93 (s, 2 H, 4-NH2), 6.37 (s, 2 H,
2-NH2), 7.38 (m, 1 H, 4′-H), 7.61 (m, 2 H, 3′,5′-H). Anal. Calcd
for (C16H15N5OCl2‚0.1H2O) C, H, N, Cl.
2,4-Dia m in o-5,6,7,8-t et r a h yd r o-7-b en zylp yr id o[4′,3′:
4,5]fu r o[2,3-d ]p yr im id in e (1). Compound 16 (0.46 g, 1.40
mmol) was suspended in 5 mL of anhydrous DMSO. To it was
added anhydrous potassium carbonate (0.48 g, 3.50 mmol) and
benzyl bromide (0.24 g, 1.40 mmol) and the mixture stirred
for 24 h at room temperature. The mixture was then diluted
with water (30 mL) and stirred for an additional 24 h at room
temperature. The resulting precipitate was collected by filtra-
tion, washed with water, acetone, and ether, and air-dried.
The crude solid was dissolved in a mixture of DMF:MeOH
(1:5), 1.20 g of silica gel was added, and the mixture was
evaporated to dryness under reduced pressure. The resulting
silica gel plug was placed on the top of a dry silica gel col-
umn (1.5 × 10 cm) and eluted with MeOH in CHCl3. The
fractions containing the desired product (TLC) were pooled and
evaporated to dryness under reduced pressure. The resulting
solid was triturated with ether and filtered to afford 0.16 g
(38%) of 1 as a white powder: mp 233-235 °C; TLC Rf
0.55 (CHCl3/MeOH, 2:1, silica gel); 1H NMR (Me2SO-d6) δ
2.70 (overlapped m, 4 H, 5-CH2, 6-CH2), 3.43 (s, 2 H, 8-CH2),
3.70 (s, 2 H, benzylic CH2), 5.91 (s, 2 H, 4-NH2), 6.35 (s, 2H,
2-NH2); 7.29 (m, 5 H, phenyl). Anal. Calcd for (C16H17N5O)
C, H, N.
2,4-Dia m in o-5,6,7,8-t et r a h yd r o-7-(3′,4′,5′-t r im et h oxy-
ben zyl)p yr id o[4′,3′:4,5]fu r o[2,3-d ]p yr im id in e (2). This
compound was prepared and purified in a manner similar to
that described for 1 using 3′,4′,5′- trimethoxybenzyl chloride,
instead of benzyl bromide, to afford 0.12 g (44%) of 2 as a
yellow solid: mp 268-270 °C; TLC Rf 0.60 (CHCl3/MeOH, 2:1,
silica gel); 1H NMR (Me2SO-d6) δ 2.70 (overlapped m, 4 H,
5-CH2, 6-CH2), 3.47 (s, 2 H, 8-CH2), 3.65 (overlapped m, 5 H,
benzylic-CH2, 4′-OCH3), 3.76 (s, 6 H, 3′,5′-OCH3), 5.91(s, 2 H,
4-NH2), 6.35 (s, 2 H, 2-NH2), 6.66 (s, 2 H, phenyl). Anal. Calcd
for (C19H23N5O4‚0.3H2O) C, H, N.
2,4-Dia m in o-5,6,7,8-t e t r a h yd r o-7-(4′-b e n zoyl-L -glu -
ta m ic a cid d ieth yl ester )p yr id o[4′,3′:4,5]fu r o[2,3-d ]p yr i-
m id in e (18). This compound was prepared and purified in a
manner similar to that described for 1 using 4′-(chloromethyl)-
benzoylglutamic acid diethyl ester 17 instead of benzyl bro-
mide to afford 0.12 g (47%) of 18: mp 280-282 °C; TLC Rf
0.62 (CHCl3/MeOH, 2:1, silica gel); 1H NMR (Me2SO-d6) δ 1.14
(m, 6 H, CH3), 2.08 (m, 2 H, Gluâ-CH2), 2.42 (m, 2 H, Gluγ-
CH2), 2.70 (m, 4 H, 5-CH2, 6-CH2), 3.46 (s, 2 H, 8-CH2), 3.76
(s, 2 H, benzyl-CH2), 4.01 (overlap m, 4 H, CH2), 4.43 (m, 1 H,
GluR-CH), 5.91 (s, 2 H, 4-NH2), 6.35 (s, 2 H, 2-NH2), 7.45 (d,
2 H, 3′-,5′-CH), 7.84 (d, 2 H, 2′,6′-CH), 8.68 (d, 1H, NH). Anal.
Calcd for (C26H32N6O6) C, H, N.
2,4-Dia m in o-5,6,7,8-t e t r a h yd r o-7-(4′-b e n zoyl-L -glu -
ta m ic a cid )-p yr id o [4′,3′:4,5]fu r o[2,3-d ]p yr im id in e (7). To
a solution of 18 (0.18 g, 0.35 mmol) in methoxyethanol (10 mL)
was added 1.5 mL of 1 N NaOH, and the solution was stirred
at room temperature for 24 h. The methoxyethanol was
evaporated under reduced pressure, the residue was dissolved
in water (10 mL), and stirring was continued for an additional
24 h. The solution was then cooled in an ice bath and the pH
adjusted to 3.5 via the dropwise addition of 1 N HCl. The
precipitate that formed was collected by filtration, washed with
water, acetone, and ether, and air-dried to afford 0.16 g (99%)
of 7 as a pale yellow solid: mp >280 °C; TLC Rf 0.70 (3% NH4-
1
HCO3, cellulose); H NMR (Me2SO-d6) δ 1.96 (m, 2 H, Gluâ-
CH2), 2.36 (m, 2 H, Gluγ-CH2), 2.70 (m, 4 H, 5-CH2, 6-CH2),
3.46 (s, 2 H, 8-CH2), 4.01 (s, 2 H, benzyl-CH2), 4.43 (m, 1 H,
GluR-CH), 5.91 (s, 2 H, 4-NH2), 6.35 (s, 2 H, 2-NH2), 7.45 (d,
2 H, 3′-,5′-CH), 7.84 (d, 2 H, 2′-,6′-CH), 8.68 (d, 1 H, CONH),
12.37 (br s, 2H, COOH). Anal. Calcd for (C22H24N6O6‚0.5H2O)
C, H, N.
4-(Ch lor om eth yl)ben zoyl-L-glu ta m ic Acid Dieth yl Es-
ter (17). 4-(Chloromethyl)benzoic acid (2.0 g, 11.70 mmol) was
dissolved in anhydrous DMF and stirred under nitrogen
at 0 °C for 10 min. To this solution was added triethylamine
(2.38 g, 23.40 mmol), and the mixture was stirred for an
additional 15 min. A solution of glutamic acid diethyl ester
(2.80 g, 11.70 mmol) in DMF (10 mL) was neutralized with
triethylamine (1.19 g, 11.70 mmol) and immediately added to
the reaction mixure. The reaction was stirred for 2 h at 0 °C,
after which the DMF was removed under reduced pressure.
2,4-Dia m in o-5,6,7,8-tetr a h yd r o-7-(3′,5′-d im eth oxyben -
zyl)p yr id o[4′,3′:4,5] fu r o[2,3-d ]p yr im id in e (3). This com-
pound was prepared and purified in a manner similar to that
described for 1 using 3′,5′-dimethoxybenzyl chloride, instead
of benzyl bromide, to afford 0.13 g (52%) of 3: mp 228-230
°C; TLC Rf 0.48 (CHCl3/MeOH, 4:1, silica gel); 1H NMR (Me2-
SO-d6) δ 2.69 (overlapped m, 4 H, 5-CH2, 6-CH2), 3.45 (s, 2 H,
8-CH2), 3.63 (s, 2 H, benzylic CH2), 3.73 (s, 6H, 3′,5′-OCH3),

Inhibitors of Dihydrofolate Reductases
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1415
(4) Schnapp, L. M.; Geaghan, S. M.; Campagna, A.; Fahy, J .; Steiger,
D.; Ng, V.; Hadley, W. K.; Hopwell, P. C.; Stansell, J . D.
Toxoplasma gondii Pneumonitis in Patients Infected with the
Human Immunodeficiency Virus. Arch. Intern. Med. 1992, 152,
1073-1077.
(5) Wanke, C.; Tuazon, C. U.; Kovacs, A.; Dina, D.; Davis, D. O.;
Barton, N.; Katz, D.; Lunde, M.; Levy, C.; Conley, F. K.; Lane,
H. C.; Fauci, A. S.; Masur, H. Toxoplasma Encephalitis in
Patients with Acquired Immunodeficiency Syndrome: Diagnosis
and Response to Therapy. Am. J . Trop. Med. Hyg. 1987, 36,
509-516.
(6) Gordin, F. M.; Simon, G. L.; Wofsy, C. B.; Mills, J . Adverse
Reactions to Trimethoprimsulfamethoxazole in Patients with the
Acquired Immunodeficiency Syndrome. Ann. Intern. Med. 1984,
100, 495-499.
(7) Allegra, C. J .; Kovacs, J . A.; Drake, J . C.; Swan, J . C.; Chabner,
B. A.; Masur, H. Activity of Antifolates against Peumocystis
carinii Dihydrofolate Reductase and Identification of a Potent
New Agent. J . Exp. Med. 1987, 165, 926-931.
(8) Queener, S. F.; Bartlett, M. S.; J ay, M. A.; Durkin, M. M.; Smith,
J . W. Activity of Lipid-soluble Inhibitors of Dihydrofolate Re-
ductase Against Pneumocystis carinii in Culture and in a Rat
Model of Infection. Antimicrob. Agents Chemother. 1987, 31,
1323-1327.
(9) Allegra, C. J .; Kovacs, J . A.; Drake, J . C.; Swan, J . C.; Chabner,
B. A.; Masur, H. Potent in vitro and in vivo Antitoxoplasma
Activity of the Lipid-soluble Antifolate Trimetrexate. J . Clin.
Invest. 1987, 79, 478-482.
(10) Kovacs, J . A.; Chabner, B. A.; Swan, J .; Drake, M.; Lunde, M.;
Parrillo, J . E.; Masur, H. Potent Effect of Trimetrexate, a Lipid-
soluble Antifolates, on Toxoplasma gondii. J . Infect. Dis. 1987,
155, 1027-1032.
(11) Kovacs, J . A.; Allegra, C. J .; Swan, J . C.; Drake, J . C.; Parrillo,
J . E.; Chabner, B. A.; Masur, H. Potent Antipneumocystis and
Antitoxoplasma Activities of Piritrexim, a Lipid- soluble Anti-
folate. Antimicrob. Agents Chemother. 1988, 32, 430-433.
(12) Allegra, C. J .; Chabner, B. A.; Tuazon, C. U.; Ogata-Arakaki,
D.; Baird, D.; Drake, J . C.; Simmons, J . T.; Lack, E. E.;
Shelhamer, J ., H.; Balis, F.; Walker, R.; Kovacs, J . A.; Lane, H.
C.; Masur, H. Trimetrexate, a Novel and Effective Agent for the
Treatment of Pneumocystis carinii Pneumonia in Patients with
the Acquired Immunodeficiency Syndrome. N. Engl. J . Med.
1987, 317, 978-983.
(13) Masur, H.; Polis, M. A.; Tuazon, C. U.; Ogata-Arakaki, D.;
Kovacs, J . A.; Katz, D.; Hilt, D.; Simmons, T.; Feuerstein, I.;
Lundgren, B.; Lane, H. C.; Chabner, B. A.; Allegra, C. J . Salvage
Trial of Trimetrexate-Leucovorin for the Treatment of Cerebral
Toxoplasmosis. J . Inefect. Dis. 1992. 167, 1422-1426.
(14) Bertino, J . R.; Sawicki, W. L.; Moroson, B. A.; Cashmore, A. R.;
Elslager, E. F. 2,4-Diamino- 5-methyl-6-[(3,4,5-trimethoxya-
nilino)-methyl]quinazoline (TMQ). A potent Non- classical Folate
Antagonist Inhibitor-I. Biochem. Pharmacol. 1979, 38, 1983-
1987.
The residue was extracted with a bilayer of ethyl ether and
water (100 mL; 50 mL), and the organic layer was washed
with 0.5 N HCl and sodium bicarbonate and dried over MgSO4.
The ether was evaporated under reduced pressure to af-
ford pure 17, 2.75 g (66%): TLC Rf 0.46 (hexane/ethyl acetate,
1
1:1, silica gel); H NMR (CDCl3) δ 1.20 (t, 3 H, CH3), 1.28 (t,
3 H, CH3), 2.13-2.51 (m, 4 H, Gluâ and Gluγ-CH2), 4.07 (q, 2
H, CH2), 4.20 (q, 2 H, CH2), 4.61 (s, 2 H, benzyl-CH2), 4.43
(m, 1 H, GluR-CH), 7.06 (d, 1 H, CONH), 7.45 (d, 2 H, 3′-,5′-
CH), 7.80 (d, 2 H, 2′-,6′-CH). Anal. Calcd for (C17H22NO5Cl)
C, H, N, Cl.
En zym es a n d En zym es Assa ys. Folylpolyglutamate syn-
thetase (FPGS) was partially purified from CCRF-CEM human
leukemia cells by (NH4)2SO4 fractionation, gel sieving, and
phosphocellulose chromatography.15 FPGS activity was as-
sayed as described;33 compound 7 was quantitatively ()98%)
recovered during the standard assay procedure, thus ensuring
that polyglutamate products would also be quantitatively
recovered. Kinetic constants were determined by the hyper-
bolic curve fitting subprogram of SigmaPlot (J andel) using a
>10-fold range of substrate concentration; activity was linear
with respect to time at the highest and lowest concentrations
tested. Assays contained ∼400 units of FPGS activity; one unit
of FPGS catalyzes incorporation of 1 pmol of [3H ]Glu/h.
CCRF-CEM dihydrofolate reductase was partially purified and
assayed as described.34 DHFR inhibitory potency as measured
by adding drug to standard assays; the drug concentration
required to reduce activity to 50% of control (IC50) was
determined graphically from the plots of residual activity
versus drug concentration. Assays contained 1.8 × 10-3 units
of DHFR activity; one unit of DHFR reduces 1 µmol dihydro-
folate/min under standard conditions.
Cell Lin es a n d Meth od s for Mea su r in g Gr ow th In h ib-
itor y P oten cy. The human T-lymphocytes leukemia cell line
CCRF-CEM35 and its MTX-resistant sublines R30dm27 and
R129 were cultured as described.27 R30dm expresses 1% of the
FPGS activity of CCRF-CEM. R1 expresses 20-fold elevated
levels of DHFR, the target enzyme of MTX. The FaDu human
squamous cell carcinoma monolayer cell line was cultured in
RPMI 1640/10% fetal calf serum in 100 mm cell culture dishes
(Falcon).15 Growth inhibition of all lines by continuous drug
exposure was measured as described.15,27 EC50 values were
determined visually from plots of percent control growth versus
the logarithm of drug concentration. Protection against
growth inhibition was assayed by including 10 µM leucovorin
(LV) simultaneously with a concentration of drug previously
determined to yield growth inhibition of 90-99%; the remain-
der of the assay was as described above. All lines were verified
to be negative for Mycoplasma contamination using the
GenProbe test kit.
(15) Gangjee, A.; Devraj, R.; McGuire, J . J .; Kisliuk, R. L.; Queener,
S. F.; Barrows, L. R.; Classicaland Nonclassical Furo[2,3-d]-
pyrimidines as Novel Antifolates: Synthesis and Biological
Activities. J . Med. Chem. 1994, 37, 1169-1176.
(16) Gangjee, A.; Mavandadi, F.; Queener, S. F.; McGuire, J . J .; Novel
2,4-Diamino-5-substituted-pyrrolo[2,3-d]pyrimidines as Classical
and Nonclassical Antifolate Inhibitors of Dihydrofolate Reduc-
tases. J . Med. Chem. 1995, 38, 2158-2165.
Ack n ow led gm en t. The authors thank Dr. Fu-Tyan
Lin, Department of Chemistry, University of Pittsburgh,
for the NOESY, COLOC, and 13C NMR, and Dr. W.
Bolanowska for performing enzyme assays with CCRF-
CEM, FPGS, and DHFR. The technical assistance of Ms.
J ill Canestrari in the mammalian cell culture assays is
greatly acknowledged. This work was supported in part
by NIGMS Grant GM40998 (A.G.), NIAID Grant
AI41743 (A.G.), NCI Grant CA43500 (J .J .M.), RPCI
Core Grant CA16056 (J .J .M.), and NIH Contracts NO1-
AI-87240 (S.F.Q.) and NO1-AI-35171-(S.F.Q.).
(17) Baker, D. J .; Bedell, C. R.; Champness, J . N.; Goodford, P.;
Novrington, F. E.; Smith, D. R.; Stammers, D. N. The Binding
of Trimethoprim to Bacterial Dihydrofolate Reductase. FEBS
Lett. 1981, 126, 49-52.
(18) Mathews, D. A.; Bolin, J . T.; Burdge, J . M.; Filman, K. W.;
Kaufman, B. T.; Bedell, C. R.; Champness, J . N.; Stammers, D.
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