2932
A. Gangjee et al. / Bioorg. Med. Chem. 9 (2001) 2929–2935
compared to 3. This suggests that although chloro sub-
stituents on the phenyl ring appear to unfavorably affect
potency, their effect on selectivity ratios is dependent on
the position of the substitutents in the phenyl ring.
hydride, triethyl amine or potassium carbonate. The
solution was stirred under nitrogen for 15 min before
intermediate 14 was added. The reaction was allowed to
warm to room temperature and stirred for 24 h under
nitrogen. The desired product precipitated out of solu-
tion. The yellow solid was filtered and washed with
water, ethanol and ether. Thin layer chromatographic
analyses were performed in two solvent systems (Sol-
ventA: 3:1, chloroform/me ht anol and SolventB: 5:1:3
drops, chloroform/methanol/ammonium hydroxide). In
the preparation of analogues 3–5, the precipitate was
analytically pure and required no further purification.
In the preparation of analogues 6–8, the crude pre-
cipitate was redissolved in warm N,N-dimethyl for-
mamide and 1 g of silica gel added to this solution and
the solvent was evaporated to afford a plug of silica gel
for column chromatographic purification. The crude
productsilica gel plug was loaded on ot a silica gel col-
umn and eluted with solvent prepared in a 5:1 ratio of
chloroform/methanol. Fractions shown by thin layer
chromatography to contain pure product were pooled
and evaporated to afford a light yellow solid. The solid
was washed with water, ethanol and ether. All solids were
dried in vacuum with the aid of phosphorous pentoxide.
In summary, the combination of the 2,4-diaminopyr-
ido[2,3-d]pyrimidine ring system and the thiolarylmethyl
moiety of compounds 3–8 resulted in increased poten-
cies againstpcDHFR for all analogues repor et d in ht is
series (with the exception of 7) compared to 1. Against
tgDHFR, analogues 3–5 also exhibited increased
potency compared to 1. The dichloro and dimethoxy
substitutions on the phenyl ring proved detrimental to
tgDHFR inhibitory activity compared to 1. However,
the substantial increase in potency against rlDHFR in
analogues 3–8 was the major contributing factor to the
loss of selectivity compared to 1.
Under the direction of the National Institute of Allergy
and Infectious Diseases (NIAID), the Tuberculosis
Antimicrobial Acquisition and Coordinating Facility at
Southern Research Institute selected compound 4 for an
in vitro evaluation of antimycobacterial activity. For
primary screening, Mycobacterium tuberculosis H Rv
3
7
cells (ATCC 27294) were treated with a drug con-
centration of 6.25 mg/mL. Compound 4 inhibited the
growth of ATCC 27294 cells by 58% at this concentration.
2,4-Diamino-6-[(phenylthio)methyl]pyrido[2,3-d]pyrimidine
(3). Compound 3 was synthesized from intermediate 14
(0.25 g, 1.0 mmol), phenylthiol (0.12 mL, 1.2 mmol) and
potassium carbonate (1.0 g) in 15 mL of N,N-dimethyl-
acetamide using Procedure A to afford a light yellow
ꢀ
Experimental
solid (0.022 g, 8%): mp >189 C dec; TLC R 0.33 in
f
1
Thin-layer chromatography was performed on silica gel
plates with fluorescent indicator purchased from
Aldrich Chemical Company, Milwaukee, WI. UV light
at254 and 365 nm was used for visualiza it on. Column
chromatography was carried out using silica gel, 200–
solventA; H NMR (DMSO-d ) d 4.28 (s, 2H, CH ),
6
2
7.19 (m, 2H, ArH ), 7.32 (m, 3H, ArH 2H, NH2,
exch), 8.40 (br s, 2H, NH , exch), 8.52 (s, 1H, 5-H), 8.62
5
5,
2
(s, 1H, 7-H). HR-MS: C H N S: calcd. mass
1
4
13
5
283.089167, found mass 283.088811.
4
00 mesh, purchased from Aldrich Chemical Company.
High resolution mass spectral analyses were obtained on
a VG 70G or micromass AUTOSPEC double focusing
mass spectrometer. Samples were introduced by direct
2,4-Diamino-6-[(1-naphthylthiol)methyl]pyrido[2,3-d]pyri-
midine (4). Compound 4 was synthesized from inter-
mediate 14 (0.12 g, 0.49 mmol), 1-naphthalenethiol
(0.088 g, 0.55 mmol) and sodium hydride, 80% disper-
sion in mineral oil (0.016 g) in 15 mL of N,N-dimethyl-
1
insertion probe. H NMR spectra were recorded on a
Brucker WH-300 (300 MHz) instrument. The chemical
shift( d) values are expressed in partper million (ppm)
relative to tetramethylsilane (TMS) as an internal stan-
dard: s=singlet, d=doublet, t=triplet, m=multiplet,
br=broad peak, exch=exchangeable by addition of
acetamide using Procedure A to afford a yellow solid
ꢀ
(0.028 g, 17%): mp >278 C dec; TLC R 0.41 in solvent
f
1
B; H NMR (DMSO-d ) d 4.35 (s, 2H, CH ), 6.58 (s,
6
2
2H, NH , exch), 7.46 (m, 3H, ArH ), 7.78 (m, 4H,
ArH 2H, NH , exch), 8.44 (s, 1H, 5-H), 8.61 (s, 1 H, 7-
7, 2
2
7
D O. Elemental analyses were performed by Atlantic
2
Microlabs, Inc., Atlanta, GA. Elemental compositions
were withinꢁ0.4% of the calculated value. Fractional
moles of solvents in the analytical samples frequently
found in antifolates could not be prevented in spite of
vigorous drying in vacuo and were confirmed, where
possible, by their presence in the NMR spectrum.
Melting points were determined on a MEL-TEMP II
melting point apparatus with a Fluke 51 K/J electronic
thermometer and are uncorrected.
H). HR-MS: C H N S: calcd. mass 333.104817, found
18 15 5
mass 333.105846.
2,4-Diamino-6-[(2-naphthylthiol)methyl]pyrido[2,3-d]pyri-
midine (5). Compound 5 was synthesized from inter-
mediate 14 (0.12 g, 0.49 mmol), 2-naphthalenethiol
(0.088 g, 0.55 mmol) and sodium hydride, 80% disper-
sion in mineral oil (0.016 g) in 15 mL of N,N-dimethyl-
acetamide using Procedure A to afford a yellow solid
ꢀ
(
0.033 g, 20%): mp >236 C dec; TLC R 0.43 in solvent
f
1
B; H NMR (DMSO-d ) d 4.27 (s, 2H, CH ), 6.31 (s,
6
2
2
H, NH , exch), 7.46 (m, 1H, ArH ), 7.56 (m, 3H,
2
7
General procedure for the synthesis of compounds 3–8.
ꢀ
ArH 2H, NH , exch), 8.79 (d, 1H, ArH ), 8.91 (d, 1H,
7, 2 7
ArH ), 8.22 (d, 1H, ArH ), 8.33 (s, 1H, 5-H), 8.50 (s, 1
7 7
(
Procedure A). To a cooled solution (0–5 C) of the
appropriate arylthiol dissolved in N,N-dimethyl acet-
amide or N,N-dimethyl formamide was added sodium
H, 7-H). HR-MS: C H N S: calcd. mass 333.104817,
18 15 5
found mass 333.105804.