Nonclassical 2,4-diamino-8-deazafolate analogues as inhibitors of dihydrofolate reductases from rat liver, Pneumocystis carinii, and Toxoplasma gondii

Article abstract of DOI:10.1021/jm950918e

The synthesis and biological activity of 42 6-substituted-2,4- diaminopyrido[3,2-d]pyrimidines (2,4-diamino-8-deazafolate analogues) are reported. The compounds were synthesized in improved yields compared to previous classical analogues using modifications of procedures reported previously by us. Specifically, the S-phenyl-; mono-, di-, and trimethoxyphenyl-; and mono-, di-, and trichlorophenyl-substituted analogues with H or CH₃ at the N10 position and methyl and trifluoromethyl phenyl ketone analogues with H, C0H₃, and CH₂C≡CH at the N10 position were synthesized. The S10 and N10 α- and β-naphthyl analogues along with the N10 CH₃ analogues were also synthesized. These compounds were evaluated as inhibitors of dihydrofolate reductases (DHFR) from Pneumocystis carinii (pc) and Toxoplasma gondii (tg); selectivity ratios were determined against rat liver (rl) DHFR as the mammalian reference enzyme. Against pcDHFR the IC₅₀ values ranged from 0.038 x 10^-6 M for 2,4-diamino-6-[(N-methyl-2'- naphthylamino)methyl]pyrido[3,2-d]pyrimidine (28) to 5.5 x 10^-6 M for 2,4- diamino-6-[(2',4'-dimethoxyanilino)methyl]pyrido[3,2-d]pyrimidine (15). N10 methylation in all instances increased potency. None of the analogues were selective for pcDHFR. Against tgDHFR the most potent analogue was 2,4- diamino-6-[(N-methylanilino)methyl]pyrido[3,2-d]pyrimidine (5) (IC₅₀ 0.0084 x 10^-6 M) and the least potent was 2,4-diamino-6-[(2'- naphthylamino)methyl]-pyrido[3,2-d]pyrimidine (37) (IC₅₀ 0.16 x 10^-6 M). N10 methylation afforded an increase in potency up to 10-fold. In contrast to pcDHFR, several of the 8-deaza analogues were significantly selective for tgDHFR, most notably 2,4-diamino-6-[(2'-chloro-N- methylanilino)methyl]pyrido[3,2-d]pyrimidine (13), 2,4-diamino-6-[(3',4',5'- trimethoxyanilino)methyl]pyrido-[3,2-d]pyrimidine (29), and 2,4-diamino-6- [(2',4',6'-trichloroanilino)methyl]pyrido[3,2-d]pyrimidine (32) which combined high potency at 10^-8 M along with selectivities of 8.0, 5.0, and 12.4, respectively. The potency of these three analogues are comparable to the clinically used agent trimetrexate while their selectivities for tgDHFR are 17-43-fold better than trimetrexate.

Full text of DOI:10.1021/jm950918e

1836
J . Med. Chem. 1996, 39, 1836-1845
Non cla ssica l 2,4-Dia m in o-8-d ea za fola te An a logu es a s In h ibitor s of Dih yd r ofola te
Red u cta ses fr om Ra t Liver , P n eu m ocystis ca r in ii, a n d Toxopla sm a gon d ii1
Aleem Gangjee,*,† Yuanming Zhu,† Sherry F. Queener,‡ Paula Francom,§ and Arthur D. Broom*,§
Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University,
Pittsburgh, Pennsylvania 15282, Department of Pharmacology and Toxicology, Indiana University School of Medicine,
Indianapolis, Indiana 46202, and Department of Medicinal Chemistry, 308 Skaggs Hall, University of Utah,
Salt Lake City, Utah 84112
Received December 13, 1995X
The synthesis and biological activity of 42 6-substituted-2,4-diaminopyrido[3,2-d]pyrimidines
(2,4-diamino-8-deazafolate analogues) are reported. The compounds were synthesized in
improved yields compared to previous classical analogues using modifications of procedures
reported previously by us. Specifically, the S-phenyl-; mono-, di-, and trimethoxyphenyl-; and
mono-, di-, and trichlorophenyl-substituted analogues with H or CH3 at the N10 position and
methyl and trifluoromethyl phenyl ketone analogues with H, CH3, and CH2CtCH at the N10
position were synthesized. The S10 and N10 R- and â-naphthyl analogues along with the N10
CH3 analogues were also synthesized. These compounds were evaluated as inhibitors of
dihydrofolate reductases (DHFR) from Pneumocystis carinii (pc) and Toxoplasma gondii (tg);
selectivity ratios were determined against rat liver (rl) DHFR as the mammalian reference
enzyme. Against pcDHFR the IC50 values ranged from 0.038 × 10-6 M for 2,4-diamino-6-[(N-
methyl-2′-naphthylamino)methyl]pyrido[3,2-d]pyrimidine (28) to 5.5 × 10-6 M for 2,4-diamino-
6-[(2′,4′-dimethoxyanilino)methyl]pyrido[3,2-d]pyrimidine (15). N10 methylation in all in-
stances increased potency. None of the analogues were selective for pcDHFR. Against tgDHFR
the most potent analogue was 2,4-diamino-6-[(N-methylanilino)methyl]pyrido[3,2-d]pyrimidine
(5) (IC50 0.0084 × 10-6 M) and the least potent was 2,4-diamino-6-[(2′-naphthylamino)methyl]-
pyrido[3,2-d]pyrimidine (37) (IC50 0.16 × 10-6 M). N10 methylation afforded an increase in
potency up to 10-fold. In contrast to pcDHFR, several of the 8-deaza analogues were
significantly selective for tgDHFR, most notably 2,4-diamino-6-[(2′-chloro-N-methylanilino)-
methyl]pyrido[3,2-d]pyrimidine (13), 2,4-diamino-6-[(3′,4′,5′-trimethoxyanilino)methyl]pyrido-
[3,2-d]pyrimidine (29), and 2,4-diamino-6-[(2′,4′,6′-trichloroanilino)methyl]pyrido[3,2-d]pyrim-
idine (32) which combined high potency at 10-8 M along with selectivities of 8.0, 5.0, and 12.4,
respectively. The potency of these three analogues are comparable to the clinically used agent
trimetrexate while their selectivities for tgDHFR are 17-43-fold better than trimetrexate.
Opportunistic infections in patients with acquired
immunodeficiency syndrome (AIDS) remain the princi-
pal cause of morbidity and mortality. Of these infec-
tions, those by Pneumocystis carinii and Toxoplasma
gondii are the most severe and the most prevalent.2,3
Though primary and secondary prophylaxis for P.
carinii with the antifolates trimethoprim-sulfamethox-
azole or pentamidine have been successful and the
period of survival for AIDS patients is prolonged by this
therapy, the persistence of immunodeficiency results in
multiple episodes of P. carinii infection resulting in
pneumonias, some of which are atypical.4-6 Thus,
current regimens are successful in 50-75% of cases with
considerable loss of effectiveness in patients with two
or more episodes. T. gondii infections are currently
treated with a combination of the antifolate pyrimeth-
amine and sulfadiazine. In both P. carinii and T. gondii
treatments, adverse reactions are common and the
relapse rates are high, and in up to 50% of these cases
the effects are severe enough to warrant discontinuation
of therapy.7,8 Thus, the search for alternate therapeutic
agents and combinations have resulted in a number of
newer agents and combinations each with its attending
drawbacks and adverse effects.9 Resistant strains of
these organisms to current therapy are certain to
develop as has been observed in other similar infections.
In addition, patients who cannot tolerate or do not
respond to current agents urgently require alternate
therapies.
Allegra et al.10,11 reported trimethoprim (TMP) and
pyrimethamine, which are currently used agents, as
comparatively weak inhibitors of P. carinii (pc) dihy-
drofolate reductase (DHFR) and T. gondii (tg) DHFR,
which must be used with sulfonamides to provide
synergistic effects. However trimetrexate (TMQ) was
100-10000 times more potent than TMP or pyrimeth-
amine against pcDHFR and tgDHFR. The pyrido[2,3-
d]pyrimidine piritrexim (PTX) was also found to have
similar potency as TMQ.12,13a Both TMQ and PTX are
more lipid soluble than classical antifolates such as
methotrexate (MTX). They do not require the transport
mechanism that MTX and other classical antifolates
require and are thus able to penetrate these organisms
by passive diffusion. This lipid solubility also allows
penetration into the CNS where T. gondii infections can
occur. In clinical trials,14 TMQ along with host rescue
with leucovorin, which provides adequate reduced folate
in host cells but not in P. carinii or T. gondii because it
is not taken up efficiently by these organisms, has
proved to be a viable alternate therapy, and has been
approved by the FDA. However, relapse within 3
† Duquesne University.
‡ Indiana University School of Medicine.
§ University of Utah.
X Abstract published in Advance ACS Abstracts, April 1, 1996.
0022-2623/96/1839-1836$12.00/0 © 1996 American Chemical Society

Inhibitors of Dihydrofolate Reductases
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9 1837
months has been noted in 60% of patients (33% in
another study15) who received TMQ alone as initial
therapy, thus lending further urgency to the develop-
ment of other DHFR inhibitors which are potent and
selective.
Though TMQ and PTX are significantly inhibitory
against pcDHFR and tgDHFR, they lack selectivity
against these DHFRs. A pronounced effort has been
made to improve the selectivity of TMQ and PTX.
Gangjee et al.,16a-d Rosowsky et al.,17a-c Piper et al.,18
and Queener et al.13a,b,19 have reported increased selec-
tivity of TMQ- and PTX-based analogues. However, the
compounds obtained so far are either not selective
enough or lack efficacy due to the lack of transport into
P. carinii and/or T. gondii cells in culture.20
pated that in addition to high potency and selectivity,
the 8-deaza analogues should retain good cell penetra-
tion similar to the 5-deaza analogues. This report
consists of the synthesis of a comprehensive series of
42 2,4-diamino-6-substituted-pyrido[3,2-d]pyrimidines
3-44 and their inhibitory activities against DHFRs
from P. carinii and T. gondii and selectivity ratios
against rat liver (rl) DHFR as the reference mammalian
enzyme.
Ch em istr y
8-Deaza folate analogues have been reported previ-
ously by DeGraw et al.23 and Broom et al.24a-c The
target compounds were synthesized adopting Broom’s
synthetic methodology24a as indicated in Scheme 1. The
synthesis of the crucial hydroxymethyl intermediate 48,
which has previously been reported by different
groups,24-26 was accomplished from 2,4-dioxo-6-meth-
ylpyrido[3,2-d]pyrimidine (45) which, in turn, was ob-
tained via the condensation of 5-aminouracil and cro-
tonaldehyde in HCl under reflux.27 The yield of 45 as
reported by Irwin and Wibberley27 was only 30%. The
low yield was possibly due to its high solubility in water
during the workup procedure. Water was removed by
evaporation after the reaction was completed (indicated
by TLC). A minimum amount of water was then added
and the mixture stirred and triturated with ammonium
hydroxide. The solid product obtained was filtered and
dried. This modified workup procedure significantly
increased the yield from 30%27 to 90%. Treatment of
45 with m-chloroperbenzoic acid afforded the intermedi-
ate N5-oxide, which underwent the Polonovski rear-
rangement in Ac2O-AcOH to afford compound 46,
which in turn was converted to the 2,4-dichloro deriva-
tive 47 with POCl3 under reflux. The key intermediate
2,4-diamino-6-hydroxymethyl derivative 48 was ob-
tained by heating 47 at 150-160 °C in liquid ammonia
in a closed vessel.
A second method for the synthesis of compound 48
was also carried out without modification of a previously
published procedure.28 The acetophenone precursors 4′-
(propargylamino)acetophenone and 4′-(propargylamino)-
2,2,2-trifluoroacetophenone required for the synthesis
of 42 and 44 were synthesized by previously published
methods.28 Synthesis of 4′-(methylamino)-2,2,2-trifluo-
roacetophenone required for the synthesis of 43 involved
the displacement of aromatic fluorine from the com-
mercially available 2,2,2,4′-tetrafluoroacetophenone and
differed from the published method of McNamara et al.28
only in the use of methyl- rather than propargylamine.
This approach increased yields of the desired compound
5-fold over the two-step procedure reported by Stewart
and Teo.29,30
Compound 1, a nonclassical pteridine analogue, has
been reported by Broughton and Queener,19 Chio and
Queener,21 and Piper et al.18 to be highly selective
against both pcDHFR and tgDHFR. The high selectiv-
ity against pcDHFR of 25.9 (vs rat liver DHFR) and
against tgDHFR of 319 (vs rat liver DHFR) has made
compound 1 one of the most selective analogues against
both pcDHFR and tgDHFR. The potency (IC50) of this
analogue was poor (9.5 × 10-6 M) against pcDHFR and
marginal (0.77 × 10-6 M) against tgDHFR. Compound
1 also lacked potency in in vitro cell culture studies18
which was attributed to its poor DHFR inhibitory
effects.18 Piper et al.18 also reported the 5-CH3-5-deaza
analogue 2 (a pyrido[2,3-d]pyrimidine) of compound 1,
which contained a 4-MeOPh moiety rather than a
phenyl. This analogue was 17-fold and 12-fold more
potent than 1 against pcDHFR and tgDHFR, respec-
tively; however, the analogue lacked the significant
selectivity exhibited by compound 1. Thus a comparison
of 1 and 2 indicates that the 5-deaza analogue is highly
conducive to potency but is detrimental to selectivity.
The activities and selectivities of compound 1 and its
5-CH3-5-deaza-4-MeOPh analogue 2 suggested that the
N5 of 1 was essential for high selectivity. It was
therefore of considerable interest to synthesize the
8-deaza pteridine analogues of 1 as potential potent and
selective nonclassical inhibitors of pcDHFR and tgDH-
FR. In addition, some of the 5-deaza classical anti-
folates are known to penetrate cells much better than
the pteridine analogues, an observation which was
confirmed for T. gondii cells in culture in the nonclas-
sical 5-CH3-5-deaza antifolates by Gangjee et al.,22 Piper
et al.,18 and Chio and Queener.21 Thus it was antici-
Compound 48 was converted to the bromomethyl
analogue 49 by bromination with PBr3 under anhydrous
conditions. Compound 49 was extremely sensitive to
moisture due to its arylmethyl bromide moiety. During
the workup procedure, the removal of the solid from
excess PBr3 by filtration followed by rapid washing of
the product 49 with cold ether was carried out under
nitrogen in order to minimize the decomposition of 49
to 48. Displacement of the bromide of 49 with ap-
propriate nucleophiles (thiophenol, naphthylamines and
substituted anilines) in anhydrous DMAc afforded the
desired target compounds in yields of 19-57% over the
last two steps.

1838 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9
Gangjee et al.
Sch em e 1. Synthetic Route of 6-Substituted-2,4-Diaminopyrido[3,2-d]pyrimidinesa
a
(a) 20% HCl, crotonaldehyde; (b) (i) MCPBA; (ii) acetic anhydride; (c) POCl3, NEt3; (d) NH3, 150-160 °C; (e) PBr3, anhydrous THF;
(f) HX-Ar, DMAC, 50-60 °C; (g) HX-Ar, CaCO3, DMAC, 100 °C; (h) NaCNBH3, HCHO, pH 1-3.
The nucleophilic displacement of the bromide 49 was
performed in an anhydrous solvent under nitrogen
which allowed for an increase in the yield. In order to
optimize the yield of the displacement reaction to the
final compounds, inorganic bases were generally avoided
(except in the synthesis of compounds 40-44) to prevent
water formation which resulted from the reaction of the
liberated HBr and the inorganic base. The water thus
formed reacts with the bromide 49 to afford the alcohol
48 which decreased the yield of the final products.
Biologica l Resu lts a n d Discu ssion
The compounds were evaluated as inhibitors of pcDH-
FR, tgDHFR, and rlDHFR. Selectivity ratios were
determined vs rlDHFR as described previously (Table
1).13b,19 The 8-deaza analogue 3 of compound 1 was a
good inhibitor of tgDHFR, and its pcDHFR inhibitory
activity was similar to 1. However, the rlDHFR activity
was high; thus compound 3 did not have the high
selectivity displayed by 1. On the basis of the reported
selectivity of 1 and 2 along with the results obtained
for 3 in this report, it is apparent that both N5 and N8
are required for high selectivity against pcDHFR and
tgDHFR, at least for a (methylthio)phenyl substitution.
After the displacement of the bromide 49 with ap-
propriate nucleophiles was complete, the reaction mix-
ture was pretreated to remove polar water soluble
impurities and excess nucleophile prior to purification
by column chromatography. The pretreatment proce-
dure involved the removal of the solvent DMAc under
reduced pressure, followed by addition of water and
chloroform. Acidification of the heterogeneous mixture,
separation of the organic layer, basification of the water
phase with ammonium hydroxide to pH 11, followed by
extraction with chloroform and evaporation of the
organic solvent afforded the crude products which were
then purified by column chromatography. Without this
pretreatment, the final compounds which were synthe-
sized directly from the displacement of bromide 49 were
extremely difficult to purify due to the retention of side
products in the reaction mixture and resulted in yields
of less than 10% of the desired products.
For compounds 4-33, the side chain S10 of 3 was
replaced by N, and a comprehensive structure-activity
relationship was developed for the phenyl substitution
and N10-methyl and desmethyl analogues. Replace-
ment of the S10 with an N (compounds 4 and 5)
enhances potency against all three DHFRs. Selectivity
ratios are, however, decreased compared to 3. N10
methylation of 4 to 5 increases potency about 10-fold
for each enzyme, with a drop in selectivity against both
pc- and tgDHFR. This indicates that for the unsubsti-
tuted phenyl ring the increase in potency against
rlDHFR on N10 methylation is greater than for pc- or
tgDHFR.
In the monomethoxy series, the most potent analogue
among the N10-H compounds (6, 8, and 10) is the 4′-
OMe compound 10. N10 methylation of the monometh-
oxy derivatives significantly increases potency against
all three DHFRs; however, the increase is most appar-
ent in the 3′-OMe analogue (compare 8 and 9) whereas
in the 4′-OMe analogue (compare 10 and 11) the
increase is not as significant. The most selective
analogue against tgDHFR was the N10-CH3 2′-OMe
compound 7. The monomethoxy analogues were not
selective for pcDHFR.
Reductive N-methylation of compounds 4, 6, 12, 23,
25, 27, 30, and 32 to afford the corresponding 5, 7, 13,
24, 26, 28, 31, and 33 was carried out by a modified
procedure previously reported by us.16a,22 N-Methyla-
tion in acetonitrile with sodium cyanoborohydride and
formaldehyde was facilitated by dropwise addition of
sufficient 1 N HCl to effect solution, as reported previ-
ously,22 and afforded excellent yields of the N-methy-
lated products up to 99% for compound 7.

Inhibitors of Dihydrofolate Reductases
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9 1839
Ta ble 1. Inhibitory Concentratons (IC50, µM) and Selectivity Ratios against pcDHFR and tgDHFR vs rlDHFR19,13b
compd
X
R
pcDHFR
rlDHFR
rl/pc
25.9
tgDHFR
rl/tg
319
8.25
4.0
3.06
2.86
3.50
4.62
2.0
2.33
1.35
1.13
1.27
8.0
1.08
3.4
2.3
1.14
2.33
2.0
0.67
0.28
1.8
1.16
2.20
1.88
2.93
1.60
0.95
0.63
5.00
5.5
0.51
2.29
12.39
1.18
3.3
1
2
3
4
5
6
7
8
9.5
0.56
2.0
1.7
0.29
2.7
246
0.52
0.52
0.26
0.024
0.42
0.12
0.77
0.063
0.13
0.085
0.0084
0.12
0.026
0.1
0.015
0.054
0.016
0.11
0.015
0.13
0.02
0.14
0.014
0.12
0.025
0.09
0.0098
0.05
0.05
0.091
0.025
0.028
0.03
0.057
0.027
0.04
0.0047
0.087
0.038
0.046
0.044
0.049
0.048
0.026
0.16
0.018
0.016
0.027
0.015
0.020
0.046
0.054
2.7
0.93
0.26
0.15
0.08
0.16
0.24
0.12
0.36
0.09
0.072
0.26
0.57
0.07
0.032
0.06
0.10
0.06
0.24
0.07
0.02
0.12
0.12
0.13
0.31
0.082
0.28
0.13
0.45
0.10
0.20
0.07
0.35
0.29
0.43
0.34
0.23
0.17
0.13
0.18
0.14
0.01
0.04
0.07
0.13
0.06
S
NH
NMe
NH
NMe
NH
NMe
NH
NMe
NH
NMe
NH
NMe
NH
NMe
NH
NMe
NH
NMe
NH
NMe
NH
NMe
NH
NMe
NH
NMe
NH
NMe
NH
H
H
H
2′-OMe
2′-OMe
3′-OMe
3′-OMe
4′-OMe
4′-OMe
0.51
1.7
0.2
9
0.097
0.85
0.25
0.53
0.21
2.0
2.1
5.5
0.16
4.4
0.21
0.90
0.091
0.73
0.5
1.6
0.15
1.0
0.17
0.41
0.038
2.0
0.13
0.66
0.25
2.0
0.12
0.47
0.38
0.23
1.6
0.04
0.052
0.41
0.13
0.22
0.25
0.12
12
0.035
0.073
0.018
0.14
0.12
0.14
0.067
0.32
0.016
0.28
0.05
0.06
0.0027
0.088
0.058
0.2
0.047
0.082
0.048
0.054
0.017
0.2
0.026
0.044
0.087
0.57
0.052
0.16
10
11
12
13
14
14a a
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
29a a
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
TMP
TMQ
PTX
2′-Cl
2′-Cl
3′-Cl
3′-Cl
2′,4′-(OMe)2
2′,4′-(OMe)2
2′,5′-(OMe)2
2′,5′-(OMe)2
3′,4′-(OMe)2
3′,4′-(OMe)2
2′,4′-Cl2
2′,4′-Cl2
2′,5′-Cl2
2′,5′-Cl2
2′,6′-Cl2
2′,6′-Cl2
3′,4′-Cl2
3′,4′-Cl2
3′,4′,5′-(OMe)3
3′,4′,5′-(OMe)3
3′,4′,5′-Cl3
3′,4′,5′-Cl3
2′,4′,6′-Cl3
2′,4′,6′-Cl3
2′,3′-C4H4
3′,4′-C4H4
2′,3′-C4H4
3′,4′-C4H4
2′,3′-C4H4
3′,4′-C4H4
4′-COCH3
4′-COCH3
4′-COCH3
4′-COCF3
4′-COCF3
NMe
NH
NMe
S
S
NH
0.086
0.04
0.21
1.8
1.54
1.31
0.41
0.45
0.09
0.35
0.74
0.69
0.14
48
NH
NMe
NMe
NH
0.0073
0.0072
0.0025
0.0051
0.015
0.032
0.0075
130
NMe
N-propargyl
NMe
N-propargyl
11
0.07
0.048
0.042
0.031
0.003
0.0015
0.01
0.017
0.30
0.088
a
Values from ref 31.
Replacing the 2′-OMe substituent with electron-
withdrawing chlorine (compound 12) increases the
inhibitory potency against pc- and rlDHFR but not
tgDHFR. This causes a drop in selectivity against
tgDHFR compared to compound 6. N10 methylation of
12 to 13 allows for an increase in tgDHFR inhibitory
potency with slight increases against pcDHFR and
rlDHFR and parallels the 2′-OMe analogues 6 and 7.
The increase in tgDHFR activity of 13 is accompanied
by a significant increase in tgDHFR selectivity which
makes 13 the second most selective compound against
tgDHFR in this study. The 3′-Cl analogue 14 was
equipotent with 12 against rlDHFR and tgDHFR but
was less potent than 12 against pcDHFR. Thus the
position of the electron-withdrawing monosubstitution
was important for both activity and selectivity.
The phenyl ring was next disubstituted. For the
isomeric diOMe analogues, the 2′,4′-; 2′,5′-; and 3′,4′-
diOMe analogues were evaluated. The 2′,4′- and 2′,5′-
diOMe N10-H analogues 15 and 17 were very similar
in inhibitory activity and selectivity. N10 methylation
of 15 and 17 to afford 16 and 18, respectively, provided
for a 5-34-fold increase in activity against all three
DHFRs with selectivity ratios remaining almost un-
changed from 15 and 17. The increase in potency
against pcDHFR on N10 methylation was similar to
that observed for the 3′-OMe analogue.
The 3′,4′-diOMe analogues 19 and 20 followed the
same trend as the previous diOMe analogues regarding
increasing potency on N10 methylation with the impor-
tant difference that this substitution pattern was most
conducive to rlDHFR inhibition (rather than pcDHFR);

1840 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9
Gangjee et al.
consequently the molecules lacked selectivity toward
both pc- and tgDHFR. On the basis of the monomethoxy
and dimethoxy analogues the 3′-OMe substitution is
most conducive for potency but not selectivity.
analogue 34. For the N10 unsubstituted analogues the
R-naphthyl analogue 36 had significantly better inhibi-
tory activity against all three DHFRs than the â-naph-
thyl analogue 37. N10 methylation of 36 and 37 to 38
and 39, respectively, provides for increased potency
against all three enzymes (except for 38 against tgDH-
FR). With the exception of 34, which had some selectiv-
ity against tgDHFR, the other analogues were either
not selective or marginally selective.
The dichloro-substituted isomers were somewhat dif-
ferent from the diOMe analogues as well as from the
monochloro analogues. The 2′,4′-diCl analogue 21 was
more potent than the 2′-Cl analogue 12 against rlDH-
FR and tgDHFR and was also more potent than the
N10-methyl 2′-Cl analogue 13 against rlDHFR. N10
methylation of 21 to 22, in contrast to the electron-
donating diOMe analogues, does not increase inhibitory
activity against any of the three enzymes. The 2′,5′-
diCl analogue 23 was less active than the 2′,4′-diCl
compound; however, the decrease for rlDHFR was
greater than for tgDHFR, providing for a slight increase
in selectivity. N10 methylation of 23 to 24 increases
inhibitory activity against pc- and rlDHFR by 5-10-
fold but less so for tgDHFR. For the 2′,6′-diCl analogues
25 and 26, the activity of the N10-H compound 25 was
similar to the 2′,5′-diCl analogue 23. N10 methylation
of 25 to 26 increases potency against both rlDHFR and
pcDHFR, but interestingly, tgDHFR inhibition remains
unchanged. The 2′,6′-diCl substitution demonstrated
the same level of selectivity as the 2′,5′-diCl analogues
indicating that conformation restriction about the N10-
C1′ bond did not increase potency or selectivity. The
3′,4′-diCl analogues 27 and 28 were remarkably similar
to the 2′,5′- and 2′,6′-diCl analogues; however, these
analogues did not show any selectivity for either en-
zyme. Therefore in the disubstituted analogues, as was
observed for the monosubstituted compounds, the na-
ture and position of the substitution were important for
both activity and selectivity.
Compounds 40-44 with electron-withdrawing carbo-
nyl groups at the 4′-position were potent inhibitors of
tgDHFR and rlDHFR. Compound 41 had IC50 values
for these two DHFRs which were comparable to those
reported previously for TMQ and PTX31 and were more
potent than TMP.31 Potency of inhibition for pcDHFR
was 10-fold lower than that observed for rl- and tgDH-
FR. Given the potent inhibition of the mammalian and
T. gondii enzymes observed with 40-44, it would
appear that incorporation of a p-carbonyl group in the
PABA moiety has a greater affect on potency of inhibi-
tors for pcDHFR than does replacement of N8 with CH
in the pteridine ring. Compounds 40-44 were not
selective for pc- or tgDHFR owing to their better
inhibition of rlDHFR.
In summary, against pcDHFR, the activities ranged
from 0.038 × 10-6 M for 28 to 5.5 × 10-6 M for 15, a
range of 100-fold, and N10 methylation increased activ-
ity in all cases, but the increase ranged from a high of
30-fold (for 37 and 39) to activities that were almost
unchanged. The extent of the increase depended upon
both the nature and position of the substitution on the
phenyl ring. None of the compounds were selective for
pcDHFR.
For tgDHFR the activity range was only 20-fold with
the most active compound 5 at 0.0084 × 10-6 M and
the least active analogue 37 at 0.16 × 10-6 M. All the
analogues had IC50 values which were submicromolar
or better. N-Methylation afforded a 10-fold or less
increase in activity with the exception of the diCl, triCl,
R-naphthyl, and 4′-OCH3 analogues where the increase
was slight or absent. For pcDHFR activity, the increase
was dependent on the nature and position of the phenyl
substitution. In contrast to a lack of selectivity for
pcDHFR, several of the 8-deaza analogues were signifi-
cantly selective for tgDHFR, most notably 13, 29, and
32 which had selectivities of 8.0, 5.0, and 12.4, respec-
tively. For tgDHFR, 3′,4′-disubstitution was detrimen-
tal to selectivity irrespective of the nature or position
of the substitution. Thus compounds 19, 20, 27, and
28 were nonselective. In addition, the R- and â-naph-
thyl N10-methyl analogues 38 and 39 and the 4′-ketone
analogues 40-44 were nonselective.
One 3′,4′,5′-triOMe analogue 29 was synthesized, and
it demonstrated low inhibitory activity against pcDHFR
similar to the corresponding diOMe N10 unsubstituted
analogues described above. However, this analogue did
show selectivity for tgDHFR with a ratio of 5. Four
isomeric triCl analogues were also evaluated in this
series. The N10-methyl 3′,4′,5′-triCl analogue 31 and
more particularly the N10-H 2′,4′,6′-triCl analogue 32
were both selective for tgDHFR with 32 being the most
selective analogue of the compounds in this report. It
should be noted that N10 methylation of the 3′,4′,5′-
triCl isomer causes an increase in tgDHFR selectivity
by almost 5-fold. Surprisingly, however, N10 methy-
lation of the 2′,4′,6′-triCl isomer decreased tgDHFR
selectivity by more than 10-fold and was attributed to
a 10-fold increase in rlDHFR inhibitory potency. The
reason for this significant increase in potency is not
known at this time.
Three analogues, compounds 13, 29, and 32, had
excellent combinations of both high potency (IC50 values
from 1.5 × 10-8 to 4.6 × 10-8 M) and good selectivity
against tgDHFR. The potencies of these analogues are
comparable to TMQ and PTX, and the analogues are
from 17- to 43-fold more selective than TMQ and from
56- to 149-fold more selective than PTX. With respect
to TMP, the analogues 13, 29, and 32 are 180-, 68-, and
59-fold more potent than TMP and compound 32 is only
3.8 times less selective than TMP.
The R- and â-naphthyl-substituted 2,4-diaminopyrido-
[3,2-d]pyrimidines 34-39 were synthesized to deter-
mine the influence on inhibitory activities and selectiv-
ity of larger than phenyl substitutions on the S10 and
N10 moieties. For the S10, both R- and â-naphthyl
substitutions (compounds 34 and 35) increase activity
against all three DHFRs compared to the phenyl
analogue 3. However, the selectivity ratios remained
almost unaltered. It was interesting to note that while
the pcDHFR and tgDHFR activities remained unaltered
in going from the R- to the â-naphthyl in analogues 34
and 35, the rlDHFR activity for the â-naphthyl substi-
tution increased 2-fold compared to the R-naphthyl
While the manuscript for this paper was in prepara-
tion and subsequent to the publication of a preliminary
account of this work,1 a report of a limited series of six

Inhibitors of Dihydrofolate Reductases
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9 1841
phase mixture was acidified to pH 2 by dropwise addition of 2
N HCl. The water phase was separated, washed with chlo-
roform (2 × 10 mL), and basified to pH 11 with ammonium
hydroxide. The resulting precipitate was filtered. The filtrate
which contained additional amounts of the desired product was
extracted with methylene chloride (3 × 30 mL). The combined
methylene chloride extracts were dried over Na2SO4, and the
solvent was removed under reduced pressure. The solid
residue, along with the previously collected precipitate, was
combined and dissolved in 10 mL of methanol, and 2 g of silica
gel was added and the methanol evaporated to form a plug
which was dried and loaded onto a ammonium hydroxide
pretreated silica gel column and eluted with 6% methanol in
chloroform. The fractions with Rf 0.28-0.35 (in 1: 4 methanol/
chloroform) were pooled, and the solvent was evaporated to
afford the desired products. The overall yield for the last two
steps combined varied from 20 to 57%.
2,4-Dia m in o-6-[(p h en ylt h io)m et h yl]p yr id o[3,2-d ]p y-
r im id in e (3). Compound 3 was synthesized from the bromide
49 obtained from 48 (0.48 g, 2.5 mmol) with thiophenol (0.55
g, 5 mmol) to afford 0.24 g (34%) of 3: TLC MeOH/CHCl3/
NH4OH (3:7:2 drops)/silica gel, Rf 0.67; mp 190-192 °C; 1H
NMR (DMSO-d6) δ 4.37 (s, 2H, 9-CH2), 6.21 (s, 2H, 2-NH2,
exch), 7.27 (t, 1H, 4′-H), 7.30 (t, 2H, 3′-H and 5′-H), 7.39 (d,
2H, 2′-H and 6′-H), 7.01-7.47 (collapsed br, 2H, 4-NH2, exch),
7.58 (dd, 2H, 7-H and 8-H); MS (EI) m/ e 283 (M+), 174, 132.
Anal. (C14H13N5S) C, H, N, S.
analogues, which included compounds 17, 28, and 29,
was submitted and later published by Rosowsky et al.31
In their report, these workers concluded that the design
of fused-ring 2,4-diaminopyrimidine systems with the
high potency of TMQ and PTX along with the high
selectivity of TMP, remained a difficult challenge.
Compound 32 of this report, a 2′,4′,6′-triCl analogue,
has a potency of 46 nM and a selectivity of 12.4 for
tgDHFR. The potency of 32 against tgDHFR is com-
parable to TMQ and PTX, and the selectivity is 43- and
149-fold better than TMQ and PTX, respectively. Com-
pared to TMP, compound 32 is almost 60 times more
potent and only 3.8 times less selective. Thus, com-
pound 32 demonstrates that the 6-substituted-2,4-
diaminopyrido[3,2-d]pyrimidines are viable lead com-
pounds with significant potency and selectivity. Further
studies of the inhibition of the growth of T. gondii cells
in culture are currently in progress with 32 as a prelude
to whole animal studies. The high lipid solubility of 32
bodes well for its activity both in in vitro cell culture
and in animal studies and will be reported elsewhere.
Exp er im en ta l Section
All evaporations were carried out in vacuo with a rotary
evaporator or by short-path distillation. Analytical samples
were dried in vacuo (0.2 mmHg) in an Abderhalden drying
apparatus over P2O5 and refluxing ethanol or toluene. Thin-
layer chromatography (TLC) was performed on Eastman
Kodak silica gel chromatogram plates with fluorescent indica-
tor. Spots were visualized by UV light (254 and 350 nm) or
using vanillin as an indicator. All analytical samples were
homogeneous on TLC in at least two different solvent systems.
Purifications by column and flash chromatography were
carried out using Merck silica gel 60 (230-400 mesh). Sepa-
rations through reversed-phase LC were achieved on a 4.6 mm
× 25 cm Microsorb-MV 5 C-18 column with a mobile phase of
20% distilled water in acetonitrile at a flow rate of 1 mL/min.
Melting points were determined by capillary method on a
Fisher-J ohns or Thomas-Hoover melting point apparatus and
are uncorrected. UV spectra were obtained on a Hewlett-
Packard 8452A diode array spectrophotometer operated in the
general scanning mode. 1H NMR spectra were recorded on a
Bruker WH-300 (300 MHz) or an IBM AF200 FT-NMR
spectrometer. The chemical shifts (δ) values are reported as
parts per million (ppm) relative to tetramethylsilane as
internal standard: s ) singlet, d ) doublet, dd ) doublet of
doublet, t ) triplet, q ) quartet, m ) multiplet, br ) broad
peak, br s ) broad singlet, exch ) protons exchangeable by
addition of D2O. Mass spectra were obtained with either a
Finnegan MAT94 or a Finnegan MAT95 mass spectrometer.
Purity of compounds submitted for high-resolution mass
analysis was verified by high-performance liquid chromatog-
raphy (HPLC) utilizing a Hitachi L6200 intelligent pump
equipped with a L3000 photo diode array detector. Elemental
analyses were performed by Atlantic Microlab, Inc., Atlanta,
GA, or M-H-W Laboratories, Phoenix, AZ. Elemental compo-
sitions were within (0.4% of the calculated values.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
3, 4, 6, 8-12, 14-23, 25, 27, 29, 30, 32, 34-39. A suspension
of compound 48 (0.48 g, 2.5 mmol) in 10 mL of anhydrous THF
was stirred with 0.67 mL of phosphorustribromide overnight.
The mixture was rapidly filtered under nitrogen and the
precipitate washed with cold ether and dried to afford crude
bromide 49 which was used without further purification. To
a suspension of freshly prepared bromide 49 in 12 mL of
anhydrous dimethylacetamide (DMAc) was added 5 mmol of
the appropriate nucleophile (aryl thiol, N-H or N-Me substi-
tuted or unsubstituted aniline, or naphthylamine). The
suspension was stirred at 50-60 °C under nitrogen for 2-3
days until all the bromide 49 had reacted as evidenced on TLC.
DMAc was removed under vacuum, and 15 mL of water and
40 mL of chloroform were then added to the flask. The two-
2,4-Dia m in o-6-(a n in ilom e t h yl)p yr id o[3,2-d ]p yr im i-
d in e (4). Using the same quantities as described for com-
pound 3, 0.22 g (34%) of compound 4 was obtained: TLC
MeOH/CHCl3
222 °C; 1H NMR (DMSO-d6) δ 4.38 (d, 2H, 9-CH2), 6.14 (s, 2H,
, exch), 6.37 (t, 1H, N10-H, exch), 6.55 (t, 1H, 4′-H), 6.70
/NH OH (3:7:2 drops)/silica gel, R 0.61; mp 220-
4
f
2-NH2
(d, 2H, 2′-H and 6′-H), 7.09 (t, 2H, 3′-H and 5′-H), 7.37 (br,
1H, 4-NH2, exch), 7.60 (br, 1H, 4- NH2, exch), 7.54 (d, 2H, 7-H
and 8-H). Anal. (C14H14N6‚0.25NH4Cl) C, H, N.
2,4-Dia m in o-6-[(2′-m eth oxya n ilin o)m eth yl]p yr id o[3,2-
d ]p yr im id in e (6). This compound was obtained using the
same quantities described for 3 and afforded 0.18 g (24%) of
6: TLC MeOH/CHCl3/NH4OH (3:7:2 drops)/silica gel, Rf 0.67;
mp 194-196 °C; 1H NMR (DMSO-d6) δ 3.81 (s, 3H, 2′-OCH3),
4.42 (d, 2H, 9-CH2), 5.78 (t, 1H, N10-H, exch), 6.16 (s, 2H,
2-NH2, exch), 6.51 (m, 2H, C6H4), 6.68 (t, 1H, C6H4), 6.81 (d,
1H, C6H4), 7.15-7.40 (br, 2H, 4-NH2, exch), 7.50 (s, 2H, 7-H
and 8-H). Anal. (C15H16N6O) C, H, N.
2,4-Dia m in o-6-[(3′-m eth oxya n ilin o)m eth yl]p yr id o[3,2-
d ]p yr im id in e (8). Using the same quantities as described
for compound 3, 0.16 g (21%) of compound 8 was obtained:
TLC MeOH/CHCl3/NH4OH (3:7:2 drops)/silica gel, Rf 0.63; mp
1
180-183 °C dec; H NMR (DMSO- d6) δ 4.37 (d, 2H, 9-CH2),
6.07 (s, 2H, 2-NH2, exch), 6.15 (dd, 1H, C6H4), 6.25-6.32 (m,
2H, C6H4), 6.40 (t, 1H, N10-H, exch), 7.00 (t, 1H, C6H4), 7.42
(br, 1H, 4-NH2, exch), 7.54 (d, 2H, 7-H and 8-H), 7.70 (br, 1H,
4- NH2, exch). Anal. (C15H16N6O‚0.2H2O) C, H, N.
2,4-Dia m in o-6-[(3′-m eth oxy-N-m eth yla n ilin o)m eth yl]-
p yr id o[3,2-d ]p yr im id in e (9). N-Methyl-3-methoxyaniline
(0.45 g, 3.3 mmol) was condensed with intermediate 49
prepared from 48 (2 mmol, 0.38 g) to afford 0.20 g (32%) of
compound 9: TLC MeOH/CHCl3/NH4OH (3:7:2 drops)/silica
gel, Rf 0.69; mp 179-180.5 °C; 1H NMR (DMSO-d6) δ 3.10 (s,
3H, N10-CH3), 3.66 (s, 3H, 3′-OCH3), 4.64 (s, 2H, 9-CH2), 6.19
(m, 2H, C6H4), 6.24 (br s, 2H, 2-NH2, exch), 6.34 (dd, 1H, C6H4),
7.03 (t, 1H, C6H4), 7.13 (br, 1H, 4-NH2, exch), 7.31 (d, 1H, 7-H
or 8-H), 7.37 (br, 1H, 4-NH2, exch), 7.49 (d, 1H, 7-H or 8-H).
Anal. (C16H18N6O‚0.2H2O) C, H, N.
2,4-Dia m in o-6-[(4′-m eth oxya n ilin o)m eth yl]p yr id o[3,2-
d ]p yr im id in e (10). Anisidine (0.73 g, 5.6 mmol) was con-
densed with the bromide 49 prepared from 48 (0.48 g, 2.5
mmol) in anhydrous DMAc at room temperature for 3 days.
After workup and separation, 0.20 g (27%) of compound 10
was obtained: TLC MeOH/CHCl3/NH4OH (3:7:2 drops)/silica
1
gel, Rf 0.63; mp 197.5-199.5 °C; H NMR (DMSO-d6) δ 3.63
(s, 3H, 4′-OCH3), 4.32 (d, 2H, 9-CH2), 6.00 (t, 1H, N10-H, exch),
6.13 (s, 2H, 2-NH2, exch), 6.70 (dd, 4H, C6H4), 7.35 (br, 1H,
4-NH2, exch), 7.59 (br, 1H 4-NH2, exch), 7.60 (s, 2H, 7-H and
8-H). Anal. (C15H16N6O‚0.05NH4Cl) C, H, N.

1842 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9
Gangjee et al.
2,4-Dia m in o-6-[(4′-m eth oxy-N-m eth yla n ilin o)m eth yl]-
p yr id o[3,2-d ]p yr im id in e (11). The bromide 49 prepared
from 48 (0.48 g, 2.5 mmol) was condensed with N-methylani-
sidine (0.68 g, 5 mmol) which was synthesized by methylation
of anisidine with 1.2 equiv of methyl iodide in the presence of
1.2 equiv of iPr2NEt at 35 °C for 10 h. The yield of compound
11 was 0.42 g (54%): TLC MeOH/CHCl3/NH4OH (3:7:2 drops)/
silica gel, Rf 0.69; mp 240-242 °C dec; 1H NMR (DMSO-d6) δ
3.00 (s, 3H, N10-CH3), 3.64 (s, 3H, 4′-OCH3), 4.56 (s, 2H, 9-CH2),
6.17 (s, 2H, 2-NH2, exch), 6.75 (m, 4H, C6H4), 7.14 (br, 1H,
4-NH2, exch), 7.31 (collapsed br, 1H, 4-NH2, exch), 7.34 (d, 1H,
7-H or 8-H), 7.49 (d, 1H, 7-H or 8-H). Anal. (C16H18N6O‚0.4H2-
O) C, H, N.
2,4-Dia m in o-6-[(2′-ch lor oa n ilin o)m eth yl]p yr id o[3,2-d ]-
p yr im id in e (12). Using the same quantities as described for
compound 3, 0.19 g (26%) of compound 12 was obtained: TLC
MeOH/CHCl3/NH4OH (3:7:2 drops)/silica gel, Rf 0.67; mp 232-
234 °C dec; 1H NMR (DMSO-d6) δ 4.51 (d, 2H, 9-CH2), 6.16 (s,
2H, 2-NH2, exch), 6.28 (t, 1H, N10-H, exch), 6.58 (t, 1H, C6H4),
6.67 (d, 1H, C6H4), 7.04 (t, 1H, C6H4), 7.27 (d, 1H, C6H4), 7.10-
7.45 (collapsed br, 2H, 4-NH2, exch), 7.52 (dd, 2H, 7-H and
8-H). Anal. (C14H13N6Cl) C, H, N, Cl.
2,4-Dia m in o-6-[(3′-ch lor oa n ilin o)m eth yl]p yr id o[3,2-d ]-
p yr im id in e (14). Using the same quantities as described for
compound 3 afforded 0.21 g (28%) of compound 14: TLC
MeOH/CHCl3/NH4OH (3:7:2 drops)/silica gel, Rf 0.64; mp 178-
180 °C; 1H NMR (DMSO-d6) δ 4.39 (d, 2H, 9-CH2), 6.15 (br s,
2H, 2-NH2, exch), 6.54-6.76 (m, 4H, N10-H, exch and C6H4),
7.10 (t, 1H, 5′-H), 7.38 (br, 1H, 4-NH2, exch), 7.63 (br, 1H,
4-NH2, exch), 7. 52 (q, 2H, 7-H and 8-H). Anal. (C14H13N6-
Cl) C, H, N, Cl.
2,4-Dia m in o-6-[(3′,4′-d im eth oxya n ilin o)m eth yl]p yr id o-
[3,2-d ]p yr im id in e (19). Using the same quantities as de-
scribed for compound 3 afforded 0.30 g (37%) of the product
19: TLC MeOH/CHCl3/NH4OH (3:7:2 drops)/silica gel, Rf 0.67;
mp 255-256 °C dec; 1H NMR (DMSO-d6) δ 3.61 (s, 3H, OCH3),
3.70 (s, 3H, OCH3), 4.33 (d, 2H, 9-CH2), 6.06 (t, 1H, N10-H,
exch), 6.15 (m, 3H, 6′-H and 2-NH2, exch), 6.47 (d, 1H, 2′-H),
6.71 (d, 1H, 5′-H), 7.36 (br, 1H, 4-NH2, exch), 7.60 (br, 1H,
4-NH2, exch), 7.52 (d, 2H, 7-H and 8-H). Anal. (C16H18N6O2‚
0.8H2O) C, H, N.
2,4-Diam in o-6-[(3′,4′-dim eth oxy-N-m eth ylan ilin o)m eth -
yl]p yr id o[3,2-d ]p yr im id in e (20). N-Methyl-3′,4′-dimethoxy-
aniline (0.66 g, 4 mmol) was prepared from 3,4-dimethoxy-
aniline (1.5 g, 10 mmol) with stirring with 1.1 equiv of MeI
and 1.1 equiv of iPr2NEt in acetonitrile and was reacted with
49 [from 48 (0.38 g, 2 mmol)] to afford 0.20 g (29%) of
compound 20: TLC MeOH/CHCl3/NH4OH (3:7:2 drops)/silica
1
gel, Rf 0.76; mp 207-209 °C dec; H NMR (DMSO-d6) δ 3.03
(s, 3H, N10-CH3), 3.62 (s, 3H, OCH3), 3.68 (s, 3H, OCH3), 4.59
(s, 2H, 9-CH2), 6.16 (br s, 2H, 2-NH2, exch), 6.20 (dd, 1H, 6′-
H), 6.47 (d, 1H, 2′-H), 6.76 (d, 1H, 5′-H), 7.10-7.35 (br, 2H,
4-NH2, exch), 7.36 (d, 1H, 7-H or 8-H), 7.51 (d, 1H, 7-H or 8-H).
Anal. (C17H20N6O2‚0.5H2O) C, H, N.
2,4-Dia m in o-6-[(2′,4′-d ich lor oa n ilin o)m et h yl]p yr id o-
[3,2-d ]p yr im id in e (21). Compound 49 [from 48 (0.19 g, 1
mmol)] was reacted with 2,4-dichloroaniline (0.40 g, 2.0 mmol)
to afford 0.12 g (37%) of 21: TLC MeOH/CHCl3/NH4OH (3:
7:2 drops)/silica gel, Rf 0.71; mp 239-241 °C dec; 1H NMR
(DMSO-d6) δ 4.49 (d, 2H, 9-CH2), 6.16 (s, 2H, 2-NH2, exch),
6.43 (t, 1H, N10-H), 6.68 (d, 1H, 6′-H), 7.10 (dd, 1H, 5′-H), 7.36
(d, 1H, 3′-H), 7.15-7.35 (collapsed br, 2H, 4-NH2, exch), 7.50
(dd, 2H, 7-H and 8-H). Anal. (C14H12N6Cl2‚0.2HCl) C, H, N,
Cl.
2,4-Dia m in o-6-[(2′,4′-d im eth oxya n ilin o)m eth yl]p yr id o-
[3,2-d ]p yr im id in e (15). Using the same quantities as de-
scribed for compound 3 afforded 0.31 g (38%) of compound 15:
TLC MeOH/CHCl3/NH4OH (3:7:2 drops)/silica gel, Rf 0.72; mp
2,4-Dia m in o-6-[(2′,4′-ch lor o-N-m eth yla n ilin o)m eth yl]-
p yr id o[3,2-d ]p yr im id in e (22). Bromide 49 prepared from
48 (0.38 g, 2 mmol) was reacted with N-methyl-2,4-dichloro-
aniline (0.70 g, 4 mmol) to afford 0.15 g (22%) of 22: TLC
MeOH/CHCl3/NH4OH (3:7:2 drops)/silica gel, Rf 0.77; mp 180-
1
>200 °C dec; H NMR (DMSO-d6) δ 3.62 (s, 3H, OCH3), 3.80
(s, 3H, OCH3), 4.36 (d, 2H, 9-CH2), 5.35 (t, 1H, N10-H, exch),
6.14 (s, 2H, 2-NH2, exch), 6.27 (dd, 1H, 5′-H), 6.39 (d, 1H, 6′-
H), 6.48 (d, 1H, 3′-H), 7.26 (br, 2H, 4-NH2, exch), 7.50 (s, 2H,
7-H and 8-H). Anal. (C16H18N6O2‚0.5H2O) C, H, N.
1
182 °C; H NMR (DMSO-d6) δ 2.70 (s, 3H, N10-CH3), 4.33 (s,
2H, 9-CH2), 6.20 (s, 2H, 2-NH2, exch), 7.08 (br, 1H, 4-NH2,
exch), 7.23 (d, 1H, 6′-H), 7.33 (m, 2H, 4-NH2, exch, and 5′-H),
7.58 (m, 3H, 3′-H, 7-H and 8-H). Anal. (C15H14N6Cl2) C, H,
N, Cl.
2,4-Diam in o-6-[(2′,4′-dim eth oxy-N-m eth ylan ilin o)m eth -
yl]p yr id o[3,2-d ]p yr im id in e (16). N-Methyl-2,4-dimethoxy-
aniline was prepared from 2,4-dimethylaniline (2 g, 13 mmol),
methyl iodide (1.1 equiv), and triethylamine (1.1 equiv) in
acetonitrile at 35 °C for 8 h (60% yield). N-Methylated aniline
(0.47 g, 2.8 mmol) reacted with 49 prepared from 48 (0.38 g,
2 mmol) afforded 0.20 g (35%) of the product 16: TLC MeOH/
CHCl3/NH4OH (3:7:2 drops)/silica gel, Rf 0.73; mp 191-193
2,4-Dia m in o-6-[(2′,5′-d ich lor oa n ilin o)m et h yl]p yr id o-
[3,2-d ]p yr im id in e (23). Compound 49 [from 48 (0.38 g, 2
mmol)] was reacted with 2,5-dichloroaniline (0.65 g, 4 mmol)
to afford 0.15 g (22%) of 23: TLC MeOH/CHCl3/NH4OH (3:
7:2 drops)/silica gel, Rf 0.73; mp >270 °C dec; 1H NMR (DMSO-
d6) δ 4.50 (d, 2H, 9-CH2), 6.21 (s, 2H, 2-NH2, exch), 6.55 (t,
1H, N10-H, exch), 6.60 (dd, 1H, 4′-H), 6.72 (d, 1H, 6′-H), 7.15
(br, 1H, 4-NH2, exch), 7.29 (d, 1H, 3′-H), 7.35 (br, 1H, 4-NH2,
exch), 7.52 (dd, 2H, 7-H and 8-H). Anal. (C14H12N6Cl2‚0.3H2O)
C, H, N, Cl.
1
°C; H NMR (DMSO-d6) δ 2.59 (s, 3H, N10-CH3), 3.71 (s, 3H,
OCH3), 3.82 (s, 3H, OCH3), 4.12 (s, 2H, 9-CH2), 6.16 (br s, 2H,
2-NH2, exch), 6.41 (dd, 1H, 5′-H), 6.57 (d, 1H, 3′-H), 6.86 (d,
1H, 6′-H), 7.05-7.4 (br, 2H, 4-NH2, exch), 7.57 (dd, 2H, 7-H
and 8-H). Anal. (C17H20N6O2) C, H, N.
2,4-Dia m in o-6-[(2′,6′-d ich lor oa n ilin o)m et h yl]p yr id o-
[3,2-d ]p yr im id in e (25). Using the same quantities as de-
scribed for 23 and the same procedure as described in general
procedure except that the temperature of the reaction was
raised to 100 °C afforded 0.13 g of 25 (19%): TLC MeOH/
CHCl3/NH4OH (3:7:2 drops)/silica gel, Rf 0.75; mp >190 °C dec;
1H NMR (DMSO-d6) δ 4.68 (d, 2H, 9-CH2), 5.85 (t, 1H, N10-H,
exch), 6.30 (br s, 2H, 2-NH2, exch), 6.84 (t, 1H, 4′-H), 7.03 (br,
1H, 4-NH2, exch), 7.33 (d, 2H, 3′-H and 5′-H), 7.48 (br, 1H,
4-NH2, exch), 7.55 (s, 2H, 7-H and 8-H). Anal. (C14H12N6-
Cl2‚0.35H2O) C, H, N, Cl.
2,4-Dia m in o-6-[(2′,5′-d im eth oxya n ilin o)m eth yl]p yr id o-
[3,2-d ]p yr im id in e (17). Compound 49 prepared from 48
(0.48 g, 2.5 mmol) was reacted with 2,5-dimethoxyaniline (0.61
g, 4 mmol) at 82 °C for 14 h to afford 0.23 g (28%) of 17: TLC
MeOH/CHCl3/NH4OH (3:7:2 drops)/silica gel, Rf 0.74; mp 199-
201 °C; 1H NMR (DMSO-d6) δ 3.55 (s, 3H, OCH3), 3.76 (s, 3H,
OCH3), 4.40 (d, 2H, 9-CH2), 5.92 (t, 1H, N10-H, exch), 6.06 (br
s, 2H, C6H3), 6.63 (br s, 2H, 2-NH2, exch), 6.70 (d, 1H, C6H3),
7.58 (q, 2H, 7-H and 8-H), 7.77 (br, 2H, 4-NH2, exch). Anal.
(C16H18N6O2‚0.58H2O) C, H, N.
2,4-Diam in o-6-[(2′,5′-dim eth oxy-N-m eth ylan ilin o)m eth -
yl]p yr id o[3,2-d ]p yr im id in e (18). N-Methyl-2,5-dimethoxy-
aniline (1.74 g, 79%) was prepared from 2,5-dimethoxyaniline
(2.5 g, 16 mmol) and methyl iodide (1.2 equiv) at the presence
of iPr2NEt (1.2 equiv) at 40 °C for 36 h. Then 0.67 g of it was
condensed with 49 synthesized from 48 (0.48 g, 2.5 mmol)
afforded 0.27 g (32%) of compound 18: TLC MeOH/CHCl3/NH4-
OH (3:7:2 drops)/silica gel, Rf 0.77; mp 202-204 °C dec; 1H
NMR (DMSO-d6) δ 2.67 (s, 3H, N10-CH3), 3.66 (s, 3H, OCH3),
3.75 (s, 3H, OCH3), 4.32 (s, 2H, 9-CH2), 6.17 (br s, 2H, 2-NH2,
exch), 6.45 (m, 2H, 4′-H and 6′-H), 6.86 (d, 1H, 3′-H), 6.96-
7.42 (br d, 2H, 4-NH2, exch), 7.57 (q, 2H, 7-H and 8-H). Anal.
(C17H20N6O2) C, H, N.
2,4-Dia m in o-6-[(3′,4′-d ich lor oa n ilin o)m et h yl]p yr id o-
[3,2-d ]p yr im id in e (27). Using the same quantities as de-
scribed for compound 3 afforded 0.17g (21%) of 27: TLC
MeOH/CHCl3/NH4OH (3:7:2 drops)/silica gel, Rf 0.64; mp 187-
1
189 °C dec; H NMR (DMSO-d6) δ 4.40 (d, 2H, 9-CH2), 6.18
(br s, 2H, 2-NH2, exch), 6.73 (dd, 1H, 6′-H), 6.81 (t, 1H, N10-H,
exch), 6.94 (d, 1H, 2′-H), 7.30 (d, 1H, 5′-H), 7.42 (br, 1H, 4-NH2,
exch), 7.52 (dd, 2H, 7-H and 8-H), 7.65 (br, 1H, 4-NH2, exch).
Anal. (C14H12N6Cl2‚0.4H2O‚0.4HCl) C, H, N, Cl.
2,4-Dia m in o-6-[(3′,4′,5′-tr im eth oxya n ilin o)m eth yl]p yr -
id o[3,2-d ]p yr im id in e (29). Using the same quantities as
described for compound 3 afforded 0.36 g (41%) of 29: TLC

Inhibitors of Dihydrofolate Reductases
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9 1843
MeOH/CHCl3/NH4OH (3:7:2 drops)/silica gel, Rf 0.66; mp 214-
216 °C; 1H NMR (DMSO-d6) δ 3.53 (s, 3H, OCH3), 3.71 (s, 6H,
OCH3), 4.38 (d, 2H, 9-CH2), 6.05 (s, 2H, 2′-H and 6′-H), 6.15
(br s, 2H, 2-NH2, exch), 6.24 (t, 1H, N10-H, exch), 7.39 (br s,
1H, 4-NH2, exch), 7.53 (d, 2H, 7-H and 8-H), 7.57 (br s, 1H,
4-NH2, exch). Anal. (C17H20N6O3) C, H, N.
2,4-Dia m in o-6-[(N-m eth yl-2′-n a p h th yla m in o)m eth yl]-
p yr id o[3,2-d ]p yr im id in e (39). N-Methyl-2-naphthylamine
(0.64 g, 4 mmol) was prepared from 1-naphthylamine (0.93 g,
7.5 mmol) on stirring with 1.1 equiv of MeI and 1.1 equiv of
Et3N in acetonitrile and was reacted with bromide 49 made
from 48 (0.38 g, 2 mmol) to afford 0.22 g (33%) of compound
39: TLC MeOH/CHCl3/NH4OH (3:7:2 drops)/silica gel, Rf 0.71;
mp 215-217 °C; 1H NMR (DMSO-d6) δ 3.18 (s, 3H, N10-CH3),
4.80 (s, 2H, 9-CH2), 6.84 (br s, 2H, 2-NH2, exch), 7.01 (d, 1H,
C10H7), 7.14 (br t, 2H, C10H7 and 4-NH2, exch), 7.26-7.39 (m,
4H, C10H7 and 4-NH2, exch), 7.49 (d, 2H, 7-H and 8-H), 7.63-
7.71 (m, 3H, C10H7). Anal. (C19H18N6‚0.34HCl) C, H, N.
Gen er a l P r oced u r e for th e Red u ctive N-Meth yla tion
of 4, 6, 12, 23, 25, 27, 30, a n d 32. To a suspension of 6 (120
mg, 0.40 mmol) in 10 mL of acetonitrile was added 0.15 mL of
37% HCHO (1.8 mmol), followed by the addition of NaCNBH3
(0.08 g, 1.2 mmol). The reaction mixture was stirred for 5 min
and followed by the addition of 2 N HCl dropwise over 2-3
min until the suspension dissolved (pH 2). After a while a
white solid started to precipitate out of the solution. The
reaction was continued at room temperature and monitored
by TLC (CHCl3:MeOH, 7:3). After 15 h, the starting material
had reacted, at which time the solvent (acetonitrile) was
removed using a rotary evaporator. The residue was dissolved
in 10 mL of water and 1 mL of ethyl acetate and the resulting
solution basified with ammonium hydroxide to pH 10. The
white solid which precipitated was filtered, washed with ethyl
acetate (2 × 0.5 mL), and dried in vacuum overnight to afford
0.12 g (99%) of 7.
2,4-Dia m in o-6-[(3′,4′,5′-tr ich lor oa n ilin o)m eth yl]pyr id o-
[3,2-d ]p yr im id in e (30). Using the same procedure and the
same quantities as described for 3 afforded 0.21 g (22%) of
compound 30: TLC MeOH/CHCl3/NH4OH (3:7:2 drops)/silica
1
gel, Rf 0.64; mp 248-250 °C dec; H NMR (DMSO-d6) δ 4.42
(d, 2H, 9-CH2), 6.20 (s, 2H, 2-NH2, exch), 7.00 (br s, 3H, N10
-
H, exch, 2′-H and 6′-H), 7.47 (collapsed br, 1H, 4-NH2, exch),
7.67 (br, 1H, 4-NH2, exch), 7.53 (dd, 2H, 7-H and 8-H). Anal.
(C14H11N6Cl3) C, H, N, Cl.
2,4-Dia m in o-6-[(2′,4′,6′-tr ich lor oa n ilin o)m eth yl]pyr ido-
[3,2-d ]p yr im id in e (32). Compound 32 was synthesized by
the procedure as described for 3. The yield was 0.19 g (23%):
TLC MeOH/CHCl3/NH4OH (3:7:2 drops)/silica gel, Rf 0.70; mp
1
>260 °C dec; H NMR (DMSO-d6) δ 4.67 (d, 2H, 9-CH2), 5.96
(t, 1H, N10-H, exch), 6.22 (br s, 2H, 2-NH2, exch), 6.90 (br, 1H,
4-NH2, exch), 7.36 (br, 1H, 4-NH2, exch), 7.50 (s, 2H, 3′-H and
5′-H), 7.53 (s, 2H, 7-H and 8-H). Anal. (C14H12N6Cl3‚0.19C6H6)
C, H, N, Cl.
2,4-Dia m in o-6-[(1′-n a p h th ylth io)m eth yl]p yr id o[3,2-d ]-
p yr im id in e (34). Compound 49 [from 48 (0.38 g, 2 mmol)]
was reacted with 1-naphthylthiol (0.64 g, 4 mmol) to afford
0.23 g (35%) of 34: TLC MeOH/CHCl3/NH4OH (3:7:2 drops)/
silica gel, Rf 0.68; mp 181-183 °C; 1H NMR (DMSO-d6) δ 4.44
(s, 2H, 9-CH2), 6.21 (s, 2H, 2-NH2, exch), 7.10 (br s, 1H, 4-NH2,
exch), 7.32 (br s, 1H, 4-NH2, exch), 7.38-7.60 (m, 5H, C10H7,
7-H and 8-H), 7.68 (d, 1H, C10H7), 7.78 (d, 1H, C10H7), 7.92 (d,
1H, C10H7), 8.21 (d, 1H, C10H7). Anal. (C18H15N5S‚0.15H2O)
C, H, N, S.
2,4-Diam in o-6-[(N-m eth ylan ilin o)m eth yl]pyr ido[3,2-d]-
p yr im id in e (5). Compound 4 (0.10 g, 0.38 mmol) was reacted
with 37% HCHO (0.15 mL, 1.8 mmol) and NaCNBH3 (0.080
g, 1.2 mmol) for 4.5 h using the procedure described above.
After column chromatographic purification using MeOH/CH3-
Cl/NH4OH (50:450:1) as eluent, 0.063 g (60%) of 5 was
obtained: TLC MeOH/CHCl3/NH4OH (3:7:2 drops)/silica gel,
2,4-Dia m in o-6-[(2′-n a p h th ylth io)m eth yl]p yr id o[3,2-d ]-
p yr im id in e (35). Bromide 49 prepared from 48 (0.38 g, 2
mmol) was reacted with 2-naphthylthiol (0.64 g, 4 mmol) to
afford 0.27 g (41%) of 35: TLC MeOH/CHCl3/NH4OH (3:7:2
1
Rf 0.68; mp 182-184 °C; H NMR (DMSO-d6) δ 3.10 (s, 3H,
N10-CH3), 4.66 (s, 2H, 9-CH2), 6.24 (br s, 2H, 2-NH2, exch), 6.61
(t, 1H, 4′-H), 6.75 (d, 2H, 2′-H and 6′-H), 7.14 (m, 2H, 3′-H
and 5′-H), 7.20 (br, 1H, 4-NH2, exch), 7.33 (d, 1H, 7-H or 8-H),
7.40 (br, 1H, 4-NH2, exch), 7.51 (d, 1H, 7-H or 8-H). Anal.
(C15H16N6‚0.44HCl) C, H, N.
1
drops)/silica gel, Rf 0.70; mp >200 °C dec; H NMR (DMSO-
d6) δ 4.48 (s, 2H, 9-CH2), 6.22 (br s, 2H, 2-NH2, exch), 7.36
(br, 2H, 4-NH2, exch), 7.40-8.10 (m, 9H, C10H7, 7-H and 8-H).
Anal. (C18H15N5S‚0.40H2O‚0.30AcOH) C, H, N, S.
2,4-Dia m in o-6-[(2′-m eth oxy-N-m eth yla n ilin o)m eth yl]-
p yr id o[3,2-d ]p yr im id in e (7): TLC MeOH/CHCl3/NH4OH (3:
7:2 drops)/silica gel, Rf 0.70; mp 177.5-179.6 °C; 1H NMR
(DMSO-d6) δ 2.66 (s, 3H, N10-CH3), 3.82 (s, 3H, 2′-OCH3), 4.29
(s, 2H, 9-CH2), 6.17 (s, 2H, 2-NH2, exch), 6.90 (m, 4H, C6H4),
7.09 (br s, 1H, 4-NH2, exch), 7.28 (br s, 1H, 4-NH2, exch), 7.53
(d, 1H, 7-H or 8-H), 7.62 (d, 1H, 7-H or 8-H). Anal.
(C16H18N6O‚0.5H2O) C, H, N.
2,4-Dia m in o-6-[(2′-ch lor o-N-m et h yla n ilin o)m et h yl]-
p yr id o[3,2-d ]p yr im id in e (13). Compound 12 (0.112 g, 0.37
mmol) was reacted with 37% HCHO (0.15 mL, 1.8 mmol) and
NaCNBH3 (0.080 g, 1.2 mmol) for 3 h using the procedure as
described in the general procedure. After purification through
a column eluted with 6% MeOH in chloroform (v/v), 0.102 g
(88%) of 13 was obtained: TLC MeOH/CHCl3/NH4OH (3:7:2
drops)/silica gel, Rf 0.63; mp 212-214.5 °C; 1H NMR (DMSO-
d6) δ 2.70 (s, 3H, N10-CH3), 4.33 (s, 2H, 9-CH2), 6.17 (s, 2H,
2-NH2, exch), 7.04-7.28 [m, 5H, 4-NH2 (exch), 4′-H, 5′-H and
6′-H], 7.44 (dd, 1H, 3′-H), 7.55 (d, 1H, 7-H or 8-H), 7.65 (d,
1H, 7-H or 8-H). Anal. (C15H15N6Cl) C, H, N, Cl.
2,4-Diam in o-6-[(2′,5′-dich lor o-N-m eth ylan ilin o)m eth yl]-
p yr id o[3,2-d ]p yr im id in e (24). Compound 23 (0.053 g, 0.16
mmol) was reductively N-methylated for 12 h to form 24 using
the procedure as described as in the general procedure to afford
0.043 g (78%) of 24: TLC MeOH/CHCl3/NH4OH (3:7:2 drops)/
silica gel, Rf 0.72; mp >260 °C dec; 1H NMR (DMSO-d6) δ 2.79
(s, 3H, N10-CH3), 4.46 (s, 2H, 9-CH2), 6.44 (s, 2H, 2-NH2, exch),
7.15 (dd, 1H, 4′-H), 7.33 (d, 1H, 6′-H), 7.52 (d, 1H, 4′-H), 7.60-
8.10 (m, 4H, 4-NH2, exch, 7-H and 8-H). Anal. (C15H14N6-
Cl2‚0.7HCl‚0.8H2O) C, H, N, Cl.
2,4-Dia m in o-6-[(1′-n a p h th yla m in o)m eth yl]p yr id o[3,2-
d ]p yr im id in e (36). Bromide 49 [from 48 (0.29 g, 1.5 mmol)]
was reacted with 1-naphthylamine (0.50 g, 4 mmol) to afford
0.12 g (26%) of 36: TLC MeOH/CHCl3/NH4OH (3:7:2 drops)/
silica gel, Rf 0.65; mp >200 °C; 1H NMR (DMSO-d6) δ 4.58 (d,
2H, 9-CH2), 6.16 (br s, 2H, 2-NH2, exch), 6.44 (d, 1H, C10H7),
7.02 (t, 1H, N10-H, exch), 7.07-7.20 (m, 2H, C10H7), 7.43
(collapsed br, 2H, 4-NH2, exch), 7.44 (m, 2H, C10H7), 7.53 (dd,
2H, 7-H and 8-H), 7.73 (m, 1H, C10H7), 8.21 (m, 1H, C10H7).
Anal. (C18H16N6‚0.80H2O) C, H, N.
2,4-Dia m in o-6-[(2′-n a p h th yla m in o)m eth yl]p yr id o[3,2-
d ]p yr im id in e (37). Intermediate 49 prepared from 48 (0.29
g, 1.5 mmol) was reacted with 2-naphthylamine (0.23 g, 1.5
mmol) to afford 0.10 g (22%) of 37: TLC MeOH/CHCl3/NH4-
OH (3:7:2 drops)/silica gel, Rf 0.62; mp >220 °C dec; 1H NMR
(DMSO-d6) δ 4.50 (d, 2H, 9-CH2), 6.15 (br s, 2H, 2-NH2, exch),
6.69 (t, 1H, N10-H, exch), 6.83 (s, 1H, C10H7), 7.14 (t, 1H, C10H7),
7.22 (dd, 1H, C10H7), 7.30 (t, 1H, C10H7), 7.41 (collapsed br,
2H, 4-NH2, exch), 7.55 (m, 3H, C10H7, 7-H and 8-H), 7.65 (d,
1H, C10H7). Anal. (C18H16N6) C, H, N.
2,4-Dia m in o-6-[(N-m eth yl-1′-n a p h th yla m in o)m eth yl]-
p yr id o[3,2-d ]p yr im id in e (38). N-Methyl-1-naphthylamine
(0.47 g, 3 mmol) was prepared from 1-naphthylamine (0.93 g,
7.5 mmol) on stirring with 1.1 equiv of MeI and 1.1 equiv of
Et3N in acetonitrile and was reacted with 49 [from 48 (0.38 g,
2 mmol)] to afford 0.13 g (20%) of compound 38: TLC MeOH/
CHCl3/NH4OH (3:7:2 drops)/silica gel, Rf 0.70; mp 206-208
1
°C; H NMR (DMSO-d6) δ 2.80 (s, 3H, N10-CH3), 4.41 (s, 2H,
9-CH2), 6.22 (br s, 2H, 2-NH2, exch), 7.15 (br, 1H, 4-NH2, exch),
7.20 (d, 1H, C10H7), 7.33 (br, 1H, 4-NH2, exch), 7.44 (t, 1H,
C10H7), 7.54 (m, 4H, C10H7, 7-H and 8-H), 7.58 (dd, 1H, C10H7),
7.62 (d, 1H, C10H7), 8.34 (s, 1H, C10H7). Anal. (C19H18N6‚
0.20H2O) C, H, N.
2,4-Diam in o-6-[(2′,6′-dich lor o-N-m eth ylan ilin o)m eth yl]-
p yr id o[3,2-d ]p yr im id in e (26). Compound 25 (0.093 g, 0.28
mmol) was used to synthesize compound 26 using the proce-
dure described for 7. After 12 h, the crude product was worked

1844 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9
Gangjee et al.
up and purified via a column with 6% MeOH in chloroform
(v/v) as eluent to afford 0.091 g (94%) of the 26: TLC MeOH/
CHCl3/NH4OH (3:7:2 drops)/silica gel, Rf 0.77; mp 208-209
9-CH2), 6.2 (br s, 2H, 2-NH2), 6.8 and 7.7 (q, 4H, C6H4), 7.1
(br s, 2H, 4-NH2), 7.3 and 7.5 (q, 2H, 7-H and 8-H); high-
resolution mass calcd for C19H18N6O 323.162 03, obsd
323.160 42.
1
°C; H NMR (DMSO-d6) δ 2.73 (s, 3H, N10-CH3), 4.43 (s, 2H,
9-CH2), 6.17 (br s, 2H, 2-NH2, exch), 7.22 (m, 3H, 4-NH2, exch
and 4′-H), 7.48 (d, 2H, 3′-H and 5′-H), 7.60 (d, 1H, 7-H or 8-H),
7.87 (d, 1H, 7-H or 8-H). Anal. (C15H14N6Cl2) C, H, N, Cl.
2,4-Diam in o-6-[(3′,4′-dich lor o-N-m eth ylan ilin o)m eth yl]-
p yr id o[3,2-d ]p yr im id in e (28). Using the procedure de-
scribed for compound 7, 0.091 g (75%) of compound 28 was
prepared by the reductive N-methylation of 27 (0.116 g, 0.35
mmol): TLC MeOH/CHCl3/NH4OH (3:7:2 drops)/silica gel, Rf
4-[[(2,4-Dia m in op yr id o[3,2-d ]p yr im id in -6-yl)m eth yl]-
p r op a r glyla m in o]a cetop h en on e (42). The DMAC adduct
was purified by dissolution in EtOH/0.1 N HCl, treatment with
Norit decolorizing charcoal, filtration through Celite, neutral-
ization with 0.1 N NaOH, and concentration in vacuo. The
product was collected as a light tan powder: 39.5% yield; mp
240-241 °C dec; MS (FAB) m/ e 347 (MH+); UVmax (ꢀmax) (pH
1
1) 326 (25 262); H NMR (DMSO-d6) δ 2.4 (s, 3H, 4′-COCH3),
0.73; mp 205-207 °C; 1H NMR (DMSO-d6) δ 3.12 (s, 3H, N10
-
3.2 (t, 1H, N10-HCCCH2), 4.5 (d, 2H, HCCCH2-N10), 4.8 (s, 2H,
9-CH2), 6.3 (br s, 2H, 2-NH2), 6.8 and 7.8 (q, 4H, C6H4), 7.4
(br s, 2H, 4-NH2), 7.5 (m, 2H, 7-H and 8-H); high-resolution
mass calcd for C19H18N6O 347.162 03, obsd 347.162 12.
4-[[(2,4-Dia m in op yr id o[3,2-d ]p yr im id in -6-yl)m eth yl]-
m eth yla m in o]tr iflu or oa cetop h en on e (43). The DMAC
adduct was purified as described for 42 above. The product
was collected as a pale yellow powder: 25.6% yield; mp 210-
211 °C dec; MS (FAB) m/ e 377 (MH+); UVmax (ꢀmax) (pH 1) 366
(28 878); 1H NMR (DMSO-d6) δ 3.2 (s, 3H, N10-CH3), 4.8 (s,
2H, 9-CH2), 6.2 (br s, 2H, 2-NH2), 7.0 and 7.8 (q, 4H, C6H4),
7.1 (br s, 2H, 4-NH2), 7.4 and 7.5 (q, 2H, 7-H and 8-H). Anal.
(C17H15N6F3O‚H2O) C, H, N.
CH3), 4.69 (s, 2H, 9-CH2), 6.19 (s, 2H, 2-NH2, exch), 6.74 (dd,
1H, 6′-H), 6.93 (d, 1H, 2′-H), 7.06 (br s, 1H, 4-NH2 exch), 7.33
(m, 3H, 5′-H, 4-NH2 and 7-H or 8-H), 7.52 (d, 1H, 7-H or 8-H).
Anal. (C15H14N6Cl2‚1.0H2O) C, H, N, Cl.
2,4-Diam in o-6-[(3′,4′,5′-tr ich lor o-N-m eth ylan ilin o)m eth -
yl]p yr id o[3,2-d ]p yr im id in e (31). Compound 30 (0.050 g,
0.14 mmol) was reacted with 37% HCHO (0.075 mL, 0.9 mmol)
and NaCNBH3 (0.050 g, 0.74 mmol) for 24 h using the
procedure described for 7. After purification via a column with
MeOH/CHCl3 (3:47) as eluent, 0.028 g of 31 (55%) was
obtained: TLC MeOH/CHCl3/NH4OH (3:7:2 drops)/silica gel,
1
Rf 0.69; mp >250 °C dec; H NMR (DMSO-d6) δ 3.13 (s, 3H,
4-[[(2,4-Dia m in op yr id o[3,2-d ]p yr im id in -6-yl)m eth yl]-
p r op a r gyla m in o]tr iflu or oa cetop h en on e (44). The DMAC
adduct was purified as described for 42 above. The product
was collected as a white powder: 26% yield; mp 226-227 °C
dec; MS (FAB) m/ e 401 (MH+); UVmax (ꢀmax) (pH 1) 354
N10-CH3), 4.72 (s, 2H, 9-CH2), 6.24 (br s, 2H, 2-NH2, exch), 6.98
(s, 2H, 2′-H and 6′-H), 7.05 (br s, 1H, 4-NH2, exch), 7.38 (d,
1H, 7-H or 8-H), 7.39 (br, 1H, 4-NH2, exch), 7.54 (d, 1H, 7-H
or 8-H). Anal. (C15H23N6Cl3‚0.42H2O) C, H, N, Cl.
2,4-Diam in o-6-[(2′,4′,6′-tr ich lor o-N-m eth ylan ilin o)m eth -
yl]p yr id o[3,2-d ]p yr im id in e (33). This compound was syn-
thesized from 32 (0.080 g, 0.22 mmol) using the procedure
described for compound 7. After column chromatography
using the same eluent used for 3, 0.033 g (40%) of the product
33 was obtained: TLC MeOH/CHCl3/NH4OH (3:7:2 drops)/
silica gel, Rf 0.71; mp 193-195 °C; 1H NMR (DMSO-d6) δ 2.72
(s, 3H, N10-CH3), 4.41 (s, 2H, 9-CH2), 6.20 (s, 2H, 2-NH2, exch),
7.18 (br, 1H, 4-NH2, exch), 7.33 (br, 1H, 4-NH2, exch), 7.59 (d,
1H, 7-H or 8-H), 7.67 (s, 2H, 3′-H and 5′-H), 7.82 (d, 1H, 7-H
or 8-H). Anal. (C15H14N6Cl3) C, H, N, Cl.
1
(30 030); H NMR (DMSO-d6) δ 3.3 (t, 1H, HCCCH2-N10), 4.6
(d, 2H, HCCCH2-N10), 4.9 (s, 2H, 9-CH2), 6.2 (s, 2H, 2-NH2),
7.0 and 7.8 (q, 4H, C6H4), 7.1 and 7.3 (br s, 1H ea, 4-NH2), 7.5
(collapsed m, 2H, 7-H and 8-H). Anal. (C19H15N6F3O) C, H,
N.
Ack n ow led gm en t. This work was supported in part
by NIH Grants GM 40998 (A.G.), and AI 30900 (A.G.),
Contracts NO1-AI-87240 (S.F.Q.) and NO1-AI-35171
(S.F.Q.), and an American Chemical Society Division of
Medicinal Chemistry/Bristol-Myers Squibb Fellowship
(P.F., 1990) and American Foundation for Pharmaceuti-
cal Education Fellowships (P.F., 1990, 1992-1994).
Facilities support (mass spectrometry, NMR, and DNA
synthesis) was partially provided through Cancer Cen-
ter Grant IP30 CA42014.
Gen er a l P r oced u r e for P r ep a r a tion of 40-44. Freshly
prepared 2,4-diamino-6-(bromomethyl)pyrido[3,2-d]pyrimidine
(49) (9 mmol), 2.25 equiv of N-(alkylamino)-p-aminoacetophe-
none analogue, and 2.7 equiv of dry CaCO3 were suspended
in 80 mL of dry DMAc under an atmosphere of dry argon and
stirred at 100 °C in an oil bath. The reaction was monitored
by TLC (CHCl3/MeOH, 7:3). After disappearance of the
reactive bromomethyl intermediate 49 (6-12 h), the reaction
mixture was cooled and the solvent removed under reduced
pressure on a rotary evaporator with applied heat (60 °C).
Residual DMAc was removed by coevaporation with 3-4
volumes of xylene. The dark brown residue was resuspended
in water and filtered to remove impure product as a dark
orange precipitate. The aqueous filtrate was extracted with
CHCl3, dried over Na2SO4, and concentrated in vacuo. Solid
residues were combined and applied to a silica gel column
packed with CHCl3. Unreacted starting materials and byprod-
ucts were eluted with CHCl3/MeOH (85:15). Desired products
were then eluted with CHCl3/MeOH (75:25) as DMAc adducts.
4-[[(2,4-Dia m in op yr id o[3,2-d ]p yr im id in -6-yl)m eth yl]-
a m in o]a cetop h en on e (40). The DMAC adduct was recrys-
tallized from acetone/water as yellow crystals: 57% yield; mp
255-256 °C dec; MS (FAB) m/ e 309 (MH+); UVmax (ꢀmax) (pH
1) 324 (24 696); 1H NMR (DMSO-d6) δ 2.39 (s, 3H, 4′-COCH3),
4.5 (d, 2H, 9-CH2), 6.17 (br s, 2H, 2-NH2), 6.7 and 7.7 (q, 4H,
C6H4), 7.2 (t, 1H, N10-H), 7.5 (m, 2H, 7-H and 8-H), and 7.4 to
7.5 (br s, 2H, 4-NH2); high-resolution mass calcd for C16H16N6O
309.146 38, obsd 309.147 05.
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