2,4-Diamino-5-deaza-6-substituted pyrido[2,3-d]pyrimidine antifolates as potent and selective nonclassical inhibitors of dihydrofolate reductases

Article abstract of DOI:10.1021/jm950786p

Fifteen novel nonclassical and two classical 2,4-diamino-6- (benzylamino)pyrido[2,3-d]pyrimidine antifolates were synthesized as potential inhibitors of Pneumocystis carinii, (pc) Toxoplasma gondii, (tg) rat liver (rl), and human (h) recombinant dihydrofolate reductases (DHFR). These analogues lack a 5-methyl substitution which has been shown to be important for increased hDHFR inhibitory activity. In addition, they contain a reversal of the C9-N10 bridge present in folates and most antifolates. The synthesis of the compounds involved the reaction of 2,4,6-triaminopyrimidine with the sodium salt of nitromalonaldehyde to afford the key intermediate 2,4-diamino-6-nitropyrido[2,3-d]pyrimidine (7), in a single step. Reduction of 7 to the 2,4,6-triaminopyrido[2,3-d]pyrimidine (8), followed by reductive amination with the appropriate benzaldehydes or phenylacetaldehydes afforded the target compounds. N9 methylation of these analogues was carried out using formaldehyde and sodium cyanoborohydride. The analogues demonstrated significant inhibition of pcDHFR and tgDHFR. N9 methylation significantly increased DHFR inhibitory potency. Compound 11, the 3',4',5'-trimethoxy- substituted analogue with a selectivity ratio of 9.4 for tgDHFR (compared to rlDHFR) was the most selective analogue of the nonclassical series. Compound 22, the N9 methyl 2',5'-dimethoxy-substituted analogue was the most potent analogue against tgDHFR (IC₅₀ = 6.3 nM) and was the second most selective analogue for tgDHFR (compared to rlDHFR) in the nonclassical series. The naphthyl-substituted analogues 23-25 were generally more potent against rlDHFR than against pcDHFR and tgDHFR. Selected analogues were also evaluated against Streptococcus faecium (sf) DHFR, Escherichia coli (ec) DHFR, Lactobacillus casei (lc) DHFR and tgDHFR with hDHFR as the mammalian reference, under slightly different assay conditions than those employed for rlDHFR. Analogues 11 and 22 had selectivity ratios of greater than 100 for tgDHFR (compared to hDHFR). Analogue 22 in particular, was the most selective analogue of the nonclassical series against tgDHFR (selectivity ratio = 303.5) with excellent potency (28 nM). Analogue 11, also displayed significant selectivity for sfDHFR (selectivity ratio = 4902). Compound 22 was evaluated in vivo for the inhibition of the growth of T. gondii trophozoites in mice, where at 50 mg/kg orally, it demonstrated distinct prolongation of survival without toxicity. Compounds 11, 12, and 21-23 were evaluated as antitumor agents in the National Cancer Institutes preclinical in vitro screening program. Compounds 12, 22, and 23 showed GI₅₀s for tumor growth inhibition in the 10^-6-10^-7 M range.

Full text of DOI:10.1021/jm950786p

1438
J . Med. Chem. 1996, 39, 1438-1446
2,4-Dia m in o-5-d ea za -6-Su bstitu ted P yr id o[2,3-d ]p yr im id in e An tifola tes a s P oten t
a n d Selective Non cla ssica l In h ibitor s of Dih yd r ofola te Red u cta ses1,2
Aleem Gangjee,*,† Anil Vasudevan,† Sherry F. Queener,‡ and Roy L. Kisliuk§
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 Biochemistry, Tufts University School of Medicine,
Boston, Massachusetts 02111
Received October 24, 1995X
Fifteen novel nonclassical and two classical 2,4-diamino-6-(benzylamino)pyrido[2,3-d]pyrimidine
antifolates were synthesized as potential inhibitors of Pneumocystis carinii, (pc) Toxoplasma
gondii, (tg) rat liver (rl), and human (h) recombinant dihydrofolate reductases (DHFR). These
analogues lack a 5-methyl substitution which has been shown to be important for increased
hDHFR inhibitory activity. In addition, they contain a reversal of the C9-N10 bridge present
in folates and most antifolates. The synthesis of the compounds involved the reaction of 2,4,6-
triaminopyrimidine with the sodium salt of nitromalonaldehyde to afford the key intermediate
2,4-diamino-6-nitropyrido[2,3-d]pyrimidine (7), in a single step. Reduction of 7 to the 2,4,6-
triaminopyrido[2,3-d]pyrimidine (8), followed by reductive amination with the appropriate
benzaldehydes or phenylacetaldehydes afforded the target compounds. N9 methylation of these
analogues was carried out using formaldehyde and sodium cyanoborohydride. The analogues
demonstrated significant inhibition of pcDHFR and tgDHFR. N9 methylation significantly
increased DHFR inhibitory potency. Compound 11, the 3′,4′,5′-trimethoxy-substituted analogue
with a selectivity ratio of 9.4 for tgDHFR (compared to rlDHFR) was the most selective analogue
of the nonclassical series. Compound 22, the N9 methyl 2′,5′-dimethoxy-substituted analogue
was the most potent analogue against tgDHFR (IC50 ) 6.3 nM) and was the second most
selective analogue for tgDHFR (compared to rlDHFR) in the nonclassical series. The naphthyl-
substituted analogues 23-25 were generally more potent against rlDHFR than against pcDHFR
and tgDHFR. Selected analogues were also evaluated against Streptococcus faecium (sf) DHFR,
Escherichia coli (ec) DHFR, Lactobacillus casei (lc) DHFR and tgDHFR with hDHFR as the
mammalian reference, under slightly different assay conditions than those employed for
rlDHFR. Analogues 11 and 22 had selectivity ratios of greater than 100 for tgDHFR (compared
to hDHFR). Analogue 22 in particular, was the most selective analogue of the nonclassical
series against tgDHFR (selectivity ratio ) 303.5) with excellent potency (28 nM). Analogue
11, also displayed significant selectivity for sfDHFR (selectivity ratio ) 4902). Compound 22
was evaluated in vivo for the inhibition of the growth of T. gondii trophozoites in mice, where
at 50 mg/kg orally, it demonstrated distinct prolongation of survival without toxicity.
Compounds 11, 12, and 21-23 were evaluated as antitumor agents in the National Cancer
Institutes preclinical in vitro screening program. Compounds 12, 22, and 23 showed GI50s for
tumor growth inhibition in the 10-6-10-7 M range.
Infections with Pneumocystis carinii and Toxoplasma
gondii are the principal cause of death in patients with
acquired immunodeficiency syndrome (AIDS).3 One
approach to treat infections with these organisms is the
inhibition of the enzyme, dihydrofolate reductase
(DHFR).4 Trimetrexate (TMQ) and piritrexim (PTX) are
potent nonclassical DHFR inhibitors.5 TMQ has re-
cently been approved for the treatment of P. carinii
infections,6 while PTX has been evaluated in clinical
trials for the treatment of these opportunistic infections.
However, neither of these drugs are selective for P.
carinii (pc) or T. gondii (tg) DHFR, and hence their use
alone is associated with considerable host toxicity.
Thus, TMQ is administered with the reduced folate
leucovorin7 to rescue host cells. Since P. carinii and T.
gondii lack the carrier mediated uptake mechanism(s)
necessary for classical folates, they do not benefit from
the coadministration of leucovorin, which is selectively
taken up by the host cells. Recently,8 epiroprim, a
trimethoprim analogue, has been reported to be 200-
fold selective for pcDHFR (against human DHFR).
However, its potency is low and in the micromolar
range.
We9,10 along with others11 have reported the syntheses
and biological activities of a number of nonclassical
pyrido[2,3-d]pyrimidine antifolates of general structure
1 as inhibitors of pcDHFR and tgDHFR. These ana-
logues contain variations of both the substituents on the
side chain as well as the N10 nitrogen to optimize
hydrophobic interactions with the enzyme(s). These
studies afforded several analogues which display high
selectivity or high potency against pcDHFR and/or
tgDHFR. However, compounds with high selectivity
and potency that are able to significantly penetrate P.
carinii and T. gondii cells have remained a challenge.
The 5-methyl moiety of 5-deaza folates has been
shown to be important for potent inhibition of human
DHFR and of tumors and contributes favorably to the
† Duquesne University.
‡ Indiana University School of Medicine.
§ Tufts University School of Medicine.
X Abstract published in Advance ACS Abstracts, March 1, 1996.
0022-2623/96/1839-1438$12.00/0 © 1996 American Chemical Society

Pyrido[2,3-d]pyrimidine Antifolates
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1439
Ta ble 1. Structures of
2,4-Diamino-6-(benzylamino)pyrido[2,3-d]pyrimidines
OCH3
NH2 CH3
OCH3
OCH3
OCH3
NH2 CH3
N
NH2
R
N
N
H
OCH3
5
4
n
H2N
N
N
N
6
10
3
N
9
R′′
H2N
N
2
7
R′
H2N
N
1
N
8
TMQ
PTX
O
analogue no.
n
R
R′
R′′
CLGlu
NH2 CH3
NH2 CH3
R′
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
H
H
H
CH3
H
CH3
H
H
CH3
H
H
H
H
CH3
H
CH3
H
H
H
H
H
CH3
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
N
N
R
N
N
R
3′,4′,5′-OMe
3′,4′,5′-OMe
3′,4′,5′-OMe
2′,3′,4′-OMe
2′,3′,4′-OMe
2′,4′,6′-OMe
2′,4′,5′-OMe
3′,4′-OMe
3′,5′-OMe
3′,5′-OMe
2′,5′-OMe
2′,5′-OMe
H2N
N
N
H2N
N
Y
2: Y = N; R = H, CH3
3: Y = CH; R = H, CH3
1
O
NH2
N
O
N
CLGlu
NH2
N
H
N
X
Y
H2N
N
N
O
H2N
2′,3′-C4H4
4: X = Y = N
5: X = Y = CH
epiroprim
4′-OMe, 2′,3′-C4H4
6′-OMe, 2′,3′-C4H4
4′-O-C6H5
H
H
NH2
R
N
n
N
R′′
R′
4′-CONH-L-glutamic acid
4′-CONH-L-glutamic acid
H2N
N
N
CH3
6
inhibition of bacterial DHFRs as well. Piper et al.12
have synthesized 5-alkyl-5-deaza analogues of amino-
pterin (AMT) and methotrexate (MTX) represented by
the general structure 2 and reported that the introduc-
tion of a 5-methyl substituent resulted in a decrease in
the Ki (3.7 to 2.9 nM) for inhibition of DHFR from L1210
cells, indicating that a methyl group at the 5-position
was beneficial to binding to DHFR. The 5-methyl-
substituted analogues (IC50 0.1-0.3 nM) of AMT were
also reported to be superior to AMT (IC50 0.72 nM)
against the growth of L1210 cells in culture. The Km
values for unidirectional influx of these 5-deaza folates
were identical to those of AMT and MTX. This, along
with the fact that the 5-deaza analogues were poor
substrates for polyglutamylation, and hence have no
improved cellular retention over MTX or AMT, indicated
that the sole reason for the high potency of the 5-methyl
analogues is their increased ability to inhibit DHFR.
Hynes et al.13 have synthesized 5-methyl-5,8-dideaza
analogues of aminopterin and methotrexate of general
structure 3. These workers similarly reported that
5-methyl-5,8-dideaza-AMT is 4 times as potent as the
5-desmethyl analogue against rat liver (rl) DHFR.
Bertino et al.14 also reported nonclassical 2,4-diamino-
quinazolines as DHFR inhibitors and of MTX resistant
murine leukemia cell lines and found that 2,4-diamino-
5-methyl-6-[N-(9-phenanthryl)amino]quinazoline (IC50
) 1.5 nM) was 60 times more potent than the 5-des-
methyl analogue (IC50 ) 94 nM). Similarly, 2,4-di-
amino-5-methyl-6-[N-(2-fluoryl)amino] quinazoline (IC50
) 1.7 nM) was 88 times more potent than its 5-des-
methyl congener (IC50 ) 150 nM), demonstrating fur-
ther the importance of the 5-methyl group for potency
against mammalian DHFR. We9,10,15 have also reported
that 5-methyl-substituted nonclassical antifolates are
more potent as inhibitors of rlDHFR compared to their
5-desmethyl analogues.
sources, we decided to synthesize compounds of general
structure 6 which lack the 5-methyl group. The design
of these analogues was based on the premise that since
the 5-methyl moiety was important for the inhibition
of rlDHFR, L1210 DHFR, and hDHFR and its absence,
in certain instances, resulted in analogues with signifi-
cantly decreased potency for mammalian DHFRs, such
5-desmethyl analogues would be less toxic to mam-
malian cells. Removal of the 5-methyl group was
expected to also result in a decrease in potency against
pcDHFR and tgDHFR. However, if this decrease in
potency against pcDHFR and tgDHFR was less than
the decrease in the inhibition of mammalian DHFR,
selective and nontoxic inhibitors of pcDHFR and/or
tgDHFR would be at hand. To investigate this hypoth-
esis, we synthesized a series of 5-desmethyl-6-substi-
tuted, 2,4-diaminopyrido[2,3-d]pyrimidines 9-28 (Table
1), in which the usual C9-N10 bridge of folates and
most antifolates was reversed to also determine the
effect of substitutions at the 9-position of 5-deazapyrido-
[2,3-d]pyrimidines, a feature which has not been inves-
tigated in the 5-deaza series. There is precedence in
the literature for enhanced DHFR inhibition of 9-sub-
stituted antifolates. DeGraw et al.16 reported the
synthesis and biological activity of 9-methyl-10-deaza
AMT, which was as potent as MTX against DHFR.
Interestingly, it was 20 times more potent than MTX
against the growth of L1210 cells in culture. The choice
of the side chain substitutents of compounds 9-28 was
based, in part, on the results obtained from our previous
studies with other nonclassical analogues.9,10,17-19 The
nonclassical analogues 9-28 were evaluated as inhibi-
tors of pcDHFR, tgDHFR, and rlDHFR.5 Selected
analogues were also evaluated33 against Streptococcus
faecium (sf) DHFR, Escherichia coli (ec) DHFR, Lacto-
bacillus casei (lc) DHFR, and recombinant tgDHFR and
hDHFR under slightly different assay conditions. Davoll
et al.20 have reported 2,4-diamino-6-(benzylamino)-
pyrido[2,3-d]pyrimidine, 2,4-diamino-6-[(3′,4′-dichlo-
On the basis of the demonstrated importance of the
5-methyl group for inhibition of DHFR from mammalian

1440 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Gangjee et al.
Sch em e 1
with 2,4,6-triaminopyrimidine. The nitromalonalde-
hyde was synthesized according to the method of
Fanta28 by the reaction of mucobromic acid and sodium
nitrite to afford the desired â-dialdehyde in 65% yield
as a sodium salt which was used immediately following
its preparation. Thus, condensation of nitromalon-
aldehyde and 2,4,6-triaminopyrimidine in absolute eth-
anol at reflux, containing sufficient concentrated HCl
to solubilize the pyrimidine, afforded a thick red sus-
pension (Scheme 1). Due to the susceptibility of the
4-amino group of folates to hydrolysis under similar
conditions,29 the cyclocondensation reaction was closely
monitored by TLC. The 1H NMR of the product cor-
responded to that expected of 7. Thus, 7 was obtained
in a single step starting from 2,4,6-triaminopyrimidine
in 76% yield. A similar methodology, involving base-
catalyzed cyclization of 2,6-diamino-4-hydroxypyrimi-
dine and nitromalonaldehyde, has been reported by
Price et al.30 for the synthesis of the 2-amino-4-hydroxy-
6-nitropyrido[2,3-d]pyrimidine.
NH2
N
NH2
H
O
EtOH
HCl
NO2
NO2
H
N
N
O
+
1 h
76%
H2N
NH2
H2N
N
N
7
NH2
H
R′′
N
N
1. Raney Ni, H2, 35 psi
DMF
2. ArCHO, AcOH
H2N
N
N
7
9, 11, 14, 16, 17, 18,
19, 21, 23, 25, 26, 27
1. Raney Ni, H2, 35 psi
13
DMF
2. ArCOCH3, AcOH
NH2
NH2
1. ArCOCH3, AcOH
2. BH3•Et3N
Raney Ni, H2, 35 psi
DMF
N
7
H2N
N
N
8
OCH3
OCH3
Compound 7 was reduced to the corresponding 6-ami-
no analogue 8 using Raney Ni in DMF at 35 psi.
Complete reduction was obtained in 3 h, as indicated
by TLC. This intermediate was not isolated but reacted
directly with the appropriately substituted benzalde-
hydes or phenylacetaldehyde in acetic acid and hydro-
genated at 35 psi. The duration of the reductive
aminations varied from 3 to 6 h and was predicated on
the nature of the substitution on the aldehyde. This
procedure afforded analogues 9, 11, 14, 16-19, 21, 23,
and 25-28 in 60-70% yield following chromatographic
purification. These analogues displayed an intense
green fluorescence at 254 nM, probably due to the
presence of the exocyclic amino moiety. Similar at-
tempts with 3,4,5-trimethoxyacetophenone and 7 for the
synthesis of 13 did not afford the desired C10 methyl
analogue. Prolonged reaction times and increased pres-
sure conditions resulted in recovery of unreacted 8. As
a result, 8 had to be isolated, and reductive amination
with 3,4,5-trimethoxyacetophenone was carried out
in acetonitrile using a borane-triethylamine complex
to reduce the intermediate Schiff base which afforded
13.
NH2
H
N
N
OCH3
CH3
H2N
N
N
13
HCHO
9, 11, 14, 19, 21, 27
10, 12, 15, 20, 22, 28
NaCNBH3
HCl
robenzyl)amino]pyrido[2,3-d]pyrimidine, and its N9 ni-
troso derivative as antimalarial agents. However, no
DHFR inhibitory activity was reported for these ana-
logues nor were they evaluated against pcDHFR or
tgDHFR. Hence, we resynthesized one of these ana-
logues 9 and evaluated it along with all the other
analogues against pcDHFR, tgDHFR, rlDHFR, and
hDHFR.
The synthesis and DHFR inhibitory activity of the
classical analogues 29 and 30 (Table 1) of the 5-deaza
series with a N9-C10 bridge were of particular interest
in light of the potent activity of isoaminopterin 4 against
L1210 DHFR reported by Nair and Baugh21 and of the
N9 methyl analogue of 5,8-dideazaisoaminopterin 5,
synthesized by Hynes et al.,22 which was not only a
potent inhibitor of DHFR (IC50 ) 4.6 nM) but also a
potent inhibitor of thymidylate synthase (IC50 ) 33 nM).
N-Methylation of these analogues was carried out by
reductive methylation, using HCHO and NaCNBH3 as
reported by us earlier.9 It was observed that carrying
out the methylations in acetonitrile using sufficient
concentrated HCl to effect solution resulted in high
isolated yields of the pure N9 methylated products.9
Ch em istr y
The synthesis of the desired compounds was achieved
by the reaction of 2,4,6-triaminopyrido[2,3-d]pyrimidine
(8), with appropriately substituted benzaldehydes or
phenylacetaldehyde. The precursor to this compound,
2,4-diamino-6-nitropyrido[2,3-d]pyrimidine 7 (Scheme
1), had been reported by Davoll20 in 1972 via a three-
step procedure which involved the reaction of nitro-
malonaldehyde with cyanoacetamide followed by chlo-
rination and cyclization with guanidine (62% overall
yield). Since 7 was to serve as the key intermediate for
the entire series, it was important to devise a more
efficient synthesis of 7. We, along with others, have
previously reported the reaction of a variety of bis-
electrophiles such as â-keto esters,23,24 â-keto alde-
hydes,25 and â-dialdehydes26,27 with 2,4,6-triamino-
pyrimidine to yield pyrido[2,3-d]pyrimidines. Thus, we
envisioned that 7 could similarly be synthesized via the
condensation of nitromalonaldehyde (bis-electrophile)
1
The position of the alkylation was confirmed by the H
NMR in deuterated dimethyl sulfoxide which indicated
the absence of the N9 H and the presence of a sharp
singlet at around δ 2.8 ppm corresponding to the N9
CH3 group.
For the synthesis of the classical analogues 29 and
30, 4-carboxybenzaldehyde was coupled with diethyl
L-glutamate in anhydrous pyridine using diethylcarbo-
diimide by the method of Hynes and Garrett31 to afford
(4-formylbenzoyl)-L-glutamate, 31 (Scheme 2). Com-
pound 7 was reduced to 8 using Raney Ni and then
reductively aminated with 31, as described above for
the nonclassical analogues, to afford the diester 32. N9
methylation of 32 with HCHO, NaCNBH3, and HCl
afforded 33 in 72% yield. Both 32 and 33 were saponi-
fied with 1 N NaOH to afford the desired classical
analogues 29 and 30.

Pyrido[2,3-d]pyrimidine Antifolates
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1441
Sch em e 2
COOEt
COOEt
H2N CH
DCC
OHC
CONH
CH
+
OHC
COOH
pyridine
CH2
CH2
CH2
CH2
COOEt
COOEt
31
COOH
CH
CONH
NH2
H
N
CH2
N
1 N NaOH
CH2
H2N
N
N
COOH
COOEt
29
CH
CONH
NH2
COOEt
CH
H
CH2
1. Raney Ni,
H2, 35 psi
N
N
CONH
NH2
N
CH3
N
7 + 31
CH2
DMF
H2N
N
N
CH2
HCHO
N
COOEt
NaCNBH3
CH2
32
H2N
N
COOEt
33
1 N NaOH
COOH
CH
CONH
NH2
N
CH3
CH2
CH2
COOH
N
N
H2N
N
30
Ta ble 2. Inhibition Concentrations (IC50, in nM) of
Biologica l Acitivity a n d Discu ssion
Dihydrofolate Reductases from P. carinii, T. gondii, and Rat
The nonclassical analogues were evaluated5,32 as
inhibitors of pcDHFR, tgDHFR, and rl DHFR, and the
results (IC50) are reported in Table 2. Selectivity ratios
(IC50 rlDHFR/IC50 pcDHFR and IC50 rlDHFR/IC50 tgDH-
FR) determined using rlDHFR as the mammalian
source are also reported in Table 2. The classical
analogues along with selected nonclassical analogues
were also evaluated against ecDHFR, lcDHFR, sfDHFR,
and hDHFR33 as well as recombinant tgDHFR under
slightly different assay conditions, and the results are
reported in Table 3. In the nonclassical analogues
evaluated, N9 methylation consistently resulted in a
substantial increase in inhibition of pcDHFR, tgDHFR,
rlDHFR, and hDHFR. In some cases, N9 methylation
also provided for increased selectivity against pcDHFR
and tgDHFR compared to the N9 H analogues. As
indicated in Table 2, comparison of the unsubstituted
phenyl analogues 9 and 10 indicates that N9 methyl-
ation increases potency 15-40-fold against all three
DHFRs and also increases selectivity for both pcDHFR
and tgDHFR (vs rlDHFR). This increase on N9 methy-
lation in both potency and selectivity is significantly
greater for pcDHFR than tgDHFR. For the N9 H
trimethoxy-substituted analogues (11, 14, 16, and 17),
the effect was predicated on the position of the methoxy
groups. For the 3′,4′,5′- and 2′,3′,4′-trimethoxy ana-
logues 11 and 14, potency decreased compared to 9, the
phenyl-unsubstituted analogue, for all DHFRs except
tgDHFR. Selectivity for pcDHFR in all of the tri-
Liver and Selectivity Ratiosa
no.
pcDHFR
tgDHFR
rlDHFR
rl/pc
rl/tg
9
10
2700
68
520
32
2100
140
0.8
2.1
4.0
4.4
9.4
2.4
6.5
4.8
1.2
5.2
2.3
2.1
2.8
2.3
1.1
9.0
0.2
1.1
0.5
0.8
11
12
14100
61
350
14
3300
33
0.23
0.5
13
14
15
9200
15300
79
194
670
26
1270
3240
30
0.14
0.2
0.4
16
17
18
19
20700
5500
4800
5700
76
230
480
730
1200
31
1200
1100
1500
3400
72
0.06
0.2
0.3
0.6
0.9
20
21
22
3800
84
310
6.3
350
57
0.09
0.7
23
24
25
26
27
28
TMP
TMQ
epir.
MTX
3900
8200
15400
24300
1940
1800
12000
42
980
380
710
3700
4450
920
2700
10
240
430
370
2900
280
1400
133000
3.0
73000
2.5
0.06
0.05
0.02
0.12
0.14
0.8
11.1
0.07
28.1
1.9
0.06
1.5
49
0.3
155
0.2
2600
1.3
470
14
a
These assays were carried out at 37 °C under conditions of
substrate (90 µM dihydrofolic acid) and cofactor (119 µM NADPH)
in the presence of 150 mM KCl.5,32
methoxy N9 H analogues was less than for 9. However
for tgDHFR, the selectivity increases with trimethoxy
substitution (except for 17) with compound 11 being the

1442 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Gangjee et al.
Ta ble 3. Inhibition Concentrations (IC50, in nM) and Selectivity Ratios of Selected Analogues against DHFRs from Various Sourcesa
no.
hDHFR
tgDHFR
h/tg
ecDHFR
h/ec
sfDHFR
h/sf
lcDHFR
h/lc
9
10
11
15
19
20
21
22
6200
700
76000
530
160000
2900
13000
8500
320
nd
35
1300
26
nd
29
500
28
nd
nd
22
nd
20
192
20.4
nd
100
26
304
nd
nd
nd
760
nd
1400
nd
130
140
13
nd
nd
329
nd
114.2
nd
100
60.7
24.6
50
nd
nd
51
nd
86
nd
15
28
10
2.2
2.2
nd
nd
4902
nd
1860
nd
867
304
32
nd
nd
15000
nd
17000
nd
5100
5700
67
nd
nd
17
nd
9.4
nd
2.5
1.5
4.8
3.8
0.7
29
30
MTX
650
38
nd
1.4
13
2.2
295
17
170
55
17
a
Methotrexate was purchased from Sigma, St. Louis, MO. Recombinant hDHFR was provided by Dr. J . H. Freisheim. Recombinant
tgDHFR was provided by Dr. D. V. Santi. Recombinant ecDHFR was provided by Dr. R. L. Blakley. DHFR assay conditions:33 all enzymes
were assayed spectrophotometrically in a solution containing 50 µM dihydrofolate, 80 µM NADPH, 0.05 M Tris HCl, 0.001 M
2-mercaptoethanol, and 0.001 M EDTA at pH 7.4 and 30 °C. The reaction was initiated with an amount of enzyme yielding a change in
o.d. at 340 nM of 0.015/min.
most selective tgDHFR inhibitor in the entire series. N9
methylation of 11 and 14 to afford 12 and 15, respec-
tively, significantly increased potency from 25- to 231-
fold and from 25- to 194-fold, respectively, against all
three DHFRs and was similar to that observed for 9 and
10. Thus, while the 2′,4′,5′-trimethoxy analogue 17 was
the most potent of the N9 H trimethoxy series against
pcDHFR, the 2′,4′,6′-trimethoxy-substituted analogue
16 was the most potent against tgDHFR. The 3′,4′,5′-
trimethoxy analogue 11 on the other hand was the most
selective against tgDHFR. It was 31 times more selec-
tive than TMQ against tgDHFR and was the most
tgDHFR selective compound (vs rlDHFR). N9 methyl-
ation of 11 (analogue 12) resulted in a 230-fold increase
in potency against pcDHFR and a 25-fold increase
against tgDHFR inhibition. However the selectivity
ratio for tgDHFR decreased 4-fold. These differential
inhibitory effects of N9 methylation on pcDHFR and
tgDHFR underscore the fact that the active site of
pcDHFR and tgDHFR are different and that separate
structure-activity/selectivity relationships need to be
developed for each enzyme. C10 methylation of 11 to
afford 13 provided unexpected results. This analogue
though more potent than the nonmethylated analogue
11 is still a poor inhibitor of pcDHFR, tgDHFR, and
rlDHFR with IC50s in the micromolar range. This was
surprising since in the C9-N10 series previously re-
ported by us,15 methylation of N10 afforded an ex-
tremely potent inhibitor of pcDHFR, tgDHFR, and
rlDHFR, with IC50s in the 10-8 M range. The low
inhibitory activity of 13 could be attributed, in part, to
the intolerance of the enzyme(s) to the N9 H as a H-bond
donor or a hydrophilic substitution at the 9-position and
is reflected in the low activity of all the N9 H analogues.
Further studies are currently underway in our labora-
tory to determine the cause of this apparent anomaly
in the SAR. The N9 methyl 2′,3′,4′-trimethoxy analogue
15 displayed increased activity against all the three
DHFRs to a similar extent as did 12. Thus, 15 was 195-
fold and 25-fold more active than the N9 H analogue
14 against pcDHFR and tgDHFR, respectively. As a
result, the pcDHFR selectivity for 15 increased 2-fold,
and decreased 4-fold for tgDHFR.
20 and 22 allows for a significant increase in potency
for all three DHFRs. N9 methylation of 19 results in a
75-fold increase in pcDHFR inhibition and a 47-fold
increase in tgDHFR inhibition. Selectivity for pcDHFR
is somewhat increased for 20 with a slight decrease in
selectivity for tgDHFR. The 2′,5′-dimethoxy substitu-
tion pattern, as we have previously reported for the
5-CH3, C9-N10 analogues,9 afforded the highest po-
tency against all three DHFRs. N9 methylation of 21
to afford the N9 methyl 2′,5′-dimethoxy analogue 22
resulted in the most potent inhibitor of tgDFHR of all
the analogues tested. Compound 22 is 429 times more
potent than TMP against tgDHFR. It is also more
potent than TMQ against tgDHFR and is 30-fold more
selective. Analogue 22 afforded a 10-fold increase in
selectivity relative to TMQ against both pcDHFR and
tgDHFR. With its high potency of 6.3 nM (better than
TMQ) against tgDHFR along with the second highest
selectivity against tgDHFR of the nonclassical series,
22 was an excellent lead analogue. Its potency and
selectivity against pcDHFR was noteworthy and ranked
it among the most potent and selective analogues of the
nonclassical series. The significantly different effects
of N9 methylation on pcDHFR and tgDHFR inhibition
observed for the trimethoxy analogues was absent in
the dimethoxy analogues. Thus, deletion of a methoxy
group in the trimethoxy series provides for an increase
in potency and selectivity against both pcDHFR and
tgDHFR.
Analogues 23-25 were designed to evaluate the role
of a naphthalene moiety on the side chain in place of a
phenyl ring. Piper et al.34 have shown that the active
site of human DHFR is large enough to accommodate
such a substitution. Further, we9 have shown that
naphthalene substitution provides analogues which
have excellent cell penetration, which is an important
attribute for clinical viablility. Comparison of the
activities of 23 and 9 indicate that the additional
hydrophobic contacts provided by the naphthalene ring
are conducive to rlDHFR inhibition, but are detrimental
to pcDHFR and tgDHFR inhibition. Compound 23 is
10 times more potent than 9 against rlDHFR and is the
most potent of all the N9 H analogues against rlDHFR.
Incorporation of a methoxy group at either the ortho or
the para position of the naphthalene ring results in
analogues with decreased affinity for pcDHFR and
rlDHFR and a slight increase in tgDHFR inhibition.
Substitution of a phenoxy group at the para position of
the phenyl ring of compound 9 affords 26 which is an
In the dimethoxy-substituted N9 H analogues 18, 19
and 21, the compounds have decreased potency com-
pared to the phenyl unsubstituted analogue 9 against
all three DHFRs (except 21 for rlDHFR). Moving the
2′-methoxy group of 21 to the 3′-position as in 19, results
in a 10-fold decrease in inhibition of rlDHFR. N9
methylation in the dimethoxy series to give compounds

Pyrido[2,3-d]pyrimidine Antifolates
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1443
extremely poor inhibitor of all three DHFRs reflecting
a lack of bulk tolerance at the 4′-position.
Insertion of an extra methylene between the N9 and
the side chain phenyl ring affords the three-atom-
bridged analogue 27. A similar bridge extension has
been reported by us in the tetrahydroquinazoline se-
ries.19 This analogue 27 is more potent than 9 against
pcDHFR and rlDHFR but is 10-fold less active against
tgDHFR. Interestingly N9 methylation of 27 to afford
28 results in a significant increase in potency against
tgDHFR and a slight increase in inhibition of pcDHFR,
but the rlDHFR inhibition is decreased 5-fold.
From Table 2, the most important criteria for high
activity against pcDHFR and tgDHFR was N9 methyl-
ation as seen in compounds 10, 12, 15, 20, and 22. The
substitution on the side chain phenyl ring or their
positions were relatively unimportant except for 12 and
22 with respect to tgDHFR. Compound 12 was also the
most potent inhibitor of pcDHFR. Thus the 3′,4′,5′-
trimethoxy analogue 12 and the 2′,5′-dimethoxy ana-
logue 22 provided the most potent analogues of the
series. In addition compounds 12 and 22 were the most
potent analogues against tgDHFR, and 11 and 22 were
the most selective. Thus in the dimethoxy-substituted
compounds, N9 methylation was important for both
pcDHFR and tgDHFR selectivity.
F igu r e 1. Percent survival of mice inoculated with T. gondii
trophozoites. Each mouse received 5000 T. gondii trophozoites
from culture (DCD13); the strain was derived from a clinical
isolate from a patient at Indiana University Medical Center.
The daily dose of drug was 50 mg/kg administered in food
(compounded daily, 1.8 g of powdered rodent chow + 10 mg of
drug + 3.2 g of peanut butter). There were 10 mice in this
group. The control group consisted of 12 mice which received
no drug. The amounts of drug were reduced proportionately
as the mice died.
The nonclassical analogues 9-11, 15, and 19-22
were also evaluated against ecDHFR, lcDHFR, sfDHFR,
recombinant tgDHFR, and recombinant hDHFR, and
the results are listed in Table 3. These results were
obtained under different assay conditions than that
reported in Table 2. The tgDHFR inhibitory data in
Tables 2 and 3 are remarkably similar despite different
assay conditions. However, a significant difference in
the inhibition of rlDHFR compared to hDHFR was
observed. As a result, the selectivity ratios of these
analogues for tgDHFR vs hDHFR were quite different
and are also reported in Table 3. The corresponding
selectivity ratios for MTX are also listed for comparison
and are different for hDHFR compared to rlDHFR in
Table 2. Thus 20, which has a selectivity ratio of 2.3
(vs rlDHFR) for tgDHFR (Table 2), displays a 100-fold
selectivity for tgDHFR compared to hDHFR (Table 3).
Similarly, compound 11 which has a selectivity ratio of
9.4 (vs rlDHFR) for tgDHFR displays a 192-fold selec-
tivity for tgDHFR compared to hDHFR. This analogue
also displayed more than a 4000-fold selectivity for
sfDHFR vs hDHFR. Compound 19 was also extremely
selective for sfDHFR with a selectivity ratio of 1860. The
N9 methyl analogue of 19, compound 20, showed an
increase in inhibition of hDHFR by 2 orders of magni-
tude compared to the N9 H analogue and displayed
excellent tgDHFR selectivity, compared to hDHFR, of
100.
our initial premise that selective inhibitors of a variety
of DHFRs can be generated by removal of the 5-methyl
moiety and that this results in significantly greater
decrease in potency against hDHFR, which translates
into remarkably selective analogues against tgDHFR,
ecDHFR, and sfDHFR.
Compound 22 on the basis of its potency and selectiv-
ity was identified for further development. A pilot study
against T. gondii infection in the mouse model was
carried out. The data for eight days is shown in Figure
1. The data indicates that after 7 days only one mouse
survived in the control group (8.3% survival), while four
mice survived in the drug group (40% survival), and
after 8 days, all the mice in the control group had died
and three mice were alive in the drug group, providing
for a 30% survival. This data strongly indicates that
22 is active orally and allows a prolongation of life.
Further, the arbitrarily chosen dose produced no obvious
toxicity.
The classical analogues 29 and 30 were evaluated
against lcDHFR, ecDHFR, sfDHFR, and hDHFR. These
results are reported in Table 3. Both the N9 H and the
N9 methyl analogues were moderate inhibitors of
hDHFR with IC50s in the 10-7 M range. N9 methylation
decreased activity against hDHFR and lcDHFR, while
there was no change against ecDHFR inhibition. Against
sfDHFR, 30 was as potent as MTX.
Selected analogues were also evaluated as antitumor
agents in the National Cancer Institute preclinical in
vitro screening program.35 The improved DHFR inhibi-
tion observed on N9 methylation was corroborated by
the inhibitory activity observed against the growth of
nonsmall lung cancer cell lines, colon cancer cell lines,
melanoma cell lines, and breast cancer cell lines. Thus,
while 11 was an extremely poor inhibitor of all the cell
The N9 methyl 2′,5′-dimethoxy-substituted analogue
22 showed astounding results with respect to potency
and selectivity against tgDHFR. Compound 22 had a
selectivity ratio of 300 for tgDHFR (Table 3) and is one
of the most selective tgDHFR inhibitors reported to
date. The high inhibitory potency against tgDHFR of
22 and its outstanding selectivity for tgDHFR suggests
that this analogue could be a clinical candidate for the
treatment of T. gondii infection.
The spectacular selectivity ratios obtained with 11,
20, and 22 for tgDHFR; 11, 19, and 21 for ecDHFR; and
11, 19, 21, 22, and 30 for sfDHFR (Table 3) validates

1444 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Gangjee et al.
lines evaluated with GI50s > 10-4 M, the N9 methyl
congener displayed GI50s in the 10-6-10-7 M range
against several of the cell lines. Similarly, a 1-2 orders
of increase in the inhibition of tumor growth was
observed for 22 compared to 21. The higher rlDHFR
inhibition observed with 23, the naphthyl analogue, was
corroborated by its tumor growth inhibition (GI50s in
the 10-6-10-7 M range), which was much better than
any of the other N9 H analogues tested.
thaldehyde, or phenylacetaldehyde) was added followed by 15
mL of acetic acid, and the mixture was hydrogenated for a
further 3.5 h. The reaction mixture was treated with Norit
and filtered through a Celite mat. Silical gel (1.0 g) was added
to the filtrate which was then evaporated to dryness to afford
a dry plug. This plug was chromatographed on a 1.05 in. ×
23 in. column using CHCl3:MeOH (10:1) as the eluant. Frac-
tions containing the pure product, which showed an intense
green fluorescence, were pooled and evaporated to yield a
powder.
2,4-Dia m in o-6-(N-b en zyla m in o)p yr id o[2,3-d ]p yr im i-
d in e, 9. Compound 9 was synthesized from 7 (0.49 g, 2.42
mmol), Raney Ni (3.0 g) and benzaldehyde (0.26 g, 2.42 mmol)
to afford 0.39 g (61%) of 9: mp 267 °C; 1H NMR (Me2SO-d6) δ
4.28 (d, 2 H, CH2), 6.09 (br s, 2 H, 2-NH2), 6.29 (t, 1 H, NH),
7.23 (m, 1 H, 4′-H), 7.33 (m, 2 H, 3′-H and 5′-H), 7.39 (m, 2 H,
2′-H and 6′-H), 7.44 (d, 1 H, H-5), 8.33 (d, 1 H, H-7). Anal.
Calcd for (C14H14N6‚H2O) C, H, N.
Remarkably potent and selective inhibitors of various
DHFRs compared to hDHFR have been achieved by the
removal of the 5-methyl moiety and the reversal of the
C9-N10 bridge of nonclassical 6-substituted 2,4-di-
aminopyrido[2,3-d]pyrimidine antifolates. Whether the
selectivities obtained are solely attributable to the
removal of the 5-methyl moiety, the reversal of the C9-
N10 bridge, or both factors will be the subject of future
reports. Preliminary animal studies indicate that at 50
mg/kg orally, compound 22 is active in the prolongation
of life in mice infected with T. gondii without toxicity.
Analogue 22 with its impressive potency and selectivity
against tgDHFR is currently undergoing further studies
under the auspices of The National Institute of Allergy
and Infectious Diseases, Division of AIDS. In addition,
the crystal structures of this analogue with pcDHFR
and hDHFR is in progress and will be the subject of
future communications.
2,4-Dia m in o-6-[N-(3′,4′,5′-t r im et h oxyb en zyl)a m in o]-
p yr id o[2,3-d ]p yr im id in e, 11. Compound 11 was synthe-
sized from 7 (0.50 g, 2.43 mmol), Raney Ni (3.0 g), and 3,4,5-
trimethoxybenzaldehyde (0.48 g, 2.43 mmol) to afford 0.54 g
1
(62%) of 11: mp 250 °C; H NMR (Me2SO-d6) δ 3.62 (s, 3 H,
4′-OCH3), 3.76 (s, 6 H, 3′-OCH3 and 5′-OCH3), 4.20 (d, 2 H,
CH2), 6.31 (br s, 3H, 2-NH2 and NH), 6.77 (s, 2 H, 2′H and
6′H), 7.50 (d, 1 H, 5-H), 7.63 (s, 2H, 4-NH2), 8.32 (d, 1 H, 7-H).
Anal. Calcd for (C17H20N6O3‚0.5H2O) C, H, N.
2,4-Dia m in o-6-[N-(2′,3′,4′-t r im et h oxyb en zyl)a m in o]-
p yr id o[2,3-d ]p yr im id in e, 14. Compound 14 was synthe-
sized from 7 (0.49 g, 2.42 mmol), Raney Ni (3.0 g), and 2,3,4-
trimethoxybenzaldehyde (0.48 g, 2.42 mmol) to afford 0.52 g
1
(61%) of 14: mp 234 °C; H NMR (Me2SO-d6) δ 3.76 (s, 3 H,
2′-OCH3), 3.77 (s, 3 H, 4′-OCH3), 3.83 (s, 3 H, 3′-OCH3), 4.18
(d, 2 H, CH2), 5.933 (t, 1 H, NH), 6.02 (br s, 2H, 2-NH2), 6.75
(d, 1 H, 5′-H), 7.05 (d, 1 H, 6′-H), 7.44 (m, 3 H, 4-NH2 and
5-H), 8.34 (d, 1 H, 7-H). Anal. Calcd for (C17H20N6O3‚H2O)
C, H, N.
Exp er im en ta l Section
Melting points were determined on a Mel-Temp II melting
point apparatus and are uncorrected. Infrared spectra (IR)
were recorded with a Perkin-Elmer Model 1430, in Nujol
mulls. Nuclear magnetic resonance spectra for proton (1H
NMR) were recorded on a Bruker WH-300 (300 MHz) with
internal standard TMS; s ) singlet, br s ) broad singlet, d )
doublet, t ) triplet, q ) quartet, m ) multiplet. Low-
resolution mass spectra were obtained on an LKB-9000
instrument. Thin layer chromatography was performed on
silica gel plates with fluorescent indicator and were visualized
with light at 254 and 366 nm. Column chromatography was
performed with 230-400 mesh silica gel purchased from
Aldrich Chemical Company, Milwaukee, WI. All anhydrous
solvents were purchased from Aldrich Chemical Co. and were
used without further purification. Samples for microanalysis
were dried in vacuo over phosphorus pentoxide at 70 °C or
110 °C. Microanalyses were performed by Atlantic Microlabs,
Norcoss, GA, and are within (0.4% of the theoretical value.
2,4-Dia m in o-6-n itr op yr id o[2,3-d ]p yr im id in e, 7. To a
suspension of 2,4,6-triaminopyrimidine (2.00 g, 0.02 mol) in
refluxing ethanol was added concentrated HCl dropwise to
effect solution. To this solution was added the sodium salt of
nitromalonaldehyde (2.24 g, 0.02 mol) all at once, and the
reaction mixture was stirred at reflux under nitrogen. Within
15 min, a thick red precipitate started to form. After 1 h, TLC
analysis indicated trace amounts of starting material and a
major spot for the product. The suspension was cooled,
basified to a pH of 8, and filtered. The precipitate was washed
with 100 mL of cold water to remove unreacted triamino-
pyrimidine and the precipitate dried to yield 2.51 g of pure 7
as a red solid (76%). A small amount was crystallized from
2,4-Dia m in o-6-[[N-(2′,4′,6′-t r im et h oxyb en zyl)a m in o]-
m eth yl]p yr id o[2,3-d ]p yr im id in e, 16. Compound 16 was
synthesized from 7 (0.75 g, 3.64 mmol), Raney Ni (4.00 g), and
2,4,6-trimethoxybenzaldehyde (0.71 g, 3.64 mmol) to afford
0.82 g (63%) of 16: mp 213 °C; 1H NMR (Me2SO-d6) δ 3.78 (s,
3 H, 4′-OCH3), 3.79 (s, 6 H, 2′-OCH3 and 6′-OCH3), 4.04 (d, 2
H, CH2), 5.39 (br s, 1H, NH), 6.12 (br s, 2 H, 2-NH2), 6.27 (s,
2 H, 3′-H and 5′-H), 7.46 (br s, 3 H, 4-NH2 and 5-H), 8.30 (d,
1 H, 7-H). Anal. Calcd for (C17H20N6O3‚0.5H2O) C, H, N.
2,4-Dia m in o-6-[N-(2′,4′,5′-t r im et h oxyb en zyl)a m in o]-
p yr id o[2,3-d ]p yr im id in e, 17. Compound 17 was synthe-
sized from 7 (0.45 g, 2.18 mmol), Raney Ni (3.0 g), and 2,4,5-
trimethoxybenzaldehyde (0.43 g, 2.18 mmol) to afford 0.50 g
1
(65%) of 17: mp 235 °C; H NMR (Me2SO-d6) δ 3.66 (s, 3 H,
2′-OCH3), 3.78 (s, 3 H, 4′-OCH3), 3.81 (s, 3 H, 5′-OCH3), 4.17
(d, 2 H, CH2), 6.00 (t, 1 H, NH), 6.49 (br s, 2 H, 2-NH2), 6.71
(s, 1 H, 3′-H), 7.03 (s, 1 H, 6′-H), 7.55 (d, 1 H, 5-H), 7.79 (s, 2
H, 4-NH2), 8.34 (d, 1 H, 7-H). Anal. Calcd for (C17H20N6O3‚
0.75H2O) C, H, N.
2,4-Diam in o-6-[N-(3′,4′-dim eth oxyben zyl)am in o]pyr ido-
[2,3-d ]p yr im id in e, 18. Compound 18 was obtained from 7
(0.50 g, 2.43 mmol), Raney Ni (3.0 g), and 3,4-dimethoxy-
benzaldehyde (0.40 g, 0.50 mmol) to afford 0.46 g (58%) of 18:
mp 257 °C; 1H NMR (Me2SO-d6) δ 3.72 (s, 3 H, 4′-OCH3), 3.75
(s, 3 H, 3′-OCH3), 4.20 (d, 2 H, CH2), 6.36 (br s, 3 H, 2 NH2
and H), 6.91 (m, 2 H, 5′-H and 6′-H), 7.05 (s, 1 H, 2′-H), 7.51
(d, 1 H, H-5), 7.74 (br s, 2 H, 4-NH2), 8.32 (d, 1 H, H-7). Anal.
Calcd for (C16H18N6O2‚H2O) C, H, N.
2,4-Diam in o-6-[N-(3′,5′-dim eth oxyben zyl)am in o]pyr ido-
[2,3-d ]p yr im id in e, 19. Compound 19 was obtained from 7
(0.35 g, 1.70 mmol), Raney Ni (1.50 g), and 3, 5-dimethoxy-
benzaldehyde (0.28 g, 1.70 mmol) to afford 0.33 mg (60%) of
19: mp 264 °C; 1H NMR (Me2SO-d6) δ 3.71 (s, 6 H, 3′,5′-OCH3),
4.24 (d, 2 H, CH2), 5.72 (s, 1 H, 4′-H), 5.86 (s, 2H, 2′,6′-H),
6.01 (t, 1 H, NH), 6.14 (br s, 2 H, 2-NH2), 7.14 (s, 2 H, 4-NH2),
7.91 (d, 1 H, H-5), 8.31 (d, 1 H, H-7). Anal. Calcd for
(C16H18N6O2‚0.5H2O) C, H, N.
1
water to yield analytically pure compound: mp >370 °C; H
NMR (Me2SO-d6) δ 7.25 (br s, 2H, 2-NH2), 8.23 (br s, 2H,
4-NH2), 9.32 (m, 2H, H-5 and H-7). Anal. Calcd for
(C7H6N6O2‚H2O) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
9, 11, 14, 16-19, 21, a n d 23-27. To a solution of 7 in 50 mL
of warm (40 °C) N,N-dimethylformamide (DMF) was added
Raney Ni. The mixture was hydrogenated in a Parr shaker
at 35 psi for 3 h. TLC analysis (CHCl3:MeOH, 5:2) indicated
the presence of a blue fluorescent spot (Rf ) 0.27). At the end
of this period, the appropriate aldehyde (substituted or un-
substituted benzaldehyde, substitued or unsubstituted naph-
2,4-Diam in o-6-[N-(2′,5′-dim eth oxyben zyl)am in o]pyr ido-
[2,3-d ]p yr im id in e, 21. Compound 21 was obtained from 7
(0.49 g, 2.42 mmol), Raney Ni (3.0 g), and 2,5-dimethoxy-

Pyrido[2,3-d]pyrimidine Antifolates
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1445
benzaldehyde (0.40 g, 2.42 mmol) to afford 0.54 g (68%) of 21:
mp 216 °C; 1H NMR (Me2SO-d6) δ 3.66 (s, 3 H, 2′-OCH3), 3.71
(s, 3 H, 5′-OCH3), 4.22 (d, 2 H, CH2), 5.97 (br s, 3H, 2-NH2
and NH), 6.79 (m, 1H, 6′-H), 6.92 (m, 2H, 3′-H and 4′-H), 7.32
(s, 2 H, 4-NH2), 7.43 (d, 1 H, 5-H), 8.35 (d, 1 H, 7-H). Anal.
Calcd for (C16H18N6O2‚H2O) C, H, N.
2,4-Diam in o-6-[N-(1′-n aph th ylm eth ylen e)am in o]pyr ido-
[2,3-d ]p yr im id in e, 23. Compound 23 was synthesized from
7 (0.25 g, 1.21 mmol), Raney Ni (3.0 g), and 1-naphthaldehyde
(0.19 g, 1.21 mmol) to afford 0.23 g (61%) of 23: mp 259 °C;
1H NMR (Me2SO-d6) δ 4.61 (d, 2 H, CH2), 6.11 (t, 1 H, NH),
6.71 (br s, 2 H, 2-NH2), 7.41 (m, 2 H, 2′-H and 3′-H), 7.56 (m,
2 H, 4′-H and 5′-H), 7.75 (d, 1 H, H-5), 7.90 (m, 5-H, 6′-H, 7′-
H, 8′-H and 4-NH2), 8.33 (d, 1 H, H-7). Anal. Calcd for
(C18H16N6‚H2O) C, H, N.
2,4-Diam in o-6-[N-[(4′-m eth oxy-1′-n aph th yl)m eth ylen e]-
a m in o]p yr id o[2,3-d ]p yr im id in e, 24. Compound 24 was
obtained from 7 (0.49 g, 2.42 mmol), Raney Ni (3.0 g), and
4-methoxy-1-naphthaldehyde (0.45 g, 2.42 mmol) to afford 0.49
g (59%) of 24: mp 297 °C; 1H NMR (Me2SO-d6) δ 3.91 (s, 3 H,
4′-OCH3), 4.61 (d, 2 H, CH2), 6.11 (t, 1 H, NH), 6.60 (br s, 2 H,
2-NH2), 7.31 (d, 1 H, 3′-H), 7.37 (d, 1 H, 2′-H), 7.56 (m, 1 H,
5′-H), 7.79 (d, 1 H, H-5), 7.96 (m, 5 H, 6′, 7′, 8′ H and 4-NH2),
8.37 (d, 1 H, H-7). Anal. Calcd for (C19H18N6O‚0.5H2O) C, H,
N.
2,4-Diam in o-6-[N-[(2′-m eth oxy-1′-n aph th yl)m eth ylen e]-
a m in o]p yr id o[2,3-d ]p yr im id in e, 25. Compound 25 was
obtained from 7 (0.60 g, 2.91 mmol), Raney Ni (3.0 g), and
2-methoxy-1-naphthaldehyde (0.54 g, 2.91 mmol) to afford 0.54
g (54%) of 25: mp 288 °C; 1H NMR (Me2SO-d6) δ 3.98 (s, 3 H,
2′-OCH3), 4.58 (d, 2 H, CH2), 6.03 (t, 1 H, NH), 6.65 (br s, 2 H,
2-NH2), 7.39 (m, 1 H, 3′-H), 7.52 (m, 2 H, 4′ and 5′-H), 7.75 (d,
1 H, H-5), 7.90 (m, 5 H, 6′, 7′, 8′-H and 4-NH2), 8.33 (d, 1 H,
H-7). Anal. Calcd for (C19H18N6O‚H2O) C, H, N.
2,4-Dia m in o-6-[N-(4′-p h en oxyben zyl)a m in o]pyr ido[2,3-
d ]p yr im id in e, 26. Compound 26 was synthesized from 7
(0.40 g, 1.94 mmol), Raney Ni (3.0 g), and 4-phenoxybenz-
aldehyde (0.36 g, 1.94 mmol) to afford 0.38 g (55%) of 26: mp
290 °C; 1H NMR (Me2SO-d6) δ 4.26 (d, 2 H, CH2), 5.87 (br s, 2
H, 2-NH2), 6.23 (t, 1 H, NH), 6.38 (s, 2 H, 4-NH2), 6.96-7.4
(m, 5 H), 7.95 (d, 2 H, 2′-H), 8.31 (d, 1 H, H-7), 8.69 (d, 1 H),
8.75 (d, 1 H). Anal. Calcd for (C20H18N6O‚H2O) C, H, N.
2,4-Dia m in o-6-[N-(2-p h en yleth yl)a m in o]p yr id o[2,3-d ]-
p yr im id in e, 27. Compound 27 was synthesized from 7 (0.60
g, 2.91 mmol), Raney Ni (3.0 g), and phenylacetaldehyde (0.35
g, 2.91 mmol) to afford 0.49 g (60%) of 27: mp 251 °C; 1H NMR
(Me2SO-d6) δ 2.86 (t, 2 H, CH2CH2), 3.24 (t, 2 H, CH2CH2),
5.80 (t, 1 H, NH), 5.87 (br s, 2 H, 2-NH2), 7.32 (m, 5 H, C6H5),
7.37 (d, 1 H, H-5), 8.25 (d, 1 H, H-7). Anal. Calcd for
(C15H16N6‚0.5H2O) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
10, 12, 15, 20, 22, a n d 28. To a suspension of the correspond-
ing N9 H compound in 15 mL of acetonitrile were added HCHO
and NaCNBH3, followed by concentrated HCl dropwise to
effect solution. TLC analyses after 1 h indicated the presence
of a new spot corresponding to the product. The reaction
mixture was stirred for 3 h, and the acetonitrile was evapo-
rated. The residue was dissolved in water and brought to pH
of 8 with concentrated NH4OH. The resulting precipitate was
filtered and washed with 25 mL of cold water to afford the
pure N9 methyl compound.
2,4-Diam in o-6-(N-ben zyl-N-m eth ylam in o)pyr ido[2,3-d]-
p yr im id in e, 10. Compound 10 was synthesized from 9 (0.10
g, 0.38 mmol), NaCNBH3 (0.07 g, 1.12 mmol), and HCHO (0.10
mL) to afford 0.07 g (72%) of 10: mp 255 °C; 1H NMR (Me2SO-
d6) δ 2.97 (s, 3 H, N9 CH3), 4.31 (s, 2 H, CH2), 6.17 (br s, 2-H,
2-NH2), 7.32 (m, 1 H, 4′-H), 7.36 (m, 2 H, 3′-H and 5′-H), 7.43
(m, 2 H, 2′-H and 6′-H), 7.59 (d, 1 H, H-5), 8.41 (d, 1 H, H-7).
Anal. Calcd for (C15H16N6‚0.5H2O) C, H, N.
7.82 (d, 1 H, 5-H), 8.25 (s, 2 H, 4-NH2), 8.46 (d, 1 H, 7-H).
Anal. Calcd for (C18H22N6O3‚0.5H2O) C, H, N.
2,4-Dia m in o-6-[N-(2′,3′,4′-t r im et h oxyb en zyl)-N-m et h -
yla m in o]p yr id o[2,3-d ]p yr im id in e, 15. Compound 15 was
obtained from 14 (0.09 g, 0.25 mmol), HCHO (0.10 mL), and
NaCNBH3 (0.05 g, 0.76 mmol) to afford 0.07 g (80%) of 15:
1
mp 236 °C; H NMR (Me2SO-d6) δ 2.81 (s, 3 H, CH3), 3.75 (s,
3 H, 2′-OCH3), 3.77 (s, 3 H, 4′-OCH3), 3.81 (s, 3 H, 3′-OCH3),
4.21 (s, 2 H, CH2), 6.11 (br s, 3 H, 2 NH2 and NH), 6.77 (d, 1
H, H-5), 7.11 (d, 1 H, 6′-H), 7.53 (m, 3 H, 4-NH2 and H-5),
8.36 (d, 1 H, H-7). Anal. Calcd for (C18H22N6O3‚H2O) C, H,
N.
2,4-Dia m in o-6-[N-(3′,5′-d im et h oxyb en zyl)-N-m et h yl-
a m in o]p yr id o[2,3-d ]p yr im id in e, 20. Compound 20 was
obtained from 19 (0.12 g, 0.37 mmol), NaCNBH3 (0.06 g, 0.92
mmol), and HCHO (0.12 mL) to afford 0.09 g (79%) of 20: mp
1
259 °C; H NMR (Me2SO-d6) δ 2.94 (s, 3 H, N9-CH3), 3.71 (s,
6 H, 3′,5′-OCH3), 4.26 (s, 2 H, CH2), 5.75 (s, 1 H, 4′-H), 5.84 (s,
2 H, 2′,6′-H), 6.19 (br s, 2 H, 2-NH2), 7.24 (s, 2 H, 4-NH2), 7.95
(d, 1 H, H-5), 8.37 (d, 1 H, H-7). Anal. Calcd for (C17H20N6O2‚
H2O) C, H, N.
2,4-Dia m in o-6-[N-(2′,5′-d im et h oxyb en zyl)-N-m et h yl-
a m in o]p yr id o[2,3-d ]p yr im id in e, 22. Compound 22 was
obtained from 21 (0.08 g, 0.25 mmol), NaCNBH3 (0.05 g, 0.74
mmol), and HCHO (0.08 mL) to afford 0.06 g (69%) of 22: mp
1
240 °C; H NMR (Me2SO-d6) δ 3.04 (s, 3 H, N9 CH3), 4.60 (s,
2 H, CH2), 6.54 (d, 1 H, 3′-H), 6.79 (d, 1 H, 4′-H), 6.96 (m, 3 H,
2-NH2 and 6′-H), 7.30 (br s, 2 H, 4-NH2), 7.87 (d, 1 H, H-5),
8.36 (d, 1 H, H-7). Anal. Calcd for (C17H20N6O2‚0.75H2O) C,
H, N.
2,4-Dia m in o-6-[N -(2-p h e n yle t h yl)-N -m e t h yla m in o]-
p yr id o[2,3-d ]p yr im id in e, 28. Compound 28 was synthe-
sized from 27 (0.20 g, 0.71 mmol), NaCNBH3 (0.14 g, 2.14
mmol), and HCHO (0.20 mL) to afford 0.14 g (66%) of 28: mp
1
258 °C; H NMR (Me2SO-d6) δ 2.86 (t, 2 H, CH2CH2), 2.92 (s,
3 H, CH3), 3.21 (t, 2 H, CH2CH2), 5.89 (br s, 2 H, 2-NH2), 7.39
(m, 5 H, C6H5), 7.47 (d, 1 H, H-5), 8.01 (br s, 2 H, 4-NH2), 8.33
(d, 1 H, H-7). Anal. Calcd for (C16H18N6‚H2O) C, H, N.
2,4-Dia m in o-6-[N-[(3′,4′,5′-tr im eth oxyben zyl)m eth yl]-
a m in o]p yr id o[2,3-d ]p yr im id in e, 13. Compound 7 (0.45 g,
2.18 mmol) was dissolved in 40 mL of warm DMF, Raney Ni
(2.5 g) added, and the mixture hydrogenated for 4 h. The
reaction mixture was filtered through a Celite mat and the
DMF evaporated under reduced pressure (40 °C) to yield a
brown solid. This solid was dissolved in 15 mL of methanol,
and 3,4,5-trimethoxyacetophenone (0.47 g, 2.18 mmol) was
added all at once. Following this, 2-3 mL of acetic acid was
added followed by BH3‚Et3N (0.48 g, 6.54 mmol). After 2 h,
another 2-3 mL of acetic acid was added and the reaction
mixture stirred overnight at room temperature. Water (15
mL) was then added, and all the solvents were evaporated.
The residue was dissolved in methanol (30 mL), 1.0 g of silica
gel added, and the methanol evaporated to yield a dry plug.
This plug was eluted with CHCl3:MeOH (5:1), and fractions
containing the product were pooled and evaporated to dryness
to yield pure 13 as a yellow powder (0.09 g, 12%): mp 283 °C;
1H NMR (Me2SO-d6) δ 1.3 (d, 3 H, CH3), 3.62 (s, 3 H, 4′-OCH3),
3.71 (s, 6 H, 3′ and 5′-OCH3), 4.17 (m, 1 H, CH), 6.29 (m, 3 H,
2-NH2 and NH), 6.61 (s, 2 H, 2′ H and 6′ H), 7.91 (d, 1 H,
H-5), 8.12 (br s, 2 H, 4-NH2), 8.41 (d, 1 H, H-7). Anal. Calcd
for (C18H22N6O3‚H2O) C, H, N.
Dieth yl 4-(F or m ylben zoyl)-L-glu ta m a te, 31. A solution
of 4-carboxybenzaldehyde (3.38 g, 0.02 mol), diethyl L-
glutamate ester hydrochloride (5.40 g, 0.02 mol), and DCC
(4.64 g, 0.02 mol) in anhydrous pyridine was stirred for 7 days.
At the end of this period, the pyridine was evaporated, and
the oil was chromatographed on a silica gel column using
hexanes:ethyl acetate (10:1) as the eluant. Fractions contain-
ing the desired product (TLC) were pooled and evaporated to
yield 3.0 g (40%) of a white solid: mp 75 °C (lit.31 mp 74-76
°C).
5-Dea za isoa m in op ter in , 29. To a solution of 7 (0.50 g,
2.42 mmol) in warm DMF was added 3.0 g of Raney Ni, and
the mixture was hydrogenated for 3 h at 35 psi. At the end of
this period, 4-(formylbenzoyl)-L-glutamate diethyl ester 31
(0.81 g, 2.42 mmol) was added followed by 15 mL of acetic acid,
and the mixture was hydrogenated for a further 3.5 h. The
2,4-Dia m in o-6-[N-(3′,4′,5′-t r im et h oxyb en zyl)-N-m et h -
yla m in o]p yr id o[2,3-d ]p yr im id in e, 12. Compound 12 was
synthesized from 11 (0.10 g, 0.28 mmol), HCHO (0.10 mL),
and NaCNBH3 (0.05 g, 0.84 mmol) to afford 0.08 g (75%): mp
1
279 °C; H NMR (Me2SO-d6) δ 3.03 (s, 3 H, N9 CH3), 3.62 (s,
3 H, 4′-OCH3), 3.71 (s, 6 H, 3′OCH3 and 5′-OCH3), 4.55 (s, 2
H, CH2), 6.57 (s, 2 H, 2′H and 6′H), 6.88 (br s, 2 H, 2-NH2),

1446 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Gangjee et al.
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reaction mixture was filtered through Celite, and the filtrate
was evaporated to dryness. Workup and purification was
carried out as described above for 16 to yield the diethyl ester,
32 (0.69 g, 58%).
The diester was dissolved in 5 mL of DMSO and saponified
using 10 mL of 1 N NaOH at room temperature over a period
of 6 h to afford 29 as a yellow powder (0.47 g, 77%): mp 280
°C; 1H NMR (Me2SO-d6) δ 1.93-1.97 (m, 2 H, Glu â-CH2),
1.99-2.03 (m, 2 H, Glu δ-CH2), 4.36 (overlapping m, 3 H, CH2
and Glu R-CH), 6.54 (br m, 3 H, 2-NH2 and NH), 7.49 (m, 3 H,
H-5 and 3′- and 5′-H), 7.67 (br s, 2 H, 4-NH2), 7.83 (d, 2 H, 2′-
and 6′-H), 8.32 (overlapping m, 2 H, H-7 and CONH). Anal.
Calcd for (C20H21N7O5‚1.5H2O) C, H, N.
9-Meth yl-5-d ea za isoa m in op ter in , 30. To a suspension
of 32 (0.5 g, 1 mmole) in 30 mL of acetonitrile was added
NaCNBH3 (0.06 g, 3 mmol) and HCHO (0.5 mL) followed by
concentrated HCl dropwise to effect solution. The reaction
mixture was stirred for 3 h, and the acetonitrile was evapo-
rated. The residue was dissolved in water and brought to pH
8 with concentrated NH4OH. The resulting precipitate was
filtered and washed with cold water to afford pure 33.
Saponification of 33 was carried out in a manner similar to
that described for 32 (in the synthesis for 29 above) to yield
30 (0.24 g, 54%): mp 276 °C; 1H NMR (Me2SO-d6) δ 1.94-
1.98 (m, 2 H, Glu â-CH2), 1.99-2.03 (m, 2 H, Glu δ-CH2), 2.89
(s, 3 H, CH3), 4.41 (overlapping m, 3 H, CH2 and Glu R-CH),
6.60 (br s, 2 H, 2-NH2), 7.52 (m, 3 H, H-5 and 3′- and 5′-H),
7.71 (br s, 2 H, 4-NH2), 7.87 (d, 2 H, 2′- and 6′-H), 8.34
(overlapping m, 2 H, H-7 and CONH). Anal. Calcd for
(C21H23N7O5‚2.0H2O) C, H, N.
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Ack n ow led gm en t. This work was supported in part
by NIH Grants GM40998 (A.G.) and CA10914 (R.L.K.)
and NIH Contracts NO1-AI-87240 (S.F.Q.) and NO1-
AI-35171 (S.F.Q.) Division of AIDS.
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(35) We thank the Developmental Therapeutics Program of the
National Cancer Institute for performing the in vitro anticancer
evaluation.
J M950786P