Rationally Designed Inhibitors of IMPDH
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 1
7
mmol) in dry triethyl phosphate (0.5 mL) was cooled to 0 °C,
phosphorus oxychloride (2 mmol) was added, and the mixture
was stirred at 0 °C for 6 h. Ether was added, and the resulting
precipitate was separated by centrifugation. Ice water (5 mL)
was added to the residual solid, and the resulting solution was
extracted with CHCl3. The aqueous layer was adjusted to pH
8 with sodium bicarbonate and the solution concentrated and
passed through a Dowex-50 column (formate form). Elution
with aqueous formic acid gave the 2-substituted inosinic acids
2 and 10. For enzyme inhibition assays the solution was
adjusted to pH 8.0 and lyophilized.
) 4.0 Hz), 8.74 (1H, s, H-8). Anal. (C11H13N4Na2O10P‚
3.5H2O): C, H; N: calcd, 11.12; found, 9.91.
P r ep a r a tion of 2-(Ch lor om eth yl)in osin e (9). 2-(Hy-
d r oxym eth yl)-2′,3′-isop r op ylid en ein osin e (6). Perchloric
acid (70%) (8 mL) was added to a suspension of 2-(hydroxy-
methyl)inosine (5) (2.98 g, 10 mmol) in acetone (150 mL) at 0
°C, the mixture was stirred overnight, and the solution was
neutralized with 30% NH3. The solvent was removed in vacuo,
and the residue was purified by column chromatography (silica
gel, MeOH/EtOAc, 4:1). Recrystallization gave 2.8 g of pure
6: 72%; mp 196-198 °C; 1H NMR (CD3OD) δ 1.39 (3H, s, CH3),
1.61 (3H, s, CH3), 3.69 (1H, dd, J gem ) 11.5 Hz, J ) 3.5 Hz,
H-5′), 3.75 (1H, dd, J gem ) 11.50 Hz, J ) 3.5 Hz, H-5′), 4.31
(1H, m, H-4′), 4.58 (2H, s, CH2O), 5.00 (1H, m, H-3′), 5.22 (1H,
m, H-2′), 6.12 (1H, d, J ) 4.0 Hz), 8.22 (1H, s, H-8).
2-(H y d r o x y m e t h y l)-2′,3′-i s o p r o p y li d e n e -5′-(4,4′-
d im eth oxytr ityl)in osin e (7). To a solution of 6 (0.1 g, 0.3
mmol) in dry pyridine (0.8 mL) was added 4,4-dimethoxytrityl
chloride (0.14 g, 0.42 mmol) at 0 °C. After 5 h the reaction
mixture was poured into ice water, and the mixture was
extracted with methylene chloride. The combined organic
extracts, after drying over Na2SO4 and chromatography, gave
0.11 g of pure 7: 58%; mp 140-142 °C; 1H NMR (CDCl3) δ
1.35 (3H, s, CH3), 1.62 (3H, s, CH3), 3.30 (2H, m, H-5′), 3.75
(6H, s, 2CH3O), 4.46 (1H, br, H-4′), 4.52 (2H, m, CH2O), 4.75
(1H, br, OH), 4.83 (1H, m, H-3′), 5.19 (1H, m, H-2′), 6.02 (1H,
d, J ) 3 Hz), 6.68-7.4 (14H, m, 3Ar), 7.82 (1H, s, H-8), 11.17
(1H, br, NH).
2-[[(Meth oxym eth ylen e)oxy]m eth yl]in osin ic a cid (2a ):
30%; 1H NMR (CD3OD) δ 3.32 (3H, s, CH3O-), 4.01 (2H, m,
H-5′), 4.13 (1H, m, H-4′), 4.31 (1H, m, H-3′), 4.52 (2H, -OCH2-
inosine), 4.58 (1H, t, H-2′), 4.70 (2H, s, -OCH2O-), 5.97 (1H, d,
J ) 5.40 HZ, H-1′), 8.32 (1H, s, H-8).
1
2-(P h en ylvin yl)in osin ic a cid (2b): 43%; H NMR (CD3-
OD) δ 4.07 (2H, m, H-5′), 4.22 (1H, m, H-4′), 4.39 (1H, t, H-3′),
4.68 (1H, t, H-2′), 6.11 (1H, d, J ) 5.0 Hz, H-1′), 6.93 (1H, d,
J ) 15.6 Hz, vinyl), 7.40-7.62 (5H, m, Ph), 7.90 (1H, d, J )
15.6 Hz, vinyl), 8.41 (1H, s, H-8). Anal. (C18H19N4O8P‚H2O):
C, H, N.
2-(Tr iflu or om eth yl)in osin ic a cid (2c): 59%; 1H NMR
(D2O) δ 4.08 (2H, m, H-5′), 4.28 (1H, br, H-4′), 4.42 (1H, m,
H-3′), 4.68 (1H, H-2′), 6.07 (1H, d, J ) 4.07 Hz, H-1′), 8.52
(1H, s, H-8). Anal. (C11H12F3N4O8P‚2H2O) C, H, N.
2-(Diflu or om eth yl)in osin ic a cid (2d ): 60%; 1H NMR
(D2O) δ 4.03 (2H, m, H-5′), 4.31 (1H, m, H-4′), 4.48 (1H, m,
H-3′), 6.10 (1H, d, J ) 4.0 Hz, H-1′), 6.68 (1H, t, CHF2, J ) 51
Hz), 8.44 (1H, s, H-8). Anal. (C11H13F2N4O8P‚H2O) C; H:
calcd, 3.63; found, 2.78. N: calcd, 13.45; found, 12.57.
2-(F lu or om eth yl)in osin ic a cid (2e): 42%; 1H NMR (D2O)
δ 4.10 (2H, m, H-5′), 4.31 (1H, br, H-4′), 4.42 (1H, m, H-3′),
5.45 (1H, d, J ) 45 Hz), 6.09 (1H, d, J ) 4.5 Hz, H-1′), 8.67
(1H, s, H-8). Anal. (C11H12FN4Na2O8P‚H2O) C, H, N.
2-(Ch lor om eth yl)-2′,3′-isop r op ylid en e-5′-(4,4′-d im eth -
oxytr ityl)in osin e (8). To a suspension of (7) (0.26 g, 0.41
mmol) in carbon tetrachloride was added tributylphosphine
(0.2 mL, 0.81 mmol), and the reaction mixture was stirred at
25 °C for 40 min. After evaporation of the solvent in vacuo at
room temperature, the residue was purified by preparative
TLC (EtOAc as eluant). The product, 0.145 g, which was
contaminated with a small amount of tributylphosphine was
used for the following reaction without further purification:
1H NMR (CDCl3) δ 1.38 (3H, s, CH3), 1.61 (3H, s, CH3), 3.31
(2H, m, H-5′), 3.75 (6H, s, 2CH3O), 4.33 (1H, d, CH2Cl, J ) 11
Hz), 4.38 (1H, d, CHCl, J ) 11.0 Hz), 4.48 (1H, br, H-4′), 4.90
(1H, m, H-3′), 5.20 (1H, m, H-2′), 6.06 (1H, d, J ) 3.0 Hz),
6.65-7.35 (13H, m, Ar), 7.87 (1H, s, H-8), 11.73 (1H, br, NH).
1
2-Vin ylin osin ic a cid (2f): 35%; H NMR (CD3OD) δ 4.20
(2H, m, H-5′), 4.33 (1H, br, H-4′), 4.61 (1H, br, H-3′), 5.85 (1H,
dd, J ) 11, <1 Hz, CHdCH2), 6.09 (1H, d, J ) 4.0 Hz), 6.55-
6.67 (2H, m, CHdCH2), 8.80 (1H, s, H-8). Anal. (C12H13
-
N4Na2O8P‚4H2O): C, N; H: calcd, 4.28; found, 3.55.
1
2-(P h en yleth yl)in osin ic a cid (2g): 45%; H NMR (D2O)
δ 3.01 (4H, m, -CH2CH2-), 3.90-4.24 (2H, m, H-5′), 4.25 (1H,
m, H-4′), 4.28 (1H, m, H-3′), 4.40 (1H, m, H-2′) 6.00 (1H, d, J
) 4.5 Hz, H-1′), 7.05-7.20 (5H, m, Ph), 8.8 (1H, s, H-8). Anal.
(C18H21N4O8P‚1.5H2O) C, H, N.
2-(Ch lor om eth yl)in osin e (9). A solution of 8 (0.18 g, 0.27
mmol) in 80% aqueous formic acid (2 mL) was stirred at 0 °C
for 10 min, warmed to room temperature, and stirred for an
additional 2.5 h. After evaporation to dryness in vacuo, the
residue was purified by column chromatography (silica gel,
EtOAc/MeOH, 5:1, as eluant) to give 9; recrystallization from
MeOH/EtOAc gave pure material (40 mg, 46.3%), which
2-(Ch lor om eth yl)in osin ic a cid (10): 46%; 1H NMR (CD3-
OD) δ 4.03 (2H, m, H-5′), 4.17 (1H, m, H-4′), 4.37 (1H, m, H-3′),
4.51 (2H, s, CH2Cl), 4.64 (1H, t, H-2′), 6.00 (1H, d, J ) 4.8 Hz,
H-1′), 8.41 (1H, s, H-8). Anal. (C11H12ClN4Na2O8P‚H2O) C,
H, N.
2-(Hyd r oxym eth yl)in osin ic Acid (3). A solution of MOM
ether 2a (24 mg, 0.056 mmol) in 50% trifluoroacetic acid (1.5
mL) was stirred at 25 °C for 36 h. After evaporation of the
solution to dryness, the residual oil was neutralized with
sodium bicarbonate to pH 7.5 and applied to a column of
Dowex 1 × 6 (formate, 100-200 mesh). After washing with
distilled water, the column was eluted with increasing con-
centrations of formic acid (0.2-1 N). The product was removed
by 0.5 N formic acid, and the combined fractions were
evaporated to give 12 mg of pure 2-(hydroxymethyl)inosinic
1
decomposed at 150 °C without melting: H NMR (CD3OD) δ
3.81 (1H, dd, J gem ) 12.5 Hz, J ) 2.8 Hz, H-5′), 3.93 (1H, dd,
J gem ) 12.5 Hz, J ) 2.8 Hz, H-5′), 4.20 (1H, m, H-4′), 4.48 (1H,
m, H-3′), 4.56 (2H, s, CH2Cl), 4.73 (1H, t, H-2′), 6.00 (1H, d, J
) 5.69 Hz, H-1′), 8.26 (1H, s, H-8).
Assa y a n d In h ibition of IMP DH Activity. IMPDH
activity was determined by a spectrophotometric assay as
described previously.8,24 The assay measures the enzyme-
catalyzed increase in absorbance of NADH at 340 nm as
NADH and XMP are formed from NAD and IMP. Reactions
to determine inhibition by the compounds were performed in
1 cm path length spectrophotometer cells. Standard reactions
contained 50 µM IMP, 100 µM NAD, and varying concentra-
tions of inhibitor in a buffer composed of 0.1 M potassium
phosphate, pH 7.4, 0.1 M potassium chloride, 3 mM EDTA, 1
mM DTT, and 100 µg/mL bovine serum albumin. The reac-
tions were initiated by the addition of enzyme to a final
concentration of 25 nM in a total volume of 1 mL. Rate data
were collected at 37 °C in a spectrophotometer equipped with
a temperature-regulated multicell transporter. IC50 values
were determined by nonlinear regression curve fit of the rate
data to the following equation (eq 1) using the Macintosh
program Systat:
1
acid (3): 49%; dec at 150 °C; H NMR (D2O) δ 4.10 (2H, m,
H-5′), 4.31 (1H, br, H-4′), 4.39 (1H, m, H-3′), 4.65 (2H, s, CH2-
OH), 6.11 (1H, br, H-1′), 8.89 (1H, s, H-8). Anal. (C11H13N4-
Na2O9P‚0.5H2O) C, H; N: calcd, 12.96; found, 12.38.
2-F or m ylin osin ic Acid H yd r a t e (4). A solution of
2-styrylinosinic acid (2b) (18 mg) in 25 mL of dry methanol
was cooled to -78 °C. Ozone was introduced with stirring at
-78 °C until the solution retained the blue color. Excess ozone
was removed with N2, and dimethyl sulfide (0.5 mL) was
added. The solution was allowed to stir at room temperature
for 3 h, the solvent was removed under reduced pressure, and
the residual material was purified by ion-exchange chroma-
tography (Dowex-50, formate); 6.7 mg of pure product 4 was
obtained: 41%; 1H NMR (D2O) δ 4.01 (1H, m, J gem ) 13.5 Hz,
H-5′), 4.14 (1H, m, J gem ) 13.0 Hz), 4.31 (1H, br, H-4′), 4.41
(1H, t, J ) 4.7 Hz, H-3′), 5.86 (1H, s, CH(OH)2), 6.13 (1H, d, J