Synthesis and dihydrofolate reductase inhibitory activities of 2,4- diamino-5-deaza and 2,4-diamino-5,10-dideaza lipophilic antifolates

Article abstract of DOI:10.1021/jm9606913

Two series of nonclassical antifolates (2,4-diamino-5-deaza compounds 2- 5 and 5,10-dideaza compounds 6-13) were synthesized as inhibitors of dihydrofolate reductase (DHFR) from Pneumocystis carinii (pc) and Toxoplasma gondii (tg) organisms that are responsible for fatal opportunistic infections in AIDS patients. Rat liver (rl) DHFR served as the mammalian reference enzyme to determine selectivity. Syntheses of the target 5-deaza compounds were achieved by initial construction of the pivaloyl-protected 2,4-diamino- 6-bromopyrido[2,3-d]-pyrimidine 17 via a cyclocondensation of 2,4,6- triaminopyrimidine with bromomalonaldehyde. Sequential Heck coupling of 17 with styrene followed by ozonolysis afforded the 6-formyl derivative 19. Reductive amination of 19 with 3,4,5-trimethoxyaniline afforded the N10-H analog. The N10-Me and N10-Et analogs were synthesized by nucleophilic displacement of the 6-bromomethyl derivative 22 (obtained from the 6-formyl derivative 19 by reduction and bromination) with the appropriate N- alkylaniline. The trans-5,10-dideaza analogs 6-8 were synthesized via a Heck coupling of the appropriate methoxystyrene with 17, and selective reduction of the resulting 9,10-double bond afforded target compounds 9-11. Further reduction to the tetrahydro derivatives afforded analogs 12 and 13. The 5- deaza N10-Me 3,4,5-trimethoxy analog 3 maintained the best balance of potency and selectivity against both tgDHFR and pcDHFR. Compared to trimethoprim, compound 3 was only slightly less selective but was 300-fold more potent against tgDHFR. The 5,10-dideaza analogs were generally less potent and selective than the 5-deaza compounds.

Full text of DOI:10.1021/jm9606913

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470
J . Med. Chem. 1997, 40, 470-478
Syn th esis a n d Dih yd r ofola te Red u cta se In h ibitor y Activities of
2,4-Dia m in o-5-d ea za a n d 2,4-Dia m in o-5,10-d id ea za Lip op h ilic An tifola tes1
Aleem Gangjee,*,† Rajesh Devraj,† and Sherry F. Queener‡
Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University,
Pittsburgh, Pennsylvania 15282, and Department of Pharmacology and Toxicology, Indiana University
School of Medicine, Indianapolis, Indiana 46202
Received October 4, 1996X
Two series of nonclassical antifolates (2,4-diamino-5-deaza compounds 2-5 and 5,10-dideaza
compounds 6-13) were synthesized as inhibitors of dihydrofolate reductase (DHFR) from
Pneumocystis carinii (pc) and Toxoplasma gondii (tg) organisms that are responsible for fatal
opportunistic infections in AIDS patients. Rat liver (rl) DHFR served as the mammalian
reference enzyme to determine selectivity. Syntheses of the target 5-deaza compounds were
achieved by initial construction of the pivaloyl-protected 2,4-diamino-6-bromopyrido[2,3-d]-
pyrimidine 17 via a cyclocondensation of 2,4,6-triaminopyrimidine with bromomalonaldehyde.
Sequential Heck coupling of 17 with styrene followed by ozonolysis afforded the 6-formyl
derivative 19. Reductive amination of 19 with 3,4,5-trimethoxyaniline afforded the N10-H
analog. The N10-Me and N10-Et analogs were synthesized by nucleophilic displacement of
the 6-bromomethyl derivative 22 (obtained from the 6-formyl derivative 19 by reduction and
bromination) with the appropriate N-alkylaniline. The trans-5,10-dideaza analogs 6-8 were
synthesized via a Heck coupling of the appropriate methoxystyrene with 17, and selective
reduction of the resulting 9,10-double bond afforded target compounds 9-11. Further reduction
to the tetrahydro derivatives afforded analogs 12 and 13. The 5-deaza N10-Me 3,4,5-trimethoxy
analog 3 maintained the best balance of potency and selectivity against both tgDHFR and
pcDHFR. Compared to trimethoprim, compound 3 was only slightly less selective but was
300-fold more potent against tgDHFR. The 5,10-dideaza analogs were generally less potent
and selective than the 5-deaza compounds.
In tr od u ction
sulfonamides or leucovorin and could result in safer and
less expensive therapy.
Opportunistic infections caused by Pneumocystis
carinii and Toxoplasma gondii remain the principal
cause of death in patients with the acquired immune
deficiency syndrome (AIDS).2-5 In recent years several
nonclassical, lipophilic dihydrofolate reductase (DHFR)
inhibitors have been used to treat the infections caused
by these organisms. Although lipophilic antifolates such
as trimethoprim (TMP),6 pyrimethamine,7 trimetrexate,
(TMQ),8,9 and piritrexim (PTX)10 are currently used for
the treatment of these opportunistic infections, they
suffer from several drawbacks. TMP is a selective but
weak inhibitor of DHFR derived from P. carinii and T.
gondii; pyrimethamine is also a weak inhibitor with no
selectivity for P. carinii (pc) DHFR and modest selectiv-
ity for T. gondii (tg) DHFR. Both these drugs must be
used with sulfonamides to provide synergistic effects.11
TMQ and PTX are potent inhibitors of P. carinii DHFR
and T. gondii DHFR; however, they have a higher
affinity for the mammalian enzyme.12,13 Thus, both
TMQ and PTX must be used in combination with the
reduced folate leucovorin (5-formyl-5,6,7,8-tetrahydro-
folate) to selectively protect the host.14,15 In addition
to the high cost and lack of selectivity, the severe side
effects frequently require cessation of treatment with
these antifolate regimens.2,16,17 Thus there has been a
considerable effort to develop more potent and more
selective inhibitors of pcDHFR and tgDHFR. A selec-
tive agent would obviate the necessity to use either
Gangjee et al.18-23 and others24-27 have reported
several bicyclic lipophilic antifolates as potent and/or
selective inhibitors of DHFR isolated from P. carinii and
T. gondii. Investigations of the 2,4-diamino-5-methyl-
6-(anilinomethyl)pyrido[2,3-d]pyrimidine (5-methyl-5-
deazapteridine, 1) class of lipophilic antifolates have
provided agents superior in both selectivity and potency
compared to TMQ and PTX against pcDHFR and
† Duquesne University.
‡ Indiana University School of Medicine.
X Abstract published in Advance ACS Abstracts, J anuary 15, 1997.
S0022-2623(96)00691-7 CCC: $14.00 © 1997 American Chemical Society

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Diaminodeaza Compounds as DHFR Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 471
tgDHFR.18,19,23,24 In particular, the 5-methyl-5-deaza-
pteridine analog with a 3,4,5-trimethoxy substitution
on the phenyl ring and a methyl group on the N10
nitrogen, compound 1a , was reported by Gangjee et al.18
tgDHFR.21 It was anticipated that comparison of activ-
ity across the series 6-13 would illustrate how potency
and/or selectivity against pcDHFR and tgDHFR can be
affected by the side chain phenyl ring orientation and
the number of methoxy groups around the 3-, 4-, and
5-positions of the phenyl ring.
to be an extremely potent inhibitor of tgDHFR (IC50
)
0.85 nM) with good selectivity for this enzyme compared
to rat liver (rl) DHFR (selectivity ratio ) IC50(rlDHFR)/
IC50(tgDHFR) ) 8.94).
Several studies have documented the importance of
the 5-methyl group of 5-deaza folates for potent inhibi-
tion of DHFR from mammalian sources and in some
instances bacterial sources as well.28-30 Thus, removal
of the 5-methyl group from the 5-deaza folates was
expected to potentially increase the selectivity of these
agents provided the decrease in potency against pcDH-
FR and tgDHFR was less than the decrease against
mammalian DHFR. The viability of this idea was
recently demonstrated by Gangjee et al.23 in the N9
methyl, reversed bridge analogue 1b. This compound
was significantly potent and highly selective for both
pcDHFR and tgDHFR and is currently undergoing
animal studies. Utilizing the 3,4,5-trimethoxy side
chain substitution pattern that provided excellent po-
tency and good selectivity in the 5-methyl-5-deaza
series, we synthesized four 5-desmethyl-5-deaza lipo-
philic antifolates (2-5) which have varied substituents
on the N10 nitrogen. It has been shown that small
substituents on the N10 position in the 5-methyl-5-
deaza series have provided for remarkably increased
potency and/or selectivity for pcDHFR and tgDHFR.18,19
Ch em istr y
The synthesis of all the analogs utilized the common
intermediate 2,4-bis(pivaloylamino)-6-bromopyrido[2,3-
d]pyrimidine (17). This critical intermediate 17 was
synthesized via reaction of 2,4,6-triaminopyrimidine
(14) and bromomalonaldehyde (15) under acidic condi-
tions to afford the cyclized compound 16 followed by
protection of the 2- and 4-amino groups with pivaloyl
anhydride and pyridine.31 The synthesis of the 5-deaza
analogs 2-5 is shown in Scheme 1. A palladium-
catalyzed Heck coupling32,33 of 17 with styrene under
the standard conditions afforded the desired product 18
in modest yield (47%). Ozonolysis34 of the styryl deriva-
tive 18 followed by reductive workup with dimethyl
sulfide gave the desired 6-formyl derivative 19 in good
yield (80%). Reductive amination of 19 with 3,4,5-
trimethoxyaniline was best carried out in two stages:
formation of the intermediate imine in glacial acetic acid
at room temperature followed by reduction of the imine
with borane-triethylamine complex. Column chro-
matographic purification and recrystallization afforded
the desired bispivaloylated compound 20 in 48% yield.
Deprotection of the pivaloyl groups under the conditions
(liquid ammonia, sealed vessel) used for the deprotection
of the 5,10-dideaza analogs (see below) did not afford
any of the desired product 2; instead an extremely polar
and water soluble material was obtained, which was not
identified. Basic hydrolysis of pivaloyl amides have
been reported in the literature,35 but concerns regarding
the lability of the 4-amino group under these conditions
prompted an investigation of alternate methods of
deprotection. Sodium methoxide has been reported to
cleave acetamido groups in related pyrido[2,3-d]pyrim-
idines.36 Utilizing this precedent the di-depivaloylation
was carried out with freshly prepared sodium methoxide
in anhydrous methanol to afford the desired 5-deaza
compound 2 in 52% yield.
In order to elucidate the importance of the 10-nitrogen
atom on DHFR inhibitory potency and selectivity in this
class, we also synthesized a series of 5,10-dideaza
lipophilic antifolates in which the 10-nitrogen is re-
placed by a carbon atom. The synthesis of the 3,4-
dimethoxyphenyl-substituted 5,10-dideaza analogs 7,
10, and 13 was reported by us in a preliminary com-
munication.31 This report describes the biological activ-
ity of analogs 7, 10, and 13 and the synthesis and
biological activity of additional 5,10-dideaza lipophilic
antifolates which varied in the number of methoxy
substituents in the 3-, 4-, and 5-positions on the phenyl
ring. The methoxy substituent present in several of
these analogs was chosen in part on the basis of
previous reports from our laboratory with other non-
classical antifolates.18-23 Three 9,10-dehydro-5,10-
dideaza analogs (6-8) were synthesized as conforma-
tionally constrained derivatives of lipophilic antifolates
9-11 where the methoxy-substituted phenyl side chain
has been locked in an extended (trans) orientation. The
reduced 5,6,7,8-tetrahydro-5,10-dideaza analogs 12 and
13 were also of interest as lipophilic DHFR inhibitors
related to 2,4-diamino-6-substituted tetrahydroquinazo-
lines which were potent inhibitors of DHFR derived
from P. carinii and T. gondii with selectivity for
A similar reductive amination procedure was also
expected to afford the N-alkylated derivatives 3 and 4.
Both N-methyl-37 and N-ethyl-3,4,5-trimethoxyaniline19
were synthesized by direct alkylation with methyl

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472 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Gangjee et al.
Sch em e 1a
a
Reagents (a) HCl, EtOH, ∆; (b) [(CH3)3CCO]2O, pyridine, reflux; (c) styrene, Pd(OAc)2, (o-tol)3P, CuI, CH3CN, reflux; (d) (i) O3, CH2Cl2/
MeOH, -78 °C, (ii) O2, Me2S; (e) (i) 3,4,5-trimethoxyaniline, glacial AcOH, rt, (ii) BH3‚Et3N; (f) NaOMe, MeOH, rt to 50 °C; (g) NaBH4,
MeOH, 0-5 °C; (h) Ph3P, Br2, CH2Cl2, rt; (i) for 23 (R ) CH3) N-methyl-3,4,5-trimethoxyaniline, DMAP, N,N-DMAc; (j) for 24 (R )
CH2CH3) N-ethyl-3,4,5-trimethoxyaniline, DMAP, N,N-DMAc; (k) for 3 (R ) CH3) NaOMe, MeOH, rt to 50 °C; (l) for 4 (R ) CH2CH3) 30%
HBr in AcOH, THF, rt to 50 °C; (m) 98% HCOOH, Ac2O, rt.
iodide. Reductive amination of 19 with either of the two
secondary amines under the conditions used for the
synthesis of 20 did not afford the desired products 23
and 24 in acceptable yields. We then turned our
attention to a nucleophilic displacement to synthesize
the N10 alkyl compounds. Reduction of the aldehyde
moiety (Scheme 1) present in 19 with sodium boro-
hydride afforded the desired 6-hydroxymethyl derivative
21 in good yield. Bromination of the alcohol 21 with
triphenylphosphine and bromine38 afforded the bromide
22 which was not stable to storage and was used
immediately in the next step.
Nucleophilic displacement of the bromide 22 (Scheme
1) with N-methyl-3,4,5-trimethoxyaniline afforded the
dipivaloylated compound 23 which was deprotected to
afford the desired N-methyl derivative 3 in 48% yield
from bromide 22. A similar nucleophilic displacement
with N-ethyl-3,4,5-trimethoxyaniline afforded the pro-
tected derivative 24. Although the di-depivaloylation
could be achieved in principle under the reported
conditions, we were interested in exploring other meth-
ods of cleavage of the 2- and 4-pivaloyl groups without
concomitant hydrolysis of the 4-amino group. This was
achieved for 24 with 30% hydrogen bromide in acetic
acid in anhydrous tetrahydrofuran. Following column
chromatographic purification the desired N-ethyl de-
rivative 4 was obtained in 42% yield from the bromide
22. Formylation of the N10 nitrogen of compound 2
with 98% formic acid and a catalytic amount of acetic
anhydride18 afforded the N-formyl compound 5 in good
yield (86%).
The syntheses of the 5,10-dideaza compounds are
shown in Scheme 2 and followed the conditions reported
in our previous communication31 for the synthesis of the
3,4-dimethoxy derivatives. 3,4,5-Trimethoxystyrene was
not commercially available and was synthesized in 86%
yield by a Wittig reaction of 3,4,5-trimethoxybenz-
aldehyde with the anion of methyltriphenylphos-
phonium bromide.39 A palladium-catalyzed Heck cou-
pling of 3,4,5-trimethoxystyrene with 17 afforded the
dipivaloylated derivative 25 in 59% yield. A similar
Heck coupling of 17 with 4-methoxystyrene afforded 27
in a lower yield (45%). The trans stereochemistry for
the 9,10-double bond of the monomethoxy derivative 27
and compound 25 was evident by the large coupling
constant between the two protons (J ) 16.0 Hz) in the
1H NMR spectrum of 27 in deuterated dimethyl sulfox-
1
ide and the H NMR spectrum of compound 25 run in
deuterated chloroform. Deprotection of the 2- and
4-pivaloyl groups of 25 and 27 in a Parr acid digestion

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Diaminodeaza Compounds as DHFR Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 473
Sch em e 2a
Ta ble 1. Inhibitory Concentrations (IC50, µM) against DHFRs
from P. carinii (pc), T. gondii (tg), and Rat Liver (rl) and
Selectivity Ratiosa
compd
pcDHFR
tgDHFR
rlDHFR
rl/pc
rl/tg
1a b
2
0.013
1.5
0.00085
0.3
0.0076
1.9
0.58
1.3
8.9
6.3
3
4
5
6
7
8
9
0.24
0.19
18.5
>5.0
2.6
5.3
5.0
1.4
0.29
61.7
7.7
12.0
0.042
0.038
0.009
0.049
1.1
1.4
1.4
1.5
0.2
0.2
0.25
0.47
1.1
2.7
0.010
0.011
0.28
0.12
7.4
12.9
2.1
11.8
1.14
0.61
0.26
6.1
1.2
0.63
0.40
31
3.2
6.7
9.2
1.5
7.9
5.7
3.0
1.0
13.0
1.9
49
0.81
2.2
0.23
0.44
0.9
10
11
12
13
TMP
TMQ
PTX
0.1
2.1
0.27
11.1
0.07
0.04
133.0
0.003
0.0015
0.3
0.14
a
IC50 values were determined as described in the Experimental
b
Section. Data from ref 18.
indicate that as a series the 5-deaza-5-desmethyl ana-
logs 2-5 of 1a were less potent against all three DHFRs
compared to compound 1a . For compound 3, the
decrease in potency against pcDHFR and tgDHFR was
less than the decrease against rlDHFR, and compound
3 had better selectivity ratios for both pcDHFR (1.2) and
tgDHFR (31.1) than 1a . Replacement of the N10
methyl of 3 with a hydrogen (compound 2) afforded a
decrease in potency against all three enzymes compared
to 3, but the selectivity against pcDHFR was main-
tained and the selectivity against tgDHFR was 5-fold
lower than 3. The N10 ethyl analog 4 was more potent
than the N10-unsubstituted analog 2, but the selectivity
for pcDHFR and tgDHFR was less than that for 2 or 3.
The N10 formyl analog 5 was the least potent of the
series (compounds 2-5). It was however reasonably
selective for tgDHFR.
Replacement of the N10 of 2 with a carbon afforded
the 5,10-dideaza analog 9 which had decreased potency
and selectivity against pcDHFR and retained selectivity
against tgDHFR compared to 2. Sequential removal of
the methoxy moieties from 9 (compounds 10 and 11)
surprisingly increased potency against pcDHFR and
rlDHFR, but potency remained unchanged against
tgDHFR. This led to a decrease in selectivity for
tgDHFR and a increase in selectivity for pcDHFR. The
trans precursors of 9-11 (compounds 6-8) were less
potent against all three DHFRs, but the trimethoxy
analog 6 showed reasonable selectivity (9.2) for tgDHFR.
Reduction of the B-ring of 9 and 10 afforded the
tetrahydropyrido[2,3-d]pyrimidines 12 and 13. Both
reduced analogs were less potent than their unreduced
precursors. In particular the decrease in potency for
12 compared to 9 was 12-fold for pcDHFR and 5-fold
for rlDHFR but only 2-fold for tgDHFR which increases
the selectivity of compound 12 to 13.0 for tgDHFR
compared to 5.7 for compound 9. Compound 13 was less
potent and less selective than 10 against all three
DHFRs.
In summary compound 3 retained significant potency
against tgDHFR and was much less potent against
rlDHFR which translated to a 3-fold increase in selec-
tivity for tgDHFR compared to 1a and a 2-fold increase
in selectivity for pcDHFR compared to 1a . Thus the
premise that the removal of the 5-methyl moiety would
a
Reagents: (a) 3,4,5-trimethoxystyrene (for 25), 3,4-dimethoxy-
styrene (for 26), or 4-methoxystyrene (for 27), Pd(OAc)2, CuI, Et3N,
CH3CN, reflux; (b) liquid NH3, MeOH/CH2Cl2, Parr bomb; (c) (i)
CF3COOH, (ii) DMF, 5% Pd-C, 25 psi H2; (d) 10% CF3COOH in
MeOH, 5% Pd-C, 50 psi H2; (e) 60% aq AcOH, 5% Pd-C, 50 psi
H2.
bomb afforded the desired diamino compounds 6 and 8
in good yields (78-80%). The trans stereochemistry for
the 9,10-double bond in compound 8 was also assigned
from the large coupling constant (J ) 16.2 Hz) in the
1H NMR spectrum. Literature evidence suggests that
in analogous pyrido[2,3-d]pyrimidines containing mix-
tures of cis and trans isomers, the aromatic 7-CH
protons occur separately, with the proton of the cis
isomer occurring more upfield due to shielding by the
side-chain phenyl group.40-42 For compounds 6-8 the
chemical shifts of the 7-CH proton were quite similar
and were within 0.1 ppm of each other, which further
supports the trans geometry.
The free bases of the diamino compounds 6 and 8 were
insoluble in conventional organic solvents; thus the
reduction of the 9,10-double bond was carried out using
the trifluoroacetate salts of 6 and 8 in N,N-dimethyl-
formamide. Hydrogenation (Scheme 2) with 5% pal-
ladium on carbon under pressure afforded the desired
5,10-dideaza compounds 9 and 11 in 51-55% yield.
Hydrogenation of 6 with 5% palladium under pressure
for 24 h afforded the desired hexahydro analog 12 in
59% yield.
Biologica l Activity a n d Discu ssion
The compounds were evaluated12,13 as inhibitors of
pcDHFR, tgDHFR, and rlDHFR, and the results are
reported as IC50 values in Table 1. Selectivity ratios
were determined using rlDHFR as the mammalian
source and are also listed in Table 1. The results

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474 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Gangjee et al.
gel column (4.2 × 58 cm, packed with CH2Cl2). The column
was eluted with CH2Cl2, collecting 10 mL fractions. Fractions
showing a single spot on TLC were pooled and evaporated to
afford a white residue. This residue was recrystallized twice
from acetone to afford 22.8 g (44%) of 17 as small needles:
mp 212-214 °C (lit.31 mp 212-214 °C); TLC Rf 0.67 (CH2Cl2/
MeOH, 15:1, silica gel); MS (EI) calculated for C17H22N5O2Br
m/ z 409 (M++ 2), 407 (M+).
allow a decrease in DHFR inhibition which was antici-
pated to be less for pcDHFR and/or tgDHFR than for
rlDHFR was realized. Compared to TMP and TMQ
(Table 1) compound 3 combines the selectivity of TMP
and potency of TMQ for tgDHFR. Compared to TMQ
and PTX compound 3 is 18- and 29-fold more selective
against pcDHFR, respectively, and 104- and 222-fold
more selective against tgDHFR. Compared to TMP
compound 3 was only 1.5-fold less selective but 300-fold
more potent against tgDHFR.
For pcDHFR all the 5-desmethyl compounds synthe-
sized (2-13) were significantly more selective than
TMQ and PTX; however, except for 2, 3, and 8, the
compounds were more potent against rlDHFR than
pcDHFR. In addition for tgDHFR, all of the 5-des-
methyl analogs were more selective than TMQ and PTX,
but only 3, 6, and 12 were more selective than 1a .
Similar to the 5-methyl series of analogs, the most
potent and selective analog in the 5-desmethyl series
of this study, the N10 methyl 3,4,5-trimethoxy deriva-
tive 3, was the most potent and most selective for
tgDHFR. Compound 3 is undergoing further biological
evaluation, and the results of these studies will be
reported in a future communication.
2,4-Bis(p iva loyla m in o)-6-(2-p h en yleth en yl)p yr id o[2,3-
d ]p yr im id in e (18). A mixture of 17 (2.86 g, 7.0 mmol),
palladium acetate (0.016 g, 0.07 mmol, 1 mol %), tri-o-
tolylphosphine (0.043 g, 0.14 mmol), cuprous iodide (0.007 g,
0.035 mmol), and triethylamine (10 mL) in acetonitrile (40 mL)
was brought to reflux under nitrogen. Styrene (0.73 g, 7.0
mmol) was added to the refluxing mixture and the reflux
continued for 24 h. A second portion of styrene (0.73 g, 7.0
mmol) was added, and reflux was continued for an additional
24 h. The mixture was then cooled and evaporated to dryness
under reduced pressure. The gummy residue was dissolved
in CH2Cl2 and chromatographed on a silica gel column (2.4 ×
32 cm, packed with CH2Cl2). The column was first eluted with
CH2Cl2 (500 mL) followed by 1% MeOH in CH2Cl2. Fractions
showing a single spot were pooled, evaporated to dryness, and
dried to afford 1.42 g (47%) of 18 as a bright yellow solid. An
analytical sample was recrystallized from a mixture of EtOAc
and hexanes: mp 228-230 °C; TLC Rf 0.64 (CH2Cl2/MeOH,
1
15:1, silica gel); H NMR (DMSO-d6, 300 MHz) δ 1.28-1.30
(2s, 18 H, 2 × C(CH3)3), 7.30-7.70 (m, 7 H, Ar-CH, and 9-,
10-CH), 8.76 (s, 1 H, 5-CH), 9.30 (s, 1 H, 7-CH), 11.42 (br s, 1
H, 4-NH), 15.45 (br s, 1 H, 2-NH). Anal. Calcd for (C25H29N5O2‚
0.5H2O) C, H, N.
Exp er im en ta l Section
Melting points were determined on a Mel-Temp or Fisher-
J ohns melting point apparatus and are uncorrected. Infrared
spectra (IR) were recorded with a Perkin Elmer 1430 or 1320
infrared spectrophotometer in Nujol mulls and are reported
in reciprocal centimeters (cm-1). The 1H NMR spectra were
recorded on a Varian EM 390 (300 MHz) or Bruker WH-300
(300 MHz) spectrometer. The 13C NMR spectra were recorded
on a Varian EM 390 instrument at 75.46 MHz, 90° pulse, 14
µs. The data were accumulated by 16K size with 0.5 s delay
time and 70° tip angle. The chemical shift (δ) values are
expressed in parts per million (ppm) relative to tetramethyl-
silane as an internal standard: s ) singlet, d ) doublet, t )
triplet, m ) multiplet; Ar-CH ) aromatic proton. Thin layer
chromatography (TLC) was performed on cellulose or silica gel
plates with fluorescent indicator and visualized with UV light
at 254 and 350 nm. Flash chromatography was performed on
230-400 mesh silica gel purchased from Aldrich, Milwaukee,
WI. Samples for microanalysis were dried in vacuo over
phosphorus pentoxide with heating over refluxing ethanol or
toluene. Elemental analyses were performed by Atlantic
Microlab, Inc., Atlanta, GA. Fractional moles of solvents in
the analytical samples frequently found in such antifolates
could not be prevented in spite of vigorous drying in vacuo
and were confirmed, where possible, by their presence in the
NMR spectrum.
2,4-Bis(p iva loyla m in o)-6-b r om op yr id o[2,3-d ]p yr im i-
d in e (17). 2,4,6-Triaminopyrimidine (14) (20.0 g, 160.0 mmol)
was dissolved in concentrated HCl (30 mL) and absolute
ethanol (20 mL) at 80 °C under nitrogen. To this vigorously
stirred solution was added bromomalonaldehyde (15) (12.06
g, 80.0 mmol). The solution was brought to reflux for 5 min,
when an orange-colored solid fell out of solution. The thick
suspension was cooled to 5 °C and diluted with water (30 mL).
The mixture was made basic with concentrated NH4OH to pH
8, with the temperature maintained below 10 °C. The
precipitate 16, was filtered, washed with water until neutral,
air-dried, and further dried in vacuo over phosphorus pentox-
ide at 70 °C. To the crude dried precipitate 16 were added
pyridine (60 mL) and pivaloyl anhydride (100 mL, 480.0
mmol). The mixture was refluxed under nitrogen for 8 h and
the reaction mixture cooled to room temperature. The excess
pyridine and pivaloyl anhydride were removed under reduced
pressure (oil pump). To the residue was added CH2Cl2 (1000
mL), and the suspension was stirred overnight at room
temperature. The undissolved material was filtered and the
filtrate evaporated to ∼70 mL and chromatographed on a silica
2,4-Bis(p iva loyla m in o)-6-for m ylp yr id o[2,3-d ]p yr im i-
d in e (19). A solution of 18 (1.10 g, 2.55 mmol) was dissolved
in a mixture of CH2Cl2 (70 mL) and MeOH (10 mL), and this
solution was cooled to -78 °C in a dry ice-acetone bath.
Ozone was passed through this solution until a blue color
persisted. A stream of oxygen was then passed through the
solution for 15 min to remove excess ozone. Dimethyl sulfide
(1 mL) was added, the reaction mixture stirred for 15 min,
and the solution was evaporated to dryness under reduced
pressure. The residue so obtained was dissolved in a minimum
amount of CH2Cl2 and chromatographed on a silica gel column
(2.4 × 22 cm, packed with CH2Cl2) eluting first with CH2Cl2
(500 mL) followed by 1% MeOH in CH2Cl2. Fractions showing
a single spot (TLC) were pooled and evaporated to dryness to
afford 0.73 g (80%) of 19 as a white solid: mp 241-242 °C;
TLC Rf 0.58 (CH2Cl2/MeOH, 15:1, silica gel); IR (Nujol) 3300,
1
1720, 1680 cm-1; H NMR (DMSO-d6, 300 MHz) δ 1.27-1.29
(s, 18 H, 2 × C(CH3)3), 9.11 (s, 1 H, 5-CH), 9.31 (s, 1 H, 7-CH),
10.18 (s, 1 H, 6-CHO), 11.63 (s, 1 H, NH), 15.43 (s, 1 H, NH).
Anal. Calcd for (C18H23N5O3) C, H, N.
2,4-Bis(p iva loyla m in o)-6-[(3′,4′,5′-tr im eth oxya n ilin o)-
m eth yl]p yr id o[2,3-d ]p yr im id in e (20). To a solution of 19
(0.36 g, 1.0 mmol) in glacial acetic acid (10 mL) was added
3,4,5-trimethoxyaniline (0.20 g, 1.10 mmol), and the mixture
was stirred at room temperature, under nitrogen, for 18 h. To
this solution was added borane-triethylamine complex (51 µL,
0.35 mmol), and the mixture was stirred for an additional 4
h. The mixture was then evaporated to dryness under reduced
pressure. The residue was dissolved in CH2Cl2 (50 mL) and
washed with a saturated solution of NaHCO3 (2 × 40 mL)
followed by back-extraction of the aqueous layers with CH2Cl2
(50 mL). The combined organic layers were washed with water
(50 mL) and brine (50 mL), dried (MgSO4), and filtered. The
filtrate was evaporated to a small volume and chromato-
graphed on a silica gel column (2.4 × 18 cm, packed with
CH2Cl2). The column was eluted with CH2Cl2 (250 mL), then
with 1% MeOH in CH2Cl2 (100 mL), and finally with 2% MeOH
in CH2Cl2 (100 mL). The fractions showing a single spot (TLC)
were pooled and evaporated to dryness. The residue so
obtained was recrystallized from a mixture of EtOAc and
hexanes to afford 0.25 g (48%) of 20 as an orange solid: mp
1
214-216 °C; TLC Rf 0.45 (CH2Cl2/MeOH, 15:1, silica gel), H
NMR (DMSO-d6, 300 MHz) δ 1.22 (s, 9 H, C(CH3)3), 1.27 (s, 9
H, C(CH3)3), 3.50 (s, 3 H, 4′-OCH3), 3.66 (s, 6 H, 3′-, 5′-OCH3),
4.44 (d, 2 H, 9-CH2, J ) 6.0 Hz), 5.95 (s, 2 H, 2′-, 6′-CH), 6.23

+
+
Diaminodeaza Compounds as DHFR Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 475
(br t, 1 H, 10-NH), 8.70 (s, 1 H, 5-CH), 8.94 (s, 1 H, 7-CH),
11.35 (br s, 1 H, NH). Anal. Calcd for (C27H36N6O5‚H2O) C,
H, N.
mmol) and methyl iodide (0.75 mL, 12.0 mmol), and the
mixture was stirred at room temperature, under nitrogen, for
5 days. The solvent was removed under reduced pressure
(pump) and the residue dissolved in CH2Cl2 (100 mL). The
CH2Cl2 layer was washed with water (50 mL), a saturated
solution of NaHCO3 (50 mL), and brine (50 mL). The organic
layers were dried (MgSO4), filtered, concentrated under re-
duced pressure, and subjected to column chromatography on
silica gel (2.4 × 24 cm, packed with toluene/EtOAc, 14:1).
Elution with the same solvent afforded fractions showing a
single spot (TLC) which were pooled and evaporated to dryness
under reduced pressure to afford 0.75 g (38%) of the desired
compound as a tan semisolid: TLC Rf 0.30 (toluene/EtOAc,
4:1, silica gel); 1H NMR (CDCl3, 300 MHz) δ 2.82 (s, 3 H,
N-CH3), 3.60 (s, 1 H, NH), 3.80 (s, 3 H, 4′-OCH3), 3.86 (s, 6 H,
3′-, 5′-OCH3), 5.88 (s, 2 H, 2′-, 6′-CH).
2,4-Dia m in o-6-[(3′,4′,5′-t r im e t h oxya n ilin o)m e t h yl]-
p yr id o[2,3-d ]p yr im id in e (2). To a solution of sodium hy-
dride (0.05 g, 2.10 mmol) in anhydrous methanol (15 mL) was
added 20 (0.37 g, 0.70 mmol), and the mixture was stirred
under nitrogen at room temperature for 24 h. Another portion
of sodium hydride (0.05 g, 2.1 mmol) in anhydrous methanol
(5 mL) was added to the reaction mixture, and it was stirred
at 50 °C for 12 h. Silica gel (1 g) was added to this mixture
and the suspension evaporated under reduced pressure to
dryness. The solid plug obtained was loaded on a silica gel
column (2.4 × 20 cm) and the column flushed with chloroform
(300 mL). Stepwise elution with 100 mL portions of 1-14%
MeOH in CHCl3 afforded fractions which showed a spot
corresponding to that of the desired product at Rf 0.34 (CHCl3/
MeOH, 3:1, silica gel) along with a minor trailing impurity.
These fractions were pooled and evaporated to dryness under
reduced pressure. The residue so obtained was dissolved in a
minimum amount of glacial acetic acid, filtered, and evapo-
rated to dryness. This oily residue was redissolved in MeOH
and the solution stored at 0 °C for 72 h to deposit a solid which
was filtered, washed with ether, and dried to afford 0.13 g
(52%) of 2 as a light brown solid: mp 249-252 °C dec; TLC
(a) Rf 0.34 (CHCl3/MeOH, 3:1, silica gel), (b) Rf 0.17 (CHCl3/
MeOH/NH4OH, 14:2:1, silica gel); 1H NMR (DMSO-d6, 300
MHz) δ 3.51 (s, 3 H, 4′-OCH3), 3.67 (s, 6 H, 3′-, 5′-OCH3), 4.20
(d, 2 H, 9-CH2, J ) 3.8 Hz), 5.90-5.93 (overlapping peaks, 3
H, 10-NH, 2′-, 6′-CH), 6.28 (br s, 2 H, 4-NH2), 7.47 (br s, 2 H,
2-NH2), 8.38 (s, 1 H, 5-CH), 8.64 (s, 1 H, 7-CH). Anal. Calcd
for (C17H20N6O3‚0.5CH3COOH‚0.75H2O) C, H, N.
2,4-Bis(p iva loyla m in o)-6-(h yd r oxym eth yl)p yr id o[2,3-
d ]p yr im id in e (21). To a cooled (ice bath) solution of 19 (0.375
g, 1 mmol) in MeOH (8 mL) was added a solution of sodium
borohydride (0.038 g, 1 mmol) in MeOH (2 mL), and the
mixture stirred for 30 min under nitrogen. TLC (EtOAc, silica
gel) indicated the disappearance of starting material (Rf 0.40)
and formation of a product spot (Rf 0.08). The reaction mixture
was diluted with water (50 mL) and the pH adjusted to 4 with
dropwise addition of 0.5 N HCl. This mixture was extracted
with CH2Cl2 (2 × 75 mL), the combined organic layers were
dried (MgSO4) and filtered, and the filtrate was evaporated
under reduced pressure to dryness. The residue so obtained
was chromatographed on a silica gel column (2.4 × 16 cm)
eluting with a mixture of EtOAc/CHCl3 (2:1). Fractions which
showed a single spot (TLC) were pooled and evaporated to
afford 0.27 g (75%) of 21 as a white solid: mp 166-168 °C;
TLC Rf 0.19 (EtOAc/MeOH, 19:1, silica gel); IR (Nujol) 3480,
3400, 3200, 1670 cm-1, 1H NMR (DMSO-d6, 300 MHz) δ 1.26-
1.28 (s, 18 H, 2 × C(CH3)3), 4.66 (d, 2 H, 6-CH2OH, J ) 5.7
Hz), 5.50 (t, 1 H, 6-CH2OH, J ) 5.7 Hz), 8.67 (d, 1 H, 5-CH, J
) 2.4 Hz), 8.86 (d, 1 H, 7-CH, J ) 2.4 Hz), 11.36 (s, 1 H, NH);
MS (FAB) calcd for C18H25N5O3 m/ z 360 (MH+); high-resolu-
tion FABMS calcd MH+ 360.4388, found 360.4398.
N-Eth yl-3,4,5-tr im eth oxya n ilin e. This compound was
prepared in a manner similar to that described above for the
preparation of N-methyl-3,4,5-trimethoxyaniline in 39% yield
from 3,4,5-trimethoxyaniline and ethyl iodide, dark brown
oil: TLC Rf
0.33 (toluene/EtOAc, 4:1, silica gel); 1H NMR
(CDCl3, 300 MHz) δ 1.23 (t, 3 H, CH2CH3), 3.15 (q, 2 H,
CH2CH3), 3.76 (s, 3 H, 2′-OCH3), 3.83 (s, 6 H, 3′-, 5′-OCH3),
5.86 (s, 2 H, 2′-, 6′-CH).
2,4-Dia m in o-6-[(3′,4′,5′-t r im et h oxy-N-m et h yla n ilin o)-
m eth yl]p yr id o[2,3-d ]p yr im id in e (3). To a solution of 22
(0.21 g, 0.50 mmol) and N-methyl-3,4,5-trimethoxyaniline (0.20
g, 1.0 mmol) in anhydrous N,N-DMAc (5 mL) was added
4-(dimethylamino)pyridine (0.12 g, 1.0 mmol), and the mixture
was stirred under nitrogen at room temperature for 4 days.
TLC (CH2Cl2/MeOH, 15:1, silica gel) at this time indicated the
formation of the product (23) spot at Rf 0.47. The solvent was
then removed under reduced pressure (pump) and the residue
dissolved in CH2Cl2 (50 mL). This solution was washed with
water (50 mL) and a saturated solution of NaHCO3 (50 mL),
and the organic layers were dried (MgSO4) and filtered. The
filtrate was evaporated under reduced pressure to afford crude
23 as a dark brown residue which was dissolved in anhydrous
MeOH (20 mL). Sodium hydride (dry 95%, 60 mg, 2.5 mmol)
was carefully added to this solution and the mixture stirred
at room temperature for 24 h and then at 50 °C for 12 h. Silica
gel (1.0 g) was then added and the suspension evaporated to
dryness under reduced pressure. This plug was loaded on a
dry silica gel column (2.4 × 18 cm) and flushed with CHCl3
(500 mL). Stepwise elution with 100 mL portions of 1-14%
MeOH in CHCl3 afforded fractions showing a single spot on
TLC, which were pooled and evaporated under reduced pres-
sure to dryness to afford 0.09 g (48%) of 3 as a yellow solid:
mp 248-250 °C dec; TLC (a) Rf 0.50 (CHCl3/MeOH, 3:1, silica
1
gel), (b) Rf 0.28 (CHCl3/MeOH/NH4OH, 14:2:1, silica gel); H
NMR (DMSO-d6, 300 MHz) δ 2.97 (s, 3 H, N-CH3), 3.53 (s, 3
H, 4′-OCH3), 3.71 (s, 6 H, 3′-, 5′-OCH3), 4.50 (s, 2 H, 9-CH2),
6.04 (s, 2 H, 2-NH2), 8.30 (s, 1 H, 5-CH), 8.58 (s, 1 H, 7-CH).
Anal. Calcd for (C18H22N6O3‚0.75H2O) C, H, N.
2,4-Dia m in o-6-[(3′,4′,5′-t r im e t h oxy-N -e t h yla n ilin o)-
m eth yl]p yr id o[2,3-d ]p yr im id in e (4). To a solution of 22
(0.21 g, 0.5 mmol) and N-ethyl-3,4,5-trimethoxyaniline (0.21
g, 1.0 mmol) in anhydrous N,N-DMAc (5 mL) was added
4-(dimethylamino)pyridine (0.12 g, 1.0 mmol), and the mixture
was stirred under nitrogen at room temperature for 4 days.
The TLC (CH2Cl2/MeOH, 15:1, silica gel) at this time indicated
the formation of the product (24) spot at Rf 0.48. The solvent
was then removed under reduced pressure (pump) and the
residue dissolved in CH2Cl2 (50 mL). This solution was
washed with water (50 mL) and a saturated solution of
NaHCO3 (50 mL), and the organic layers were dried (MgSO4)
and filtered. The filtrate was evaporated to dryness under
reduced pressure. The crude product 24 was obtained as a
dark residue which was dissolved in anhydrous THF (30 mL);
30% HBr in acetic acid (2 mL) was added to this solution and
the mixture stirred at room temperature under nitrogen for
24 h and then at 50 °C for 3 h. The reaction mixture was
evaporated to dryness and the oily residue coevaporated twice
with 50 mL portions of absolute MeOH. The residue was
dissolved in MeOH, silica gel (1.2 g) was added, and the
suspension was evaporated to afford a plug which was loaded
2,4-Bis(p iva loyla m in o)-6-(br om om eth yl)p yr id o[2,3-d ]-
p yr im id in e (22). To a solution of triphenylphosphine (0.32
g, 1.22 mmol) in CH2Cl2 was added liquid bromine until the
solution just turned yellow in color. Alcohol 21 (0.22 g, 0.61
mmol) was then added as a solid and the mixture stirred for
1 h at room temperature until TLC (EtOAc, silica gel) indicated
disappearance of starting material (Rf 0.08) and formation of
product (Rf 0.27). The solution was diluted with CH2Cl2 (70
mL) and washed with water (2 × 50 mL), and the combined
organic layers were dried (MgSO4) and filtered. The filtrate
was evaporated to a small volume and flash chromatographed
on a silica gel column (2.4 × 10 cm) eluting with a mixture of
EtOAc/CHCl3 (2:1). Fractions showing a single spot (TLC)
were pooled and evaporated to dryness under reduced pressure
to afford 0.20 g (78%) of 22 as a pale yellow solid: mp >150
°C dec; TLC Rf 0.27 (EtOAc, silica gel); MS (FAB) calcd for
C18H24N5O2 Br m/ z 424 (MH++ 2), 422 (MH+). This compound
was immediately used in the next step.
N-Meth yl-3,4,5-tr im eth oxya n ilin e. To a solution of 3,4,5-
trimethoxyaniline (1.83 g, 10.0 mmol) in anhydrous N,N-DMAc
(10 mL) were added N,N-diisopropylethylamine (2.09 mL, 12.0

+
+
476 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Gangjee et al.
on a dry silica gel column (2.4 × 18 cm) and flushed with
CHCl3 (500 mL). The column was then eluted stepwise with
100 mL portions of 1-14% MeOH in CHCl3. Fractions
showing a single spot on TLC were pooled and evaporated to
dryness under reduced pressure. The residue obtained was
stirred in anhydrous ether (30 mL) for 4 h and filtered to afford
0.08 g (42%) of 4 as a light brown solid: mp 220-223 °C dec;
TLC (a) Rf 0.54 (CHCl3/MeOH, 3:1, silica gel), (b) Rf 0.33
(CHCl3/MeOH/NH4OH, 14:2:1, silica gel); 1H NMR (DMSO-
d6, 300 MHz) δ 1.11 (t, 3 H, N-CH2CH3), 3.48-3.52 (over-
lapping q and s, 5 H, N-CH2CH3, 4′-OCH3), 3.67 (s, 6 H, 3′-,
5′-OCH3), 4.45 (s, 2 H, 9-CH2), 5.98 (s, 2 H, 2′-, 6′-CH), 6.43
(br s, 2 H, 4-NH2), 7.64 (br s, 2 H, 2-NH2), 8.31 (s, 1 H, 5-CH),
sponding to the product were pooled and evaporated under
reduced pressure to afford 2.0 g (59%) of 25 as an orange solid.
An analytical sample was recrystallized from a mixture of
ethyl acetate and hexanes: mp 207-208 °C; TLC Rf 0.58
(CH2Cl2/MeOH, 15:1, silica gel); 1H NMR (DMSO-d6, 300 MHz)
δ 1.28-1.30 (2s, 18 H, 2 × C(CH3)3), 3.69 (s, 3 H, 4′-OCH3),
3.86 (s, 6 H, 3′-, 5′-OCH3), 7.01 (s, 2 H, 2′-, 6′-CH), 7.43 (s, 2
H, 9-, 10-CH), 8.65 (s, 1 H, 5-CH), 9.27 (s, 1 H, 7-CH), 11.60
(br s, NH), 15.45 (br s, NH). Anal. Calcd for (C28H35N5O5‚H2O)
C, H, N.
2,4-Bis(pivaloylam in o)-6-[2-(4′-m eth oxyph en yl)eth en yl]-
p yr id o[2,3-d ]p yr im id in e (27). To a mixture of 17 (3.06 g,
7.50 mmol), palladium acetate (0.084 g, 0.375 mmol, 5 mol %),
tri-o-tolylphosphine (0.228 g, 0.75 mmol), and cuprous iodide
(0.036 g, 0.19 mmol) in acetonitrile (50 mL) was added
triethylamine (15 mL), and the reaction mixture was brought
to reflux under nitrogen. To this mixture was added 4-meth-
oxystyrene (1.0 g, 7.5 mmol), and the reflux was continued for
a period of 36 h. A second portion of 4-methoxystyrene (1.0 g,
7.5 mmol) and triethylamine (5 mL) was added to the hot
mixture and reflux continued for an additional 36 h. The
reaction mixture was cooled and evaporated to dryness under
reduced pressure. The dark gummy residue was dissolved in
a mixture of CH2Cl2/MeOH (4:1), filtered through Celite, and
washed with the same solvent. The combined filtrates were
evaporated to dryness, and the residue was dissolved in a
minimum amount of CH2Cl2/MeOH (96:4) and subjected to
column chromatography on silica gel (2.4 × 40 cm, packed with
CH2Cl2). The column was eluted with CH2Cl2 (500 mL) and
then with 1% MeOH in CH2Cl2. The fractions showing a single
spot (TLC) corresponding to that of the product were pooled
and evaporated under reduced pressure to a small volume. The
solution was stored at 0 °C for 24 h to deposit a solid which
was filtered, washed with cold CH2Cl2 (20 mL), and dried to
afford 1.55 g (45%) of 27 as an orange-yellow solid: mp 240-
242 °C; TLC Rf 0.61 (CH2Cl2/MeOH, 19:1, silica gel); 1H NMR
(DMSO-d6, 300 MHz) δ 1.28-1.30 (2s, 18 H, 2 × C(CH3)3), 3.80
(s, 3 H, 4′-OCH3), 6.98 (d, 2 H, 2′-, 6′-CH, J ) 8.6 Hz), 7.32 (d,
1 H, 9- or 10-CH, J ) 16.7 Hz), 7.44 (d, 1 H, 9- or 10-CH, J )
16.7 Hz), 7.63 (d, 2 H, 3′-, 5′-CH, J ) 8.6 Hz), 8.72 (s, 1 H,
5-CH), 9.25 (s, 1 H, 7-CH), 10.29 (br s, 1 H, NH). Anal. Calcd
for (C26H31N5O3‚1.5H2O) C, H, N.
2,4-Dia m in o-6-[2-(3′,4′,5′-t r im et h oxyp h en yl)et h en yl]-
p yr id o[2,3-d ]p yr im id in e (6). To a solution of 25 (0.55 g, 1.06
mmol) in CH2Cl2/MeOH (3:1, 20 mL) was added liquid am-
monia (40 mL), and the mixture was stirred in a Parr acid
digestion bomb for a period of 72 h. The liquid ammonia was
allowed to evaporate and the suspension filtered. The residue
was extracted with boiling MeOH (100 mL) and then with
boiling acetone (100 mL). The resulting solid was dissolved
in 25% aqueous acetic acid and the solution filtered through
glass wool. The filtrate was evaporated under reduced pres-
sure to dryness and the residue stirred in the dark in a mixture
of EtOAc/MeOH (2:1, 150 mL) and filtered. The residue was
washed with EtOAc followed by acetone and dried to afford
0.30 g (80%) of 6 as a yellow solid: mp >300 °C; TLC (a) Rf
0.21 (CHCl3/MeOH/NH4OH, 14:2:1, silica gel), (b) Rf 0.75 (50%
aqueous acetic acid, cellulose); 1H NMR (DMSO-d6, 300 MHz)
δ 3.67 (s, 3 H, 4′-OCH3), 3.83 (s, 6 H, 3′-, 5′-OCH3), 6.88 (s, 2
H, 2′-, 6′-CH), 7.26-7.36 (overlapping s, 4 H, 9-, 10-CH, and
4-NH2), 8.40 (br s, 2 H, 2-NH2), 8.83 (s, 1 H, 5-CH), 8.89 (s, 1
H, 7-CH). Anal. Calcd for (C18H19N5O3‚2H2O) C, H, N.
2,4-Dia m in o-6-[2-(4′-m et h oxyp h en yl)et h en yl]p yr id o-
[2,3-d ]p yr im id in e (8). This compound was prepared in a
manner similar to that described above for the synthesis of
the trimethoxy derivative 6. Thus ammonolysis of 27 (0.46 g,
1.0 mmol) afforded 0.23 g (78%) of the desired depivaloylated
compound 8 as a bright yellow solid: mp >300 °C; TLC (a) Rf
0.20 (CHCl3/MeOH/NH4OH, 14:2:1, silica gel), (b) Rf 0.52 (50%
aqueous acetic acid, cellulose); 1H NMR (DMSO-d6, 300 MHz)
δ 3.79 (s, 3 H, 4′-OCH3), 6.99 (d, 2 H, 2′-, 6′-CH, J ) 8.0 Hz),
7.11-7.33 (2d overlapped with br s, 4 H, 9-, 10-CH, 4-NH2, J
) 16.2 Hz), 7.54 (d, 2 H, 3′-, 5′-CH, J ) 8.0 Hz), 8.37 (br s, 2
H, 2-NH2), 8.85 (s, 2 H, 5-, 7-CH; separates on addition of D2O
to give 2s at 8.82 and 8.86 ppm). Anal. Calcd for
(C12H15N5O‚1.5H2O) C, H, N.
.
8.57 (s, 1 H, 7-CH). Anal. Calcd for (C19H24N6O3 0.25HBr‚
0.75H2O) C, H, N.
2,4-Dia m in o-6-[(3′,4′,5′-t r im et h oxy-N-for m yla n ilin o)-
m eth yl]p yr id o[2,3-d ]p yr im id in e (5). To a solution of 2
(0.06 g, 0.16 mmol) in 98% formic acid (3 mL) was added 2
drops of acetic anhydride, and the solution was stirred for 48
h at room temperature when TLC (CHCl3/MeOH/NH4OH, 14:
2:1, silica gel) indicated disappearance of starting material 2
(Rf 0.17) and formation of product (Rf 0.25). The solvent was
then evaporated under reduced pressure and the residue
dissolved in MeOH (20 mL). Silica gel (0.5 g) was added to
this solution and the suspension evaporated to dryness. This
plug was loaded on a dry silica gel column (1 × 10 cm) and
flushed with CHCl3 (100 mL). The column was then eluted
stepwise with 75 mL portions of 2-12% MeOH in CHCl3.
Fractions showing a single spot (TLC) were pooled and
evaporated to dryness. The residue was stirred in anhydrous
ether (20 mL) and filtered to afford 0.05 g (86%) of 5 as an
off-white solid: mp 239-242 °C; TLC Rf 0.25 (CHCl3/MeOH/
1
NH4OH, 14:2:1, silica gel); H NMR (DMSO-d6, 300 MHz) δ
3.60 (s, 3 H, 4′-OCH3), 3.75 (s, 6 H, 3′-, 5′-OCH3), 5.04 (s, 2 H,
9-CH2), 6.43 (br s, 2 H, 4-NH2), 6.73 (s, 2 H, 2′-, 6′-CH), 7.62
(br s, 2 H, 2-NH2), 8.19 (s, 1 H, 5-CH), 8.52 (s, 1 H, 7-CH),
8.59 (s, 1 H, N-CHO). Anal. Calcd for (C18H20N6O4‚0.75H2O)
C, H, N.
3,4,5-Tr im eth oxystyr en e. A mixture of methyltriphenyl-
phosphonium bromide (11.43 g, 32 mmol) in anhydrous THF
(120 mL) was cooled to 0 °C (ice-salt bath). To this suspen-
sion, under nitrogen, was added a 1.6 M solution of nBuLi in
hexanes (20.64 mL, 32 mmol) dropwise over a period of 20 min.
The suspension was stirred at 0 °C for 30 min, warmed to room
temperature, and stirred for an additional 1 h. A solution of
trimethoxybenzaldehyde (5.89 g, 30 mmol) in anhydrous THF
(25 mL) was added over a period of 10 min and the reaction
mixture stirred at room temperature for 5 h. The reaction
mixture was diluted with water (300 mL) and the suspension
filtered. The filtrate was washed with water (3 × 100 mL)
followed by back-extraction of the aqueous layers with ether
(100 mL). The combined organic layers were washed with
brine (150 mL), dried (MgSO4), and filtered. After evaporation
of the filtrate the resulting oil was flash chromatographed on
a silica gel column (2.4 × 38 cm) eluting with hexanes/EtOAc
(4:1) to afford 5.02 g (86%) of trimethoxystyrene as a viscous
oil: 1H NMR (CDCl3, 300 MHz) δ 3.82 (s, 3 H, 4′-OCH3), 3.86
(s, 6 H, 3′-, 5′-OCH3), 5.20 (d, 1 H, J ) 11.0 Hz, HCHdCH),
5.60 (d, 1 H, J ) 18.0 Hz, HCHdCH), 6.60 (dd, 1 H, J ) 11.0,
18.0 Hz, CH2dCH), 6.65 (s, 2 H, 2′-, 6′-CH); MS (EI) calcd for
C11H14O3 m/ z 194 (M+).
2,4-Bis(p iva loyla m in o)-6-[2-(3′,4′,5′-tr im eth oxyp h en yl)-
eth en yl]p yr id o[2,3-d ]p yr im id in e (25). A mixture of 17
(2.65 g, 6.5 mmol), palladium acetate (0.016 g, 0.07 mmol, 1
mol %), tri-o-tolylphosphine (0.043 g, 0.14 mmol), cuprous
iodide (0.007 g, 0.035 mmol), and triethylamine (10 mL) in
acetonitrile (40 mL) was brought to reflux under nitrogen.
3,4,5-Trimethoxystyrene (2.33 g, 12.0 mmol) was added to the
solution and the reaction mixture refluxed for 24 h. A second
portion of triethylamine (5 mL) was then added and reflux
continued for an additional 24 h. The reaction mixture was
cooled and evaporated to dryness under reduced pressure. The
residue was suspended in CH2Cl2, and loaded onto a silica gel
column (2.4 × 34 cm, packed with CH2Cl2), and flushed with
CH2Cl2 (500 mL). The column was then eluted with 1% MeOH
in CH2Cl2. The fractions showing a single spot (TLC) corre-

+
+
Diaminodeaza Compounds as DHFR Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 477
2,4-Dia m in o-6-[2-(3′,4′,5′-t r im e t h oxyp h e n yl)e t h yl]-
p yr id o[2,3-d ]p yr im id in e (9). In order to increase the
solubility of 6 in DMF it was converted to the trifluoroacetate
salt. Compound 6 (0.18 g, 0.51 mmol) was dissolved in
CF3COOH (20 mL), and the solution was evaporated to
dryness (<30 °C) with an oil pump. The residue was dissolved
in DMF (20 mL), and 5% Pd-C (0.20 g) was added to the
solution. The suspension was hydrogenated at 25 psi for 15
min in a Parr apparatus. TLC (cellulose, 10% aqueous acetic
acid, v/v) of the reaction mixture showed two spots, the desired
product at Rf 0.58 and unreacted starting material 6 at Rf 0.05.
The reaction mixture was filtered through Celite and the Celite
washed with DMF (20 mL). The filtrate was evaporated under
reduced pressure (<50 °C) to dryness. The residue was
dissolved in boiling MeOH containing drops of glacial AcOH.
To this solution was added silica gel (0.50 g), and the solvent
was removed under reduced pressure. This solid dispersion
was loaded on a dry silica gel column (35 g, 2.4 × 20 cm) which
was flushed with CHCl3 (500 mL) and then eluted stepwise
with 100 mL portions of 1-20% MeOH in CHCl3, collecting
10 mL fractions. Fractions showing a single spot on TLC
corresponding to the product were pooled and evaporated to
dryness. The residue was stirred overnight in anhydrous ether
in the dark and filtered. The filtered solid was dried in vacuo
at 70 °C over phosphorus pentoxide to afford 0.09 g (52%) of 9
as a cream solid: mp 286-289 °C; TLC (a) Rf 0.22 (CHCl3/
MeOH/NH4OH, 14:2:1, silica gel), (b) Rf 0.58 (10% aqueous
acetic acid, cellulose); 1H NMR (DMSO-d6, 300 MHz) δ 2.86-
2.94 (AB system, 4 H, 9-, 10-CH2, J ) 7.5 Hz), 3.59 (s, 3 H,
4′-OCH3), 3.71 (s, 6 H, 3′-, 5′-OCH3), 6.52 (s, 2 H, 2′-, 6′-CH),
7.11 (br s, 2 H, 4-NH2), 8.20 (br s, 2 H, 2-NH2), 8.41 (d, 1 H,
5-CH, J ) 2.0 Hz), 8.56 (d, 1 H, 7-CH, J ) 2.0 Hz). Anal.
DHF R In h ibitor y Con cen tr a tion s. The enyme assays
were carried out at 37 °C in the presence of 90 µM dihydrofolic
acid as the substrate and 119 µM NADPH as the cofactor;
assays for rlDHFR and tgDHFR also contained 150 mM
KCl.12,13
Ack n ow led gm en t. This work was supported, in
part, by NIH grants GM40998 (A.G.) and GM52811
(A.G.) and NIH contracts NO1-AI-87240 (S.F.Q.) and
NO1-AI-35171 (S.F.Q.).
Refer en ces
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.
Calcd for (C18H21N5O3 0.75CF3COOH‚H2O) C, H, N.
2,4-Dia m in o-6-[2-(4′-m eth oxyp h en yl)eth yl]p yr id o[2,3-
d ]p yr im id in e (11). This compound was prepared in
a
manner similar to that reported above for the synthesis of 9.
Thus conversion of compound 8 (0.15 g, 0.5 mmol) to its
trifluoroacetate salt and subsequent hydrogenation and chro-
matographic purification afforded 0.08 g (55%) of 11 as a light
brown solid: mp >300 °C; TLC (a) Rf 0.20 (CHCl3/MeOH/NH4-
OH, 14:2:1, silica gel), (b) Rf 0.32 (10% aqueous acetic acid,
cellulose); 1H NMR (DMSO-d6, 300 MHz) δ 2.87 (s, 4 H, 9-,
10-CH2), 3.71 (s, 3 H, 4′-OCH3), 6.23 (br s, 2 H, 4-NH2), 6.83
(d, 2 H, 2′-, 6′-CH, J ) 8.5 Hz), 7.13 (d, 2 H, 3′-, 5′-CH, J ) 8.5
Hz), 7.43 (br s, 2 H, 2-NH2), 8.26 (d, 1 H, 5-CH, J ) 2.1 Hz),
8.44 (d, 1 H, 7-CH, J ) 2.1 Hz). Anal. Calcd for (C16H17N5O‚
1.5CF3COOH‚H2O) C, H, N.
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(()-2,4-Dia m in o-6-[2-(3′,4′,5′-tr im eth oxyp h en yl)eth yl]-
5,6,7,8-tetr a h yd r o-8H-p yr id o[2,3-d ]p yr im id in e (12). To a
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pooled and evaporated to afford a residue which was re-
evaporated twice with ether (30 mL). The solid obtained was
triturated in anhydrous ether and filtered to afford 0.10 g
(59%) of 12 as a white solid: mp >150 °C dec; TLC (a) Rf 0.40
(CHCl3/MeOH/NH4OH, 14:2:1, silica gel), (b) Rf 0.47 (10%
aqueous acetic acid, cellulose); 1H NMR (DMSO-d6, 300 MHz)
δ 1.53-1.66 (m, 3 H, 6-CH, 9-CH2), 1.85-1.93 (dd, 1 H, 5-CH,
J ) 15.3, 9.0 Hz), 2.42-2.49 (dd, 1 H partially obscured by
DMSO peak, 5-CH, J ) 15.3, 3.4 Hz), 2.58-2.63 (br q, 2 H,
10-CH2), 2.80-2.87 (dd, 1 H, 7-CH, J ) 11.0, 9.0 Hz), 3.22-
3.26 (d, 1 H, 7-CH, J ) 11.0 Hz), 3.61 (s, 3 H, 4′-OCH3), 3.75
(s, 6 H, 3′-, 5′-OCH3), 5.91 (s, 2 H, 4-NH2), 6.09 (s, 2 H, 2-NH2),
6.52 (s, 2 H, 2′-, 6′-CH), 6.63 (s, 1 H, 8-NH). Anal. Calcd for
(C18H25N5O3‚0.25CF3COOH‚0.75H2O) C, H, N.
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1995, 38, 1778-1785.

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