Nonclassical 2,4-diamino-5-aryl-6-ethylpyrimidine antifolates: Activity as inhibitors of dihydrofolate reductase from Pneumocystis carinii and Toxoplasma gondii and as antitumor agents

Article abstract of DOI:10.1021/jm970055k

Twelve novel 2,4-diamino-5-(4'-benzylamino)- and 2,4-diamino-5-[4'-(N- methylbenzylamino)-3'-nitrophenyl]-6-ethylpyrimidines bearing 4-substituents on the benzylamino or N-methylbenzylamino aryl ring were synthesized and evaluated as nonclassical inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase (DHFR). Compounds were prepared by reaction of 2,4-diamino-5-(4'-chloro-3'-nitrophenyl)-(8) or 2,4-diamino-5-(4'-fluoro-3'- nitrophenyl)-6-ethylpyrimidine (15) with the appropriate 4-substituted (CO₂H, CO₂Me, SO₂NH₂, dioxolan-2-yl, CHO, dimethyloxazolin-2-yl) benzylamine or N-methylbenzylamine derivative. Compounds 25-29 were synthesized from 2,4-diamino-5-{4'-[N-(4''-carboxybenzyl)amino]-3'- nitrophenyl}-6-ethylpyrimidine (10) and the corresponding amine (NH₃, MeNH₂, Me₂NH, piperidine, diethyl L-glutamate) via isobutyl mixed anhydride coupling; hydrolysis of the diethyl L-glutamate 29 afforded the L-glutamate analogue 30. The compounds exhibited potent inhibitory activity against T. gondii (IC₅₀ values 0.0018-0.14 μM) and rat liver (IC₅₀ values 0.0029- 0.27 μM) DHFR, with a 4-substituent invariably enhancing binding to both enzymes relative to the unsubstituted benzoprim (5) or methylbenzoprim (6). Modest selectivity for T. gondii enzyme was observed with several analogues, whereas all of the compounds were relatively weak inhibitors of P. carinii DHFR and exhibited no selectivity. Selected analogues were evaluated for in vivo antitumor activity against the methotrexate-resistant M5076 murine reticulosarcoma, with 2,4-diamino-5-{4'-[N-[4''-(N'- methylcarbamoyl)benzyl]-N-methylamino]-3'-nitrophenyl}-6-ethylpyrimidine (14) (K(i) for rat liver DHFR = 0.000 35 ± 0.000 29 nM) combining significant antitumor activity with minimal toxicity.

Full text of DOI:10.1021/jm970055k

3040
J . Med. Chem. 1997, 40, 3040-3048
Non cla ssica l 2,4-Dia m in o-5-a r yl-6-eth ylp yr im id in e An tifola tes: Activity a s
In h ibitor s of Dih yd r ofola te Red u cta se fr om P n eu m ocystis ca r in ii a n d
Toxopla sm a gon d ii a n d a s An titu m or Agen ts
Claire Robson,† Michelle A. Meek,‡ J an-Dierk Grunwaldt,†,| Peter A. Lambert,‡ Sherry F. Queener,§
Dirk Schmidt,†,| and Roger J . Griffin*,†
Department of Chemistry, Bedson Building, The University, Newcastle upon Tyne NE1 7RU, U.K., Pharmaceutical Sciences
Institute, Department of Pharmaceutical Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, U.K., and
Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202
Received J anuary 27, 1997X
Twelve novel 2,4-diamino-5-(4′-benzylamino)- and 2,4-diamino-5-[4′-(N-methylbenzylamino)-
3′-nitrophenyl]-6-ethylpyrimidines bearing 4-substituents on the benzylamino or N-methyl-
benzylamino aryl ring were synthesized and evaluated as nonclassical inhibitors of Pneumocystis
carinii and Toxoplasma gondii dihydrofolate reductase (DHFR). Compounds were prepared
by reaction of 2,4-diamino-5-(4′-chloro-3′-nitrophenyl)- (8) or 2,4-diamino-5-(4′-fluoro-3′-nitro-
phenyl)-6-ethylpyrimidine (15) with the appropriate 4-substituted (CO2H, CO2Me, SO2NH2,
dioxolan-2-yl, CHO, dimethyloxazolin-2-yl) benzylamine or N-methylbenzylamine derivative.
Compounds 25-29 were synthesized from 2,4-diamino-5-{4′-[N-(4′′-carboxybenzyl)amino]-3′-
nitrophenyl}-6-ethylpyrimidine (10) and the corresponding amine (NH3, MeNH2, Me2NH,
piperidine, diethyl L-glutamate) via isobutyl mixed anhydride coupling; hydrolysis of the diethyl
L-glutamate 29 afforded the L-glutamate analogue 30. The compounds exhibited potent
inhibitory activity against T. gondii (IC50 values 0.0018-0.14 µM) and rat liver (IC50 values
0.0029-0.27 µM) DHFR, with a 4-substituent invariably enhancing binding to both enzymes
relative to the unsubstituted benzoprim (5) or methylbenzoprim (6). Modest selectivity for T.
gondii enzyme was observed with several analogues, whereas all of the compounds were
relatively weak inhibitors of P. carinii DHFR and exhibited no selectivity. Selected analogues
were evaluated for in vivo antitumor activity against the methotrexate-resistant M5076 murine
reticulosarcoma, with 2,4-diamino-5-{4′-[N-[4′′-(N′-methylcarbamoyl)benzyl]-N-methylamino]-
3′-nitrophenyl}-6-ethylpyrimidine (14) (Ki for rat liver DHFR ) 0.000 35 ( 0.000 29 nM)
combining significant antitumor activity with minimal toxicity.
In tr od u ction
not DDMP or the antimalarial antifolate pyrimethamine
(PYM), are substrates for the membrane-bound P-
glycoprotein (GP-170)14,15 which effluxes a diverse range
of unrelated antitumor agents from cells and is respon-
sible for the multidrug-resistance (MDR) phenotype.16
Baccanari et al. have demonstrated that TMQ and PTX
do not attain appreciable CSF/plasma ratios, and the
endothelial cells of the blood-brain barrier are known
to overexpress GP-170.17 By denying access of drug to
CNS tumors, MDR represents a potential resistance
mechanism for this class of antitumor agent, and a
number of novel lipophilic antifolates which are non-
substrates for GP-170 have reportedly been identified.2
The antimetabolite drug methotrexate (MTX), which
acts principally via inhibition of folate-dependent en-
zymes, is the prototype of the antifolate drugs used in
cancer chemotherapy.1-3 Problems associated with the
antitumor activity of MTX, a potent nonselective inhibi-
tor of dihydrofolate reductase (DHFR), include a limited
spectrum of activity and the development of resis-
tance.4,5 Lipophilic DHFR inhibitors, exemplified by the
first-generation antifolates metoprine (DDMP) (1) and
etoprine (2) lack a polar glutamate side chain and differ
from MTX in not requiring a carrier-mediated active
transport mechanism to gain ingress to cells, entering
by passive or facilitated diffusion.6,7 As a consequence,
these agents exhibit activity against MTX-resistant
tumors and also central nervous system (CNS) malig-
nancies inaccessible to the more hydrophilic MTX.8 The
lipophilic antifolates piritrexim (PTX) (3)9,10 and trime-
trexate (TMQ) (4)11,12 were developed to overcome
toxicity problems encountered with DDMP, attributed
to the prolonged biological half-life7 and inhibition of
histamine metabolism observed for this highly lipid
soluble compound.13 Interestingly, PTX and TMQ, but
Lipophilic DHFR inhibitors, including PTX and TMQ,
have also enjoyed a more recent resurgence in interest
as agents for the treatment of infection by opportunistic
pathogens, including Candida albicans,18 Toxoplasma
gondii,19 and Pneumocystis carinii,20 in immunocom-
promised patients. Indeed, TMQ has recently gained
clinical approval for the treatment of P. carinii infec-
tions in patients with acquired immune deficiency
syndrome (AIDS).21 Unfortunately, unlike PYM or the
antibacterial DHFR inhibitor trimethoprim, these an-
tifolates exhibit no selectivity for pathogen DHFR and
are more potent inhibitors of the mammalian enzyme.
As a consequence, the concomitant administration of
leucovorin is necessary in order to ameliorate host
antifolate toxicity.22 Clearly, there is a requirement for
novel and potent lipophilic antifolates with improved
* Author to whom correspondence should be addressed.
† The University.
‡ Aston University.
§ Indiana University School of Medicine.
| ERASMUS Chemistry exchange student from University of Ham-
burg.
X Abstract published in Advance ACS Abstracts, August 1, 1997.
S0022-2623(97)00055-1 CCC: $14.00 © 1997 American Chemical Society

2,4-Diamino-5-aryl-6-ethylpyrimidine Antifolates
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19 3041
Ch a r t 1
and activity has been observed in vitro and in vivo for
both diaminopyrimidines against a panel of MTX-
sensitive and -resistant human tumor xenografts.25
The potent DHFR-inhibitory activity of MBP and
related compounds was originally ascribed to a resem-
blance of the N-methylbenzylamino substituent to the
4-(N-methylamino)benzoyl moiety of MTX. This sug-
gested that a carboxylate or carboxamide function in the
para position of the benzyl ring should enhance binding
to the enzyme. In this paper we describe the results of
these studies and also the synthesis of an L-glutamate
derivative, 30, in the expectation that this “classical”
analogue might also exhibit some interesting biological
properties. The comparative DHFR-inhibitory activity
of these diaminopyrimidines was determined against
enzyme from P. carinii, T. gondii, and rat liver. Se-
lected compounds were also evaluated for in vivo
antitumor activity against the M5076 reticulosarcoma
in mice.
selectivity for pathogen DHFR, and a large number of
compounds have been prepared and evaluated to this
end.23
Ch em istr y
The synthetic procedures employed for the prepara-
tion of the target compounds are outlined in Scheme 1.
We have previously described the use of m-nitro-
pyrimethamine (8) as a key starting material for the
synthesis of diaminopyrimidines bearing amine sub-
stituents in the 5-aryl ring, where simply heating a
solution of 8 in the appropriate amine generally afforded
the target antifolates in excellent yields.24,26 Unfortu-
nately, analogous reactions of 8 with 4-(aminomethyl)-
or 4-[(N-methylamino)methyl]benzoic acid, which would
furnish carboxylic acids 9 and 10 and enable subsequent
elaboration to other derivatives, were precluded by the
lack of reactivity and very poor solubility of these
amines. Copper(II) chloride catalysis of the amination
In an earlier paper we reported on the synthesis and
biological properties of a series of 2,4-diamino-5-aryl-
6-ethylpyrimidines encompassing secondary and ter-
tiary amine groups on the 5-aryl ring.24 Of particular
interest were analogues bearing a benzylamino (5) or
N-alkylbenzylamino substituent (e.g., 6), as these proved
to be potent inhibitors of mammalian DHFR and
exhibited in vivo antitumor activity superior to that of
DDMP against the MTX-resistant M5076 murine retic-
ulosarcoma. Methylbenzoprim (MBP, 6) and the closely
related dichlorobenzoprim (DCB, 7) were selected for
more extensive evaluation on the basis of promising
biological activity combined with relatively low toxicity,
Sch em e 1a
a
Reagents and conditions: (a) CuCl2‚2H2O, NaOAc, DMF, reflux; (b) amine, EtO(CH2)2OH, reflux; (c) NaOH, MeOH, reflux; (d) amine,
NEt3, NMP, 100 °C; (e) 1. i-BuOCOCl, NEt3, DMF, 0 °C, 2. amine, 25 °C; (f) 1. NBu4HSO4, NaOCl, EtOAc, 25 °C, 2. NaOH, MeOH, 25
°C; (g) AcOH (aq), 25 °C; (h) 1. Ba(OH)2‚8H2O, EtOH (aq), 25 °C, 2. Na2SO4, H2O.

3042 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19
Robson et al.
Sch em e 2a
a
Reagents and conditions: (a) NEt3, NMP, 100 °C.
of chloronitroarenes has been reported previously,27,28
and this approach was investigated for the reaction of
8 with 4-(aminomethyl)benzoic acid in DMF. Interest-
ingly, while none of the required product 9 was obtained,
the (dimethylamino)pyrimidine 11 was isolated in excel-
lent yield, as an N-formyl-4-(aminomethyl)benzoate salt.
We have previously utilized DMF-ethylenediamine, as
reported by Yamamoto et al.,29 for the dimethylamina-
tion of nitropyrimethamine (8), and this reaction is
clearly catalyzed by copper(II) salts, as no reaction was
observed when a mixture of 8 and 4-(aminomethyl)-
benzoic acid was heated in DMF, in the absence of a
copper salt.
Reaction of 8 with the more soluble methyl 4-[(N-
methylamino)methyl]benzoate, prepared from 4-(chlo-
romethyl)benzoic acid by standard methodology, af-
forded the corresponding diaminopyrimidine methyl
ester 12 in low yield, together with the 2-ethoxyethyl
ester 13 arising from transesterification by the 2-ethoxy-
ethanol employed as solvent. Conversion of the mixture
of esters into the required 12 was achieved with
methanol-sulfuric acid, and subsequent base-catalyzed
hydrolysis of 12 with methanolic sodium hydroxide gave
the target carboxylic acid derivative 10, which was
isolated as an ethanesulfonic acid salt. An authentic
sample of 13 was also prepared from 8 and 2-ethoxy-
ethyl 4-[(N-methylamino)methyl]benzoate under identi-
cal reaction conditions. The analogous reaction of 8
with N-methyl-4-[(N-methylamino)methyl]benzamide
furnished the required N-methylcarboxamide 14, albeit
in moderate yield.
in excellent yield, and similar reactions with 4-(ami-
nomethyl)benzoic acid and 4-(aminomethyl)benzene-
sulfonamide gave good yields of the benzoic acid 9 and
sulfonamide 16, respectively. 2,4-Diamino-5-{4′-[N-[4′′-
(4,4-dimethyloxazolin-2-yl)benzyl]amino]-3′-nitrophenyl}-
6-ethylpyrimidine (17), prepared analogously from 15
and 2-[4-[(N-methylamino)methyl]phenyl]-4,4-dimethyl-
oxazoline (19), was a potential precursor to the acid 10.31
Unfortunately, conversion of 17 to the required acid 10
proved problematical owing to degradation of the di-
aminopyrimidine, although treatment with sodium hy-
pochlorite-sodium hydroxide under phase-transfer con-
ditions did give 10 in poor yield.32 The aldehyde 23 was
obtained by acid hydrolysis of acetal 22, which was
synthesized by the reaction of 15 with 2-[4-(amino-
methyl)phenyl]dioxolane (21). This amine was prepared
from 4-cyanobenzaldehyde,33 with lithium triethylboro-
hydride proving to be the most satisfactory reagent for
reduction of the nitrile group without concomitant
cleavage of the acetal function.
Surprisingly, the reaction of 15 with 4-[(N-methyl-
amino)methyl]benzoic acid did not give 10 as expected
but rather the (N-methylamino)pyrimidine 24 arising,
presumably, from debenzylation of the initially formed
10. In an earlier paper we have described the acid-
catalyzed debenzylation of methylbenzoprim (6) to give
24.34 Although the exact mechanism of this reaction
remains to be elucidated, debenzylation of 10 by nu-
cleophilic attack of unsolvated fluoride ion (NEt3+F-)
in the polar aprotic solvent (NMP) employed would
afford 24 and 4-(fluoromethyl)benzoic acid (Scheme 2).
The poor product yields arising from the relatively
vigorous conditions necessary to effect the reaction of 8
with amines led us to consider an alternative, more
reactive pyrimidine starting material. The increased
reactivity, to nucleophilic substitution, of fluoro- over
chloronitroarenes is well established, and we have
previously reported the preparation of 2,4-diamino-5-
(4′-fluoro-3′-nitrophenyl)-6-ethylpyrimidine (15), the flu-
oro analogue of 8.30 Reaction of 15 with N-methyl-4-
[(N-methylamino)methyl]benzamide in 2-ethoxyethanol
at 80 °C afforded the required N-methylcarboxamide 14
Amides 25-28 were prepared from the diaminopyri-
midinecarboxylic acid 9 in excellent yields via the mixed
anhydride, and the analogous coupling reaction with
diethyl L-glutamate gave the diester 29 in good yield.35
The use of sodium hydroxide to effect ester hydrolysis
resulted in extensive decomposition, and conversion into
the target folate analogue 30 was eventually achieved
using barium hydroxide,36 the dibarium salt precipitat-
ing from solution as the reaction proceeded. Dissolution
in water and addition of sodium sulfate afforded the

2,4-Diamino-5-aryl-6-ethylpyrimidine Antifolates
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19 3043
Ta ble 1. Inhibition Concentration (IC50, in µM) of
Dihydrofolate Reductase from P. carinii, T. gondii, and Rat
Liver and Selectivity Ratiosa
mammalian enzyme over that from P. carinii, and this,
combined with the poor inhibitory activity observed
against pathogen DHFR, mitigates against this class
of inhibitor offering any advantages over existing agents.
no.
rlDHFR
pcDHFRb tgDHFR
rl/pc
rl/tg
5
6
8
9
0.025
0.0032
0.015
0.0029
0.0038
0.0068
0.0031
0.015
0.016
0.0075
0.006
0.0089
0.27
0.0067
0.013
1.03c
1.6c
0.019
0.091
0.013
0.0018
0.0031
0.0074
0.0058
0.014
0.015
0.0037
0.0043
0.0079
0.14
0.024
0.002
0.02
0.01
0.01
0.01
0.01
0.02
0.02
0.01
0.01
0.01
0.12
0.01
0.13
0.048
0.071
1.3
0.04
1.1
The diaminopyrimidine derivatives exhibited potent
inhibitory activity against T. gondii DHFR with IC50
values ranging from 0.0018 µM (9) to 0.14 µM (28). In
contrast to pcDHFR, N-methylation of the benzylamino
side chain marginally reduced binding to the T. gondii
enzyme; for example, 9 and 26 are approximately 2-fold
more potent than their N-methyl counterparts 10 and
14, and this is consistent with the relative activities of
benzoprim (5) and methylbenzoprim (6) against tgDH-
FR. Again, the introduction of a para-substituent on
the aryl ring (9, 10, 14, 16, 27, 29, 30) enhanced binding
compared to 5 and 6. Para-substitution with a dim-
ethyloxazoline (17) or dioxolane (22) ring had little effect
on activity against tgDHFR, but a piperidino substitu-
ent (28) reduced activity, and this trend was also
observed against mammalian DHFR. Indeed, with the
exception of 6, which was some 28 times more active
against mammalian enzyme (IC50 ) 0.0032 µM) than
that from T. gondii (IC50 ) 0.091 µM), inhibitory activity
against rlDHFR closely paralleled that against tgDHFR,
as is evident from the rlDHFR/tgDHFR selectivity
indices. Consequently, the compounds exhibited little
selectivity for the T. gondii over mammalian enzyme,
although 25, 28, and the classical analogue 30 were
approximately 2-fold more active against the tgDHFR.
0.85c
0.27
0.27
0.46
0.46
0.95
0.93
0.53
0.58
1.3
1.61
1.23
0.92
0.53
1.07
1.07
2.03
1.40
1.13
1.93
0.94
2.10
0.088
0.29
10
14
16
17
22
25
26
27
28
29
30
2.2
0.45
0.1
0.031
0.042
12.0
0.0071
0.0062
0.017
0.010
2.7
PTX (3)
TMQ (4)
TMP
epiroprim
MTX
0.0015
0.003
133.0
33.2
0.0025
11.1
12.8
1.9
49.0
70.6
0.18
2.6
0.0013
0.47
0.014
a
Assays were conducted at 37 °C under conditions of substrate
(90 µM dihydrofolic acid) and cofactor (119 µM NADPH) in the
b
presence of 150 mM KCl.19,20 Recombinant P. carinii DHFR
employed unless indicated otherwise. c DHFR isolated from P.
carinii.
disodium salt, although only a poor yield of the free acid
was isolated on acidification with aqueous hydrochloric
acid.
Biology
The potent inhibitory activity observed against mam-
malian DHFR was studied in more detail by conducting
inhibition constant (Ki) determinations on three selected
compounds (10, 12, and 14). Inhibitory activity was
determined spectroscopically at 340 nm against rat liver
DHFR as described previously, employing the zone B
analysis method appropriate for tight-binding inhibitors
(Table 2).39 Ki values for DDMP (1) and MBP (6) are
included for comparative purposes. All three com-
pounds were more potent than 1, and in comparison
with MBP (6), inhibitory activity was of the order 10 ≡
14 > 6 > 12, with the carboxylic acid 10 (Ki ) 0.0004 (
0.0003 nM) and N-methylcarboxamide 14 (Ki ) 0.000 35
( 0.000 29 nM) proving to be among the most potent
lipophilic inhibitors of mammalian DHFR reported to
date. The in vivo antitumor activity of 10 and 14 was
determined against the M5076 reticulosarcoma, a mu-
rine solid tumor intrinsically resistant to MTX as a
result of impaired transport,40 in order to evaluate the
potential of these novel lipophilic antifolates against
MTX-resistant malignancies. The results are sum-
marized in Table 2, and antitumor activity is expressed
as the inhibition of tumor volume (T/C) of test (T) versus
control (C) animals, where a T/C value of 8% represents
maximal activity in this system. DDMP (1) exhibited
activity in this tumor model but was highly toxic at the
lowest dose level utilized, whereas the N-methylcar-
boxamide 14 combined good antitumor activity with no
host toxicity at 12.5 mg‚kg-1. Some toxicity was ob-
served with both 10 and 14 at the higher dose of 25
mg‚kg-1. The promising activity of 14 as an agent for
the treatment of MTX-resistant malignancies is cur-
rently being investigated against a broader spectrum
of tumors, including those expressing the MDR pheno-
type, and the results of these studies will be published
subsequently.
The DHFR-inhibitory activity of the benzoprim ana-
logues was determined against enzyme from P. carinii
(pc), T. gondii (tg), and rat liver (rl) as described
previously,20,37 and the results are summarized in Table
1. Published data20,37 for benzoprim (5), MBP (6),
nitropyrimethamine (8), and the reference compounds
TMP, epiroprim, PTX (3), and TMQ (4) are included for
comparison purposes. Selectivity ratios (IC50(rlDHFR)/
IC50(pcDHFR) and IC50(rlDHFR)/IC50(tgDHFR)) are also
presented in Table 1. Potency against pcDHFR was
generally weak, although all of the novel compounds
were more potent than TMP and at least equipotent
with epiroprim, with IC50 values varying from 0.1 µM
(30) to 2.2 µM (28). The differential inhibition of the
various DHFRs by compounds 6, 27, and 30 may reflect
subtle differences in the ability of the active sites of the
respective DHFRs to accommodate larger substituents
such as p-aminobenzoyl L-glutamate versus smaller
substituents (e.g., N-methylbenzylamino) as suggested
previously.38
Where a comparison was possible, replacement of a
benzylamino by an N-methylbenzylamino substituent
had no significant effect on binding to P. carinii DHFR
(compare 5 with 6, 9 with 10, and 26 with 14). The
introduction of a para-substituent (CO2H, CONH2,
CONHMe, SO2NH2) on the benzylamino or N-methyl-
benzylamino ring enhanced binding 2-5-fold compared
to the unsubstituted inhibitors 5 and 6. By contrast, a
dimethyloxazoline (17) or dioxolane (22) ring or a
disubstituted carboxamide (27, 28) at this position
reduced potency, and these compounds were approxi-
mately equipotent with 5 and 6 against pcDHFR.
Unfortunately, from the rlDHFR/pcDHFR selectivity
indices, it is evident that all of the new compounds
evaluated exhibit a 50-100-fold higher affinity for

3044 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19
Robson et al.
Ta ble 2. Dihydrofolate Reductase-Inhibitory Activity (Ki)a and in Vivo Antitumor Activity against the M5076 Reticulum Cell
Sarcoma in Miceb for Selected 2,4-Diamino-5-aryl-6-ethylpyrimidines
M5076 reticulum cell sarcoma
optimum treatment,
mg/kg/dayc
survivors
no.
MTX
DDMP (1)
6
10
12
14
solvent
Ki (nM)
T/C, %d
(test/control)e
3.125
3.125
25
12.5
NDg
12.5
92
5/5
1/5
5/5
5/5
ND
5/5
A
A
B
C
A
0.12 ( 0.04
<8f
37
0.009 ( 0.002
0.0004 ( 0.0003
0.014 ( 0.011
0.00035 ( 0.00029
100
ND
15
a
Partially purified rat liver DHFR (EC 1.5.1.3) was prepared by the method of Bertino and Fischer.45 Inhibitory activity was determined
spectrophotometrically in duplicate at a minimum of five inhibitor concentrations, Ki values being obtained by the zone B analysis method,39
assuming a Km value of 0.2 µM for dihydrofolate.46 Solvents: (A) 0.1 M hydrochloric acid; (B) 95% ethanol; (C) DMSO. 106 cells implanted
b
im into the left leg of BDF1 female mice (groups of 5) on day 0. Drug as a solution in 10-20% DMSO in arachis oil was administered ip
daily on days 1-17, and tumor volumes were measured on days 12, 16, 20, and 24 after tumor implantation. Control animals received
vehicle only. c Drug administered at dose levels of 12.5, 6.25, 3.125, 1.5, and 0.75 mg‚kg-1, the optimum treatment dose being that nearest
d
in value to the LD10
.
Antitumor activity expressed as a ratio of tumor volumes of test (T) animals compared to control (C) animals ×
100. e On day 24 when all animals were sacrificed. f Maximal activity for this system. Not determined.
g
2-[(4-Ch lor om eth yl)ph en yl]-4,4-dim eth yloxazolin e (18).
The introduction of carboxylate or carboxamide sub-
stituents onto the benzylamino ring of 5 and 6 was
initially predicted to enhance inhibitory activity by
facilitating binding to the putative PABA binding site
of DHFR. However, subsequent X-ray analysis41 and
molecular modeling studies42 conducted with 6 have
indicated that the antifolate exhibits very different
binding characteristics compared with other lipophilic
antifolates and MTX. Interestingly, the preferred bind-
ing orientation positions the central nitrophenyl ring
of 6 into the NAD+ binding domain, such that competi-
tion with the cofactor occurs. In addition, the pendant
benzylamino substituent occupies a large hydrophobic
pocket which will clearly accommodate much bulkier
substituents. In light of these observations, more
detailed kinetic studies are in progress with 14 and
related compounds, with a view to establishing the
interaction of these compounds with DHFR at the
molecular level.
To a stirred solution of 4-(chloromethyl)benzoic acid (5 g, 29
mmol) in dry THF (40 mL) was added thionyl chloride (2.3
mL, 32 mmol) and DMF (0.1 mL), and the reaction mixture
was stirred at ambient temperature for 48 h. Evaporation of
solvents in vacuo gave a green solid which was redissolved in
dichloromethane (10 mL), and added dropwise to a stirred
solution of 2-amino-2-methylpropan-1-ol (7.63 g, 57 mmol) in
dichloromethane (10 mL) at 0 °C. The mixture was allowed
to warm to room temperature and stirred for a further 12 h,
and the white solid which remained on removal of the solvent
was triturated with H2O and collected. Thionyl chloride (5
mL) was added dropwise to the solid with stirring, and when
the vigorous reaction had subsided, the mixture was triturated
with cold ether (100 mL). The white crystals which deposited
were collected and dissolved in H2O, and the solution was
neutralized with 5 M NaOH solution. The solution was
extracted with ether (3 × 20 mL), the combined organic
extracts were dried (MgSO4), and the solvent was evaporated
to furnish a white solid. Recrystallization from EtOAc gave
1
18 (3.62 g, 55%): mp 73-75 °C; H-NMR (200 MHz, DMSO-
d6) δ 1.39 (6H, s, CH3 × 2), 4.22 (2H, s, OCH2), 4.93 (2H, s,
CH2Cl), 7.61 (2H, d, J ) 8.2 Hz, 2,6-ArH), 7.98 (2H, d, J ) 8.2
Hz, 3,5-ArH); MS (EI) m/z 223 [M]+. Anal. (C12H14NOCl) C,
H, N.
Exp er im en ta l Section
Melting points were obtained on an electrothermal melting
point apparatus and are uncorrected. Infrared spectra (IR)
were recorded on a Unicam SP200 infrared spectrometer or a
Nicolet 205X spectrometer. Mass spectra were determined on
a AE1 MS9 or Kratos MS80 spectrometer in electron impact
(EI) mode or on a Kratos instrument using a m-nitrobenzyl
alcohol matrix in fast atom bombardment (FAB) mode. UV
spectra were recorded in EtOH on a Pye Unicam SP8000 or a
UVIKON 810 recording spectrophotometer. 1H-NMR spectra
were obtained on either a Bruker Spectrospin AC 200E (200
MHz) or a Bruker WH 400 (400 MHz) spectrometer, employing
TMS as internal standard. Unless indicated otherwise, spectra
were recorded in [2H6]DMSO as solvent. NH signals appeared
as broad singlets (br s) exchangeable with D2O. Key: t )
triplet, s ) singlet, q ) quartet, d ) doublet, dd ) double
doublet, m ) multiplet. The TLC systems employed aluminum
sheets precoated with Kieselgel 60F254 (0.2 mm) as the
adsorbent and were visualized with light at 254 and 366 nm.
Column chromatography was conducted on silica gel (Kieselgel
60, 240-400 mesh). Elemental analyses were performed by
Butterworth Laboratories, Middlesex, U.K., and are within
(0.4% of theory unless otherwise specified. Reagents were
purchased from Aldrich Chemical Co., Gillingham, U.K., and
used as received unless otherwise stated. Ethanol and metha-
nol were dried using Mg/I2 and stored over 3 Å molecular
sieves. Diethyl ether and tetrahydrofuran were predried over
CaCl2 and distilled from sodium/benzophenone. Benzene was
static dried over alumina. Petroleum ether refers to that
fraction in the boiling range 60-80 °C.
2-[4-[(N-Met h yla m in o)m et h yl]p h en yl]-4,4-d im et h yl-
oxa zolin e (19). A suspension of the oxazoline 18 (2 g, 8.95
mmol) in 33% methylamine in EtOH (30 mL) was stirred at
room temperature overnight, the solvent was removed under
reduced pressure, and H2O (15 mL) was added. The mixture
was extracted with EtOAc (3 × 25 mL), and the combined
organic layers were dried (MgSO4). The crude product was
purified by chromatography using 3:2 (v/v) CH2Cl2/MeOH as
eluent, to give a colorless oil (1.46 g, 75%): 1H-NMR (200 MHz,
DMSO-d6) δ 1.37 (6H, s, CH3 × 2), 2.34 (3H, s, NHCH3), 3.77
(2H, s, OCH2), 4.19 (2H, s, CH2NHCH3), 5.30 (1H, br s, NH),
7.51 (2H, d, J ) 8.1 Hz, 2,6-ArH), 7.86 (2H, d, J ) 8.2 Hz,
3,5-ArH); MS (EI) m/z 218 [M]+. Anal. (C13H18N2O‚0.3H2O)
C, H, N.
2-(4-Cya n op h en yl)d ioxola n e (20).43 A mixture of 4-cy-
anobenzaldehyde (1 g, 7.63 mmol), ethane-1,2-diol (2 g, 30.53
mmol), and p-toluenesulfonic acid (0.2 g) in benzene (10 mL)
was stirred under reflux for 21 h, with the continuous removal
of H2O (Dean-Stark). The solvent was removed by distilla-
tion, and the residue was redissolved in EtOAc (15 mL) and
washed successively with saturated sodium bicarbonate solu-
tion (10 mL) and water (10 mL), and the organic layer was
dried (MgSO4). Removal of the solvent gave a yellow solid,
and pure 20 was obtained following recrystallization from
petroleum ether (0.98 g, 73%): mp 59-61 °C; IR (KBr) 2959,
1
2229, 1284 cm-1; H-NMR (200 MHz, DMSO-d6) δ 4.18 (4H,
m, OCH2CH2O), 5.95 (1H, s, OCHO), 7.77 (2H, d, J ) 8.1 Hz,
2,6-ArH), 8.09 (2H, d, J ) 8.0 Hz, 3,5-ArH); MS (EI) m/z 175
[M]+. Anal. (C10H9NO2) C, H, N.

2,4-Diamino-5-aryl-6-ethylpyrimidine Antifolates
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19 3045
2-[4-(Am in om eth yl)p h en yl]d ioxola n e (21).44 To a solu-
tion of the acetal 20 (0.25 g, 1.45 mmol) in THF (10 mL) was
added lithium triethylborohydride (1 M solution in THF, 7.15
mL, 7.15 mmol), and the mixture was stirred under a nitrogen
atmosphere at ambient temperature for 12 h. H2O (5 mL) was
added cautiously, and after extraction with ether (3 × 10 mL),
the organic layers were combined and dried (MgSO4), and the
solvent was removed to give a clear oil (0.24 g, 92%): 1H-NMR
(200 MHz, DMSO-d6) δ 3.80 (2H, s, CH2NH2), 4.15 (4H, m,
OCH2CH2O), 5.20 (2H, br s, NH2), 5.81 (1H, s, OCHO), 7.49
(4H, s, ArH); MS (EI) m/z 179 [M]+.
(3H, t, J ) 7.0 Hz, OCH2CH3), 2.11 (2H, q, J ) 7.5 Hz, CH2-
CH3), 2.75 (3H, s, NCH3), 3.50 (2H, q, J ) 7.0 Hz, OCH2CH3),
3.69 (2H, m, CO2CH2CH2OEt), 4.38 (2H, m, CO2CH2CH2OEt),
4.51 (2H, br s, ArCH2N), 5.69 (2H, br s, NH2), 5.88 (2H, br s,
NH2), 7.28 (2H, m, 5,6-ArH), 7.46 (2H, d, J ) 8.4 Hz, 2′,6′-
ArH), 7.56 (1H, m, 2-ArH), 7.95 (2H, d, J ) 8.4 Hz, 3′,5′-ArH);
HRMS (EI) m/z 494.2273 [M]+ (C25H30N6O5 requires 495.2278).
Anal. (C25H30N6O5) C, H, N.
2,4-Dia m in o-5-{4′-[N-[4′′-(N′-m eth ylca r ba m oyl)ben zyl]-
N-m eth yla m in o]-3′-n itr op h en yl}-6-eth ylp yr im id in e (14).
a . F r om Nitr op yr im eth a m in e (8). A mixture of N-methyl-
4-[(methylamino)methyl]benzamide (2.56 g, 14.4 mmol) and
nitropyrimethamine (8) (2.1 g, 7.2 mmol) in 2-ethoxyethanol
(25 mL) was heated under reflux for 6 h, cooled, and poured
into ice-H2O (100 mL). The solid which precipitated was
collected and washed thoroughly with H2O. Recrystallization
from EtOAc-petroleum ether (bp 60-80 °C) furnished 14 as
an amorphous orange powder (0.8 g, 26%): mp 235-237 °C;
1H-NMR (200 MHz, DMSO-d6) δ 0.97 (3H, t, J ) 7.5 Hz,
CH2CH3), 2.12 (2H, q, J ) 7.5 Hz, CH2CH3), 2.73 (3H, s, NCH3),
2.78 (3H, d, CONHCH3), 4.47 (2H, br s, ArCH2N), 5.70 (2H,
br s, NH2), 5.89 (2H, br s, NH2), 7.28 (2H, m, 5,6-ArH), 7.38
(2H, d, J ) 8.2 Hz, 2′,6′-ArH), 7.56 (1H, m, 2-ArH), 7.81 (2H,
d, J ) 8.2 Hz, 3′,5′-ArH); HRMS (EI) m/z 435.2104 [M]+
(C22H25N7O3 requires 435.2019). Anal. (C22H25N7O3) C, H, N.
b. F r om F lu or on itr op yr im eth a m in e (15). A solution
of 15 (0.23 g, 0.84 mmol), N-methyl-4-[(methylamino)methyl]-
2,4-Diam in o-5-[4′-(N,N-dim eth ylam in o)-3′-n itr oph en yl]-
6-eth ylp yr im id in e (11). A mixture of 8 (5.0 g, 17 mmol),
sodium acetate (9 g, 80 mmol), copper(II) chloride dihydrate
(0.05 g, 0.03 mmol), and 4-(aminomethyl)benzoic acid (6.8 g,
80 mmol) in DMF (20 mL) was heated under reflux for 6 h.
The DMF was removed in vacuo, and the orange residue was
triturated with H2O and collected. Recrystallization from
EtOH afforded 11 as the N-formyl-4-(aminomethyl)benzoate
salt (5.41 g, 66%): mp 237-239 °C; IR (mineral oil) 3385, 3050,
1
2830, 1680, 1640, 1541 cm-1; H-NMR (400 MHz, DMSO-d6)
δ 1.04 (3H, t, J ) 7.2 Hz, CH2CH3), 2.20 (2H, q, J ) 7.2 Hz,
CH2CH3), 2.97 (6H, s, N(CH3)2), 4.48 (2H, d, 4-CHOHNC6H4CH2-
CO2-), 6.12 (2H, br s, NH2), 6.54 (2H, br s, NH2), 7.28 (1H, d,
J ) 8.7 Hz, 5-ArH), 7.35 (1H, dd, J ) 8.7 Hz, J ′ ) 2.1 Hz,
6-ArH), 7.38 (2H, d, J ) 7.5 Hz, 3,5-ArH of 4-CHOHNC6H4-
CH2CO2-), 7.67 (1H, d, J ) 2.1 Hz, 2-ArH), 8.01 (2H, d, J )
7.7 Hz, 2,6-ArH of 4-CHOHNC6H4CH2CO2-), 8.21 (1H, s,
ArNHCHO), 8.66 (1H, t, ArNHCHO); MS (EI) m/z 302 [M]+.
Dissolution of salt 11 in H2O and addition of 1 M NaOH
furnished orange crystals of the pyrimidine free base, which
were identical (1H-NMR, MS, mp) to an authentic sample of
2,4-diamino-5-[4-(N,N-dimethylamino)-3-nitrophenyl]-6-eth-
ylpyrimidine.24
benzamide (0.36 g, 1.68 mmol), and NEt3
(0.36 g, 3.6 mmol)
in 2-ethoxyethanol (15 mL) was stirred at 80 °C for 16 h. The
solvent was removed in vacuo, the residue was suspended in
H2O (30 mL) and extracted with EtOAc (3 × 15 mL), and the
combined organic layers were dried (MgSO4). Evaporation of
the solvent and recrystallization from MeOH afforded 14 (0.28
g, 76%), which was identical (TLC, MS, NMR) to that produced
by method a above.
2,4-Dia m in o-5-[4′-[N-[4′′-(m eth oxyca r bon yl)ben zyl]-N-
m eth yla m in o]-3′-n itr op h en yl]-6-eth ylp yr im id in e (12). A
solution of methyl 4-[(methylamino)methyl]benzoate (1.06 g,
6 mmol), 8 (0.86 g, 3 mmol), and NEt3 (0.8 mL, 9 mmol) in
2-ethoxyethanol (10 mL) was heated under reflux for 72 h.
Evaporation of the solvent gave a red syrup, and chromatog-
raphy on silica gel with 95:5 (v/v) CHCl3/MeOH as eluent
afforded a yellow oil characterized (1H-NMR, MS) as compris-
ing a mixture of the methyl (12) and 2-ethoxyethyl (13) esters.
The mixture was redissolved in MeOH (30 mL) containing
sulfuric acid (0.5 mL) and boiled for 6 h, and the solvent was
evaporated under reduced pressure. The remaining orange
solid was dissolved in EtOAc (50 mL), washed with 10%
aqueous sodium carbonate solution, and filtered, and the
solvent was removed to furnish the methyl ester 12 (0.16 g,
6.0%): mp 198-200 °C; IR (mineral oil) 3490, 3320, 3150, 1720
2,4-Dia m in o-5-{4′-[N-(4′′-ca r boxyben zyl)-N-m eth yla m i-
n o]-3′-n itr op h en yl}-6-eth ylp yr im id in e (10). A mixture of
methyl ester 12 (0.92 g, 2.11 mmol) and NaOH (0.5 g, 12.5
mmol) in MeOH (25 mL) was refluxed for 16 h. After cooling,
the solution was acidified to pH 6.0 with concentrated HCl,
and the solid which deposited was collected and washed with
cold MeOH. The orange powder (0.72 g, 80%) was dissolved
in hot H2O containing ethanesulfonic acid (0.21g 1.88 mmol),
and the precipitate which formed on cooling was collected and
recrystallized from H2O to afford the benzoic acid monohydrate
1
(0.41 g, 44%): mp 244-246 °C; H-NMR (400 MHz, DMSO-
d6) δ 0.96 (3H, t, J ) 7.5 Hz, CH2CH3), 2.12 (2H, q, J ) 7.5
Hz, CH2CH3), 2.74 (3H, s, NCH3), 4.49 (2H, s, ArCH2N), 5.86
(2H, br s, NH2), 6.10 (2H, br s, NH2), 7.27 (2H, m, 5,6-ArH),
7.40 (2H, d, J ) 8.1 Hz, 2′,6′-ArH), 7.56 (1H, s, 2-ArH), 7.91
(2H, d, J ) 8.1 Hz, 3′,5′-ArH); MS (FAB) m/z 423 [M + 1]+.
Anal. (C21H22N6O4‚1H2O) C, H, N.
cm-1 1H-NMR (400 MHz, DMSO-d6) δ 0.96 (3H, t, J ) 7.5
;
Hz, CH2CH3), 2.11 (2H, q, J ) 7.5 Hz, CH2CH3), 2.74 (3H, s,
NCH3), 3.84 (3H, s, CO2Me), 4.50 (2H, br s, ArCH2N), 5.75 (2H,
br s, NH2), 5.92 (2H, br s, NH2), 7.28 (2H, s, 5,6-ArH), 7.45
(2H, d, J ) 8.2 Hz, 2′,6′-ArH), 7.56 (1H, s, 2-ArH), 7.95 (2H,
d, J ) 8.2 Hz, 3′,5′-ArH); MS (EI) m/z 436 [M]+. Anal.
(C22H24N6O4) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
9, 16, 17, a n d 22. To a solution of fluoronitropyrimethamine
(15) in N-methyl-2-pyrrolidone (15 mL) was added NEt3
. The
solution was purged with N2, and after addition of the
appropriate benzylamine derivative, the reaction mixture was
stirred under N2 at 100 °C until TLC analysis indicated the
absence of 15 (8-10 h). The solvent was evaporated in vacuo,
and the remaining solid was triturated with H2O, collected
by filtration, and purified by recrystallization.
2,4-Dia m in o-5-{4′-[N-[4′′-[(2-et h oxyet h oxy)ca r b on yl]-
ben zyl]-N-m eth yla m in o]-3′-n itr op h en yl}-6-eth ylp yr im i-
d in e (13). Thionyl chloride (6 mL, 83 mmol) was added
dropwise cautiously to a solution of 4-[(methylamino)methyl]-
benzoic acid in 2-ethoxyethanol (100 mL). After the mixture
stirred for 16 h at 50 °C, the solvent was removed under
reduced pressure, the remaining cream solid was dissolved in
CHCl3 (50 mL) and washed with 10% aqueous potassium
carbonate (2 × 50 mL), and the organic layer was dried (Na2-
SO4) and filtered. Evaporation of the solvent gave 2-ethoxy-
ethyl 4-[(N-methylamino)methyl]benzoate as a colorless oil (3.5
g, 54%). Without further purification the ester (3.5 g, 15 mmol)
was added to a solution of 8 (3.5 g, 12 mmol) in 2-ethoxyethanol
(10 mL), and the mixture was stirred under reflux for 20 h.
After evaporation of the solvent, the residual red oil was
purified by chromatography on silica gel, employing 9:1 (v/v)
CHCl3/MeOH as eluent, to furnish the 2-ethoxyethyl ester 13
as a yellow powder (0.18 g, 3%): mp 114-116 °C; 1H-NMR
(400 MHz, DMSO-d6) δ 0.97 (3H, t, J ) 7.5 Hz, CH2CH3), 1.11
2,4-Dia m in o-5-{4′-[N-(4′′-ca r b oxyb en zyl)a m in o]-3′-n i-
tr op h en yl}-6-eth ylp yr im id in e (9). Compound 9 was syn-
thesized from 15 (0.1 g, 0.36 mmol), 4-(aminomethyl)benzoic
acid (0.11 g, 0.72 mmol), and NEt3 (0.36 g, 3.6 mmol) and
purified by recrystallization from EtOH (0.10 g, 69%): mp
292-294 °C; 1H-NMR (400 MHz, DMSO-d6) δ 1.09 (3H, t, J )
7.4 Hz, CH2CH3), 2.23 (2H, q, J ) 7.3 Hz, CH2CH3), 4.85 (2H,
d, NHCH2), 6.19 (2H, br s, NH2), 6.49 (2H, br s, NH2), 7.08
(1H, d, J ortho ) 8.9 Hz, 5-ArH), 7.41 (1H, dd, J ortho ) 8.8 Hz,
J meta ) 1.9 Hz, 6-ArH), 7.63 (2H, d, J ortho ) 8.1 Hz, 2′,6′-ArH),
7.96 (1H, d, J meta ) 1.9 Hz, 2-ArH), 8.01 (2H, d, J ortho ) 8.1
Hz, 3′,5′-ArH), 8.94 (1H, t, NH); high-resolution EIMS 408.1545
(calcd 408.1546). Anal. (C20H20N6O4‚0.5H2O) C, H, N.
2,4-Dia m in o-5-{4′-[N-(4′′-su lfon a m id oben zyl)a m in o]-3′-
n itr op h en yl}-6-eth ylp yr im id in e (16). Compound 16 was

3046 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19
Robson et al.
synthesized from 15 (1.0 g, 3.61 mmol), 4-(aminomethyl)-
benzenesulfonamide (0.84 g, 3.76 mmol), and NEt3 (1.02 g, 10.2
mmol) and purified by recrystallization from MeOH (1.12 g,
69.9%): mp 266-268 °C; 1H-NMR (200 MHz, DMSO-d6) δ 1.09
(3H, t, J ) 7.5 Hz, CH2CH3), 2.25 (2H, q, J ) 7.5 Hz, CH2-
CH3), 4.86 (2H, d, NHCH2), 5.90 (2H, br s, NH2), 6.10 (2H, br
s, NH2), 7.03 (1H, d, J ortho ) 8.9 Hz, 5-ArH), 7.41 (1H, dd, J ortho
) 8.8 Hz, J meta ) 1.9 Hz, 6-ArH), 7.63 (2H, d, J ortho ) 8.1 Hz,
2′,6′-ArH), 7.96 (1H, d, J meta ) 1.9 Hz, 2-ArH), 8.01 (2H, d,
J ortho ) 8.1 Hz, 3′,5′-ArH), 8.94 (1H, t, NH); MS (FAB) m/z 408
[M + 1]+. Anal. (C19H21N7O4S) C, H, N.
microcrystals: mp 263-264 °C (lit.24 mp 262-265 °C); 1H-
NMR (200 MHz, DMSO-d6) δ 1.01 (3H, t, J ) 7.5 Hz, CH2CH3),
2.22 (2H, q, J ) 7.5 Hz, CH2CH3), 3.09 (3H, d, J ) 4.8 Hz,
NHCH3), 5.71 (2H, br s, NH2), 5.86 (2H, br s, NH2), 7.23 (1H,
d, J ortho ) 9.0 Hz, 5-ArH), 7.53 (1H, dd, J ortho ) 8.9 Hz, J meta
)
2.0 Hz, 6-ArH), 8.02 (1H, d, J meta ) 1.9 Hz, 2-ArH), 8.43 (1H,
q, J ) 4.9 Hz, NHCH3); MS (EI) m/z 288 [M]+.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
25-28. A stirred solution of the carboxylic acid 9 in anhydrous
DMF (15 mL) was cooled to 0 °C, and NEt3 was added. After
the addition of isobutyl chloroformate, the solution was stirred
under N2 for 1 h at 0 °C, the appropriate amine was added,
and stirring was continued at 0 °C for a further 1 h and then
for 12 h at ambient temperature. The solvent was removed
in vacuo, and the product was purified by chromatography on
silica gel, using 5:1 (v/v) CH2Cl2/MeOH as eluent.
2,4-Dia m in o-5-{4′-[N-[4′′-(4,4-d im et h yloxa zolin -2-yl)-
ben zyl]a m in o]-3′-n it r op h en yl}-6-et h ylp yr im id in e (17).
Compound 17 was synthesized from 15 (0.1 g, 0.361 mmol),
19 (0.16 g, 0.72 mmol), and NEt3 (0.36 g, 3.6 mmol) and
purified by recrystallization from EtOAc-petroleum ether
1
(0.14 g, 83%): mp 173-175 °C; H-NMR (200 MHz, DMSO-
2,4-Dia m in o-5-{4′-[N-(4′′-ca r b a m oylb en zyl)a m in o]-3′-
n itr op h en yl}-6-eth ylp yr im id in e (25). Compound 25 was
synthesized from 9 (0.2 g, 0.49 mmol), isobutyl chloroformate
(0.08 mmol), ammonium hydroxide (30% (w/v) in H2O, 0.5 mL),
and NEt3 (0.12 g, 1.1 mmol) and recrystallized from MeOH
(0.16 g, 79%): mp 201-203 °C (0.58 mmol); 1H-NMR (200
MHz, DMSO-d6) δ 1.11 (3H, t, J ) 7.6 Hz, CH2CH3), 2.25 (2H,
q, J )7.5 Hz, CH2CH3), 2.80 (3H, d, J ) 4.4 Hz, NHCH3), 4.81
(2H, d, J ) 5.8 Hz, NHCH2), 6.65 (4H, br s, 2 × NH2), 7.07
(1H, d, J ortho ) 8.9 Hz, 5-ArH), 7.39 (1H, dd, J ortho ) 8.7 Hz,
J meta ) 2.0 Hz, 6-ArH), 7.42 (1H, br s, NH), 7.56 (2H, d, J )
8.1 Hz, 2′/6′-ArH), 7.95 (2H, d, J ) 8.2 Hz, 3′/5′-ArH), 8.01
(1H, d, J meta ) 2.0 Hz, 2-ArH), 8.10 (1H, br s, NH), 8.54 (1H,
m, NHCH3), 8.99 (1H, m, J ) 5.8 Hz, NHCH2); high-resolution
EIMS 407.1715 (calcd 407.1706). Anal. Calcd (C20H21N7O3‚
0.85DMF): C, 51.16; H, 4.48; N, 20.89. Found: C, 51.88; H,
4.95; N, 20.16.
d6) δ 1.08 (3H, t, J ) 7.5 Hz, CH2CH3), 1.39 (6H, s, C(CH3)2),
2.21 (2H, q, J ) 7.6 Hz, CH2CH3), 2.85 (3H, s, NCH3), 4.21
(2H, s, OCH2), 5.85 (2H, d, NMeCH2), 5.85 (2H, br s, NH2),
6.04 (2H, br s, NH2), 7.39 (2H, m, 5,6-ArH), 7.48 (2H, d, J )
8.0 Hz, 2′,6′-ArH), 7.68 (1H, d, J meta ) 1.8 Hz, 2-ArH), 7.92
(2H, d, J ) 8.1 Hz, 3′,5′-ArH); MS (FAB) m/z 476 [M + 1]+.
Anal. (C25H29N7O3‚0.5H2O) C, H, N.
2,4-Dia m in o-5-{4′-[N-(4′′-d ioxola n -2-ylb en zyl)a m in o]-
3′-n itr op h en yl}-6-eth ylp yr im id in e (22). Compound 22 was
synthesized from 15 (0.2 g, 0.72 mmol), 21 (0.26 g, 1.44 mmol),
and NEt3 (0.72 g, 7.2 mmol) and purified by recrystallization
from EtOAc-petroleum ether (0.26 g, 82%): mp 283-285 °C;
1H-NMR (400 MHz, DMSO-d6) δ 1.07 (3H, t, J ) 7.4 Hz,
CH2CH3), 2.20 (2H, q, J ) 7.4 Hz, CH2CH3), 4.16 (4H, m,
OCH2CH2O), 4.80 (2H, d, J ) 5.8 Hz, NHCH2), 5.85 (1H, s,
OCHO), 5.93 (2H, br s, NH2), 6.02 (2H, br s, NH2), 7.06 (1H,
d, J ortho ) 8.9 Hz, 5-ArH), 7.39 (1H, dd, J ortho ) 8.8 Hz, J meta
)
2,4-Dia m in o-5-{4′-[N-[4′′-(N′-m eth ylca r ba m oyl)ben zyl]-
a m in o]-3′-n it r op h en yl}-6-et h ylp yr im id in e (26). Com-
pound 26 was synthesized from 9 (0.2 g, 0.49 mmol), isobutyl
chloroformate (0.08 g, 0.58 mmol), methylamine (33% (w/v)
solution in EtOH; 1 mL, 10.6 mmol), and NEt3 (0.12 g, 1.1
mmol) and recrystallized from MeOH (0.16 g, 76%): mp 209-
2.0 Hz, 6-ArH), 7.55 (4H, s, 2′,3′,5′,6′-ArH), 7.94 (1H, d, J meta
) 2.0 Hz, 2-ArH), 8.88 (1H, t, J ) 6.0 Hz, NHCH2); high-
resolution EIMS 436.1867 (calcd 436.1859).
Anal.
(C22H24N6O4‚0.4H2O) C, H, N.
2,4-Dia m in o-5-{4′-[N-(4′′-for m ylben zyl)a m in o]-3′-n itr o-
p h en yl}-6-eth ylp yr im id in e (23). The acetal derivative 22
(80 mg, 0.183 mmol) was suspended in aqueous acetic acid
(1.5 M, 0.8 mL), and glacial acetic acid was added dropwise
until a clear solution was obtained. The reaction mixture was
stirred at room temperature for 12 h, and evaporation of the
solvents under reduced pressure gave the title compound as a
red solid (69.5 mg, 97%): mp 271-273 °C; 1H-NMR (200 MHz,
DMSO-d6) δ 1.10 (3H, t, J ) 7.4 Hz, CH2CH3), 2.22 (2H, q, J
) 7.4 Hz, CH2CH3), 4.89 (2H, m, CH2NH), 5.91 (2H, br s, NH2),
6.23 (2H, br s, NH2), 7.08 (1H, d, J ortho ) 8.9 Hz, 5-ArH), 7.43
(1H, dd, J ortho ) 8.9 Hz, J meta ) 1.8 Hz, 6-ArH), 7.60 (2H, d,
J ortho ) 8.1 Hz, 2′,6′-ArH), 7.76 (2H, d, J ortho ) 8.0 Hz, 3′,5′-
ArH), 7.99 (1H, d, J meta ) 2.3 Hz, 2-ArH), 8.97 (1H, t, NHCH2),
10.012 (1H, s, CHO); MS (FAB) m/z 393 [M + 1]+. Anal. Calcd
(C20H20N6O2‚0.4H2O): C, 59.56; H, 5.42; N, 18.95. Found: C,
60.07; H, 5.71; N, 18.29.
1
211 °C; H-NMR (200 MHz, DMSO-d6) δ 1.07 (3H, t, J ) 7.5
Hz, CH2CH3), 2.23 (2H, q, J ) 7.3 Hz, CH2CH3), 2.80 (3H, d,
J ) 4.4 Hz, NHCH3), 4.84 (2H, d, NHCH2), 6.65 (2H, br s,
NH2), 6.84 (2H, br s, NH2), 7.06 (1H, d, J ortho ) 8.9 Hz, 5-ArH),
7.42 (1H, dd, J ortho ) 8.8 Hz, J meta ) 1.9 Hz, 6-ArH), 7.62 (2H,
d, J ) 7.3 Hz, 2′/6′-ArH), 7.87 (2H, d, J ) 7.3 Hz, 3′/5′-ArH),
8.01 (1H, d, J meta ) 1.9 Hz, 2-ArH), 8.54 (1H, m, NHCH3), 8.95
(1H, m, NHCH2); high-resolution EIMS 421.1860 (calcd
421.1862). Anal. Calcd (C21H23N7O3‚2H2O): C, 55.13; H, 5.95;
N, 21.43. Found: C, 54.67; H, 5.30; N, 20.71.
2,4-Dia m in o-5-{4′-[N-[4′′-(N′,N′-d im et h ylca r b a m oyl)-
ben zyl]a m in o]-3′-n it r op h en yl}-6-et h ylp yr im id in e (27).
Compound 27 was synthesized from 9 (0.1 g, 0.25 mmol),
isobutyl chloroformate (0.10 g, 0.70 mmol), dimethylamine
(33% (w/v) solution in EtOH; 2 mL, 15 mmol), and NEt3 (0.06
g, 0.55 mmol) and recrystallized from MeOH (75 mg, 70%):
mp 166-168 °C; 1H-NMR (200 MHz, DMSO-d6) δ 1.07 (3H, t,
J ) 7.4 Hz, CH2CH3), 2.23 (2H, q, J ) 7.5 Hz, CH2CH3), 3.017
(6H, s, N(CH3)2), 4.83 (2H, d, NHCH2), 5.90 (4H, br s, 2 × NH2),
7.19 (1H, d, J ortho ) 8.9 Hz, 5-ArH), 7.41 (1H, dd, J ortho ) 8.8
Hz, J meta ) 2.0 Hz, 6-ArH), 7.52 (2H, d, J ) 8.3 Hz, 2′/6′-ArH),
7.61 (2H, d, J ) 8.3 Hz, 3′/5′-ArH), 7.95 (1H, d, J meta ) 2.0 Hz,
2-ArH), 8.951 (1H, m, NHCH2); high-resolution EIMS 435.2012
(calcd 435.2019). Anal. Calcd (C22H25N7O3‚1H2O): C, 58.25;
H, 6.00; N, 21.63. Found: C, 58.74; H, 5.75; N, 21.03.
2,4-Diam in o-5-{4′-[N-[4′′-(1-piper idin ocar bon yl)ben zyl]-
a m in o]-3′-n it r op h en yl}-6-et h ylp yr im id in e (28). Com-
pound 28 was synthesized from 9 (0.2 g, 0.49 mmol), isobutyl
chloroformate (0.14 g, 1.0 mmol), piperidine (50 mg, 0.59
mmol), and NEt3 (0.12 g, 1.1 mmol) and recrystallized from
MeOH (0.18 g, 77%): mp 192-195 °C; 1H-NMR (200 MHz,
DMSO-d6) δ 1.15 (3H, t, J ) 7.5 Hz, CH2CH3), 1.74 (6H, m,
C3H6), 2.31 (2H, q, J ) 7.5 Hz, CH2CH3), 3.20 (4H, m, CH2-
NCH2), 4.88 (2H, d, J ) 5.3 Hz, NHCH2), 6.45 (4H, br s, 2 ×
NH2), 7.20 (1H, d, J ortho ) 8.9 Hz, 5-ArH), 7.54 (5H, m, 6-ArH,
2′/3′/5′/6′-ArH), 8.03 (1H, d, J meta ) 1.9 Hz, 2-ArH), 8.99 (1H,
2,4-Dia m in o-5-{4′-[N-(4′′-ca r b oxyb en zyl)a m in o]-3′-n i-
tr op h en yl}-6-eth ylp yr im id in e (9) fr om 17. To a mixture
of sodium hypochlorite solution (0.2 M, 25 mL) and EtOAc (5
mL) were added 17 (0.2 g, 0.42 mmol) and tetrabutylammo-
nium hydrogen sulfate (14.3 mg, 0.042 mmol), and the mixture
was stirred at 25 °C for 48 h. The EtOAc layer was separated,
and the solvent was removed in vacuo to give a yellow oil,
which was redissolved in MeOH. NaOH solution (1.25 M, 6
mL) was added, and the reaction mixture was stirred at room
temperature for 48 h. After extraction with EtOAc (3 × 15
mL), the combined organic layers were dried (MgSO4), and the
solvent was removed to give an orange solid (9 mg, 5%)
identical (TLC, MS) to an authentic sample of 9.
2,4-Dia m in o-5-[4′-(N-m eth yla m in o)-3′-n itr op h en yl]-6-
eth ylp yr im id in e (24). To a suspension of 15 (0.2 g, 0.72
mmol) in N-methyl-2-pyrrolidone (15 mL) were added [(N-
methylamino)methyl]benzoic acid (0.24 g, 1.44 mmol) and NEt3
(0.72 g, 7.2 mmol). The reaction mixture was stirred at 100
°C for 2 h, and the solvent was removed under reduced
pressure. Trituration of the residue with H2O and recrystal-
lization from EtOH gave 0.18 g (87%) of 24 as orange

2,4-Diamino-5-aryl-6-ethylpyrimidine Antifolates
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19 3047
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t, J ) 5.3 Hz, NHCH2); high-resolution EIMS 475.2325 (calcd
475.2332). Anal. (C25H29N7O3‚0.6H2O) C, H, N.
Dieth yl N-{4-[[N′-[2-Nitr o-4-(2,4-d ia m in o-6-eth ylp yr i-
m id in -5-yl)a n ilin o]]m eth yl]ben zoyl}-L-glu ta m a te (29). To
a stirred solution of 9 (90 mg, 0.22 mmol) in anhydrous DMF
(10 mL) at 0 °C was added NEt3 (70 mg, 0.70 mmol) followed
by isobutyl chloroformate (36 mg, 0.26 mmol). After the
solution stirred for 1 h, L-glutamic acid diethyl ester hydro-
chloride (63.5 mg, 0.26 mmol) was added. Stirring was
continued at 0 °C for 4 h and then overnight at ambient
temperature. Evaporation of the solvent under reduced pres-
sure furnished an orange solid which was purified by chro-
matography on silica gel, employing 4:1 (v/v) CH2Cl2/MeOH
as the eluting solvent. Recrystallization from EtOH gave the
title compound (101 mg, 79%): mp 119-121 °C; 1H-NMR (200
MHz, DMSO-d6) δ 1.08 (3H, t, J ) 7.5 Hz, CH2CH3), 1.37 (6H,
m, 2 × CO2CH2CH3), 2.20 (2H, q, J ) 7.5 Hz, CH2CH3), 2.30
(4H, m, CH2CH2CO2Et), 4.23 (4H, m, 2 × CO2CH2CH3), 4.54
(1H, m, NHCHCO2Et), 4.86 (2H, d, J ) 5.5 Hz, NHCH2), 6.68
(2H, br s, NH2), 6.87 (2H, br s, NH2), 7.09 (1H, d, J ortho ) 9.0
Hz, 5-ArH), 7.42 (1H, dd, J ortho ) 8.9 Hz, 6-ArH), 7.61 (2H, d,
J ) 8.1 Hz, 2′/6′-ArH), 7.96 (2H, d, J ) 8.3 Hz, 3′/5′ArH), 8.02
(1H, d, J meta ) 2.1 Hz, 2-ArH), 8.85 (1H, d, J ) 7.4 Hz, CONH),
9.00 (1H, t, J ) 5.4 Hz, NHCH2); MS (FAB) m/z 594 [M + 1]+.
Anal. (C29H35N7O7‚0.5H2O) C, H, N.
N-{4-[[N′-[2-Nit r o-4-(2,4-d ia m in o-6-et h ylp yr im id in -5-
yl)a n ilin o]]m eth yl]ben zoyl}-L-glu ta m a tic Acid (30). The
diethyl ester 29 (30 mg, 0.05 mmol) was dissolved in EtOH (3
mL), and a solution of barium hydroxide octahydrate (31.9 mg,
0.101 mmol) in H2O (2.5 mL) was added. The reaction mixture
was stirred for 12 h at room temperature, and the solid which
precipitated was collected and redissolved in H2O (20 mL).
Sodium sulfate (14 mg, 0.101 mmol) in H2O (1 mL) was added,
and the precipitate which formed after 15 min was removed
by filtration through Celite. The filtrate was evaporated to
dryness under reduced pressure, the remaining solid was
redissolved in a minimum of H2O, and the pH was adjusted
to 2.0 with 1 M HCl. The resulting yellow precipitate was
collected, washed with water, and recrystallized from propan-
2-ol (8.5 mg, 32%): mp 136-138 °C; 1H-NMR (200 MHz,
DMSO-d6) δ 1.10 (3H, t, J ) 7.5 Hz, CH2CH3), 2.20 (2H, q, J
) 7.5 Hz, CH2CH3), 2.29 (4H, m, CH2CH2CO2Et), 4.48 (1H,
m, NHCHCO2H), 4.84 (2H, d, J ) 5.5 Hz, NHCH2), 6.88 (4H,
br s, 2 × NH2), 7.10 (1H, d, J ortho ) 8.9 Hz, 5-ArH), 7.38 (1H,
dd, J ortho ) 9.0 Hz, 6-ArH), 7.63 (2H, d, J ) 8.1 Hz, 2′/6′-ArH),
7.95 (2H, d, J ) 8.1 Hz, 3′/5′-ArH), 8.04 (1H, d, J meta ) 2.0 Hz,
2-ArH), 8.67 (1H, d, J ) 7.5 Hz, CONH), 8.90 (1H, t, J ) 5.5
Hz, NHCH2); MS (FAB) m/z 520 [M + 1 - H2O]+, 406 [M -
CH(CO2H)CH2CH2CO2H]+, 391 [M - NHCH(CO2H)CH2CH2-
CO2H]+.
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selective inhibitors of Toxoplasma gondii dihydrofolate reduc-
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Structural studies on bioactive compounds. Part 8. Synthesis,
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Ack n ow led gm en t. We thank the Cancer Research
Campaign and North of England Cancer Research
Campaign for generous support and the Engineering
and Physical Sciences Research Council, U.K., for the
award of studentships to M.A.M. and C.R. We also
thank Professor B. T. Golding, Department of Chemis-
try, University of Newcastle upon Tyne, for helpful
discussions and Dr. A. L. J ackman of the Institute of
Cancer Research, Sutton, Surrey, for assistance with
the DHFR assays. This work was supported in part by
NIH DAIDS contracts NO1-AI-87240 and NO1-AI-
35171 (S. F. Queener, PI).
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