Synthesis and in vitro biological activity of 4α(2-propenyl)-5α- cholest-24-en-3α-ol: A 24,25-dehydro analog of the hypocholesterolemic agent 4α-(2-propenyl)-5α-cholestan-3α-ol

Article abstract of DOI:10.1016/S0039-128X(98)00083-X

4α-(2-Propenyl)-5α-cholestan-3α-ol (LY295427) was previously identified from a Chinese hamster ovary (CHO) cell-based low density lipoprotein receptor/luciferase (LDLR/Luc) assay to be a potent transcriptional activator of the LDL receptor promoter in the presence of 25- hydroxycholesterol. To investigate the effect of the 24,25-unsaturation in the D-ring side chain (desmosterol D-ring side chain) on antagonizing the repressing effect of 25-hydroxycholesterol, 4α-(2-propenyl)-5α-cholest-24- en-3α-ol (17), a 24,25-dehydro analog of LY295427, was thus synthesized from lithocholic acid via the formation of 3α-[[(1,1- dimethylethyl)dimethylsilyl]oxy]-4α-(2-propenyl)5α-cholan-24-al (15). Test results showed that 17 had an EC(30) value of 2.6 (max)M, comparable to 2.9 (max)M of LY295427, in the CHO cell-based LDLR/Luc assay in the presence of 25-hydroxycholesterol. Apparently, the built-in 24,25-unsaturation in the D- ring side chain of 17 had added little effect to antagonizing the repressing effect of 25-hydroxycholesterol. In the [1-¹⁴C-acetate]cholesterol biosynthesis inhibition assay, 17 at 10 μg/ml (23 μM) has been shown to inhibit the cholesterol biosynthesis in CHO cells by 38% relative to the vehicle control; whereas LY295427 showed no inhibition in the same assay in our previous studies. In contrast to LY295427, the built-in 24,25- unsaturation in the D-ring side chain of 17 has conferred an inhibitory effect on cholesterol biosynthesis in CHO cells. In summary, the observed LDL receptor promoter activity of 17 is related to its ability to prevent 25- hydroxycholesterol from exerting the repressing effect via an undetermined mechanism and, in part, to inhibit the cholesterol biosynthesis.

Full text of DOI:10.1016/S0039-128X(98)00083-X

Steroids 64 (1999) 217–227
Synthesis and in vitro biological activity of 4␣-(2-propenyl)-5␣-cholest-
24-en-3␣-ol: A 24,25-dehydro analog of the hypocholesterolemic agent
4␣-(2-propenyl)-5␣-cholestan-3␣-ol
Ho-Shen Lin*, Ashraff A. Rampersaud, Michael E. Richett, Richard W. Harper,
Lisa S. Beavers, Don B. McClure, Anthony J. Gardner, Patrick I. Eacho,
Patricia S. Foxworthy, Robert A. Gadski
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA
Received 11 February 1998; accepted 24 September 1998
Abstract
4␣-(2-Propenyl)-5␣-cholestan-3␣-ol (LY295427) was previously identified from a Chinese hamster ovary (CHO) cell-based low density
lipoprotein receptor/luciferase (LDLR/Luc) assay to be a potent transcriptional activator of the LDL receptor promoter in the presence of
25-hydroxycholesterol. To investigate the effect of the 24,25-unsaturation in the D-ring side chain (desmosterol D-ring side chain) on
antagonizing the repressing effect of 25-hydroxycholesterol, 4␣-(2-propenyl)-5␣-cholest-24-en-3␣-ol (17), a 24,25-dehydro analog of
LY295427, was thus synthesized from lithocholic acid via the formation of 3␣-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4␣-(2-propenyl)-
5␣-cholan-24-al (15). Test results showed that 17 had an EC30 value of 2.6 ␮M, comparable to 2.9 ␮M of LY295427, in the CHO cell-based
LDLR/Luc assay in the presence of 25-hydroxycholesterol. Apparently, the built-in 24,25-unsaturation in the D-ring side chain of 17 had
added little effect to antagonizing the repressing effect of 25-hydroxycholesterol. In the [1-14C-acetate]cholesterol biosynthesis inhibition
assay, 17 at 10 ␮g/ml (23 ␮M) has been shown to inhibit the cholesterol biosynthesis in CHO cells by 38% relative to the vehicle control;
whereas LY295427 showed no inhibition in the same assay in our previous studies. In contrast to LY295427, the built-in 24,25-unsaturation
in the D-ring side chain of 17 has conferred an inhibitory effect on cholesterol biosynthesis in CHO cells. In summary, the observed LDL
receptor promoter activity of 17 is related to its ability to prevent 25-hydroxycholesterol from exerting the repressing effect via an
undetermined mechanism and, in part, to inhibit the cholesterol biosynthesis. © 1999 Elsevier Science Inc. All rights reserved.
Keywords: Oxidation; Reduction; Reductive alkylation; Sterol; LDL receptor promoter
Elevated levels of plasma cholesterol are associated with
an increased risk for the development of coronary heart
disease [1–3]. Clinical results have demonstrated that the
lowering of plasma cholesterol levels in patients with cor-
onary heart disease is beneficial for preventing and retarding
the development of atherosclerosis and coronary heart dis-
ease [4–6].
Oxysterols metabolically derived from cholesterol were
originally proposed to play a key role, via binding to the
putative cytosolic oxysterol-binding protein, in down-regu-
lating the expression of low density lipoprotein receptor
(LDLR) and the synthesis of endogenous cholesterol [7–9].
However, the most recent studies by Brown and Goldstein
reveal that a novel family of membrane-bound transcription
factors, called sterol regulatory element-binding proteins
(SREBPs), regulate genes involved in cholesterol biosyn-
thesis as well as the LDL receptor gene [10–13]. Under
sterol-deficient conditions, cells may activate the binding of
the SREBP cleavage-activating protein (SCAP) [14,15] to
the COOH-terminal domain of SREBP. The binding com-
plex allows the proteolytic cleavage to occur at site-1, lead-
ing to the subsequent cleavage at site-2 and the release of
the NH2-terminal domain of SREBP for gene regulation in
the nucleus [16,17]. Nevertheless, the mechanism of inhib-
iting the SCAP-initiated site-1 cleavage by sterols is not
clear, and the investigation is still undergoing.
* Corresponding author. Tel.: ϩ1-317-276-4182; fax: ϩ1-317-433-
Recently, we reported that a new series of 3␣-sterols, ex-
emplified by 4␣-(2-propenyl)-5␣-cholestan-3␣-ol (LY295427,
1685.
E-mail address: lin_ho-shen@lilly.com (H.-S. Lin)
0039-128X/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(98)00083-X

218
H.-S. Lin et al. / Steroids 64 (1999) 217–227
Fig. 1.
Fig. 1), could increase the LDL receptor promoter activity in
the presence of 25-hydroxycholesterol in a Chinese hamster
ovary (CHO) cell-based LDLR/Luc assay [18]. More impor-
tantly, these compounds have also shown efficacy in lowering
the total serum cholesterol levels in hypercholesterolemic
hamsters [18]. Eight compounds, including LY295427, se-
lected from this new series showed no inhibition in the [1-14C-
acetate]cholesterol biosynthesis inhibition assay [18]. Presum-
ably, the observed derepressing activity of this new series of
3␣-sterols is effected by preventing 25-hydroxycholesterol
from exerting its repressing effect; the mechanism of inhibition
has not been determined yet.
the cholesterol biosynthesis. 4␣-(2-Propenyl)-5␣-cholest-
24-en-3␣-ol (17), a 24,25-dehydro analog of LY295427, is
thus synthesized from lithocholic acid via the formation of
3␣-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4␣-(2-propenyl)-
5␣-cholan-24-al (15) (Schemes 1–4).
1. Experimental
1.1. Materials
In our previous studies [18], we had extensively explored
the structure-activity relationships (SAR) in the A-ring of
LY295427. However, few SAR studies had been done in the
D-ring side chain. Nonetheless, we observed that compound
1 (Fig. 1), a 25-hydroxyl analog of LY295427, exerted a
weaker effect on derepressing the transcriptional activity of
the LDL receptor promoter in the presence of 25-hydroxy-
cholesterol in the LDLR/Luc assay with an EC30 value of
9.4 ␮M versus 2.9 ␮M of LY295427. It appears that the
25-hydroxyl functionality in 1 may somewhat confer the
repressing effect as does the 25-hydroxyl functionality in
25-hydroxycholesterol. Consequently, 1 turns out to be less
effective in antagonizing the repressing effect caused by
25-hydroxycholesterol.
Reagents were used as supplied from Aldrich, unless
otherwise noted. Reactions were run under an 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. Reactions were monitored,
and the homogeneity of the products was checked, by thin-
layer chromatography on silica gel 60 F254 plates (E.
Merck). 1H nuclear magnetic resonance (NMR) spectra
were recorded on a General Electric QE-300 instrument.
Chemical shifts are expressed as parts per million downfield
from internal tetramethylsilane. Infrared spectra were deter-
mined on a Nicolet MX-1 FT-IR. Field desorption mass
spectra (FDMS) were recorded on a VG Analytical ZAB-3F
spectrometer. Elemental analyses were determined on a
(Perkin-Elmer Model 240C) elemental analyzer. Melting
points were determined on a Thomas-Hoover melting point
apparatus, but are uncorrected.
As part of our continued SAR studies in the D-ring side
chain of LY295427, we also investigated the effect of the
24,25-unsaturation (desmosterol D-ring side chain) on the
transcriptional activity of the LDL receptor promoter in the
presence of 25-hydroxycholesterol as well as the effect on

H.-S. Lin et al. / Steroids 64 (1999) 217–227
219
Scheme 1.
Scheme 2.

220
H.-S. Lin et al. / Steroids 64 (1999) 217–227
Scheme 3.
1.2. Syntheses
heated in an oil bath at 100°C for 4 h. After concentration in
vacuo at 40°C, the resultant sludge was diluted with ethyl
acetate (650 ml), washed with water (200 ml ϫ 2), dried,
filtered, and concentrated to give a solid mixture of 4 and 5 in
a 1:10 ratio. Following recrystallization from CH2Cl2/CH3CN,
5 (30.0 g, 59%) was obtained as a white solid. Mixture of 4 and
5: 1H NMR (CDCl3) ␦ 5.72 (s, 1H, OCHA of 5), 5.89 (d, J ϭ
10.2 Hz, 1H, OCHA of 4), 6.83 (d, J ϭ 10.2 Hz, 1H, OCHA
of 4). Five: mp 183.5–185.0°C; IR (CHCl3) 3300–2600 (br),
1709, 1661 cmϪ1; 1H NMR (CDCl3) ␦ 0.71 (s, 3H, C-18 CH3),
0.93 (d, J ϭ 6.4 Hz, 3H, C-21 CH3), 1.17 (s, 3H, C-19 CH3),
0.92–2.08 (m, 19H), 2.23–2.50 (m, 6H, two C-2, two C-6, and
two C-23 protons), 5.72 (s, 1H, OCHA); FDMS m/e 372
(Mϩ); Analysis Calculated for C24H36O3: C, 77.38; H, 9.74.
Found: C, 77.18; H, 9.64.
1.2.1. Synthesis of a mixture of 2␤-bromo-3-oxo-5␤-
cholan-24-oic acid (2) and 4␤-bromo-3-oxo-5␤-cholan-
24-oic acid (3)
Synthesis of 3 by using N-bromosuccinimide was re-
ported in the literature [19]. A mixture of lithocholic acid
(50.0 g, 133 mmol) and N-bromosuccinimide (54.4 g, 305
mmol) in water (96 ml) and dioxane (864 ml) was stirred at
50°C for 3 h. The mixture was concentrated in vacuo at
35°C to give a solid residue. It was dissolved in CH2Cl2
(500 ml), washed with water (500 ml ϫ 2), dried, filtered,
and concentrated to give an inseparable mixture of 2 and 3
(60.0 g, 100%) in a 1:5 ratio as a white solid. It was
recrystallized from EtOAc/CH3CN. mp 173.0–176.0°C; IR
(CHCl3) 3350–2550 (br), 1720, 1710 cmϪ1 1H NMR
;
(CDCl3) ␦ 0.69 (s, 3H, C-18 CH3 of 3), 0.92 (d, J ϭ 6.3 Hz,
3H, C-21 CH3 of 3), 1.08 (s, 3H, C-19 CH3 of 3), 2.62 (dd,
J ϭ 14.0, 5.6 Hz, 1H, C-4 equatorial proton of 2), 2.83 (t,
J ϭ 14.0 Hz, 1H, C-4 axial proton of 2), 4.69 (dd, J ϭ
14.0, 5.4 Hz, 1H, C-2 proton of 2), 4.96 (d, J ϭ 12.1 Hz,
1H, C-4 proton of 3); FDMS m/e 453 (Mϩ ϩ 1, 79Br), 455
(Mϩ ϩ 1, 81Br); Analysis calculated for C24H37BrO3: C,
63.57; H, 8.22. Found: C, 63.73; H, 8.32.
1.2.3. Methyl 3-oxochol-4-en-24-oate (6)
Synthesis of 6 was reported in the literature [19]. A
suspension of 5 (14.4 g, 38.7 mmol) in anhydrous CH3OH
(200 ml) was bubbled with HCl gas under ice-cooling; the
resultant mixture was stirred at ambient temperature for 8 h.
After concentration, the solid residue was dissolved in
EtOAc (150 ml); the solution was washed sequentially with
saturated aqueous NaHCO3 (50 ml) and water (50 ml),
dried, filtered, and concentrated to give a yellowish solid.
The solid was dissolved in a small amount of CH2Cl2 and
passed through a short pad of silica (eluded with 40% ethyl
acetate in hexane) to provide 6 (14.6 g, 98%) as a white
1.2.2. Syntheses of 3-oxo-5␤-chol-1-en-24-oic acid (4)
and 3-oxochol-4-en-24-oic acid (5) using lithium chloride
as a dehydrobrominating agent.
Lithium chloride (26.0 g, 615 mmol) was added to a stirred
solution of 2 and 3 (1:5, 62.0 g, 137 mmol) in anhydrous
dimethylformide (DMF) (650 ml); the resultant mixture was
solid. mp 122.0–124.0°C; IR (KBr) 1728, 1674 cmϪ1; H
NMR (CDCl3) ␦ 0.73 (s, 3H, C-18 CH3), 0.94 (d, J ϭ 6.3
Hz, 3H, C-21 CH3), 1.19 (s, 3H, C-19 CH3), 0.90–2.10 (m,
1

H.-S. Lin et al. / Steroids 64 (1999) 217–227
221
Scheme 4.
19H), 2.18–2.52 (m, 6H, two C-2, two C-6, and two C-23
protons), 3.68 (s, 3H, OCH3), 5.74 (s, 1H, C-4 proton);
FDMS m/e 386 (Mϩ); Analysis Calculated for C25H38O3:
C, 77.68; H, 9.91. Found: C, 77.65; H, 9.77.
suspension of activated MnO2 (3.37 g, 38.9 mmol) in an-
hydrous 1,2-dichloroethane (100 ml). The resultant suspen-
sion was sonicated for 5 h. The suspension was filtered
through a short pad of celite and washed with CH2Cl2 (10
ml). The filtrate was concentrated to give a solid. After
recrystallization from CH2Cl2/C6H14, 7 (2.74 g, 98%) was
obtained as a white solid. mp 131.0–132.5°C; IR (KBr)
1.2.4. 3-Oxochol-4-en-24-ol (7)
Diisobutylaluminum hydride (41.4 ml, 1.0 M in toluene)
was added to a stirred solution of 6 (5.00 g, 13.0 mmol) in
anhydrous CH2Cl2 (50 ml) at Ϫ10°C. Upon the completion
of the addition, the cold bath was removed, then the mixture
was stirred for 1 h. The mixture was cooled to Ϫ10°C, when
it was treated with 1 N HCl (30 ml). The two-layered
mixture was stirred vigorously at ambient temperature for
40 min before it was extracted with EtOAc/THF (1:1; 100
ml ϫ 2). The combined organic layers were dried, filtered,
and concentrated to give a solid. After recrystallization from
CH2Cl2/CH3CN, a mixture of diols (4.61 g, 99%) was
obtained as a white solid.
1
3379 (br), 1656 cmϪ1; H NMR (CDCl3) ␦ 0.70 (s, 3H,
C-18 CH3), 0.92 (d, J ϭ 6.4 Hz, 3H, C-21 CH3), 1.17 (s,
3H, C-19 CH3), 0.85–2.05 (m, 22H), 2.20–2.50 (m, 4H, two
C-2 and two C-6 protons), 3.61 (br t, J ϭ 6.0 Hz, 2H,
OCH2OO), 5.71 (s, 1H, C-4 proton); FDMS m/e 358
(Mϩ); Analysis Calculated for C24H38O2: C, 80.39; H,
10.68. Found: C, 80.57; H, 10.94.
1.2.6. 24-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]chol-4-en-
3-one (8)
A stirred solution of 7 (1.80 g, 5.02 mmol) and imidazole
(479 mg, 7.03 mmol) in anhydrous DMF (20 ml) was
The above diols (2.80 g, 7.77 mmol) were added to a

222
H.-S. Lin et al. / Steroids 64 (1999) 217–227
treated with tert-butyldimethylsilyl chloride (1.06 g, 7.03
mmol) at 0°C; the resultant mixture was stirred at ambient
temperature for 1.5 h. The mixture was concentrated in
vacuo, and the residue was chromatographed on silica (gra-
dient 0–15% ethyl acetate/toluene) to give 8 (2.26 g, 95%)
as a white solid. mp 75.0–77.5°C; IR (KBr) 1679 cmϪ1; 1H
NMR (CDCl3) ␦ 0.04 [s, 6H, -Si(CH3)Ϫ2 ], 0.70 (s, 3H, C-18
CH3), 0.88 (s, 9H, tert-butyl), 0.92 (d, J ϭ 6.4 Hz, 3H,
C-21 CH3), 1.17 (s, 3H, C-19 CH3), 0.75–2.07 (m, 21H),
2.18–2.48 (m, 4H, two C-2 and two C-6 protons), 3.56 (t,
J ϭ 6.4 Hz, 2H, OCH2OO), 5.71 (s, 1H, C-4 proton);
FDMS m/e 472 (Mϩ); Analysis Calculated for C30H52O2:
C, 76.21; H, 11.09. Found: C, 76.45; H, 11.16.
(CDCl3) ␦ 0.67 (s, 3H, C-18 CH3), 0.91 (d, J ϭ 6.4 Hz, 3H,
C-21 CH3), 1.04 (s, 3H, C-19 CH3), 0.68–2.05 (m, 25H),
2.16–2.50 (m, 5H, two C-2, one C-4, and two allylic pro-
tons), 3.60 (br t, J ϭ 6.0 Hz, 2H, OCH2OO), 4.93–5.03
(m, 2H, ACH2), 5.69–5.83 (m, 1H, OCHA); FDMS m/e
400 (Mϩ); Analysis Calculated for C27H44O2: C, 80.94; H,
11.07. Found: C, 81.19; H, 11.19.
1.2.9. 3-Oxo-4␣-(2-propenyl)-5␣-cholan-24-al (11)
Pyridinium chlorochromate (733 mg, 3.40 mmol) was
added to a stirred suspension of 10 (545 mg, 1.36 mmol)
and 4Å molecular sieve powder (222 mg) in anhydrous
CH2Cl2 (22 ml) at ambient temperature, and the resultant
suspension was stirred for 1.5 h. After filtration through a
short pad of silica (30% ethyl acetate/hexane), the filtrate
was concentrated, and the residue was chromatographed on
silica (gradient 10–20% ethyl acetate/hexane) to give 11
(450 mg, 83%) as a white solid. mp 133.0–135.0°C; IR
1.2.7. 24-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]-4␣-(2-
propenyl)-5␣-cholan-3-one (9)
Lithium chips (23.0 mg, 3.31 mmol) and a glass-coated stir
bar were placed in a flame-dried, three-necked, round-bot-
tomed flask fitted with a dry ice condenser under argon. Liquid
ammonia (15 ml) was collected in the flask at Ϫ78°C to form
a deep blue solution, then followed by the addition of anhy-
drous tetrahydrofuran (THF) (15 ml). A solution of 8 (614 mg,
1.30 mmol) and t-BuOH (0.122 ml, 1.30 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
(0.25 ml) to quench the excess lithium. After 15 min, allyl
iodide (0.238 ml, 2.60 mmol) was added to the white suspen-
sion, and the resultant mixture was stirred at Ϫ78°C for 2.5 h.
Saturated aqueous NH4Cl (15 ml) was cautiously added to the
white suspension. The cold bath was removed, and the mix-
ture, with the evaporation of ammonia, was allowed to warm to
ambient temperature. After extraction with EtOAc (30 ml ϫ
2), the combined organic layers were washed with saturated
aqueous NaCl (15 ml), dried, filtered, and concentrated. After
flash chromatography on silica (gradient 0–5% ethyl acetate/
toluene), 9 (402 mg, 60%) was obtained as an oil. IR (film)
1711 cmϪ1; 1H NMR (CDCl3) ␦ 0.04 [s, 6H, -Si(CH3)Ϫ2 ], 0.66
(s, 3H, C-18 CH3), 0.88 (s, 9H, tert-butyl), 0.90 (d, J ϭ 6.4 Hz,
3H, C-21 CH3), 1.04 (s, 3H, C-19 CH3), 0.68–2.06 (m, 24H),
2.15–2.52 (m, 5H, two C-2, one C-4, and two allylic protons),
3.56 (t, J ϭ 6.4 Hz, 2H, OCH2OO), 4.93–5.04 (m, 2H,
ACH2), 5.68–5.84 (m, 1H, OCHA); FDMS m/e 514 (Mϩ);
Analysis Calculated for C33H58O2Si: C, 76.97; H, 11.35.
Found: C, 77.09; H, 11.30.
1
(KBr) 1722, 1705 cmϪ1; H NMR (CDCl3) ␦ 0.69 (s, 3H,
C-18 CH3), 0.93 (d, J ϭ 6.5 Hz, 3H, C-21 CH3), 1.07 (s,
3H, C-19 CH3), 0.70–2.08 (m, 22H), 2.18–2.55 (m, 7H,
two C-2, one C-4, two C-23, and two allylic protons),
4.95–5.06 (m, 2H, ACH2), 5.70–5.87 (m, 1H, OCHA),
9.78 (s, 1H, CHO); FDMS m/e 398 (Mϩ); Analysis Calcu-
lated for C27H42O2: C, 81.35; H, 10.62. Found: C, 81.76; H,
10.65.
1.2.10. 4␣-(2-Propenyl)-5␣-cholest-24-en-3-one (12)
Potassium bistrimethylsilylamide (0.5 M in toluene, 1.00
ml) was added to a stirred suspension of isopropyltriph-
enylphosphonium iodide (233 mg, 0.540 mmol) in anhy-
drous THF (3 ml) at Ϫ15°C, and the resultant red suspen-
sion was stirred for 15 min. A solution of 11 (181 mg, 0.455
mmol) in anhydrous THF (2 ml) was added dropwise to the
red suspension at Ϫ15°C, then the mixture was stirred at
0°C for 1 h. Acetone (0.1 ml) was added to the mixture to
quench the excess phosphonium ylide, also followed by the
addition of hexane (10 ml). The mixture was filtered
through a short pad of silica (10% ethyl acetate/hexane).
The filtrate was concentrated, and the residue was chro-
matographed on silica (gradient 2–10% ethyl acetate/hex-
ane) to give 12 (90.0 mg, 47%) as a white solid. mp
1
134.0–135.0°C; IR (KBr) 1705 cmϪ1; H NMR (CDCl3) ␦
0.67 (s, 3H, C-18 CH3), 0.92 (d, J ϭ 6.3 Hz, 3H, C-21
CH3), 1.05 (s, 3H, C-19 CH3), 1.60 and 1.68 (both s, 6H,
C-26 and -27 CH3), 0.75–2.10 (m, 24H), 2.20–2.53 (m, 5H,
two C-2, one C-4, and two allylic protons), 4.95–5.12 (m,
3H, ACH2 and C-24 protons), 5.72–5.85 (m, 1H, OCHA);
FDMS m/e 424 (Mϩ); Analysis Calculated for C30H48O: C,
84.84; H, 11.39. Found: C, 84.61; H, 11.32.
1.2.8. 3-Oxo-4␣-(2-propenyl)-5␣-cholan-24-ol (10)
A stirred solution of 9 (907 mg, 1.77 mmol) in THF (9
ml) was treated with tetrabutylammonium fluoride (1 M in
THF, 3.54 ml), and the resultant mixture was stirred at
ambient temperature for 2.5 h. After dilution with ethyl
acetate (40 ml), the solution was washed with water (20
ml ϫ 2), dried, filtered, and concentrated. The residue was
chromatographed on silica (gradient 10–30% ethyl acetate/
toluene) to give 10 (650 mg, 92%) as a white solid. mp
1.2.11. 24-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]-4␣-(2-
propenyl)-5␣-cholan-3␣-ol (13)
K-Selectride (1M in THF, 1.17 ml) was added to a stirred
solution of 9 (400 mg, 0.779 mmol) in anhydrous THF (10
ml) at Ϫ10°C. Upon completion of the addition, the result-
1
136.0–138.0°C; IR (KBr) 3549 (br), 1706 cmϪ1; H NMR

H.-S. Lin et al. / Steroids 64 (1999) 217–227
223
ant yellowish solution was allowed to warm to ambient
temperature, when it was stirred for 1.5 h. The reaction
mixture was then cooled to Ϫ10°C and sequentially treated
with MeOH (1 ml), 5 N NaOH (0.93 ml), and 30% H2O2
(0.477 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 (60 ml)
were added to the mixture. The organic layer was separated,
washed with half-saturated aqueous NaCl (10 ml), dried,
filtered, and concentrated. After flash chromatography on
silica (gradient 5–10% ethyl acetate/hexane), 13 (369 mg,
92%) was obtained as a white solid. mp 101.0–102.0°C; IR
bient temperature. The resultant suspension was stirred for
1 h. After filtration through a short pad of silica (10% ethyl
acetate/hexane), the filtrate was concentrated, and the resi-
due was chromatographed on silica (gradient 0–7% ethyl
acetate/hexane) to give 15 (160 mg, 89%) as a white solid.
mp 119.0–120.0°C; IR (KBr) 1730 cmϪ1 1H NMR
;
(CDCl3) ␦ 0.02 and 0.04 [both s, 6H, -Si(CH3)Ϫ2 ], 0.65 (s,
3H, C-18 CH3), 0.79 (s, 3H, C-19 CH3), 0.90 (s, 9H,
tert-butyl), 0.91 (d, J ϭ 6.4 Hz, 3H, C-21 CH3), 0.60–1.95
(m, 26H), 2.08–2.15 (m, 1H, allylic proton), 2.25–2.52 (m,
2H, C-23 protons), 3.87 (br s, 1H, C-3 proton), 4.93–5.03
(m, 2H, ACH2), 5.72–5.85 (m, 1H, OCHA), 9.76 (s, 1H,
CHO); FDMS m/e 458 (Mϩ-C4H8); Analysis Calculated for
C33H58O2Si ⅐ 0.2C6H14: C, 77.19; H, 11.52. Found: C,
77.19; H, 11.84.
1
(KBr) 3486 (br) cmϪ1; H NMR (CDCl3) ␦ 0.04 [s, 6H,
-Si(CH3)Ϫ2 ], 0.64 (s, 3H, C-18 CH3), 0.81 (s, 3H, C-19
CH3), 0.89 (s, 9H, tert-butyl), 0.90 (d, J ϭ 6.5 Hz, 3H,
C-21 CH3), 0.55–2.05 (m, 29H), 2.22–2.35 (m, 1H, allylic
proton), 3.56 (t, J ϭ 6.5 Hz, 2H, OCH2OO), 3.90 (br s,
1H, C-3 proton), 4.98–5.15 (m, 2H, ACH2), 5.78–5.95 (m,
1H, OCHA); FDMS m/e 516 (Mϩ); Analysis Calculated
for C33H60O2Si: C, 76.68; H, 11.70. Found: C, 76.49; H,
11.51.
1.2.14. 3␣-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]-4␣-(2-
propenyl)-5␣-cholest-24-ene (16)
Potassium bistrimethylsilylamide (0.5 M in toluene,
0.815 ml) was added to a stirred suspension of isopropyl-
triphenylphosphonium iodide (189 mg, 0.437 mmol) in an-
hydrous THF (4 ml) at Ϫ15°C, and the resultant red sus-
pension was stirred for 15 min. A solution of 15 (150 mg,
0.291 mmol) in anhydrous THF (2 ml) was added dropwise
to the red suspension at Ϫ15°C, and the mixture was stirred
at 0°C for 1 h. Acetone (0.1 ml) was added to the mixture
to quench the excess phosphonium ylide, then followed by
the addition of hexane (10 ml). The mixture was filtered
through a short pad of silica (5% ethyl acetate/hexane), then
the filtrate was concentrated, and the residue was chromato-
graphed on silica (hexane) to give 16 (150 mg, 95%) as a
1.2.12. 3␣-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]-4␣-(2-
propenyl)-5␣-cholan-24-ol (14)
tert-Butyldimethylsilyl trifluoromethanesulfonate (0.130
ml, 0.560 mmol) was added to a stirred solution of 13 (192
mg, 0.373 mmol) and anhydrous pyridine (0.060 ml, 0.75
mmol) in anhydrous CH2Cl2 (4 ml) at 0°C. The resultant
mixture was then allowed to stir at ambient temperature for
2 h. The crude mixture was filtered through a short pad of
silica (5% ethyl acetate/hexane), then the filtrate was con-
centrated to give an oily residue.
1
white solid. mp 88.5–89.0°C; IR (KBr) 2932 cmϪ1; H
The above disilyl product was diluted in THF (4 ml) and
treated with tetrabutylammonium fluoride (1 M in THF,
0.933 ml); the resultant mixture was stirred at ambient
temperature for 2 h. After dilution with ethyl acetate (50
ml), the solution was washed with water (20 ml ϫ 2), dried,
filtered, and concentrated. The residue was chromato-
graphed on silica (gradient 0–25% ethyl acetate/hexane) to
give 14 (190 mg, 99%) as a white solid. mp 107.5–109.0°C;
IR (KBr) 3355 (br) cmϪ1; 1H NMR (CDCl3) ␦ 0.02 and 0.04
[both s, 6H, -Si(CH3)Ϫ2 ], 0.65 (s, 3H, C-18 CH3), 0.79 (s,
3H, C-19 CH3), 0.90 (s, 9H, tert-butyl), 0.91 (d, J ϭ 6.5
Hz, 3H, C-21 CH3), 0.60–2.00 (m, 29H), 2.06–2.19 (m, 1H,
allylic proton), 3.61 (t, J ϭ 6.5 Hz, 2H, OCH2OO), 3.87
(br s, 1H, C-3 proton), 4.92–5.05 (m, 2H, ACH2), 5.72–
5.90 (m, 1H, OCHA); FDMS m/e 459 (Mϩ-C4H9); Anal-
ysis Calculated for C33H60O2Si ⅐ 0.5C6H14: C, 77.21; H,
12.06. Found: C, 77.59; H, 11.68.
NMR (CDCl3) ␦ 0.02 and 0.04 [both s, 6H, -Si(CH3)Ϫ2 ],
0.64 (s, 3H, C-18 CH3), 0.79 (s, 3H, C-19 CH3), 0.90 (s, 9H,
tert-butyl), 0.91 (d, J ϭ 6.5 Hz, 3H, C-21 CH3), 1.60 and
1.68 (both s, 6H, C-26 and -27 CH3), 0.60–2.18 (m, 29H),
3.87 (br s, 1H, C-3 proton), 4.90–5.03 (m, 2H, ACH2), 5.09
(t, J ϭ 6.4 Hz, 1H, C-24 proton), 5.74–5.88 (m, 1H,
OCHA); FDMS m/e 541 (Mϩ ϩ 1); Analysis Calculated
for C36H64OSi: C, 79.93; H, 11.92. Found: C, 79.87; H,
12.10.
1.2.15. 4␣-(2-Propenyl)-5␣-cholest-24-en-3␣-ol (17)
Tetrabutylammonium fluoride (1 M in THF, 2.22 ml)
was added to a stirred solution of 16 (120 mg, 0.222 mmol)
in THF (2 ml), and the resultant mixture was heated to
reflux for 1 d. At ambient temperature, the mixture was
diluted with EtOAc (50 ml), washed with water (20 ml ϫ
3), dried, filtered, and concentrated. The residue was chro-
matographed on silica (gradient 0–10% ethyl acetate/hex-
ane) to give 17 (92.7 mg, 98%) as a white solid. mp
83.0–84.0°C; IR (KBr) 3453 (br) cmϪ1; 1H NMR (CDCl3)
␦ 0.64 (s, 3H, C-18 CH3), 0.81 (s, 3H, C-19 CH3), 0.91 (d,
J ϭ 6.4 Hz, 3H, C-21 CH3), 1.60 and 1.68 (both s, 6H,
C-26 and -27 CH3), 0.65–2.06 (m, 29H), 2.22–2.32 (m, 1H,
allylic proton), 3.89 (br s, 1H, C-3 proton), 4.98–5.15 (m,
1.2.13. 3␣-[[(1,1-Dimethylethyl)dimethylsilyl]oxy]-4␣-(2-
propenyl)-5␣-cholan-24-al (15)
Pyridinium chlorochromate (150 mg, 0.696 mmol) was
added to a stirred suspension of 14 (180 mg, 0.348 mmol),
sodium acetate (57.0 mg, 0.696 mmol), and 4Å molecular
sieve powder (57 mg) in anhydrous CH2Cl2 (5 ml) at am-

224
H.-S. Lin et al. / Steroids 64 (1999) 217–227
3H, ACH2 and C-24 protons), 5.78–5.94 (m, 1H, OCHA);
FDMS m/e 426 (Mϩ); Analysis Calculated for C30H50O: C,
84.44; H, 11.81. Found: C, 84.24; H, 11.54.
regression analysis method using Prism (Graphpad, San
Diego, CA, USA).
2. Results and Discussion
1.3. In vitro LDL receptor/luciferase assay
2.1. Synthesis
Compound 17 was evaluated for its derepressing capa-
bility against 25-hydroxycholesterol in the LDLR/Luc as-
say, in which stably transfected, stationary CHO cells were
exposed to 25-hydroxycholesterol (0.5 ␮g/ml) and 17 at
different concentrations (0, 0.6, 1.3, 2.5, 5.0, and 10.0 ␮g/
ml) in serum-free medium for 24 h. Two ethanol stock
solutions of 25-hydroxycholesterol (0.5 mg/ml) and 17 (4.0
mg/ml) were prepared and diluted in the assay medium to
achieve the previously indicated concentrations. The one
containing 25-hydroxycholesterol, but without 17, is a re-
pressed control. 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 [18]. EC30 refers to the effective con-
centration of 17 required to increase the specific light in-
duction by 30% over the repressed control.
Synthesis of the target 3␣-sterol 17 was envisioned to
proceed through Stork’s reductive alkylation of enone 8
[24–26], then followed by the stereoselective K-selectride
reduction of the C-3 keto group [18,27]. Enone 8 could be
derived from compound 6, which in turn could be synthe-
sized from lithocholic acid according to known procedures
[19].
Following these procedures [19], lithocholic acid was
treated with 2.3 equivalents of N-bromosuccinimide in
aqueous dioxane at 50°C for 3 h to give an inseparable
mixture of 2␤-bromoketone 2 and 4␤-bromoketone 3 in a
1
1:5 ratio in quantitative yield (Scheme 1). In the H NMR
spectrum, the signal, because of the axial proton at C-2 in 2,
appears at ␦ 4.69 as a doublet of doublets ( J ϭ 14.0, 5.4
Hz), whereas the axial proton at C-4 in 3 appears at ␦ 4.96
as a doublet ( J ϭ 12.1 Hz). In the literature, compound 3
was reported to be the only isolated product [19].
Subsequent dehydrobromination of the mixture of 2 and
3 (1:5) using Li2CO3 in DMF at 100°C for 4 h delivered an
inseparable mixture of enones 4 and 5 in a 1:4.5 ratio. The
ratio was improved to 1:10 by replacing Li2CO3 with LiCl
as the dehydrobrominating agent [28,29], and pure 5 was
isolated in a 59% yield after recrystallization from CH2Cl2/
1.3. [1-14C-Acetate]cholesterol biosynthesis
inhibition assay
Replicate, 100 mm dishes of confluent CHO cells were
incubated with 8 ml assay media (without the 25-hydroxy-
cholesterol supplement) containing the ethanol vehicle
(0.5% v/v), 17 (10 ␮g/ml or 23 ␮M), or mevinolin (10 ␮M).
After a 20-h incubation, [1-14C]-acetic acid, sodium salt (8
␮l) was added directly to each dish (final concentration of 1
␮Ci/ml), and the dishes were incubated for an additional
4 h. Cell monolayers were then harvested by scraping, and
the cell pellets were suspended in water and sonicated for
10 s. After an aliquot was removed for protein determina-
tion [20], lipids were extracted with chloroform/methanol
(2:1 v/v) [21]. After normalizing for protein content, the
lipid fraction was resuspended using chloroform/methanol
(4;1, v/v) in a small volume (100 ␮l or less), and lipids were
separated by high performance thin-layer chromatography
on Kieselgel 60 silica plates with a mobile phase consisting
of hexane/heptane/diethyl ether/acetic acid (63:18.5:18.5:1,
v/v). Lipids were visualized by dipping the plate for 3 s in
a solution of hexane/ethanol/sulfuric acid (64:35:1, v/v),
then placed for 15 min in a 110°C oven [22]. The choles-
terol band was scraped and counted for radioactivity on a
Beckman LS6500 Scintillation Counter.
1
CH3CN (Scheme 1). In the H NMR spectrum of the mix-
ture of 4 and 5, the signals, because of the olefinic protons
in 4, appear at ␦ 5.89 and 6.83 as an AB pattern ( J ϭ 10.2
Hz), whereas the olefinic proton in 5 appears at ␦ 5.72 as a
singlet, which is in agreement with the literature [19].
As depicted in Scheme 2, compound 5 was readily con-
verted to its methyl ester 6. Subsequent reduction of 6 with
diisobutylaluminum hydride in CH2Cl2 at Ϫ10°C gave a
mixture of diols, which were then oxidized with activated
MnO2 in 1,2-dichloroethane to provide enone 7 in an overall
97% yield. The reaction rate of this heterogeneous oxidation
was greatly accelerated by sonication. Utilizing the C-24
primary alcohol as an internal proton source, an attempt to
perform Stork’s reductive alkylation of 7 with allyl iodide
gave a complex mixture, which only yielded 14% of the
desired allylated product. Thus, the C-24 hydroxyl in 7 was
protected as a tert-butyldimethylsilyl ether in a 95% yield.
The resultant protected enone 8 was reductively alkylated
following Stork’s protocols to deliver the ketone 9 in a
respectable 60% yield.
1.4. Statistics
As shown in Scheme 3, there appeared to be only four
steps starting from compound 9 to the synthesis of the target
3␣-ol 17. Compound 9 was thus treated with tetrabutylam-
monium fluoride to give the primary alcohol 10 in a 92%
yield; then, in turn, it was oxidized with pyridinium chlo-
Data were analyzed by ANOVA, and differences be-
tween means of the repressed control and 17-treated CHO
cells were determined by the statistical method of Dunnett
[23]. The EC30 value was determined by the nonlinear

H.-S. Lin et al. / Steroids 64 (1999) 217–227
225
rochromate in CH2Cl2 in the presence of 4Å molecular
sieves to provide the aldehyde 11 in an 83% yield preparing
for the installation of the isopropylidene functionality [30].
The initial attempt to selectively couple the aldehyde group
in 11 with the Wittig reagent, generated from isopropyltri-
phenylphosphonium iodide and potassium tert-butoxide in
THF, only afforded the desired compound 12 in a Ͻ10%
yield. We were able to improve the coupling yield to 47%
by employing potassium bistrimethylsilylamide as the base.
At this juncture, we decided to go back to compound 9
and adopt a different synthetic approach toward the synthe-
sis of the target 3␣-ol 17. The new synthetic route would
allow for the synthesis of 3␣-[[(1,1-dimethylethyl)dimeth-
ylsilyl]oxy]-4␣-(2-propenyl)-5␣-cholan-24-al (15), which
could serve as a versatile intermediate that was suitable for
future SAR studies in the D-ring side chain (Scheme 4). It
is known that K-selectride, because of its bulkiness, almost
exclusively delivers its hydride via the equatorial course to
the carbonyl group in cyclohexanone to provide the cyclo-
hexanol with the hydroxyl group in the axial position [18,
27]. As anticipated, the desired 3␣-ol 13 in a 92% yield was
obtained as the sole product after the reduction of 9 with
1
K-selectride. In its H NMR spectrum, the C-3 equatorial
Fig. 2. Activation of LDL receptor transcriptional activity by 17 in the
presence of 25-hydroxycholesterol. CHO cells, transfected with a LDL
receptor promoter/luciferase gene construct, were treated for 24 h with
25-hydroxycholesterol (0.5 ␮g/ml) and 17 (0–10 ␮g/ml). The column
containing no 17 represents the repressed control. The EC30 value was
determined to be 1.1Ϯ0.1 ␮g/ml (2.6Ϯ0.3 ␮M) by the nonlinear regression
analysis method. Values are meansϮSE for triplicates run at three different
times. Asterisks denote a significant difference from the repressed control,
P Ͻ 0.05.
proton appears at ␦ 3.90 as a singlet. The hindered 3␣-
hydroxyl in 13 was then protected as a tert-butyldimethyl-
silyl ether, whereas the less hindered tert-butyldimethylsilyl
ether of the C-24 hydroxyl was selectively deprotected with
tetrabutylammonium fluoride in THF to give 14 in a 99%
yield. Subsequent pyridinium chlorochromate oxidation of
14 in CH2Cl2 in the presence of sodium acetate and 4Å
molecular sieves provided the target intermediate 15 in an
89% yield [30,31]. Wittig coupling of 15 with (isopropyli-
dene)triphenylphosphorane in THF at Ϫ15°C went
smoothly and delivered the compound 16 in a 95% yield,
compared to a 47% yield for the formation of 12. The
hindered tert-butyldimethylsilyl ether of the 3␣-hydroxyl
was deprotected with tetrabutylammonium fluoride in THF
at reflux to complete the synthesis of our target 3␣-ol 17 in
a 98% yield.
As shown in Fig. 2, there is a good dose-response rela-
tionship between the LDL receptor transcriptional activity
versus the concentration of 17 in the LDLR/Luc assay. EC30
refers to the effective concentration of 17 required to in-
crease the specific light induction by 30% over the repressed
control; therefore, the value was determined to be 2.6 ␮M,
comparable to 2.9 ␮M of LY295427, by the nonlinear
regression analysis method. Apparently, the built-in 24,25-
unsaturation in the D-ring side chain had conferred, on 17,
little effect on antagonizing the repressing effect of 25-
hydroxycholesterol in transfected CHO cells.
2.2. In vitro biology
A LDLR/Luc assay [18] was set up by stably transfecting
a gene construct in CHO cells in which a DNA segment
containing the promoter and regulatory control elements of
the LDL receptor gene was fused to the firefly luciferase
reporter gene. Compound 17 was evaluated for its dere-
pressing capability in the presence of 25-hydroxycholesterol
in the CHO cell-based LDLR/Luc assay. In the assay stably
transfected, stationary CHO cells were exposed to 25-hy-
droxycholesterol (0.5 ␮g/ml) and 17 at different concentra-
tions (0, 0.6, 1.3, 2.5, 5.0, and 10.0 ␮g/ml) in serum-free
medium for 24 h. The one containing 25-hydroxycholes-
terol, but without 17, is a repressed control. Cells were then
washed and lysed, and homogenates were evaluated for
luciferase-catalyzed light production.
A [1-14C-acetate]cholesterol biosynthesis inhibition as-
say was also set up to investigate the inhibition of choles-
terol biosynthesis by 17 in CHO cells. Confluent CHO cells
were incubated with assay media containing either the eth-
anol vehicle or 17 (10 ␮g/ml or 23 ␮M) or mevinolin (10
␮M) for 20 h, then they were treated with [1-14C]-acetic
acid and incubated for an additional 4 h. The [14C]-choles-
terol was isolated and counted for radioactivity according to
the procedure described in the Experimental section.
As indicated in Fig. 3, 17 shows a 38% inhibition of the
cholesterol biosynthesis compared to the control medium,
whereas LY295427 did not show any inhibition in the same
assay in our previous studies [18]. Mevinolin showed a 90%

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H.-S. Lin et al. / Steroids 64 (1999) 217–227
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duplicates with Ͻ10% variations.
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has not been identified.
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Acknowledgments
Physical chemistry data were furnished by the staff of the
physical chemistry division. We thank Dr Michael Statnick
for helpful discussions.
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