Oxysterols: 27-hydroxycholesterol and its radiolabeled analog

Article abstract of DOI:10.1016/S0039-128X(00)00099-4

We describe a convenient and stereoselective route to the synthesis of 27-hydroxycholesterol. Also its radiolabeled analog, 22, 23 di [³H]-27-hydroxycholesterol with high specific radioactivity (55 Ci/mmol) was synthesized by this method. Julia

Full text of DOI:10.1016/S0039-128X(00)00099-4

Steroids 65 (2000) 401–407
Oxysterols: 27-hydroxycholesterol and its radiolabeled analog૾
Thomas E. D’Ambraa, Norman B. Javittb, James Lacyc, Puliyur Srinivasanc,
Tadeusz Warchold,*,1
aAlbany Molecular Research, Inc., 21 Corporate Circle, Albany, NY 12203, USA
bNew York University, Medical Center, 550 First Avenue, New York, NY 10016, USA
cE. I. DuPont De Numerous & Co., Medical Products Department, 549 Albany Str., Boston, MA 02118, USA
dColumbia University in the City of New York, Department of Chemistry, New York, NY 10027, USA
Received 9 December 1999; received in revised form 1 February 2000; accpeted 7 February 2000
Abstract
We describe a convenient and stereoselective route to the synthesis of 27-hydroxycholesterol. Also its radiolabeled analog, 22, 23 di
[3H]-27-hydroxycholesterol with high specific radioactivity (55 Ci/mmol) was synthesized by this method. Julia condensation of steroidal
22-sulfone with aldehyde, led to the addition of the 23–27 carbon side chain building block to the steroid backbone. Formed in this reaction
␤-hydroxysulfone moiety was reduced by sodium amalgam generate 22–23 unsaturated bond. Further reduction either by hydrogen or
tritium furnished substrates for the synthesis of title compounds. © 2000 Elsevier Science Inc. All rights reserved.
Keywords: Oxysteroids; Hydroxycholesterol; Kryptogenin; Diosgenin; Condensation; Reductive elimination
1. Introduction
and optically pure 1 and its radiolabeled (tritiated) analog
were required.
Due to their unique properties, oxysteroids attract much
attention in their preparation and biologic evaluation. The
title2,3 compound 1 is the intermediate in the metabolic
pathway from cholesterol to the bile acids [3]. In addition to
its role as a metabolic intermediate, other important prop-
erties have been described. 27-Hydroxycholesterol (1) was
found to be an effective inhibitor of HMG-CoA reductase
[4,5], an inhibitor of DNA synthesis [6] and was shown to have
good anitumor properties (D’Ambra et al., in preparation).
Recent data also suggest important role of this type of com-
pounds in the development of atherosclerosis [7] (Fig. 1).
For our study of cholesterol metabolism, the chemically
Because the side chain of 1 is degraded during the
metabolic process to the shorter 24 carbons metabolite,
tritium should be introduced at positions before C-24.
The metabolically modified C-2, C-3, C-5, C-6, and C-7
positions should also be excluded. Previous routes to the syn-
thesis of 1 utilized the two readily available natural isopre-
noids, kryptogenin (2), and diosgenin (3). In the first approach
kryptogenin (2) was converted [8–10] to the desired 27-hy-
droxycholesterol (1) through the two-step reduction of the
C-16 and C-22 oxo functions. Overall yield was reported as
high as 40%. The second method, starting from diosgenin (3)
[11–14], utilized the Clemmensen, Wolf–Kishner reduction
sequence. However this approach requires blocking of the C-3
and C-26 hydroxyls. Also this method allowed preparation
[15] of the 25S epimer starting from yamogenin (4), which is
present in natural material in concentration of up to 15%.
These past approaches suffer from several faults. The most
important is the potential for epimerization at the C-25 stereo-
center, which leads to the contamination of the final product. It
has been shown [16–18], by HPLC analysis, that samples of
27-hydroxycholesterol (1) previously claimed as ‘pure’ con-
tained significant amounts of the 25S epimer. The mechanism
for such an epimerization is the steroidal [1,5]-H shift [9,19].
૾ This work was partially supported by N.I.H. Grant
No1R43NS33813-01 (to T.E.D.) and DK-32995 (to N.B.J.).
* Corresponding author. Tel.: ϩ1-781-274-8200; fax: ϩ1-781-274-
8228.
E-mail address: tad@sbrco.com (T. Warchol).
1 Present address: Shionogi BioResearch Corp., 45 Hartwell Av., Lex-
ington, MA 02173, USA.
2 Preliminary communication was published, see ref. [1].
3 According to the recent nomenclature (25R)-26-hydroxycholesterol is
referred as 27-hydroxycholesterol, whereas 25S isomer as a 26-hydroxy-
cholesterol, see ref [2].
0039-128X/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(00)00099-4

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T.E. D’Ambra et al. / Steroids 65 (2000) 401–407
Fig. 1.
In addition to the use of 2 and 3 other approaches to 1
methane). Melting points were obtained with an Electro-
chemical Melting point apparatus and are uncorrected. Op-
tical rotations were taken at the D-Line of sodium on a
Perkin-Elmer 243B Polarimeter at room temperature
(23°C). The tritiated analog was purified by HPLC using
column Zorbax ODS (250 ϫ 9.4 mm) column with meth-
anol-water mixture as a mobile phase, with detection at 205
nm. Radiochemical purity was determined by TLC. Specific
activity was determined by mass spectral analysis.
[20,21] and its 25S epimer [22,23] have been described.
However, none of these methods were deemed suitable for
the synthesis of the radiolabeled analog of 1 under the
requirements described above4.
Herein we described simple and convenient synthesis of
optically and chemically pure 1, as well as its radiolabeled
analog 5.
2. Experimental
2.1. (2S, 2ЈRS) 2-Methyl-3-(2-tetrahydropyranyloxy)-
propanol (9)
All non-aqueous reactions were performed under a dry
atmosphere of nitrogen. Solvents were dried by reflux over
drying agent (CaH2 for methylene chloride, LiAlH4 for
tetrahydrofuran and toluene) and distilled just before use.
Pyridine and other solvents were used without purification.
All the commercial reagents were purchased from Aldrich
(Milwaukee, WI, USA), Fluka (Buchs, Switzerland), or
Sigma (St. Louis, MO, USA). TLC analyses were per-
formed on pre-coated silica gel plates (Merck, West Point,
PA, USA) and visualized by spraying with a solution of
ammonium molybdate (48 g) and cerium sulfate (2 g) in
10% H2SO4 (1-l) and heating to about 200°C. Column
chromatography was done using silica gel (Merck or What-
man). Solutions were dried using anhydrous Na2SO4 or
anhydrous MgSO4. Amberlite IRN-78 (Alfa) was used as a
basic resin. All products that contain THP group were sta-
bilized with pyridine (10Ϫ4%) during storage. 1H NMR
spectra were recorded with Bruker AC 300 spectrometer,
with TMS as an internal standard with the following fre-
To a mixture of the hydroxyester 7 (4.8 g, 40.6 mmol),
dihydropyran (4.8 ml, 52.8 mmol), and CH2Cl2 (40 ml),
10-camphorsulfonic acid (cat. amount) was added and the
resultant solution was stirred at room temperature over-
night. After dilution with CH2Cl2 (100 ml) the reaction
mixture was washed with aqueous NaHCO3, dried and
evaporated. The residue was dissolved in hexane (1 l) and
filtered through a bed of silica gel (50 g). The filtrate was
evaporated to give crude THP derivative 8 (9.0 g) as a
slightly yellow oil. This oil was dissolved in THF (300 ml),
cooled to 0°C, and slowly added to a stirred at 0°C solution
of LiAlH4 (5.46 g, 0.144 mol) in THF (100 ml). After
complete addition the cooling bath was removed and the
reaction mixture was stirred at room temperature for 1 h,
then refluxed for 0.5 h. The reaction mixture was cooled to
5°C and the following were added: wet ether (115 ml), H2O
(6 ml), 15% NaOH (20 ml), and, finally, H2O (20 ml). A
white precipitate formed and was filtered off and washed
with ether (2 ϫ 200 ml). Combined filtrates were dried and
evaporated. The crude product 9 was purified by distillation
under reduced pressure. With a temperature of 86–88°C/0.5
Torr. Yield 5.8 g (82%) of colorless oil.
1
quencies: 300 MHz for H resonance, and 75 MHz for 13C
resonance. DEPT 135, DEPT 90, DEPT 45, and HETCOR
techniques were used for necessary structure determination
and signals assignment. Abbreviations used for 13C NMR
spectra descriptions: (CH3)-primary carbon, (CH2)-second-
ary carbon, (CH)-tertiary carbon, (C)-quaternary carbon. IR
spectra were obtained on a Perkin Elmer 1000 FT Infrared
Spectrophotometer. Mass spectroscopic analyses were per-
formed on a Shimadzu QP-5000 GC/mass spectrometer (CI,
1H NMR (CDCl3), ␦, ppm: 0.90, 0.91 (2d, J ϭ 6.9 Hz,
3H, CH3), 1.46–2.12 (m, 6H), 2.71 (br.s., 1H), 3.35 (dd, J ϭ
7.6 Hz, J ϭ 9.2 Hz, 1H), 3.45–3.74 (m, 5H), 3.81–3.96 (m,
1H), 4.56–4.64 (m, 1H); 13C NMR (CDCl3), ␦, ppm:
13.67, 13.76 (CH3); 19.74, 25.47, 30.72 (CH2); 35.65, 35.89
(CH); 62.59, 67.02, 71.89 (CH2); 99.20, 99.45 (CH); IR
(film) cmϪ1: 3422, 2942, 1454, 1120; [␣]D ϭ ϩ8.18o (c ϭ
1, CHCl3); MS, m/z: 175 [MϩH]ϩ.
4 For syntheses of isotope labeled 27-hydroxycholesterol see ref. [24]
and references cited therein.

T.E. D’Ambra et al. / Steroids 65 (2000) 401–407
403
2.2. (2S, 2ЈRS) 2-Methyl-3-(2-tetrahydropyranyloxy)propyl
4-toluenesulfonate (10)
mixture was stirred an additional 0.5 h at the same temper-
ature. Water (3 ml) was added to quench the reaction, and
the resulting slurry was stirred overnight at room tempera-
ture. A white precipitate formed and was filtered off and
washed with ether (2 ϫ 15 ml). Combined filtrates and
washings were dried and evaporated. The resulting colorless
oily residue was purified by column chromatography. Yield
of 12, 0.65 g, (89%), colorless oil.
To a stirred solution of alcohol 9 (5.3 g, 30.4 mmol) in
pyridine (50 ml) at 0°C, a solution of 4-toluenesulfonyl
chloride (7.5 g, 39.5 mmol) in pyridine (70 ml) was added
followed by a catalytic amount of DMAP. The cooling bath
was removed and reaction mixture was stirred at room
temperature for 4 h. A reaction was quenched with water (5
ml), and at the end of the exothermic reaction (20 min)
hexane (200 ml) was added. The mixture was washed with
water and aqueous CuSO4 until the blue-violet color disap-
peared. Drying and evaporation of solvent afforded tosylate
10 (9.5 g, 95%) as a colorless oil. An analytical sample was
purified by column chromatography using hexane/ethyl ac-
etate (95:5 v/v) as an eluent.
1H NMR (CDCl3), ␦, ppm: 1.00 (d, J ϭ 6.4 Hz, 3H),
1.43–1.89 (m, 4H), 2.20–2.65 (m, 4H), 3.14 (dd, J ϭ 7.7 Hz,
J ϭ 9.6 Hz, 1H), 3.34 (dd, J ϭ 4.8 Hz, J ϭ 9.4 Hz, 1H),
3.45–3.60 (m, 2H), 3.68–3.71 (m, 2H), 4.54–4.65 (m, 1H),
9.75, 9.80 (2t, J ϭ 5.1 Hz, 1H); 13C NMR (CDCl3), ␦, ppm:
17.20 (CH3); 19.29, 19.52, 25.55 (CH2); 29.23, 29.37 (CH);
30.60, 48.68, 48.83, 62.30, 63.34, 72.00, 72.47, 98.64, 99.25
(CH2); 202.50 (CH); IR (neat) cmϪ1: 2942, 1726, 1455, 1122,
1034; MS, m/z: 187 [MϩH]ϩ; [␣]D ϭ ϩ21.9o (c ϭ 1, CHCl3).
1H NMR (CDCl3), ␦, ppm: 0.94, 0.95 (2d, J ϭ 6.9 Hz,
3H), 1.38–1.80 (m, 6H), 2.02–2.15 (m, 1H); 2.45 (s, 3H),
3.17–3.29 (m, 1H), 3.42–3.64 (m, 2H), 3.69–3.81 (m, 1H),
3.92–4.10 (m, 2H), 4.40–4.50 (m, 1H), 7.30–7.40 (m, 2H),
7.74–7.84 (m, 2H); 13C NMR (CDCl3), ␦, ppm: 13.73,
13.80 (CH3); 19.43, 19.56 (CH2); 21.75 (CH3); 25.60, 30.60
(CH2); 33.65, 33.79, 62.10, 62.30, 68.12, 68.56 (CH); 72.38
(CH2); 98.74, 99.26, 128.05, 129.93 (CH); 133.28, 144.78
(C); IR (film), cmϪ1: 2943, 1598, 1454, 1361, 1177; [␣]D ϭ
ϩ4.39o (c ϭ 1, CHCl3); MS m/z: 329 [MϩH]ϩ.
2.5. (2ЈRS, 22RS, 23RS, 25R) 6␤-Methoxy-22-
phenylsulfonyl-26-(2-tetrahydropyranyloxy)-3a,5-cyclo-5a-
cholestan-23-ol (14)
To the stirred Ϫ70°C solution of sulfone 13 (205 mg,
0.43 mmol) in THF (6 ml) 1.6 M BuLi in hexane (0.27 ml,
0.43 mmol) was slowly added. After 15 min, a Ϫ70°C
solution of the aldehyde 12 (54 mg, 0.29 mmol) in THF (2
ml) was added dropwise. The reaction mixture was stirred
for an additional 15 min and sat. aqueous NH4Cl (2 ml) was
added, and the reaction mixture was extracted with Et2O
(3 ϫ 10 ml). Combined extracts were dried and evaporated.
The isomeric mixture of hydroxysulfones 14 (0.120 g, 65%)
was isolated by column chromatography. Analytical sample
was purified by column chromatography. Colorless foam.
Mixture of the isomers.
2.3. (2ЈRS, 3R) 3-Methyl-4-(2-tetrahydropyranyl-
oxy)butyronitrile (11)
A mixture of tosylate 10 (3.3 g, 10 mmol), KCN (3.9 g,
60 mmol), MeOH (240 ml), DMF (100 ml), and HMPA (20
ml) was heated at 80°C for 12 h. Methanol was evaporated
under reduced pressure leaving a residue, which was dis-
solved in ether (200 ml) and washed several times with
water and aqueous CuSO4. Drying and evaporation of the
solvents afforded crude product, which was purified by
short column chromatography. Yield 1.49 g (81%) of the
colorless oil.
1H NMR (CDCl3), ␦, ppm: 0.42 (dd, J ϭ 5.2 Hz, J ϭ 7.8
Hz, 1H), 0.57 (d, J ϭ 4.6 Hz, 3H), 0.64 (t, J ϭ 4.7 Hz, 1H),
0.70–2.12 (m, 37H), 2.18–2.33 (m, 1H), 2.70–2.77 (m, 1H),
3.30 (s, 3H), 3.04–3.36 (m, 2H), 3.92–3.95 (m, 4H), 4.30–
4.43 (m, 1H), 4.51–4.60 (m, 1H), 7.52–7.67 (m, 3H), 7.88–
7.95 (m, 2H); 13C NMR (CDCl3), ␦, ppm: 12.06 (CH3), 13.29
(CH2), 15.00, 15.15, 18.68 (CH), 19.42 (CH3), 19.77 (CH2),
21.62 (CH3), 22.90, 24.05, 25.14, 25.66, 28.31 (CH2), 30.46
(CH3), 30.74 (CH), 30.90, 33.55 (CH2), 35.17 (C), 35.39,
40.32, 41.98, 42.31 (CH2), 43.36, 43.55 (C), 46.40 (CH2),
48.07, 54.27, 56.64 (CH), 56.76 (CH3), 62.49 (CH2), 67.43,
71.62 (CH), 72.09, 72.56 (CH2), 82.45, 99.25, 99.54, 128.23,
128.74, 129.21, 129.51, 133.60 (CH), 140.55, 140.79, 140.92
(C). IR (KBr) cmϪ1: 2942, 1447, 1295, 1141, 1080, 1032; MS,
m/z: 657 [MϩH]ϩ; [␣]D ϭ ϩ17.6o (c ϭ 1, CHCl3).
1H NMR (CDCl3), ␦, ppm: 1.09, 1.10 (2d, J ϭ 6.8 Hz,
3H), 1.45–1.90 (m, 4H), 2.08–2.24 (m, 1H), 2.32–2.58 (m,
2H), 3.21 (dd, J ϭ 8.1 Hz, J ϭ 9.8 Hz, 1H), 3.38 (dd, J ϭ
4.8 Hz, J ϭ 9.9 Hz, 1H), 3.49–3.61 (m, 2H), 3.72–3.92 (m,
2H), 4.56–4.61 (m, 1H); 13C NMR (CDCl3), ␦, ppm:
16.10, 16.18 (CH3); 19.23, 19.39, 21.27, 25.28, 30.38, 30.95
(CH2); 61.95, 62.23 (CH); 70.06, 70.51 (CH2); 98.44, 98.17
(CH); 118.56 (C); IR (neat) cmϪ1: 2245; MS, m/z: 184
[MϩH]ϩ; [␣]D ϭ ϩ38.9o (c ϭ 1, CHCl3).
2.4. (2ЈRS, 3R) 3-Methyl-4-(2-tetrahydropyranyl-
oxy)butanal (12)
2.6. (2ЈRS, 25R) 6␤-Methoxy-26-(2-tetrahydro-
pyranyloxy)-3␣,5-cyclo-5␣-cholest-22-(E)-ene (15)
To a stirred solution of cyanide 11 (0.73 g, 4.0 mmol) in
toluene (20 ml) at Ϫ70°C, 1.5 M DIBAL in toluene (2.8 ml,
4.2 mmol) was slowly added over 0.5 h and the reaction
To a stirred mixture of hydroxysulfones 14 (320 mg,
0.49 mmol), THF (10 ml), and saturated solution of
Na2HPO4 in MeOH (10 ml) at 0°C, 5% sodium amalgam

404
T.E. D’Ambra et al. / Steroids 65 (2000) 401–407
[25] (2.25 g, 4.9 mmol) was added in small portions at 10
min intervals. When TLC showed no substrate liquid was
removed and mercury was washed with ether (2 ϫ 20 ml).
To the combined liquids, ether (40 ml), followed by water
(30 ml) were added. Layers were separated. Organic was
dried (MgSO4) and evaporated. Residue was chromato-
graphed to afford 15 (124 mg, 51%) as a colorless oil.
Mixture of isomers.
hexane/methylene chloride to give 1.81 g (81%) of colorless
crystals. m.p. ϭ 128–130°C; H NMR (␦, ppm): 0.67 (s,
1
3H), 0.91 (d, J ϭ 6.6 Hz, 3H), 1.01 (s, 3H), 0.95–2.10 (m,
H), 2.03 (s, 3H), 2.05 (s, 3H), 2.31 (d, J ϭ 7.8 Hz, H),
3.80–3.96 (m, 2H), 4.53–4.67 (m, 1H), 5.34–5.40 (m, 1H);
13C NMR (CDCl3) ␦, ppm: 12.14, 17.06, 18.93, 19.57
(CH3); 21.24, 21.28, 21.68, 23.49, 24.53, 28.01, 28.49
(CH2); 32.09, 32.12 (CH3); 32.73, 35.92, 36.26 (CH); 36.60
(C); 36.81, 37.22, 39.94 (CH2); 42.45 (C); 42.53 (CH2);
50.22, 56.27, 56.81 (CH); 69.76 (CH2); 74.12, 122.68 (CH);
139.66, 170.48, 171.26 (C); IR (KBr) cmϪ1: 2943, 1737,
1239, 1034; MS, m/z: 487 [MϩH]ϩ; [␣]D ϭ Ϫ41.1o (c ϭ
1, CHCl3).
1H NMR (CDCl3), ␦, ppm: 0.42 (dd, J ϭ 5.9 Hz, J ϭ 8.8
Hz, 1H), 0.66 (t, J ϭ 4.4 Hz, 1H), 0.74, 0.76 (2s, 3H), 0.90,
0.92 (2d, J ϭ 6.2 Hz, 3H), 1.00, 1.02, 1.04 (3s, 3H),
0.76–2.20 (m, 31H), 2.75–2.81 (m, 1H), 3.16–3.27 (m, 2H),
3.33 (s, 3H), 3.46–3.64 (m, 2H), 3.82–3.94 (m, 1H), 4.54–
4.62 (m, 1H), 5.17–5.34 (m, 2H); 13C NMR (CDCl3), ␦,
ppm: 12.67 (CH3); 13.29 (CH2); 17.09,19.51 (CH3); 19.76
(CH2); 20.86; 21.02 (CH); 21.72 (CH3); 22.99; 24.42;
25.19; 25.79; 28.99 (CH2); 30.71 (CH); 30.95; 33.60 (CH2);
34.04, 34.15 (CH); 35.30 (CH2); 35.55 (C); 36.94, 40.41
(CH2); 42.93 (C); 43.64 (C); 48.27, 56.22, 56.77 (CH);
56.86 (CH3); 62.29, 72.74 (CH2); 82.65, 99.03, 99.16,
125.52, 125.62, 138.95 (CH); MS, m/z: 499 [MϩH]ϩ.
2.9. (25R) Cholest-5-ene-3␤, 26-diol (1)
Diacetate 17 (265 mg, 0.54 mmol), methanol (15 ml) and
basic resin (50 mg) were stirred at room temperature, over-
night. The resin was filtered off and washed with methanol.
Filtrate and washings were evaporated to give 27-hydroxy-
cholesterol (1) (195 mg, 89%) as a white solid. m.p. ϭ
172–174°C; [␣]D ϭ Ϫ37.7o (c ϭ 1, CHCl3).
2.7. (2ЈRS, 25R) 6␤-Methoxy-26-(2-tetrahydro-
pyranyloxy)- 3␣,5-cyclo-5␣-cholestan (16)
2.10. (25R) [22,23 -3H]-Cholest-5-ene-3␤, 26-diol (5)
The mixture of olefins 15 (3.45 g, 6.92 mmol), 10% Pd-C
(300 mg), ethyl acetate (50 ml), and triethylamine (0.1 ml)
was stirred in a H2 atmosphere for 4 h. Catalyst was filtered
off, filtrate was evaporated and dried in vacuo to give 16
(3.39 g, 98%) as a white foam.
A mixture of the olefin 15 (25 mg, 0.05 mmol), anhy-
drous 1,4-dioxane (2.0 ml), and Pd/C (25 mg) was stirred in
tritium atmosphere for 2 h. After the catalyst was filtered
off, the filtrate was diluted with anhydrous 1,4-dioxane to
30 ml of final volume, to give 1800 mCi of tritium labeled
crude product (18). To 2.0 ml (120 mCi) of above solution,
p-TsOH.H2O (cat. amount) was added and resulted solution
was heated at 80°C for 5 h. At the end reaction mixture was
evaporated. The residue was dissolved in Et2O (5 ml) and
washed with NaHCO3 aq. and water. Further drying and
evaporation gave 100 mCi of 5, which was found to have
95% of the radiochemical purity. Final purification (50 mCi
scale) was done using HPLC.
1H NMR (CDCl3) ␦, ppm: 0.39 (t, J ϭ 5 Hz, 1H), 0.62
(t, J ϭ 5 Hz, 1H), 0.69 (s, 3H, CH3), 0.75–2.06 (m, 45 H),
0.88, 0.89 (2d, J ϭ 6.2 Hz, 3H), 0.99 (s, 3H, CH3), 2.70–
2.80 (m, 1H), 3.06–3.28 (m, 2H), 3.29 (s, 3H, OCH3),
3.41–3.52 (m, 2H), 3.78–3.90 (m, 1H), 4.52–4.58 (m, 1H);
13C NMR (CDCl3), ␦, ppm: 12.65 (CH3); 13.29 (CH2);
17.32, 19.44 (CH3); 19.80 (CH2); 20.86, 21.12 (CH); 21.83
(CH3); 23.05, 23.95, 24.40, 25.31, 25.70, 28.80 (CH2);
30.91 (CH); 30.99 (CH2); 33.22 (CH2); 33.99, 34.20 (CH);
35.20 (CH2); 35.55 (C); 36.88, 37.15 (CH2); 40.41 (CH2);
43.10 (C); 43.84 (C); 49.07, 56.31, 56.76 (CH); 56.90
(CH3); 63.11, 71.94, 72.46 (CH2); 82.60, 99.20, 99.22
(CH); MS, m/z: 501 [MϩH]ϩ.
3. Discussion
In 1994 Schroepfer’s group reported [24] the preparation
of 22, 23 di-(3H)-27-hydroxycholesterol (5) from compound
2.8. (25R) Cholest-5-ene-3␤,26-diol, diacetate (17)
The mixture of i-steroids 16 (2.4 g, 4.79 mmol), ZnCl2
dihydrate (1.58 g, 7.2 mmol), acetic anhydride (5 ml), and
acetic acid (30 ml) was heated at reflux for 2 h. The solvents
were evaporated; the residue was portioned between water
and CH2Cl2. The organic layer was separated, washed with
NaHCO3 aq., dried and evaporated. The residue was dis-
solved in a mixture of hexane/methylene chloride (8:1 v/v),
filtered through the bed of silica gel and eluted with mixture
of hexane/methylene chloride (30:1 v/v). Eluted fractions
were evaporated and the residue was recrystallized from
Fig. 2.

T.E. D’Ambra et al. / Steroids 65 (2000) 401–407
405
Fig. 3.
6. The olefin 6 was obtained in low yield from a byproduct
formed during preparation of 1 from 3 (Fig. 2).
Methods for introducing the 22,23 double bond by partial
synthesis are well described [26]. The method developed by
Julia and co-workers [27] utilized the readily available sul-
fones and carbonyl compounds (aldehydes or ketones).
Based on this approach we designed synthesis of the key
intermediate 6. Main features of our project are outlined in
Fig. 3.
Condensation of aldehyde iv with sulfone iii will effect the
formation of the hydroxysulfone ii. Subsequent reductive
elimination will provide the desired 22, 23-unsaturation.
For the synthesis of the C-23 through C-27 fragment, we
started with the commercially (Aldrich) available methyl
(S) (ϩ) 3-hydroxy-2-methyl propionate (7). The alcohol
function in 7 was protected as THP acetals (equimolar
mixture of diastereomers), then the resulting crude mixture
8 was subjected to LiAlH4 reduction to afford a mixture of
the two diastereomeric alcohols 9 in high overall yield
(82%)5 (Fig. 4).
Reaction of 9 with TsCl in pyridine provided the mixture
of diastereomeric tosylates 10 in excellent yield (95%).
Heating of 10 with excess of KCN in MeOH-DMF-HMPA
solution gave a mixture of cyanides 11 in 81% yield. The IR
spectrum of 11 showed an absorption corresponding to a
CN group at 2245 cmϪ1. Reduction of the cyanide 11 by
DIBAL-H provided mixture of the two aldehydes 12 (Fig. 5).
As a steroidal building block, we used sulfone 13 that
was prepared by known procedure from ergosterol [23,29].
To give the mixture of steroidal hydroxysulfones 14 in
65% yield, 13 was condensed with aldehydes 12 under Julia
1
conditions. H as well as 13C NMR spetra of 14 indicated
complex mixture of the products. Theoretically 8 diastero-
mers could be expected to form. TLC analysis showed three
main products. This mixture was partially resolved by chro-
matography and was shown (1H NMR, CI MS) to be in each
case a mixture of diastereomers. The main part of this mixture
was fully characterized (see Section 2). Reductive elimination
of the ␤-hydroxysulfone moiety in 14 by sodium amalgam
afforded a mixture of two olefins6 15 in moderate yield
(51%); the absorption corresponding to the formed double
bond was observed as a multiplets at 5.17–5.24 ppm.
One carbon homologation was achieved by replacement
of alcohol function by cyano group.
6 Such reductive elimination reactions of ␤-hydroxysulfones have been
known to produce predominantly or exclusively trans-olefin, see Ref. [30].
5 Opposite enantiomers of compounds 8–11 were described, see ref.
[28].
Fig. 4.

406
T.E. D’Ambra et al. / Steroids 65 (2000) 401–407
Fig. 5.
The synthesis of 1 from 15 was accomplished in three
Acknowledgments
steps. The alkene 15 was efficiently reduced by hydrogen
with Pd-C as a catalyst, in the presence of triethylamine,
to afford i-steroid 16, as a mixture of the THP acetals
(98% yield). Subsequent acid catalyzed (ZnCl2) ring
opening of 16 restored the original cholesterol policyclic
ring system to give diacetate 17 in 81% yield. Base
catalyzed (basic resin) methanolysis of 17 afforded 1 in
89% yield.
We would like to thank Professor Nina Berova and
Professor Koji Nakanishi of Columbia University for their
support for this work.
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