Synthesis of 4α-(2-propenyl)-5,6-secocholestan 3α-ol, a novel B-ring seco analog of the hypocholesterolemic agent 4α-(2-propenyl)-5α-cholestan- 3α-ol

Article abstract of DOI:10.1016/S0039-128X(98)00004-X

4α-(2-Propenyl)-5α-cholestan-3α-ol (LY295427) was previously identified from a CHO cell-based assay to be a potent LDL receptor up- regulator and had demonstrated to be an effective agent in lowering plasma cholesterol levels in hypercholesterolemic hamsters. In order to investigate the effect of flexibility of the 3α-hydroxy-bearing A-ring on the activity, 4α-(2-propenyl)-5,6-secocholestan-3α-ol (11), a B-ring seco analog of LY295427, is thus synthesized from cholest-4-en-3-one. Test results indicate that 11 is not active in the CHO cell-based LDL receptor/luciferase assay at concentrations up to 20 μg/mL. The result underlines the importance of maintaining the A-B-C-D ring rigidity of the 3α-sterols in terms of binding to the putative oxysterol receptor.

Full text of DOI:10.1016/S0039-128X(98)00004-X

Synthesis of 4␣-(2-propenyl)-5,6-
secocholestan-3␣-ol, a novel
B-ring seco analog of the
hypocholesterolemic agent
4␣-(2-propenyl)-5␣-cholestan-3␣-ol
Ho-Shen Lin, Ashraff A. Rampersaud, Michael E. Richett, Lisa S. Beavers,
Don B. McClure, and Robert A. Gadski
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana, USA
4␣-(2-Propenyl)-5␣-cholestan-3␣-ol (LY295427) was previously identified from a CHO cell-based assay to be a
potent LDL receptor up-regulator and had demonstrated to be an effective agent in lowering plasma cholesterol
levels in hypercholesterolemic hamsters. In order to investigate the effect of flexibility of the 3␣-hydroxy-bearing
A-ring on the activity, 4␣-(2-propenyl)-5,6-secocholestan-3␣-ol (11), a B-ring seco analog of LY295427, is thus
synthesized from cholest-4-en-3-one. Test results indicate that 11 is not active in the CHO cell-based LDL
receptor/luciferase assay at concentrations up to 20 ␮g/mL. The result underlines the importance of maintaining
the A-B-C-D ring rigidity of the 3␣-sterols in terms of binding to the putative oxysterol receptor. (Steroids
63:202–207, 1998) © 1998 by Elsevier Science Inc.
Keywords: secosterol; reductive alkylation; reduction, oxidative cleavage
interesting to investigate the effect of increasing the flexi-
bility of the 3␣-hydroxy-bearing A-ring, by disconnecting
the B-ring of LY295427, on the activity in the CHO cell-
based LDL receptor/luciferase assay. 4␣-(2-Propenyl)-5,6-
Introduction
Hypercholesterolemia is a well-known risk factor for the
development of coronary heart disease.1–3 The lowering of
plasma cholesterol levels in patients with coronary heart
disease has been known for some time to be beneficial.4
Clinical results from the Scandinavian Simvastatin Survival
Study5 and the West of Scotland Coronary Prevention
Study6 further reaffirm the cholesterol-lowering strategy for
preventing and retarding the development of atherosclerosis
and coronary heart disease.
secocholestan-3␣-ol (11),
a B-ring seco analog of
LY295427, is thus synthesized from cholest-4-en-3-one
(Schemes 1–3).
Experimental
Materials
Recently we reported that a new series of 3␣-sterols,
Reagents were used as supplied from Aldrich unless otherwise
noted. Reactions were run under anhydrous nitrogen atmosphere
unless otherwise noted. Powdered magnesium sulfate was used for
drying the organic extract. Silica gel (E. Merck, 230–400 mesh
ASTM) was used for flash column chromatography. Ozone was
generated from an ozone generator manufactured by Griffin Tech-
nics Corporation. Reactions were monitored, and the homogeneity
of the products was checked by TLC on silica gel 60 F254 plates
(E. Merck). 1H NMR spectra were recorded on a General Electric
QE-300 instrument. Chemical shifts are expressed as parts per
million downfield from internal tetramethylsilane. Infrared (IR)
spectra were determined on a Nicolet MX-1 FT-IR. Low
resolution-field desorption mass spectra (FDMS) were recorded on
an VG Analytical ZAB-3F spectrometer. High resolution-fast atom
bombardment mass spectra (HRMS) were recorded on an VG
Analytical ZAB-2-SE spectrometer. Elemental analyses were de-
termined on a (Perkin-Elmer Model 240C) elemental analyzer and
exemplified
by
4␣-(2-propenyl)-5␣-cholestan-3␣-ol
(LY295427, Figure 1), could derepress the transcription of
the LDL receptor promoter in the presence of 25-
hydroxycholesterol in a CHO cell-based LDL receptor/lu-
ciferase assay.7 More importantly, these compounds have
also shown efficacy in lowering the total serum cholesterol
levels in hypercholesterolemic hamsters.7 In our structure-
activity relationships (SAR) studies, we found that 3␣-
hydroxy group in the A-ring is essential to the activity,
presumably through binding to the putative cytoplasmic
oxysterol receptor. Therefore, as shown in Figure 1, it was
Address reprint requests to H-S Lin, Eli Lilly and Company, Lilly Research
Laboratories, Lilly Corporate Center, Indianapolis, IN 46285.
Received 24 October 1997; accepted 8 January 1998.
Steroids 63:202–207, 1998
© 1998 by Elsevier Science Inc. All rights reserved.
655 Avenue of the Americas, New York, NY 10010
0039-128X/98/$19.00
PII S0039-128X(98)00004-X

Synthesis of 4␣-(2-propenyl)-5,6-secocholestan-3␣-ol: Lin et al.
were dried, filtered and concentrated in vacuo. After chromatog-
raphy on silica (gradient 0–15% ethyl acetate/hexane), 4 (2.30 g,
76%) and 4؅ (400 mg, 13%) were obtained as white solids. 4: m.p.
101.0–102.5°C; IR (KBr) 3521 (br), 2946 cmϪ1 1H NMR
;
(CDCl3) ␦ 0.67 (s, 3H, C-18 CH3), 0.86 (d, J ϭ 6.6 Hz, 6H, C-26
and -27 CH3), 0.90 (d, J ϭ 6.5 Hz, 3H, C-21 CH3), 1.09 (s, 3H,
C-19 CH3), 0.90–1.65 (m, 24H), 1.72–1.90 (m, 3H, two C-2 and
one C-7 protons), 1.99 (br d, J ϭ 14.0 Hz, 1H, C-4 proton), 2.11
(d, J ϭ 14.0 Hz, 1H, C-4 proton), 2.80 (br s, 1H, OH), 3.52 (s, 1H,
C-6 proton), 3.90–4.00 (m, 4H, -OCH2CH2O-); FDMS m/e 462
(Mϩ). Analysis calculated for C29H50O4: C, 75.28; H, 10.89.
Found: C, 75.40; H, 10.61. 4؅: m.p. 174.0–176.0°C; IR (KBr) 3495
Figure 1
1
(br), 2936 cmϪ1; H NMR (CDCl3 ϩ D2O) ␦ 0.66 (s, 3H, C-18
CH3), 0.88 (d, J ϭ 6.6 Hz, 6H, C-26 and -27 CH3), 0.91 (d, J ϭ
6.6 Hz, 3H, C-21 CH3), 0.95 (s, 3H, C-19 CH3), 0.90–1.90 (m,
26H), 1.90–2.10 (m, 2H, C-4 protons), 3.46 (dd, J ϭ 11.3, 5.1 Hz,
1H, C-6 proton), 3.90–4.05 (m, 4H, -OCH2CH2O-); FDMS m/e
462 (Mϩ). Analysis calculated for C29H50O4: C, 75.28; H, 10.89.
Found: C, 75.31; H, 10.64.
are within 0.4% of theory unless otherwise noted. Melting points
were determined on a Thomas-Hoover melting point apparatus and
are uncorrected.
Syntheses
3,3-Ethylenedioxycholest-5-ene (1). Compound 1 was synthe-
sized according to the reported procedure:8,9 m.p. 128.0–131.0°C;
IR (KBr) 2942, 1458 cmϪ1; 1H NMR (CDCl3) ␦ 0.69 (s, 3H, C-18
CH3), 0.87 (d, J ϭ 6.6 Hz, 6H, C-26 and -27 CH3), 0.92 (d, J ϭ
6.5 Hz, 3H, C-21 CH3), 1.04 (s, 3H, C-19 CH3), 0.95–2.08 (m,
26H), 2.12 (dd, J ϭ 14.1, 2.7 Hz, 1H, C-4 proton), 2.58 (br d, J ϭ
14.1 Hz, 1H, C-4 proton), 3.92–3.98 (m, 4H, -OCH2CH2O-),
5.34–5.38 (m, 1H, –CH¢); FDMS m/e 429 (Mϩ ϩ 1).
Synthesis of 3 from oxidative cleavage of diols 4 and 4؅. Solid
sodium periodate (27.8 g, 130 mmol) was added to a stirred
suspension of diols 4 and 4؅ (7.50 g, 16.2 mmol) in 45% aqueous
THF (180 mL) at ambient temperature, the resultant mixture was
stirred vigorously for 3 h. The suspension was diluted with EtOAc
(300 mL), washed with brine (100 mL), dried and filtered. After
concentration and subsequent flash chromatography on silica (gra-
dient 20–30% ethyl acetate/hexane), 3 (6.72 g, 90%) was obtained
as an oil.
5␤,6␤-Epoxy-3,3-ethylenedioxy-5␤-cholestane (2) and 3,3-
ethylenedioxy-5-oxo-5,6-secocholestan-6-al (3). A small stream
of ozone was bubbled through a stirred solution of 1 (515 mg, 1.20
mmol) in CH2Cl2 (15 mL)/MeOH (5 mL) at Ϫ78°C until the
solution turned blue and the color remained blue for 5 min. While
at Ϫ78°C, the excessive ozone was evaporated off by bubbling
through the solution a small stream of nitrogen. Dimethyl sulfide
(4 mL) was added to the solution, the cold bath was removed, and
the solution was allowed to stir overnight. After concentration and
subsequent chromatographic purification on silica (gradient
5–25% ethyl acetate/hexane), 5␤,6␤-epoxide 2 (296 mg, 56%) was
obtained as a solid and seco aldehyde 3 (140 mg, 25%) was
obtained as an oil. 2: m.p. 126.5–127.5°C; IR (KBr) 2951, 1467
3,3-Ethylenedioxy-5-oxo-5,6-secocholestan-6-ol (5). Sodium
borohydride (280 mg, 7.39 mmol) was added to a stirred solution
of 3 (3.40 g, 7.39 mmol) in THF (40 mL)/MeOH (10 mL) at
Ϫ10°C, the resultant mixture was stirred for 1.5 h. After being
quenched with acetic acid (0.65 mL), the mixture was allowed to
warm to ambient temperature. It was then diluted with EtOAc (100
mL), washed with brine (50 mL), dried, filtered and concentrated.
The oily residue was chromatographed on silica (gradient 20–50%
ethyl acetate/toluene) to give 5 (3.20 g, 94%) as an oil. IR (neat)
3467 (br), 1705 cmϪ1 1H NMR (CDCl3) ␦ 0.66 (s, 3H, C-18
;
CH3), 0.87 (d, J ϭ 6.6 Hz, 6H, C-26 and -27 CH3), 0.91 (d, J ϭ
6.5 Hz, 3H, C-21 CH3), 1.08 (s, 3H, C-19 CH3), 0.90–2.10 (m,
27H), 2.59 (br d, J ϭ 12.8 Hz, 1H, C-4 proton), 3.20 (d, J ϭ 12.8
Hz, 1H, C-4 proton), 3.44–3.52 (m, 2H, -CH2O-), 3.90–4.05 (m,
4H, -OCH2CH2O-); FDMS m/e 463 (Mϩ ϩ 1). Analysis calcu-
lated for C29H50O4 ⅐ 0.3C7H8: C, 76.18; H, 10.77. Found: C,
76.30; H, 11.02.
1
cmϪ1; H NMR (CDCl3) ␦ 0.65 (s, 3H, C-18 CH3), 0.87 (d, J ϭ
6.6 Hz, 6H, C-26 and -27 CH3), 0.90 (d, J ϭ 6.6 Hz, 3H, C-21
CH3), 1.00 (s, 3H, C-19 CH3), 0.75–1.87 (m, 25H), 1.97 (br d, J ϭ
12.5 Hz, 1H, C-2 proton), 2.08 (br d, J ϭ 14.1 Hz, 1H, C-4
proton), 2.34 (d, J ϭ 14.1 Hz, 1H, C-4 proton), 3.06 (d, J ϭ 1.6
Hz, 1H, C-6 proton), 3.89–3.97 (m, 4H, -OCH2CH2O-); FDMS
m/e 444 (Mϩ). Analysis calculated for C29H48O3: C, 78.33; H,
10.88. Found: C, 78.59; H, 11.00. 3: IR (CHCl3) 1719, 1700 cmϪ1
;
3,3-Ethylenedioxy-5,6-secocholest-6-en-5-one (6). A solution of
5 (2.17 g, 4.70 mmol) in anhydrous THF (45 mL) was treated
sequentially with 2-nitrophenyl selenocyanate (2.13 g, 9.40 mmol)
and tributyl phosphine (2.34 mL, 9.40 mmol), the resultant mixture
was heated in an oil bath at 50°C for 2 h. At ambient temperature
the mixture was diluted with EtOAc (160 mL) before it was
filtered. After concentration and subsequent flash chromatography
on silica (gradient 0–10% ethyl acetate/toluene), a selenated prod-
uct (3.00 g, 98%) was obtained as a yellow foam. FDMS m/e 644
(Mϩ-1, 78Se), 646 (Mϩ-1, 80Se). Analysis calculated for
C35H53NO5Se: C, 65.00; H, 8.26; N, 2.17. Found: C, 65.19; H,
8.17; N, 2.29.
A stirred suspension of the selenated product (2.90 g, 4.49
mmol) and powdered calcium carbonate (2.25 g, 22.4 mmol) in
anhydrous CH2Cl2 (40 mL) at 0°C was treated with a solution of
m-chloroperoxybenzoic acid (852 mg, 4.20 mmol) in anhydrous
CH2Cl2 (5.0 mL), then the resultant mixture was allowed to stir at
ambient temperature for 45 min. The mixture was diluted with
1H NMR (CDCl3) ␦ 0.67 (s, 3H, C-18 CH3), 0.84 (d, J ϭ 6.6 Hz,
6H, C-26 and -27 CH3), 0.89 (d, J ϭ 6.5 Hz, 3H, C-21 CH3), 1.01
(s, 3H, C-19 CH3), 0.90–2.40 (m, 26H), 2.45 (br d, J ϭ 12.9 Hz,
1H, C-4 proton), 3.11 (d, J ϭ 12.9 Hz, 1H, C-4 proton), 3.85–4.00
(m, 4H, -OCH2CH2O-), 9.60 (s, 1H, –CHO); FDMS m/e 460
(Mϩ). Analysis calculated for C29H48O4 ⅐ 0.5EtOAc: C, 73.77; H,
10.38. Found: C, 73.78; H, 10.10.
3,3-Ethylenedioxy-5␤,6␤-dihydroxy-5␤-cholestane (4) and its
5␣,6␣-dihydroxy isomer (4؅). Solid osmium tetroxide (2.00 g,
7.87 mmol) was added to a stirred solution of 1 (2.80 g, 6.54
mmol) in anhydrous pyridine (40 mL), the resultant dark solution
was stirred at ambient temperature for 2 days. Water (50 mL) and
pyridine (33 mL) were sequentially added to the solution, which
was then treated with solid sodium hydrogensulfite (3.0 g). The
mixture was stirred vigorously for 2.5 h before it was extracted
with ethyl acetate (100 mL ϫ 3). The combined organic layers
Steroids, 1998, vol. 63, April 203

Papers
EtOAc (80 mL) before it was filtered and concentrated. The oily
residue was chromatographed on silica (gradient 15–20% ethyl
acetate/hexane) to give 6 (1.43 g, 71%) as an oil. IR (CHCl3) 1706
on silica (gradient 2–5% ethyl acetate/hexane), 9 (545 mg, 49%)
was obtained as an oil and 10 (328 mg, 30%) was obtained as a
1
white solid. 9: IR (CHCl3) 1707 cmϪ1; H NMR (CDCl3) ␦ 0.69
1
cmϪ1; H NMR (CDCl3) ␦ 0.68 (s, 3H, C-18 CH3), 0.86 (d, J ϭ
(s, 3H, C-18 CH3), 0.80 (t, J ϭ 7.4 Hz, 3H, -CH2CH3), 0.86 (d,
J ϭ 6.6 Hz, 6H, C-26 and -27 CH3), 0.90 (d, J ϭ 6.5 Hz, 3H, C-21
CH3), 0.95 (s, 3H, C-19 CH3), 0.90–2.40 (m, 29H), 2.46–2.70 (m,
2H, C-2 protons), 4.95–5.05 (m, 2H, ¢CH2), 5.72–5.88 (m, 1H,
-CH¢). HRMS calculated for C30H52O (Mϩ): 428.4018. Found:
6.6 Hz, 6H, C-26 and -27 CH3), 0.90 (d, J ϭ 6.5 Hz, 3H, C-21
CH3), 1.06 (s, 3H, C-19 CH3), 0.90–2.10 (m, 24H), 2.47 (br d, J ϭ
12.9 Hz, 1H, C-4 proton), 3.05 (d, J ϭ 12.9 Hz, 1H, C-4 proton),
3.90–4.00 (m, 4H, -OCH2CH2O-), 4.80–4.96 (m, 2H, ¢CH2), 5.23
(dt, J ϭ 17.2, 9.7 Hz, 1H, -CH¢); FDMS m/e 444 (Mϩ). Analysis
calculated for C29H48O3: C, 78.33; H, 10.88. Found: C, 78.39; H,
11.02.
428.4025. 10: m.p.: 65.0–66.0°C; IR (CHCl3) 1705 cmϪ1 1H
;
NMR (CDCl3) ␦ 0.66 (s, 3H, C-18 CH3), 0.77 (t, J ϭ 6.8 Hz, 3H,
-CH2CH3), 0.87 (d, J ϭ 6.8 Hz, 6H, C-26 and -27 CH3), 0.89 (d,
J ϭ 6.8 Hz, 3H, C-21 CH3), 1.19 (s, 3H, C-19 CH3), 0.90–1.98 (m,
26H), 2.17–2.32 (m, 2H, allylic protons), 2.42–2.60 (m, 3H, two
C-2 and one C-4 protons), 4.97–5.03 (m, 2H, ¢CH2), 5.67–5.81
(m, 1H, -CH¢); FDMS m/e 428 (Mϩ). Analysis calculated for
C30H52O: C, 84.04; H, 12.23. Found: C, 83.80; H, 12.08.
3,3-Ethylenedioxy-5,6-secocholestan-5-one (7). Platinum oxide
(140 mg) was added to a stirred solution of 6 (1.82 g, 4.10 mmol)
in EtOAc (18 mL) at ambient temperature, the resultant suspension
was stirred under a hydrogen atmosphere for 4 h. After filtration
and subsequent concentration, the oily residue was chromato-
graphed on silica (gradient 0–5% ethyl acetate/toluene) to give 7
(1.72 g, 95%) as an oil. IR (CHCl3) 1700 cmϪ1; 1H NMR (CDCl3)
␦ 0.63 (t, J ϭ 7.0 Hz, 3H, -CH2CH3), 0.66 (s, 3H, C-18 CH3), 0.86
(d, J ϭ 6.6 Hz, 6H, C-26 and -27 CH3), 0.90 (d, J ϭ 6.5 Hz, 3H,
C-21 CH3), 1.02 (s, 3H, C-19 CH3), 0.90–2.08 (m, 26H), 2.53 (br
d, J ϭ 12.7 Hz, 1H, C-4 proton), 3.24 (d, J ϭ 12.7 Hz, 1H, C-4
proton), 3.90–4.05 (m, 4H, -OCH2CH2O-). HRMS calculated for
C29H50O3 (Mϩ): 446.3760. Found: 446.3772.
Synthesis of a mixture of 4␣-(2-propenyl)-5,6-secocholestan-
3␣-ol (11) and its 3␤ epimer (12) from DIBALH reduction of
9. Diisobutylaluminum hydride (0.263 mL, 1M in toluene) was
added to a stirred solution of 9 (75.0 mg, 0.175 mmol) in anhy-
drous CH2Cl2 (2 mL) at Ϫ10°C. The cold bath was removed and
the resultant solution was allowed to stir for 30 min. After being
cooled to 0°C the solution was treated with 1 N HCl (2 mL) and
the resultant two-layered mixture was stirred vigorously at ambient
temperature for 45 min. The mixture was diluted with EtOAc (30
mL), the organic layer was separated, washed sequentially with
saturated aqueous NaHCO3 (5 mL) and brine (5 mL), dried,
filtered and concentrated. The residue was chromatographed on
silica (gradient 0–10% ethyl acetate/hexane) to afford an insepa-
rable mixture of 11 and 12 (60.0 mg, 80%) as an oil in a 1:2 ratio.
5,6-Secocholest-4-en-3-one (8). A solution of 7 (1.64 g, 3.69
mmol) in anhydrous THF (10 mL) was added to a stirred suspen-
sion of lithium aluminum hydride (140 mg, 3.69 mmol) in anhy-
drous THF (5 mL) at Ϫ10°C, the resultant mixture was allowed to
warm to ambient temperature where it was stirred for 1 h. The
mixture was cooled to Ϫ10°C and cautiously quenched with 2.5 N
HCl (9.0 mL), the resultant two-layered solution was stirred vig-
orously at ambient temperature for 1 h. The mixture was extracted
with EtOAc (150 mL), the organic layer was sequentially washed
with brine (25 mL) and saturated aqueous NaHCO3 (25 mL), dried
and filtered. After concentration, the residue was chromatographed
on silica (gradient 5–20% ethyl acetate/hexane) to give enone 8
(1.36 g, 95%) as an oil. IR (CHCl3) 1670 cmϪ1; 1H NMR (CDCl3)
␦ 0.66 (s, 3H, C-18 CH3), 0.76 (t, J ϭ 6.8 Hz, 3H, -CH2CH3), 0.86
(d, J ϭ 6.8 Hz, 6H, C-26 and -27 CH3), 0.89 (d, J ϭ 6.8 Hz, 3H,
C-21 CH3), 1.20 (s, 3H, C-19 CH3), 0.92–1.90 (m, 22H), 1.94 (dt,
J ϭ 12.7, 3.1 Hz, 1H, C-1 proton), 2.08–2.20 (m, 1H, C-1 proton),
2.40–2.46 (m, 2H, C-2 protons), 5.78 (d, J ϭ 10.3 Hz, 1H, C-4
vinylic proton), 6.91 (d, J ϭ 10.3 Hz, 1H, C-5 vinylic proton).
HRMS calculated for C27H46O (Mϩ): 386.3549. Found: 386.3563.
1
IR (CHCl3) 3620 (br) cmϪ1; H NMR (CDCl3) ␦ 3.29 (td, J ϭ
10.4, 4.8 Hz, 1H, -CH(OH)- of 12), 3.91 (s, 1H, -CH(OH)- of 11);
FDMS m/e 430 (Mϩ). Analysis calculated for C30H54O: C, 83.65;
H, 12.64. Found: C, 83.50; H, 12.76.
Synthesis of 11 from K-selectride reduction of 9. K-Selectride
(0.120 mL, 1M in THF) was added to a stirred solution of 9 (40
mg, 0.093 mmol) in anhydrous THF (1.5 mL) at Ϫ10°C. Upon
completion of addition, the resultant yellowish solution was al-
lowed to warm to ambient temperature where it was stirred for 2 h.
The reaction mixture was then cooled to Ϫ10°C and sequentially
treated with MeOH (0.2 mL), 5 N NaOH (0.11 mL), and 30%
H2O2 (0.055 mL). After stirring for 30 min, the cold bath was
removed and the mixture was stirred vigorously for 2 h. Half-
saturated aqueous NaCl (10 mL) and EtOAc (40 mL) were added
to the mixture. The organic layer was separated, washed with
half-saturated aqueous NaCl (10 mL ϫ 2), dried, filtered and
concentrated. After flash chromatography on silica (gradient
5–10% ethyl acetate/hexane), 11 (34 mg, 85%) was obtained as an
oil. IR (CHCl3) 3619 (br) cmϪ1; 1H NMR (CDCl3) ␦ 0.66 (s, 3H,
C-18 CH3), 0.74 (t, J ϭ 7.2 Hz, 3H, -CH2CH3), 0.86 (d, J ϭ 6.7
Hz, 6H, C-26 and -27 CH3), 0.88 (s, 3H, C-19 CH3), 0.89 (d, J ϭ
6.6 Hz, 3H, C-21 CH3), 0.90–2.18 (m, 32H), 3.91 (br s, 1H, -CH
(OH)-), 4.98–5.11 (m, 2H, ¢CH2), 5.78–5.92 (m, 1H, -CH¢);
FDMS m/e 430 (Mϩ).
4␣-(2-Propenyl)-5,6-secocholestan-3-one (9) and its 4␤ epimer
(10). Lithium chips (54.0 mg, 7.77 mmol) and a glass-coated stir
bar were placed in a flame-dried, three-necked, round-bottomed
flask fitted with a dry ice condenser under argon. Liquid ammonia
(18 mL) was collected in the flask at Ϫ78°C to form a deep blue
solution, then followed by the addition of dry THF (25 mL). A
solution of 8 (1.00 g, 2.59 mmol) and t-BuOH (0.195 mL, 2.07
mmol) in anhydrous THF (15 mL) was added dropwise to the deep
blue solution. Upon completion of the addition, the resultant blue
solution was stirred for 15 min before it was treated with 1,3-
pentadiene (0.5 mL) to quench the excess lithium. After 15 min,
allyl iodide (0.710 mL, 7.77 mmol) was added to the white
suspension and the resultant mixture was stirred at Ϫ78°C for 3h.
Saturated aqueous NH4Cl (10 mL) was cautiously added to the
white suspension. The cold bath was removed and the mixture,
with the evaporation of ammonia, was allowed to warm to ambient
temperature. After extraction with EtOAc (70 mL ϫ 2), the com-
bined organic layers were washed with saturated aqueous NaCl (30
mL), dried, filtered and concentrated. After flash chromatography
4␤-(2-Propenyl)-5,6-secocholestan-3␣-ol (13) and its 3␤
epimer (14). The procedure used for the synthesis of the mixture
of 11 and 12 was repeated with 10 (100 mg, 0.233 mmol) and
diisobutyl aluminum hydride (0.350 mL, 1 M in toluene) to pro-
vide 13 (49 mg, 49%) as an oil and 14 (51 mg, 51%) as a white
solid respectively after chromatography on silica (gradient 2–7%
1
ethyl acetate/hexane). 13. IR (CHCl3) 3619 (br) cmϪ1; H NMR
(CDCl3) ␦ 0.65 (s, 3H, C-18 CH3), 0.79 (t, J ϭ 7.3 Hz, 3H,
-CH2CH3), 0.86 (d, J ϭ 6.7 Hz, 6H, C-26 and -27 CH3), 0.89 (d,
204 Steroids, 1998, vol. 63, April

Synthesis of 4␣-(2-propenyl)-5,6-secocholestan-3␣-ol: Lin et al.
spectrum, in which the signal due to the equatorial proton at
C-6 appears at ␦ 3.06 as a doublet (J ϭ 1.6 Hz). Similar
observation of the 5␤,6␤-epoxide formation obtained from
ozonation of other ⌬5-steroids was also reported in the
literature.9 Generally, the methyl at C-19 directs chemistry
to occur on the ␣-face; but in the case of 1, reaction on the
␣-face is sterically hindered due to the axial-down blocking
by the ethylenedioxy protecting group.
Because of the low yield of 3, we turned to a two-step
process. As shown in Scheme 2, olefin 1 was treated with
osmium tetroxide in the presence of pyridine to give the
5␤,6␤-dihydroxy ketal 4 and its 5␣,6␣-dihydroxy isomer 4؅
1
in 76% and 13% yields respectively. In the H NMR spec-
trum the signal due to the equatorial proton at C-6 in 4
appears at ␦ 3.52 as a singlet, whereas the axial proton at
C-6 in 4؅ appears at ␦ 3.46 as a doublet of doublets (J ϭ
11.3, 5.1 Hz). A mixture of 4 and 4؅ was then oxidatively
cleaved with sodium periodate to afford the seco aldehyde 3
in a 90% yield. Compound 3 is not stable and tends to
undergo intramolecular Aldol condensation; therefore, it
was immediately subject to selective reduction of the alde-
hyde group with sodium borohydride to provide the keto
alcohol 5 in a 94% yield. By following Robins’ method,13,14
our attempt at deoxygenation of the thiocarbonate derivative
of 5 in the presence of tributyltin hydride resulted in reclo-
Scheme 1 Reagents: (a) ethylene glycol, PTSA, toluene, reflux;
(b) O3, MeOH/CH2Cl2, Ϫ78°C; dimethyl sulfide.
J ϭ 6.8 Hz, 3H, C-21 CH3), 0.92 (s, 3H, C-19 CH3), 0.90–1.99 (m,
31H), 2.07–2.17 (m, 1H, allylic proton), 3.82 (s, 1H, -CH(OH)-),
4.98–5.08 (m, 2H, ¢CH2), 5.72–5.88 (m, 1H, -CH¢); FDMS m/e
430 (Mϩ). Analysis calculated for C30H54O: C, 83.65; H, 12.64.
Found: C, 83.63; H, 12.73. 14: m.p. 83.0–84.0°C; IR (KBr) 3464
1
(br) cmϪ1; H NMR (CDCl3) ␦ 0.64 (s, 3H, C-18 CH3), 0.77 (t,
J ϭ 7.3 Hz, 3H, -CH2CH3), 0.86 (d, J ϭ 6.8 Hz, 6H, C-26 and -27
CH3), 0.89 (d, J ϭ 6.8 Hz, 3H, C-21 CH3), 0.93 (s, 3H, C-19 CH3),
0.90–1.96 (m, 31H), 2.38–2.48 (m, 1H, allylic proton), 3.19 (dt,
J ϭ 10.4, 4.6 Hz, 1H, -CH(OH)-), 4.99–5.07 (m, 2H, ¢CH2),
5.74–5.87 (m, 1H, -CH¢). HRMS calculated for C30H55O (Mϩ ϩ
1): 431.4253. Found: 431.4265.
In vitro LDL receptor/luciferase assay
Compounds were evaluated for their upregulating or derepressing
capability against 25-hydroxycholesterol in the LDL receptor/
luciferase assay, in which stably transfected, stationary CHO cells
were exposed to 25-hydroxycholesterol (0.5 ␮g/mL) and com-
pound (0–20 ␮g/mL) in serum free medium for 24 h. Cells were
then washed and lysed and homogenates were evaluated for
luciferase-catalyzed light production. The procedure is described
in detail in our previous publication.7
Results and discussion
It is known that ketalization of the 3-oxo functionality of
cholest-4-en-3-one is accompanied with concurrent migra-
tion of the double bond to the B-ring, where it could be
oxidatively cleavaged to deliver the seco analog in a low
yield.8,9 Therefore, as shown in Scheme 1, the olefin in 1
was subjected to ozonolysis conditions in 30% methanol in
dichloromethane followed by reduction with dimethyl sul-
fide. The isolated products were the 5␤,6␤-epoxide 2 and
the desired seco aldehyde 3 in 56% and 25% yields respec-
tively, both compounds were separable by flash chromatog-
raphy (5–15% ethyl acetate/hexane). ␤-Epoxide 2 is a
Scheme 2 Reagents: (a) OsO4, pyridine; (b) NalO4, THF/H2O; (c)
NaBH4, THF/CH3OH, Ϫ10°C; (d) 2-nitrophenyl selenocyanate,
Bu3P, THF, 50°C; MCPBA, CaCO3, CH2Cl2, O°C; (e) PtO2, H2,
EtOAc; (f) LiAIH4, THF, Ϫ10°C; 2.5 N HCl, Ϫ10°C; (g) Li, NH3,
t-BuOH, THF, Ϫ78°C; allyl iodide, Ϫ78°C.
1
known compound and its H NMR spectrum is identical to
the one reported in the literature.10–12 The ␤ orientation of
1
the 5,6-epoxide moiety in 2 is supported by its H NMR
Steroids, 1998, vol. 63, April 205

Papers
sure of the B-ring, presumably, via a radical addition to the
carbonyl group. Attention was then directed towards
Grieco’s protocol.15 Therefore, 5 was treated sequentially
with o-nitrophenylselenocyanate and tri-n-butylphosphine
to give a yellow selenide, which was readily converted to
the olefin 6 in an overall 70% yield after treatment with
m-chloroperoxybenzoic acid. Catalytic hydrogenation of 6
delivered 7 in a 95% yield.
At this juncture, we were ready to install the enone
functionality in the A-ring preparing for the reductive alky-
lation with allyl iodide. Lithium aluminum hydride reduc-
tion of the keto group of 7 and subsequent acid-catalyzed
deprotection of the ethylenedioxy ketal, accompanied by the
concurrent dehydration, furnished the enone 8. Following
Stork’s procedure,16–18 8 was subject to the reductive alky-
lation with allyl iodide to yield a separable mixture of
allylated ketones 9 and 10 in 49% and 30% yields respec-
tively. It is noteworthy to mention that only one spot,
corresponding to 9, was shown on the TLC plate prior to
workup at Ϫ78°C. Presumably, owing to the steric hin-
drance imposed by the pseudo-axial methyl group at C-19
(steroidal numbering), allylation occurred on the less hin-
dered ␣-face of the lithium enolate to deliver the ␣-allyl
ketone 9. This result is consistent with our previous obser-
vation in the reductive alkylation of cholest-4-en-3-one with
allyl iodide.7 The formation of the ␤-allyl ketone 10 prob-
ably was derived from the epimerization of 9 caused by the
ammonia at higher temperature during the workup.
K-selectride, due to its bulkiness, almost exclusively deliv-
ers its hydride via the equatorial course to the carbonyl
group in cyclohexanone to provide the cyclohexanol with
the hydroxy group in the axial position.7,19 As anticipated,
the desired 11 was obtained as the sole product after the
reduction of 9 with K-selectride in an 85% yield; its H
NMR spectrum shows no signal around ␦ 3.29 correspond-
ing to the axial proton at C-3 of 12.
1
During our previous synthetic efforts,7 we exclusively
obtained the 4␣-allyl-5␣-cholestan-3-one after the reductive
allylation of cholest-4-en-3-one, no 4␤-allyl epimer was
isolated due to the steric hindrance imposed by the axial
methyl group at C-19 and the conformational rigidity of the
A-B-C-D ring. Since in the B-ring seco analog, in addition
to the desired ␣-allyl ketone 9, we also isolated the ␤-allyl
ketone 10; we therefore proceeded to reduce 10 with the
diisobutyl aluminum hydride to furnish a separable mixture
of alcohols 13 and 14 in 49% and 51% yields respectively.
1
In the H NMR spectrum the signal due to the equatorial
proton at C-3 in 13 appears at ␦ 3.82 as a singlet, whereas
the axial proton at C-3 in 14 appears at ␦ 3.19 as a doublet
of triplets (J ϭ 10.4, 4.6 Hz).
Test results indicated that none of 11, mixture of 11 and
12, 13, and 14 turned out to be active in the CHO cell-based
LDL receptor/luciferase assay at concentrations up to 20
␮g/mL.7 Apparently, the disconnection of the B-ring in
LY295427 increases the flexibility of the 3␣-hydroxy-
bearing A-ring, but results in a loss of activity. The result
indicates the importance of maintaining the A-B-C-D ring
rigidity of the 3␣-sterols in terms of binding to the putative
oxysterol receptor.
As shown in Scheme 3, our first attempt at reducing the
␣-allyl ketone 9 with diisobutylaluminum hydride produced
an inseparable mixture of alcohols 11 and 12 in an 80%
1
yield with a 1:2 ratio. The H NMR spectrum shows two
distinct signals that are characteristic of the epimeric pro-
tons at C-3; the equatorial proton in 11 appears at ␦ 3.91 as
a singlet, whereas the axial proton in 12 appears at ␦ 3.29 as
a triplet of doublets (J ϭ 10.4, 4.8 Hz). It is known that
Acknowledgments
Physical chemistry data were furnished by the staff of the
Physical Chemistry Department of Lilly Research Labora-
tories.
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