4504 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 22
Sicinski et al.
δ 0.864 and 0.867 (3H, 3H, each d, J ) 6.6 Hz, 26-, 27-H3),
0.879 (3H, d, J ) 6.7 Hz, 21-H3), 3.33 (1H, dm, J ) 14.4 Hz,
9â-H), 8.19 (1H, br m, OH); 13C NMR (CDCl3) δ 18.1 (q, C-21),
22.5 and 22.7 (each q, C-26, C-27), 23.9 (t, C-15), 24.2 (t, C-9),
25.4 (t, C-23), 25.9 (t, C-16), 26.0 (t, C-11), 28.0 (d, C-25), 31.0
(t, C-12), 33.5 (t, C-22), 35.2 (d, C-20), 39.3 (t, C-24), 49.6 (d,
C-13), 49.8 (d, C-17), 50.5 (d, C-14), 162.1 (s, C-8); MS m/ z
(relative intensity) 265 (M+, 8), 248 (100), 231 (12), 206 (30);
exact mass calcd for C17H31ON 265.2405, found 265.2404.
(b) Oximation of Grundmann’s ketone 6 (22 mg, 0.08 mmol)
with hydroxylamine hydrochloride (20 mg) in pyridine (0.45
mL) according to the above procedure gave the crystalline
oxime 15 (22 mg, 95%): Rf 0.27 (10% ethyl acetate in hexane);
IR (CHCl3) 3588 and 3286 (OH) cm-1; 1H NMR (CDCl3) δ 0.649
(3H, s, 18-H3), 0.866 and 0.870 (3H, 3H, each d, J ) 6.6 Hz,
26-, 27-H3), 0.935 (3H, d, J ) 6.5 Hz, 21-H3), 2.09 (1H, dd, J
) 11.6, 7.5 Hz, 14R-H), 3.24 (1H, dm, J ) 13.2 Hz, 9â-H), 9.05
(1H, br m, OH); 13C NMR (CDCl3) δ 12.2 (q, C-18), 18.8 (q,
C-21), 20.7 (t, C-15), 21.8 (t, C-11), 22.5 and 22.8 (each q, C-26,
C-27), 23.8 (overlapped C-9 and C-23), 27.9 (t, C-16), 28.0 (d,
C-25), 35.9 (d, C-20), 36.1 (t, C-22), 39.3 (t, C-24), 39.5 (t, C-12),
46.5 (s, C-13), 54.1 (d, C-14), 56.0 (d, C-17), 160.6 (s, C-8); MS
m/ z (relative intensity) 279 (M+, 2), 262 (100), 245 (18), 219
(13); exact mass calcd for C18H33ON 279.2562, found 279.2562.
Anal. (C18H33ON) C, H, N.
25-[(Tr ie t h ylsilyl)oxy]-d e s-A,B-18-n or ch ole st a n -8-
on e (4c). A solution of the ketone 4b (5 mg, 19 µmol) and
imidazole (15 mg, 220 µmol) in anhydrous DMF (150 µL) was
treated with triethylsilyl chloride (15 µL, 90 µmol). The
mixture was stirred at room temperature under argon for 4
h. Ethyl acetate was added and water, and the organic layer
was separated. The ethyl acetate layer was washed with water
and brine, dried (MgSO4), filtered, and evaporated. The
residue was passed through a silica Sep-Pak cartridge in 10%
ethyl acetate in hexane and, after evaporation, purified by
HPLC (9.4 mm × 25 cm Zorbax-Sil column, 4 mL/min) using
hexane/ethyl acetate (9:1) solvent system. Pure protected
ketone 4c (3.6 mg, 50%) was eluted at RV 25 mL as a colorless
oil: 1H NMR (CDCl3) δ 0.559 (6H, q, J ) 7.9 Hz, 3 × SiCH2),
0.896 (3H, d, J ) 7.6 Hz, 21-H3), 0.939 (9H, t, J ) 7.9 Hz, 3 ×
SiCH2CH3), 1.183 (6H, s, 26-, 27-H3).
1r,25-Dih ydr oxy-18-n or vitam in D3 (2b). [2-(1Z)-[(3S,5R)-
3,5-Bis[(tert-butyldimethylsilyl)oxy]-2-methylenecyclohexylide-
ne]ethyl]diphenylphosphine oxide (5a )10 (13.9 mg, 24 µmol)
was dissolved in anhydrous THF (200 µL) and cooled to -78
°C, and n-BuLi (1.5 M in hexanes, 16 µL, 24 µmol) was added
under argon with stirring. The mixture turned deep orange.
After stirring for 5 min at -78 °C the protected ketone 4c (1.20
mg, 3 µmol) was added in anhydrous THF (200 + 100 µL).
The mixture was stirred under argon at -78 °C for 1 h and at
0 °C for 16 h. Ethyl acetate was added and the organic phase
washed with saturated NH4Cl, 10% NaHCO3, and brine, dried
(MgSO4), and evaporated. The residue was passed through a
silica Sep-Pak cartridge in 10% ethyl acetate in hexane and,
after evaporation, purified by HPLC (9.4 mm × 25 cm Zorbax-
Sil column, 4 mL/min) using 10% ethyl acetate in hexane. Pure
compound 2c (1.16 mg, 49%) was obtained as a colorless oil:
1H NMR (CDCl3) δ 0.055, 0.060, and 0.067 (3H, 3H, 6H, each
s, 4 × SiCH3), 0.556 (6H, q, J ) 7.9 Hz, 3 × SiCH2), 0.85-0.88
(21H, 21-H3, 2 × Si-t-Bu), 0.939 (9H, t, J ) 7.9 Hz, 3 ×
SiCH2CH3), 1.178 (6H, br s, 26-, 27-H3), 2.21 (1H, dd, J ) 12.8,
6.8 Hz, 4â-H), 2.44 (1H, dd, J ) 12.8, 3.6 Hz, 4R-H), 2.86 (1H,
br d, J ) 13.2 Hz, 9â-H), 4.18 (1H, m, 3R-H), 4.38 (1H, m,
1â-H), 4.89 (1H, d, J ) 2.4 Hz, 19Z-H), 5.19 (1H, br s, 19E-H),
6.09 and 6.22 (1H, 1H, each d, J ) 11.6 Hz, 7-, 6-H).
Protected vitamin 2c (1.10 mg) was dissolved in benzene
(40 µL), and the resin (AG 50W-X4, 10 mg; prewashed with
methanol) in methanol (200 µL) was added. The mixture was
stirred at room temperature under argon for 18 h, filtered
through a silica Sep-Pak cartridge, and washed with 2-pro-
panol. The solvent was evaporated, and a crude vitamin 2b
was purified by HPLC (10 mm × 25 cm Zorbax-Sil column, 4
mL/min) using hexane/2-propanol (7:3) solvent system. Ana-
lytically pure compound 2b (449 µg, 76%) was collected at RV
31.5 mL [1R,25-(OH)2D3 (2a ) was eluted at RV 31 mL in the
same system] as a white solid: UV (in EtOH) λmax 263, λmin
227 nm, Amax/Amin ) 1.9; 1H NMR (CDCl3) δ 0.887 (3H, d, J )
6.6 Hz, 21-H3), 1.210 (6H, s, 26-, 27-H3), 2.30 (1H, dd, J ) 13.3,
7.2 Hz, 4â-H), 2.61 (1H, dd, J ) 13.3, 3.5 Hz, 4R-H), 2.88 (1H,
br d, J ) 13.4 Hz, 9â-H), 4.22 (1H, m, 3R-H), 4.43 (1H, m,
1â-H), 5.03 (1H, br s, 19Z-H), 5.33 (1H, br s, 19E-H), 6.09 and
6.38 (1H, 1H, each d, J ) 11.4 Hz, 7-, 6-H); MS m/ z (relative
intensity) 402 (M+, 11), 384 (74), 366 (44), 348 (14), 152 (33),
134 (100); exact mass calcd for C26H42O3 402.3134, found
402.3142.
1r,25-Dih ydr oxy-18,19-din or vitam in D3
(3b). [2-[(3R,5R)-
3,5-Bis[(tert-butyldimethylsilyl)oxy]cyclohexylidene]ethyl]-
diphenylphosphine oxide (5b)5a (12 mg, 21 µmol) was dissolved
in anhydrous THF (200 µL) and cooled to -78 °C, and n-BuLi
(1.4 M in hexanes, 15 µL, 21 µmol) was added under argon
with stirring. The solution turned deep orange. After stirring
for 5 min at -78 °C the protected ketone 4c (3.0 mg, 7.9 µmol)
was added in anhydrous THF (200 + 100 µL). The mixture
was stirred under argon at -78 °C for 1 h and at 0 °C for 16
h. Ethyl acetate was added and the organic phase washed
with saturated NH4
Cl, 10% NaHCO3, and brine, dried
(MgSO4), and evaporated. The residue was passed through a
silica Sep-Pak cartridge in 10% ethyl acetate in hexane and,
after evaporation, purified by HPLC (9.4 mm × 25 cm Zorbax-
Sil column, 4 mL/min) using hexane/ethyl acetate (9:1) solvent
system. Pure protected vitamin 3c (1.7 mg, 29%) was obtained
as a colorless oil: 1H NMR (CDCl3) δ 0.045 and 0.054 (6H,
6H, each s, 4 × SiCH3), 0.557 (6H, q, J ) 7.9 Hz, 3 × SiCH2),
0.86-0.87 (21H, 21-H3, 2 × Si-t-Bu), 0.939 (9H, t, J ) 7.9 Hz,
3 × SiCH2CH3), 1.178 (6H, br s, 26-, 27-H3), 2.84 (1H, br d, J
) 13.5 Hz, 9â-H), 4.07 (2H, br m, 1â-, 3R-H), 5.90 and 6.14
(1H, 1H, each d, J ) 11.1 Hz, 7-, 6-H).
Protected vitamin 3c (850 µg, 1.2 µmol) was dissolved in
benzene (40 µL), and cation exchange resin (AG 50W-X4, 17
mg; prewashed with methanol) in methanol (200 µL) was
added. The mixture was stirred at room temperature under
argon for 18 h, filtered through a silica Sep-Pak cartridge, and
washed with 2-propanol. The solvent was evaporated, and a
crude vitamin 16 was purified by HPLC (10 mm × 25 cm
Zorbax-Sil column, 4 mL/min) using hexane/2-propanol (7:3)
solvent system. Analytically pure compound 3b (366 µg, 81%)
was eluted at RV 37 mL [1R,25-dihydroxy-19-norvitamin D3
(3a ) was eluted at RV 36 mL in the same system] as a white
solid: UV (in EtOH) λmax 243, 251.5, 261 nm; 1H NMR (CDCl3)
δ 0.879 (3H, d, J ) 6.5 Hz, 21-H3), 1.208 (6H, s, 26-, 27-H3),
4.07 and 4.11 (1H, 1H, each m, 1â-, 3R-H), 5.94 and 6.30 (1H,
1H, each d, J ) 11.2 Hz, 7-, 6-H); MS m/ z (relative intensity)
390 (M
+, 39), 372 (62), 354 (23), 259 (42), 231 (84), 175 (25),
133 (53), 121 (64), 69 (100); exact mass calcd for C25H42O3
390.3134, found 390.3139.
Biologica l Stu d ies. Mea su r em en t of In testin a l Ca l-
ciu m Tr a n sp or t a n d Bon e Ca lciu m Mobiliza tion . Wean-
ling male rats from the low-vitamin D colony were purchased
from the Sprague-Dawley Co. (Indianapolis, IN) and fed the
vitamin D-deficient diet,34 containing 0.47% calcium and 0.3%
phosphorus, for 1 week. They were then switched to the
reduced calcium diet (0.02% Ca) for an additional 2 weeks.
These animals have no detectable levels of 25-OH-D3 or
1R,25-(OH)2D3 in their plasma as measured by methods
described previously.35 For this first experiment, the indicated
rats received a single intravenous dose of the indicated
compound in 0.05 mL of ethanol (data not shown). In the other
experiment, the rats were given the indicated doses of com-
pounds in 0.1 mL of (95:5) 1,2-propanediol/ethanol by intra-
peritoneal injection each day for 7 days. In the first experi-
ment, the rats were euthanized at various times after the dose
(data not shown). In the second experiment, they were
sacrificed 24 h after the last dose. The rats were sacrificed
under ether anesthesia by decapitation. Serum and intestines
were collected and used immediately to determine intestinal
calcium transport and serum calcium concentration. Calcium
was determined using the Calcette automatic calcium titrator
(Precision Systems, Inc., Natick, MA) and intestinal calcium
transport by the everted intestinal sac method using the
proximal 10 cm of intestine as described earlier.34 Statistical
analysis was by the Student’s t-test.36 Intestinal calcium
transport is expressed as serosal:mucosal ratio of calcium in