Pyrimidines with Antifolate Activity
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1485
mol). Toluene was slowly distilled off until 20 mL (40% of the
original volume) had been collected. The reaction was then
quenched with 5% NaHCO3, the rest of the toluene was
removed by rotary evaporation, and the product was purified
by chromatography (“Flash” silica gel, 20 g, 2 cm × 17 cm, 2:1
isooctane-EtOAc) to obtain alkyne ester 42 (421 mg, 29%) as
an oil that was used directly in the next step.
J ) 8 Hz, 2H, 2′′-H, 6′′-H), 7.93 (d, J ) 8 Hz, 2H, 3′′-H, 5′′-H).
Anal. (C22H20N4O3) C, H, N.
Step 4. A stirred solution of 47 (146 mg, 0.376 mmol) in
slightly warm DMSO (4 mL) was treated dropwise with 2 N
NaOH (0.5 mL), then diluted with H2O (40 mL) and acidified
with 10% AcOH. The mixture was chilled and the solid was
collected and dried in a lyophilizer to obtain 29 as a white
powder (118 mg, 84%): mp >300 °C dec; HPLC, 17 min (C18
silica gel, 20% MeCN in 0.1 M NH4OAc, pH 7.4); IR (KBr) ν
3400, 3120, 1680, 1620, 1600, 1580, 1505, 1445, 1405, 1365,
Step 2. A mixture of 42 (240 mg, 1.5 mmol), iodide 31 (356
mg, 1.0 mmol), (Ph3P)2PdCl2 (10 mg), (Ph3P)3CuBr (10 mg),
and Et3N (10 mL) in DMF (3 mL) was heated at 65 °C for 2
days. The solvent was evaporated, and the residue was swirled
successively with isooctane and H2O, each of which was
decanted in turn. The residue left after trituration with H2O,
consisting of ester 43, was collected, freeze-dried, and taken
up in DMSO (4 mL). The solution was swirled and treated
dropwise with 1 N NaOH (1.5 mL), then diluted with 10
volumes of H2O and adjusted to pH 8-9 with 10% AcOH. A
small amount of precipitate was filtered off, and the filtrate
was purified by preparative HPLC on a C18 silica gel using a
98:2 mixture of 20% MeCN in 0.1 M NH4OAc, pH 8.5, and
50% MeCN in H2O as the eluent. Appropriately pooled
fractions were reduced in volume by rotary evaporation and
then freeze-dried. The residue was redissolved in dilute NaOH,
the solution was acidified with 10% AcOH, and the precipitate
was collected and dried on a lyophilizer to obtain 28 as a white
solid (54 mg, 13% combined yield for the Sonogashira reaction
and saponification): mp >250 °C dec; IR (KBr) ν 3420, 3330,
3110, 1675, 1615, 1585, 1560, 1505, 1465, 1425, 1375, 1320,
1285, 1250, 1215 cm-1 1H NMR (DMSO-d6) δ 3.52 (s, 2H,
;
bridge CH2), 3.82 (s, 3H, OMe), 5.91 (br s, NH2), 6.21 (br s,
NH2), 7.03 (d, J ) 8 Hz, 1H, 3′-H), 7.20 (s, 1H, 6′-H), 7.46 (m,
2H, 4′-H, pyrimidine 6-H), 7.56 (d, J ) 8 Hz, 2H, 2′′-H, 6′′-H),
7.89 (d, J ) 8 Hz, 2H, 3′′-H, 5′′-H). Anal. (C21H18N4O3‚0.8H2O)
C, H, N.
2,4-Dia m in o-5-[2′-(2-(4-ca r boxyp h en yl)eth yl)-5′-m eth -
oxyben zyl]p yr im id in e (30). A solution of ester 47 (140 mg,
0.361 mmol) in DMF (20 mL) was shaken with H2 and 5%
Pd-C (25 mg) in a Parr apparatus at 3 atm pressure for 18 h.
The catalyst was filtered off, the filtrate was concentrated to
dryness by rotary evaporation, the residue was taken up in
DMSO (3 mL), and the solution was swirled and treated
dropwise with 2 N NaOH (0.5 mL). The solution was then
diluted with H2O (30 mL), acidified with 10% AcOH, chilled
in ice, and filtered. The collected solid was purified by
preparative HPLC on C18 silica gel (20% MeCN in 0.1 M NH4-
OAc, pH 8.5), and appropriately pooled fractions were evapo-
rated to dryness. The residue was taken up in dilute NaOH,
the solution was acidified with 10% AcOH and chilled in ice,
and the precipitate was collected and dried in a lyophilizer to
obtain 30 as a white powder (51 mg, 35%): mp >250 °C dec;
IR (KBr) ν 3330, 2930, 1655, 1610, 1505, 1460, 1390, 1250
cm-1; 1H NMR (DMSO-d6) δ 2.79 (br s, 4H, CH2CH2), 3.46 (s,
2H, bridge CH2), 3.73 (s, 3H, OMe), 5.87 (br s, NH2), 6.16 (br
s, NH2), 6.84 (d, J ) 8 Hz, 1H, 3′-H), 6.97 (m, 2H, 4′-H, 6′-H),
7.16 (d, J ) 8 Hz, 2H, 2′′-H, 6′′-H), 7.28 (s, 1H, pyrimidine
6-H), 7.77 (d, J ) 8 Hz, 2H, 3′′-H, 5′′-H). Anal. (C21H22N4O3‚
1.7H2O) C, H, N.
1
1300, 1285, 1250, 1210 cm-1; H NMR (DMSO-d6) δ 3.57 (s,
2H, bridge CH2), 3.86 (s, 3H, OMe), 6.36 (br s, NH2), 6.52 (br
s, NH2), 7.07 (d, J ) 8 Hz, 1H, 3′-H), 7.25-7.55 (m, 4H, 4′-H,
6′-H, 5′′-H, pyrimidine 6-H), 7.70 (d, J ) 8 Hz, 1H, 6′′-H), 7.92
(d, J ) 8 Hz, 1H, 4′′-H), 8.02 (s, 1H, 2′′-H). Anal. (C21H18N4O3‚
0.9AcOH) C, H, N.
2,4-Dia m in o-5-[2′-(4-ca r boxyp h en yl)eth yn yl)-5′-m eth -
oxyben zyl]p yr im id in e (29). Step 1. To a solution of 44 (4.97
g, 0.019 mol) and 2-methyl-3-butyn-2-ol (2.24 mL, 1.93 g, 0.023
mol) in Et3N (50 mL) under N2 were added CuI (20 mg), Ph3P
(40 mg), and (Ph3)2PdCl2 (20 mg). The mixture was stirred
under reflux for 68 h, then cooled to room temperature and
partitioned between EtOAc and H2O. Evaporation of the
organic layer and recrystallization of the solid from hexane
afforded the alkyne ester 45 as a light-brown solid (4.14 g, ca.
100%): mp 74-76 °C (lit.7 83.5-84.5 °C after recrystallization
from EtOH-H2O instead of hexane). Anal. (C13H14O3) C, H.
Step 2. A stirred solution of 45 (2.18 g, 0.01 mol) in dry
toluene (50 mL) was treated with NaH (0.4 g of 60% dispersion
in mineral oil, calculated to contain 0.24 g, 0.01 mol) was
slowly heated in a distillation apparatus until the head
temperature reached 110 °C and toluene began to collect in
the receiver. After 15 mL had been removed, the reaction
mixture was cooled and quenched with 5% NaHCO3 (caution:
gas evolution). This resulted in an emulsion that was difficult
to separate into two layers. Two phases formed readily upon
addition of EtOAc and 10% citric acid, the organic layer was
separated and evaporated to a brown solid, and the latter was
purified by chromatography (“Flash” silica gel, 20 g, 2 cm ×
18 cm, 2:1 isooctane-EtOAc) to obtain 46 (1.43 g, 98%): mp
87-88 °C (lit.7 91-93 °C, purification by sublimation instead
of chromatography).
Ack n ow led gm en t. This work was supported by
Research Grants RO1-AI29904 (A.R.) and RO1-AI55064
(C.H.S.) and Research Contract NO1-AI25140 (C.B.I.)
from the National Institute of Allergy and Infectious
Diseases (NIAID), U.S. Department of Health and
Human Services. The help and advice of Dr. Christopher
Lambros of the AIDS Opportunistic Infections Program,
NIAID, Bethesda, MD, are likewise acknowledged.
Refer en ces
(1) Klepser, M. W.; Klepser, T. B. Drug treatment of HIV-related
opportunistic infections. Drugs 1997, 3, 40-73.
(2) See ref 3 for a selected list of our publications in this area since
1993.
(3) Rosowsky, A.; Forsch, R. A.; Queener, S. F. Further studies on
2,4-diamino-5-(2′,5′-disubstituted benzyl)pyrimidines as potent
and selective inhibitors of dihydrofolate reductases from three
major opportunistic pathogens of AIDS. J . Med. Chem. 2003,
46, 1726-1736.
(4) Rosowsky, A.; Forsch, R. A.; Queener, S. F. Inhibition of
Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium
avium dihydrofolate reductases by 2,4-diamino-[2-methoxy-5-
(ω-carboxyalkyloxy)benzyl]pyrimidines: Marked improvement
in potency relative to trimethoprim and species selectivity
relative to piritrexim. J . Med. Chem. 2002, 45, 233-241.
(5) Kuyper, L. F.; Roth, B.; Baccanari, D. P.; Ferone, R.; Beddell,
C. R.; Champness, J . N.; Stammers, D. K.; Dann, J . G.;
Norrington, F. E.; Baker, D. J .; Goodford, P. J . Receptor-based
design of dihydrofolate reductase inhibitors: comparison of
crystallographically determined enzyme binding with enzyme
affinity in a series of carboxy-substituted trimethoprim ana-
logues. J . Med. Chem. 1985, 28, 303-311.
(6) Calas, M.; Barbieri, A.; Giral, L.; Balmayer, B.; Despaux, E.
Synthesis of new trimethoprim analogs. Antibacterial structure-
activity relationship. Eur. J . Med. Chem. 1982, 17, 407-504.
(7) Havens, S. J .; Hergenrother, P. M. Synthesis of arylacetylenes
by the sodium hydride catalyzed cleavage of 4-aryl-2-methyl-3-
butyn-2-ols. J . Org. Chem. 1985, 50, 1763-1765.
Step 3. A stirred mixture of 46 (240 mg, 1.5 mmol), iodide
31 (356 mg, 1.0 mmol), (Ph3P)2PdCl2 (10 mg), (Ph3P)3CuBr (10
mg), and Et3N (3 mL) in DMF (3 mL) was heated at 60 °C for
18 h. A homogeneous solution formed within 10 min, followed
a few minutes later by the appearance of a solid. The reaction
mixture was chilled, and the solid was collected and washed
with Et2O to obtain the methyl ester 47 (292 mg, 78%): mp
239-240 °C; IR (KBr) ν 3480, 3370, 3160, 2950 w, 2830 w,
2200, 1700 (CdO), 1675, 1615, 1595, 1565, 1510, 1485, 1455,
1430, 1400, 1310, 1305, 1290, 1280, 1245 cm-1 1H NMR
;
(DMSO-d6) δ 3.52 (s, 2H, bridge CH2), 3.84 (s, 6H, two OMe),
5.71 (br s, NH2), 6.09 (br s, NH2), 7.04 (d, J ) 8 Hz, 1H, 3′-H),
7.20 (s, 1H, 6′-H), 7.42 (m, 2H, 4′-H, pyrimidine 6-H), 7.62 (d,