Preparation of (25R)- and (25S)-26-functionalized steroids as tools for biosynthetic studies of cholic acids

Article abstract of DOI:10.1016/j.steroids.2005.02.014

A new synthesis of both epimeric forms of 26-cholestanoic acids and 26-alcohols containing a 3β-hydroxy-Δ⁵- or a Δ⁴-3-keto-functionality in ring A is described starting from stigmasterol or (20S)-3β-acetoxy-pregn-5-en-20-carboxylic a

Full text of DOI:10.1016/j.steroids.2005.02.014

Steroids 70 (2005) 551–562
Preparation of (25R)- and (25S)-26-functionalized steroids
as tools for biosynthetic studies of cholic acids
Vladimir A. Khripacha,∗, Vladimir N. Zhabinskiia, Olga V. Konstantinovaa,
Natalya B. Khripacha, Alexey V. Antonchicka, Andrey P. Antonchicka,
Bernd Schneiderb
a
Laboratory of Chemistry of Steroids, Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus,
Kuprevich Str. 5/2, 220141 Minsk, Belarus
Max-Planck-Institute for Chemical Ecology, Beutenberg Campus, Hans Kno¨ll Str. 8, D-07745Jena, Germany
b
Received 2 November 2004; received in revised form 31 January 2005; accepted 9 February 2005
Available online 26 March 2005
Abstract
A new synthesis of both epimeric forms of 26-cholestanoic acids and 26-alcohols containing a 3␤-hydroxy-ꢀ5- or a ꢀ4-3-keto-functionality
in ring A is described starting from stigmasterol or (20S)-3␤-acetoxy-pregn-5-en-20-carboxylic acid. The obtained compounds are useful
as standards for studies of cholic acids. Construction of the side chain was achieved by linkage of steroidal 23-iodides to sulfones prepared
from (2R)- and (2S)-3-hydroxy-2-methylpropanoates. Oxidation of intermediate 26-alcohols into the corresponding carboxylic acids ensuring
preservation of stereochemistry at C-25 and functional groups in the cyclic part was achieved with sodium chlorite catalyzed by TEMPO and
bleach.
© 2005 Elsevier Inc. All rights reserved.
Keywords: Cholic acids; Cholestenoic acids; Biosynthesis; 26-Hydroxycholesterol
1. Introduction
in Zellweger syndrome is attributed to overproduction
and accumulation of cholestenoic acids [14]. Deficiency
of 3␤-hydroxy-ꢀ5-C27-steroid dehydrogenase/isomerase
results in chronic liver injury due to an overproduction or a
low clearance of cholenoic acids [15–17]. 3-Oxo-ꢀ4-steroid
5␤-reductase deficiency may result in severe neonatal
cholestasis associated with hypertyrosinemia [18,19].
An important part in the biosynthetic pathway is the
transformation of C26-cholestanoic acids by peroxisomal
␤-oxidation into the corresponding C24-acids [20]. The avail-
ability of a set of differently functionalized C26-cholestanoic
acids is a necessary prerequisite for analytical determination
of potential intermediates or metabolites [21–23]. We became
interested in the stereoselective synthesis of both epimeric
C26-acids containing a 3␤-hydroxy-ꢀ5- or ꢀ4-3-keto-
functionality in ring A (cholestenoic acid derivatives like 1
and 2) (Scheme 1). (25R)-Cholestenoic acid 1 itself is an
intermediate compound for elimination of excessive choles-
terol [24,25] and a signaling molecule for the regulation of
The past years have witnessed the growth in research
of various aspects of bile acids [1–5]. Although the main
intermediates of bile acid biosynthesis and metabolism were
established quite a long time ago [6,7], many subtle details
are still a matter of investigations. Deficiency of certain en-
zymes responsible for the transformation of cholesterol into
bile acids may lead to accumulation of intermediate products
having toxic effects and/or to the formation of unusual
compounds, which are normally absent [8–12]. For instance,
accumulation of 26-hydroxycholesterol in human atheroma
may reflect reduced levels of oxysterol 7␣-hydroxylase
activity in human macrophages. The same enzyme de-
termines the proportion of mono-, di-, and tri-hydroxy
bile acids synthesized in the liver [13]. Liver dysfunction
∗
Corresponding author. Tel.: +375 172 648 647; fax: +375 172 648 647.
E-mail address: khripach@iboch.bas-net.by (V.A. Khripach).
0039-128X/$ – see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2005.02.014

552
V.A. Khripach et al. / Steroids 70 (2005) 551–562
Scheme 1.
lipid metabolism via LXR␣ [26]. C26-acids with an enone
function in ring A are not only intermediates of bile acid
biosynthesis [27], but similar compounds of the stigmastane
series were obtained from products of microbiological
oxidation of ␤-sitosterol, e.g., with the genetically modified
Mycobacterium sp. [28]. Earlier syntheses of a number of
functionalized 26-cholestanoic acids should be mentioned in
this connection [29–34], but the demand for new ones or the
improved preparation of known compounds still remains.
(s, 3H, 18-H), 1.02 (s, 3H, 19-H), 2.04 (s, 3H, OAc), 3.36
(m, 2H, 22-H), 4.50–4.72 (m, 1H, 3-H), 5.37 (d, J = 4.5 Hz,
1H, 6-H). 13C NMR δ: 11.9, 16.8, 19.3, 21.0, 21.4, 24.4,
27.7, 31.9, 36.6, 37.0, 38.1, 38.7, 39.6, 42.4, 50.0, 52.4,
56.3, 56.4, 67.9, 74.0, 122.6, 139.7, 170.6. HRMS calc.
for C22H35O ([M − AcOH]+) 315.2688; found: 315.2687;
EIMS m/z: 328 (12), 314 ([M − AcOH]+, 100), 299 (10), 255
([M − AcOH C3H7O (fission C-17/C-20)]+, 5), 213 (6), 193
(12), 159 (5), 147 (12), 133 (7), 119 (7), 107 (10), 93 (7), 81
(9).
2. Experimental
2.2. 6β-Methoxy-3α,5-cyclo-5α-23,24-
bisnorcholan-22-ol (10)
Melting points were taken on a Boetius micro-melting
point apparatus and are uncorrected. IR spectra were recorded
on an UR-20 spectrophotometer in KBr tablets. Optical ro-
tations were measured on a JASCO J-20 polarimeter. 1H and
13C NMR spectra were taken on Bruker AC-200 (200 MHz
for 1H, 50 MHz for 13C) and Bruker AVANCE 400 (400 MHz
Starting with (20S)-6β-methoxy-3α,5-cyclo-5α-pregnan-
20-carboxylic acid 8 [48], the title compound was isolated
as an oil according to the procedure described above in 95%
yield. 1H NMR δ: 0.38 (dd, J = 7.9, 4.9 Hz, 1H, 3-H), 0.61 (t,
J = 4.8 Hz, 1H, 4-H), 0.69 (s, 3H, 18-H), 0.82 (d, J = 6.1 Hz,
3H, 21-H), 0.98 (s, 3H, 19-H), 2.73 (t, J = 2.4 Hz, 1H, 6-H),
3.28 (s, 3H, OMe), 3.29 (dd, J = 10.0, 7.5 Hz, 1H, 22-H), 3.60
(dd, J = 10.0, 3.0 Hz, 1H, 22-H). 13C NMR δ: 12.1, 12.9, 16.5,
19.0, 21.2, 22.5, 24.0, 24.9, 27.5, 30.2, 33.1, 34.5, 35.0, 38.5,
39.9, 42.6, 43.1, 47.7, 52.4, 56.0, 56.3, 67.6, 82.1.
1
for H, 100 MHz for 13C) spectrometers using TMS as an
internal standard in CDCl3 (if not stated otherwise). The ex-
act mass measurements were carried out on a Micromass
MasSpec mass spectrometer, operating in the 70 eV-EI mode.
Samples were introduced by direct probe for accurate mass
measurement by peak matching. (20S)-3␤-Acetoxy-pregn-5-
en-20-carboxylic acid was a gift from Steraloids Inc. (New-
port, RI, USA). Reactions were monitored by TLC using
aluminium or plastic sheets precoated with silica gel 60 F254
(Merck Art. 5715). Column chromatography was carried out
on silica gel 60 (Merck Art. 7734).
2.3. Tosylation of (9)
2.3.1. Variant A
A
mixture of alcohol 9 (21 g, 56 mmol), p-
toluensulfonylchloride (19 g, 100 mmol), and pyridine
(100 ml) was kept at room temperature for 12 h. Then, water
(450 ml) was slowly added to the reaction mixture, and it
was extracted with CHCl3. The combined organic fractions
were dried (Na2SO4), and the solvent was evaporated in
vacuo. The residue was chromatographed on SiO2 to give:
(a) 3β-acetoxy-22-chloro-23,24-bisnorchol-5-ene 13 (4.8 g,
22%). Mp 143–144 ◦C (hexane–EtOAc). IR (cm−1): 2950,
1755, 1480, 1450, 1380, 1385, 1260, 1045. 1H NMR δ: 0.71
(s, 3H, 18-H), 1.02 (s, 3H, 19-H), 1.10 (d, J = 6.5 Hz, 3H,
21-H), 2.04 (s, 3H, OAc), 2.32 (d, J = 8.0 Hz, 2H, 4-H),
3.42 (dd, J = 11.0, 6.0 Hz, 1H, 22-H), 3.60 (dd, J = 11.0,
2.5 Hz, 1H, 22-H), 4.61 (m, 1H, 3-H), 5.39 (d, J = 5.0 Hz,
1H, 6-H). 13C NMR δ: 12.0, 17.6, 19.3, 21.0, 21.4, 24.2,
27.5, 27.7, 31.8, 31.9, 36.5, 37.0, 38.1, 38.3, 39.4, 42.3,
49.9, 52.4, 52.8, 56.3, 73.9, 122.5, 139.6, 170.5. HRMS
calc. for C24H39ClO2 (M + 2H) 394.2638; found: 394.2630;
2.1. 3β-Acetoxy-23,24-bisnorchol-5-en-22-ol (9)
BH3·Me2S (2 M solution in THF, 200 ml) was added
dropwise to a solution of (20S)-3␤-acetoxy-pregn-5-en-20-
carboxylic acid 7 (130 g, 335 mmol) in THF (700 ml) at
−78 ◦C under argon. The reaction mixture was then stirred
at −78 ◦C for 30 min. After addition of methanol (150 ml),
the reaction mixture was allowed to warm to room temper-
ature, and the solvents were removed in vacuo. The residue
was dissolved in EtOAc, and the solution was passed rapidly
through a pad of SiO2 with EtOAc as eluent. The filtrate was
chromatographed on SiO2 to give alcohol 9 (80 g, 64%). Mp
155–157 ◦C (EtOAc) (lit. mp 143–148 ◦C [35], 154–155 ◦C
[36]). [α]D = −56.4(c0.004, CHCl3). IR(cm−1):2955, 1755,
1490, 1460, 1390, 1380, 1280, 1050, 990. 1H NMR δ: 0.70

V.A. Khripach et al. / Steroids 70 (2005) 551–562
553
EIMS m/z: 394 ([M + 2H]+, 0.6), 332 ([M − AcOH]+, 100),
317 ([M − AcOH CH3]+, 12), 224 (11), 211 (14), 159 (6),
147 (14), 107 (12); (b) 3β-acetoxy-22-toluenesulfonyloxy-
23,24-bisnorchol-5-ene 11 (19.6 g, 57 %). Mp 126–128 ◦C
(MeOH) (lit. mp 108–110 ◦C [36]). [α]D = −10.1 (c 0.005,
CHCl3). IR (cm−1): 2950, 2915, 1750, 1615, 1375, 1260,
in vacuo, and the residue was purified by column chromatog-
raphy on SiO2 to give nitrile 14 (56 g, 96%). Mp 180–182 ◦C
(hexane–EtOAc)(lit. mp186–187 ◦C[37], 179–181 ◦C[38]).
1
IR (cm−1): 2945, 2910, 1740, 1475, 1375, 1260, 1045. H
NMR δ: 0.68 (s, 3H, 18-H), 1.00 (s, 3H, 19-H), 1.15 (d,
J = 6.5 Hz, 3H, 21-H), 2.02 (s, 3H, OAc), 4.58 (m, 1H, 3-H),
5.35 (d, J = 5 Hz, 1H, 6-H). 13C NMR δ: 11.9, 19.7, 20.9, 21.4,
24.1, 24.8, 27.7, 28.0, 31.7, 33.6, 36.5, 36.9, 38.0, 39.3, 42.4,
49.8, 54.8, 56.4, 73.8, 119.0, 122.4, 139.6, 170.5. HRMS
calc. for C25H39NO2 (M + 2H) 385.2980; found: 385.2979.
EIMS m/z: 385 ([M + 2H]+, 0.4), 323 ([M − AcOH]+, 100),
308 ([M − AcOH CH3]+, 15), 215 (11), 202 (9), 145 (9),
121 (13), 107 (9), 91 (8).
1
1200, 1190, 1110, 1045. H NMR δ: 0.66 (s, 3H, 18-H),
1.00 (s, 3H, 19-H), 2.04 (s, 3H, OAc), 2.46 (s, 3H, OTs),
3.78 (dd, J = 9.5, 6.5 Hz, 1H, 22-H), 3.98 (dd, J = 9.5, 3 Hz,
1H, 22-H), 4.60 (m, 1H, 3-H), 5.37 (d, J = 4.0 Hz, 1H, 6-H),
7.34 (d, J = 8.0 Hz, 2H, OTs), 7.80 (d, J = 8.0 Hz, 2H, OTs).
13C NMR δ: 11.8, 16.9, 19.3, 20.9, 21.4, 21.7, 24.3, 27.4,
27.7, 31.8, 36.2, 36.6, 37.00, 38.1, 39.4, 42.4, 49.9, 51.8,
56.3, 73.9, 75.6, 122.5, 127.9, 129.8, 133.1, 139.7, 144.6,
170.6. HRMS calc. for C29H41O3S (M − AcOH) 469.2776;
found: 469.2788. EIMS m/z: 468 ([M − AcOH]+, 100), 453
([M − AcOH CH3]+, 6), 296 ([M − AcOH TsOH]+, 24),
281 (16), 255 ([C13H18O3S (C/D ring fission) + H]+, 7), 213
([C10H13O3S (fission C-17/C-20)]+, 11), 188 (10), 175 (13),
145 (18).
2.6. Synthesis of (14) from chloride (13)
According to the procedure described above for the trans-
formation of tosylate 11, but under more severe conditions
(100 ◦C, 10 h), the nitrile 14 was prepared from chloride 13
in 84% yield.
2.3.2. Variant B
2.7. 6β-Methoxy-3α,5-cyclo-5α-23,24-bisnorcholan-
22-nitrile (15)
A
mixture of alcohol
9
(70 g, 0.19 mol), p-
toluenesulfonylchloride (95 g, 0.5 mol), CHCl3 (100 ml),
and pyridine (70 ml) was kept at room temperature for 2
days. Then, water was added, and the mixture was extracted
with CHCl3. The combined chloroform fractions were dried
(Na2SO4), and the solvent was evaporated. The residue was
purified by column chromatography on SiO2 to give tosylate
11 (80 g, 81%).
The title compound was prepared in 89% yield as an oil
starting from 12 according to the procedure described for the
transformation of tosylate 11 to nitrile 14. 1H NMR δ: 0.43
(dd, J = 7.9, 4.9 Hz, 1H, 3-H), 0.64 (t, J = 4.7 Hz, 1H, 4-H),
0.73 (s, 3H, 18-H), 1.01 (s, 3H, 19-H), 1.15 (d, J = 6.7 Hz,
3H, 21-H), 2.20 (dd, J = 16.5, 6.7 Hz, 1H, 22-H), 2.32 (dd,
J = 16.5, 4.3 Hz, 1H, 22-H), 2.73 (t, J = 2.4 Hz, 1H, 6-H), 3.32
(s, 3H, OMe). 13C NMR δ: 12.3, 13.1, 19.2, 19.3, 21.4, 22.6,
24.0, 24.8, 24.9, 28.1, 30.5, 33.3, 33.5, 35.0, 35.1, 39.8, 42.8,
43.3, 47.8, 54.9, 56.2, 56.6, 82.3, 119.0.
2.4. 6β-Methoxy-22-toluenesulfonyloxy-
3α,5-cyclo-5α-23,24-bisnorcholane (12)
The title compound was isolated as an oil in 76% yield
starting with alcohol 10 according to the procedure described
above for tosylation of alcohol 9. 1H NMR δ: 0.42 (dd, J = 7.9,
4.9 Hz, 1H, 3-H), 0.63 (t, J = 4.8 Hz, 1H, 4-H), 0.66 (s, 3H,
18-H), 0.97 (d, J = 6.7 Hz, 3H, 21-H), 1.00 (s, 3H, 19-H),
2.44 (s, 3H, OTs), 2.75 (t, J = 2.4 Hz, 1H, 6-H), 3.30 (s, 3H,
OMe), 3.78 (dd, J = 9.2, 6.1 Hz, 1H, 22-H), 3.95 (dd, J = 9.2,
3.0 Hz, 1H, 22-H), 7.39 (d, 2H, J = 8.5 Hz, OTs), 7.77 (d, 2H,
J = 8.5 Hz, OTs). 13C NMR δ: 12.2, 13.0, 16.8, 19.2, 21.4,
21.6, 22.7, 24.1, 24.9, 27.5, 30.5, 33.3, 35.0, 35.2, 36.2, 39.9,
42.8, 43.3, 47.9, 51.9, 56.1, 56.5, 75.7, 82.3, 127.9, 129.7,
133.1, 144.5.
2.8. 3β-Hydroxy-23,24-bisnornorchol-5-en-
22-carbaldehyde (16)
DIBAL-H (1 M solution in toluene, 170 ml) was added
dropwise to a stirred solution of nitrile 14 (28 g, 73 mmol)
in absolute toluene (300 ml) at −78 ◦C under argon. After
30 min, EtOAc (50 ml) and water (100 ml) were added to
the reaction mixture, and it was allowed to warm to room
temperature. The reaction mixture was diluted with water
(200 ml) and concentrated HCl (80 ml) and extracted with
EtOAc. The combined organic fractions were washed with
saturated Na2CO3 solution and water, dried (Na2SO4), and
evaporated to give the crude aldehyde 16 (28 g) as an oil. IR
(cm−1): 2945, 1735, 1470, 1380, 1055. 1H NMR δ: 0.72 (d,
J = 6.0 Hz, 3H, 21-H), 1.02 (s, 3H, 18-H), 3.52 (m, 1H, 3-H),
5.35 (d, J = 5.0 Hz, 1H, 6-H), 9.76 (d, J = 2.0 Hz, 1H, CHO).
13C NMR δ: 11.9, 19.4, 20.0, 21.0, 24.2, 28.5, 31.6, 31.8,
36.5, 37.2, 39.6, 42.3, 42.5, 50.0, 50.9, 55.8, 56.7, 71.7, 76.4,
77.2, 96.1, 121.5, 140.8, 203.5.
2.5. 3β-Acetoxy-23,24-bisnorchol-5-en-22-nitrile (14)
A mixture of tosylate 11 (80 g, 0.15 mol) and NaCN (11 g,
0.2274 mol) in DMF (400 ml) was heated at 80 ◦C for 5 h.
After cooling to room temperature, the reaction mixture was
diluted with water and extracted with CHCl3. The combined
organic fractions were partly evaporated in vacuo and filtered
through a short column of SiO2. The filtrate was evaporated

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V.A. Khripach et al. / Steroids 70 (2005) 551–562
2.9. 6β-Methoxy-3α,5-cyclo-5α-23,24-bisnorcholan-
22-carbaldehyde (17)
with EtOAc. The combined organic extracts were dried
(Na2SO4) and evaporated in vacuo. The residue was pu-
rified by column chromatography on SiO2 to give: (a)
24-norchol-5-en-3␤,23-diol 3␤,23-ditosylate 20 (6.6 g, 16
%). Mp 119–120 ◦C (hexane–EtOAc). 1H NMR δ: 0.61
(s, 3H, 18-H), 0.84 (d, J = 6.5 Hz, 3H, 21-H), 0.96 (s, 3H,
19-H), 2.46 (s, 6H, OTs), 4.08 (m, 2H, 23-H), 4.32 (m,
1H, 3-H), 5.30 (brs, 1H, 6-H), 7.34 (dd, J = 8.0, 2.0 Hz,
4H, OTs), 7.80 (d, J = 8.0 Hz, 4H, OTs). 13C NMR δ:
11.7, 18.4, 19.1, 20.9, 21.6, 24.1, 28.0, 28.6, 31.7, 32.6,
34.8, 36.3, 36.8, 38.8, 39.5, 42.4, 49.8, 55.9, 56.5, 63.9,
68.9, 82.3, 123.4, 127.6, 127.8, 129.76, 129.80, 133.3,
134.7, 138.8, 144.4, 144.6. HRMS calc. for C30H42O3S
(M − TsOH): 482.2855; found: 482.2859. EIMS m/z: 482
([M − TsOH]+, 100), 467 ([M − TsOH CH3]+, 19), 361
(29), 310 ([M − 2TsOH]+, 14), 295 ([M − 2TsOH CH3]+,
16), 255 ([M − TsOH C11H15O3S (fission C-17/C-20)]+,
40), 213 (32), 189 (47), 172 ([TsOH]+, 73), 145 (59),
107 (74), 91 ([C6H4CH3]+, 99); (b) 24-norchol-5-en-
3␤,23-diol 23-tosylate 21 (18.5 g, 58%). Mp 114–118 ◦C
(MeOH–EtOAc). [α]D = −33.4 (c 0.004, CHCl3). IR (cm−1):
1050, 1060, 1105, 1180, 1195, 1365, 1470, 1605, 2945. 1H
NMR δ: 0.64 (s, 3H, 18-H), 0.84 (d, J = 6.5 Hz, 3H, 21-
H), 0.98 (s, 3H, 19-H), 2.45 (s, 3H, OTs), 3.53 (m, 1H,
3-H), 4.08 (m, 2H, 23-H), 5.34 (d, J = 5.0 Hz, 1H, 6-H),
7.36 (d, J = 8.0 Hz, 2H, OTs), 7.80 (d, J = 8.0 Hz, 2H, OTs).
13C NMR δ: 11.8, 18.4, 19.4, 21.0, 21.6, 24.2, 28.1, 31.6,
31.8, 32.6, 34.8, 36.4, 37.2, 39.7, 42.2, 42.4, 50.0, 55.9,
56.6, 69.0, 71.7, 121.5, 127.9, 129.8, 133.2, 140.8, 144.6.
HRMS calc. for C30H42O3S (M − H2O): 482.2839; found:
482.2855. EIMS m/z: 501 ([M + H]+, 99), 482 ([M − H2O]+,
100), 467 ([M − H2O CH3]+, 24), 415 (18.5), 389 (31), 328
([M − TsOH]+, 21), 295 (26), 255 ([M − H2O C11H15O3S
(fission C-17/C-20)]+, 29), 246 (36), 213 (50), 172 ([TsOH]+,
36), 159 (39), 145 (46).
The title compound was prepared in 82% yield as an oil
starting from nitrile 15 according to the procedure described
for the transformation of nitrile 14 into aldehyde 16. 1H NMR
δ: 0.40 (dd, J = 7.9, 4.9 Hz, 1H, 3-H), 0.62 (t, J = 4.7 Hz, 1H,
4-H), 0.74 (s, 3H, 18-H), 0.99 (d, J = 6.5 Hz, 3H, 21-H), 1.00
(s, 3H, 19-H), 2.15 (dd, J = 13.4, 3.1 Hz, 1H, 22-H), 2.44 (dd,
J = 13.4, 1.3 Hz, 1H, 22-H), 2.75 (t, J = 2.4 Hz, 1H, 6-H), 3.30
(s, 3H, OMe), 9.72 (dd, J = 3.1, 1.3 Hz, 1H, CHO). 13C NMR
δ: 12.3, 13.1, 19.3, 20.0, 21.5, 22.7, 24.1, 25.0, 28.6, 30.5,
31.9, 33.4, 35.1, 35.2, 40.1, 43.0, 43.4, 48.0, 50.9, 56.0, 56.5,
56.6, 82.3, 203.6.
2.10. 24-Norchol-5-en-3β,23-diol (18)
LiAlH4 (5.7 g, 149.88 mmol) was added to a stirred
solution of aldehyde 16 (28 g) in THF (400 ml), and the
stirring was continued for 30 min. The excess of LiAlH4
was decomposed with 15% NaOH aqueous solution, and
the obtained precipitate was filtered off. The filtrate was
evaporated, and the residue was purified by column chro-
matography on SiO2 to give diol 18 (22 g, 87% from 14).
Mp 144–147 ◦C (MeOH). IR (cm−1): 2945, 1480, 1390,
1
1065. H NMR δ: 0.69 (s, 3H, 18-H), 0.96 (d, J = 6.5 Hz,
3H, 21-H), 1.01 (s, 3H, 19-H), 3.44–3.80 (m, 3H, 3␣- and
23-H), 5.36 (d, J = 5.0 Hz, 1H, 6-H). 13C NMR δ: 11.8, 18.9,
19.4, 21.1, 24.3, 28.4, 31.6, 31.9, 32.9, 36.5, 37.3, 39.0, 39.8,
42.3, 42.4, 50.1, 56.4, 56.8, 60.9, 71.8, 121.7, 140.8. HRMS
calc. for C23H38O2: 346.2874; found: 346.2872. EIMS m/z:
346 ([M]+, 100), 331 ([M − CH3]+, 21), 328 ([M − H2O]+,
38), 313 ([M − H2O CH3]+, 21), 273 ([M − C4H9O (fission
C-17/C-20)]+, 15), 261 (27), 255 ([M − C4H9O (fission
C-17/C-20)-H2O]+, 16), 235 (36), 213 (24), 161 (19), 145
(24), 107 (32), 81 (27).
2.13. 6β-Methoxy-3α,5-cyclo-5α-24-norcholan-23-ol
23-tosylate (22)
2.11. 6β-Methoxy-3α,5-cyclo-5α-24-norcholan-23-ol
(19)
The title compound was prepared in 71% yield as an oil
The title compound was prepared in 86% yield as an oil
starting from aldehyde 17 according to the procedure de-
scribed for the transformation of aldehyde 16 into alcohol
starting from alcohol 19 according to the procedure described
for the tosylation of diol 18. H NMR δ: 0.42 (dd, J = 8.0,
1
4.9 Hz, 1H, 3-H), 0.63 (t, J = 4.8 Hz, 1H, 4-H), 0.66 (s, 3H,
18-H), 0.97 (d, J = 6.7 Hz, 3H, 21-H), 1.00 (s, 3H, 19-H),
2.44 (s, 3H, OTs), 2.75 (t, J = 2.4 Hz, 1H, 6-H), 3.31 (s, 3H,
OMe), 4.05 (m, 2H, 22-H), 7.34 (d, J = 8.5 Hz, 2H, OTs),
7.78 (d, J = 8.5 Hz, 2H, OTs). 13C NMR δ: 12.1, 13.0, 18.4,
19.2, 21.4, 21.8, 22.9, 24.0, 24.9, 28.1, 30.4, 32.8, 33.3, 34.8,
35.0, 35.2, 40.1, 42.8, 43.2, 47.9, 56.1, 56.4, 56.5, 67.0, 82.3,
127.9, 129.8, 133.3, 144.6.
1
18. H NMR δ: 0.42 (dd, J = 7.9, 4.9 Hz, 1H, 3-H), 0.63 (t,
J = 4.7 Hz, 1H, 4-H), 0.72 (s, 3H, 18-H), 0.92 (d, J = 6.1 Hz,
3H, 21-H), 1.01 (s, 3H, 19-H), 2.76 (t, J = 2.4 Hz, 1H, 6-H),
3.31 (s, 3H, OMe), 3.65 (m, 2H, 23-H). 13C NMR δ: 12.2,
13.0, 18.8, 19.3, 21.5, 22.7, 24.1, 24.9, 28.4, 30.4, 32.9, 33.3,
35.0, 35.2, 39.0, 40.2, 42.8, 43.3, 48.0, 56.5, 56.5, 60.9, 82.4.
2.12. Tosylation of (18)
2.14. 23-Iodo-24-norchol-5-en-3β-ol (23)
A mixture of diol 18 (22 g, 64 mmol), CHCl3 (200 ml),
pyridine (10 ml), and p-toluenesulfonylchloride (20 g,
105 mmol) was kept for 2 days at room temperature. Then,
the reaction mixture was diluted with water and extracted
A mixture of tosylate 21 (18.5 g, 37 mmol), KI (53 g,
319 mmol), and acetone (200 ml) was refluxed for 20 h.
After cooling, the reaction mixture was partly evaporated

V.A. Khripach et al. / Steroids 70 (2005) 551–562
555
1310, 1210, 1150, 1085, 1070, 1040. 1H NMR δ: 3.08–3.82
(m, 3H, 26-H and OTHP), 4.36–4.68 (m, 1H, OTHP), 5.35
(d, J = 4.5 Hz, 1H, 6-H), 7.58 (m, 3H, Ph), 7.90 (m, 2H, Ph).
HRMS calc. for C33H50O4S ([M − THP + 1H]+): 542.3430;
found: 542.3448. EIMS m/z: 542 ([M − THP + 1H]+, 22),
524 ([M − THPOH]+, 76), 509 (M − THPOH CH3]+,
20), 431 (15), 383 ([M − THPOH SO2Ph]+, 16), 352
([C19H29O4S (fission C-17/C-20)-1H]+, 7), 271 (20), 255
([M − C19H29O4S (fission C-17/C-20)-H2O]+, 38), 225
(30), 213 (45), 143 ([SO2Ph + 2H]+, 56), 84 ([THP H]+,
64%).
in vacuo, diluted with water, and extracted with CHCl3.
The combined extracts were dried (Na2SO4), and the sol-
vents were evaporated in vacuo. The residue was puri-
fied by column chromatography on SiO2 to give iodide 23
(15 g, 89%). Mp 130–132 ◦C (MeOH). IR (cm−1): 2945,
2910, 2870, 2855, 1645, 1480, 1390, 1230, 1200, 1065. 1H
NMR δ: 0.69 (s, 3H, 18-H), 0.93 (d, J = 6.0 Hz, 3H, 21-H),
1.00 (s, 3H, 19-H), 3.10 (m, 1H, 23-H), 3.31 (m, 1H, 23-
H), 3.52 (m, 1H, 3-H), 5.36 (d, J = 5.0 Hz, 1H, 6-H). 13C
NMR δ: 5.1, 11.9, 17.9, 19.4, 21.0, 24.2, 28.1, 31.6, 31.8,
36.5, 36.9, 37.2, 39.7, 40.3, 42.3, 42.4, 50.0, 55.6, 56.7,
71.7, 121.6, 140.7. HRMS calc. for C23H37OI: 456.1889;
found: 456.1891. EIMS m/z: 456 ([M]+, 100), 442 (25), 438
([M − H2O]+, 31), 423 ([M − H2O CH3]+, 22), 371 (21),
345 (27), 331 (7), 273 ([M − C4H8I (fission C-17/C-20)]+, 5),
255 ([M − H2O C4H8I (fission C-17/C-20)]+, 8), 213 (11),
145 (15), 107 (19).
2.17. (25R)-6β-Methoxy-24-phenylsulfonyl-3α,5-cyclo-
5α-cholestan-26-ol 26-(2ꢀ-tetrahydropyranyl) ether
(27)
The title compound was prepared in 78% yield as an
oil from iodide 24 and sulfone 25 according to the pro-
cedure described above for the preparation of sulfone 26.
IR (cm−1): 1030, 1080, 1145, 1200, 1300, 1380, 1445,
2875, 2945. 1H NMR δ: 0.42 (dd, J = 8.0, 5.5 Hz, 1H,
4-H), 2.76 (s, 1H, 6-H), 3.30 (s, 3H, OMe), 3.36–3.80
(m, 4H, 26-H and OTHP), 4.34–4.66 (m, 1H, OTHP),
7.48–7.68 (m, 3H, Ph), 7.90 (m, 2H, Ph). HRMS calc. for
C39H60O5S [M]+: 640.4163; found: 640.4161. EIMS m/z:
640 ([M]+, 12), 625 ([M − CH3]+, 9), 608 ([M − CH3OH]+,
6), 585 (16), 556 ([M − THP + H]+, 16), 539 (10), 524
([M − THP CH3O]+, 35), 498 ([M − SO2Ph H]+, 8), 383
(8), 255 ([M − CH3OH C19H29O4S (fission C-17/C-20)]+,
21), 213 (12), 143 (12), 95 (15), 85 ([THP H]+, 100).
2.15. 23-Iodo-6β-methoxy-3α,5-cyclo-5α-
24-norcholane (24)
The title compound was prepared in 98% yield starting
from tosylate 22 according to the procedure described for the
transformation of tosylate 21 into iodide 23. Mp 93–95 ◦C
(hexane–EtOAc) (lit. mp 104–105 ◦C [39]). IR (cm−1): 2950,
2870, 1475, 1455, 1385, 1330, 1200, 1160, 1100, 1015. 1H
NMR δ: 0.44 (dd, J = 8.0, 5.5 Hz, 1H, 3-H), 0.66 (t, J = 4.5 Hz,
1H, 4-H), 0.74 (s, 3H, 18-H), 0.93 (d, J = 6.0 Hz, 3H, 21-H),
1.03 (s, 3H, 19-H), 2.78 (t, J = 2.7 Hz, 1H, 6-H), 3.02–3.38
(m, 2H, 23-H), 3.33 (s, 3H, OMe). 13C NMR δ: 5.1, 12.2,
13.0, 17.8, 19.3, 21.4, 22.7, 24.1, 24.9, 28.2, 30.4, 33.3,
35.0, 35.2, 37.2, 40.2, 40.4, 42.9, 43.3, 47.9, 55.8, 56.4,
56.5, 82.3. HRMS calc. for C24H39OI: 470.2047; found:
470.2046. EIMS m/z: 470 ([M]+, 70), 455 ([M − CH3]+, 57),
438 ([M − MeOH]+, 100), 423 ([M − MeOH CH3]+, 13),
415 (96), 317(11), 255 (7), 213 (8), 145 (18), 95 (22).
2.18. (25S)-Cholest-5-en-3β,26-diol
26-(2ꢀ-tetrahydropyranyl) ether (28)
A solution of sulfone 26 (2.9 g, 7.3 mmol) in THF (35 ml)
was added to the blue colored mixture of liquid ammo-
nia (150 ml), lithium (0.5 g, 73.17 mmol) and THF (30 ml)
at −78 ◦C and stirred for 1.5 h. The excess of lithium was
quenched by addition of NH4Cl. When the reaction mixture
became white, the cooling bath was removed, and the re-
action mixture was allowed to warm to room temperature
and left until the ammonia completely evaporated. Then, wa-
ter was added to the residue, and the mixture was extracted
with CHCl3. The organic fractions were dried (Na2SO4) and
evaporated in vacuo. The residue was purified by column
chromatography on SiO2 to give ether 28 (1.5 g, 67%) as
an oil. IR (cm−1): 1040, 1065, 1085, 1130, 1210, 1360,
1385, 1450, 1475. 1H NMR δ: 0.68 (s, 3H, 18-H), 1.02
(s, 3H, 19-H), 3.06–3.30 (m, 1H, 26-H), 3.42–3.68 (m,
3H, OTHP and 26-H), 3.80–3.94 (m, 1H, OTHP), 4.58 (d,
J = 3.0 Hz, 1H, 3-H), 5.36 (d, J = 5.0 Hz, 1H, 6-H). HRMS
calc. for C32H54O3: 486.4073; found: 486.4067. EIMS m/z:
486([M]+, 11), 468([M − H2O]+, 71), 402([M − THP + H]+,
21), 384 ([M − THPOH]+, 48), 366 ([M − THPOH H2O]+,
21), 351 (12), 271 ([M − C13H25O2 (fission C-17/C-27)-
2.16. (25R)-24-Phenylsulfonylcholest-5-en-3β,26-diol
26-(2ꢀ-tetrahydropyranyl) ether (26)
A 2.5 M BuLi (11.4 ml, 28.5 mmol) solution was added
i
to a stirred solution of Pr2NH (4 ml, 28.5 mmol) in THF
(15 ml) at −10 ◦C. The mixture was warmed to 0 ◦C
and stirred for 5 min. A solution of 2-[(2R)-2-methyl-
3-phenylsulfonylpropyloxy]tetrahydro-2H-pyran 25 (5.1 g,
17.1 mmol) (prepared according to [40]) in THF (20 ml) was
i
added to the obtained solution of Pr2NLi at −78 ◦C. This
was followed by the addition of a solution of steroidal io-
dide 23 (2.6 g, 5.7 mmol) in THF (15 ml) and HMPA (5.8 ml)
over 10 min. After stirring at −78 to −65 ◦C for 20 min, the
reaction mixture was allowed to warm to room temperature
and left overnight. Then, saturated NH4Cl was added, and the
mixture was extracted with CHCl3. The organic extracts were
dried (Na2SO4) and evaporated in vacuo. The residue was pu-
rified by column chromatography on SiO2 to give sulfone 26
(2.9 g, 82%) as an oil. IR (cm−1): 2945, 2875, 1460, 1390,

556
V.A. Khripach et al. / Steroids 70 (2005) 551–562
2H]+, 34), 255 (16), 213 ([C13H25O2 (fission C-17/C-27)]+,
19), 159 (21), 145 (23), 107 (23), 85 ([THP-H)+, 100).
was neutralized with a saturated solution of Na2CO3 and
extracted with EtOAc. The combined organic fractions were
dried (Na2SO4) and evaporated in vacuo. The residue was
purified by column chromatography on SiO2 to give acetate
33 (1.1 g, 39%). Mp 129–131 ◦C (MeOH). 1H NMR δ: 0.61
(s, 3H, 18-H), 0.95 (s, 3H, 19-H), 1.98 (s, 3H, OAc), 2.25 (d,
J = 8.0 Hz, 2H, 4-H), 3.40 (m, 2H, 26-H), 4.54 (m, 1H, 3-H),
5.32 (d, J = 4.5 Hz, 1H, 6-H). 13C NMR δ: 12.3, 17.1, 19.1,
19.7, 21.4, 21.8, 23.9, 24.7, 28.2, 28.6, 32.3, 34.1, 36.2, 36.7,
38.5, 40.2, 42.7, 50.5, 56.3, 56.5, 57.1, 68.6, 68.8, 74.4, 123.0,
140.1, 170.9. HRMS calc. for C29H48O3 ([M]+): 444.3603;
found: 444.3604. EIMS m/z: 384 ([M − AcOH]+, 100), 370
([M − H2O AcOH]+, 47), 263 (8), 255 ([C19H28 (fission C-
17/C-20)-AcOH]+, 11), 213 (8), 159 (6.5), 147 (17), 133 (8),
105 (11), 81 (14).
2.19. (25S)-6β-Methoxy-3α,5-cyclo-5α-cholestan-26-ol
26-(2ꢀ-tetrahydropyranyl) ether (29)
The title compound was prepared in 72% yield as an oil
from 27 according to the procedure described above for the
preparation of 28 from 26. IR (cm−1): 1040, 1085, 1105,
1125, 1190, 1210, 1280, 1330, 1360, 1385, 1460, 1475, 2875,
2945. 1H NMR δ: 0.44 (dd, J = 8.5, 5.5 Hz, 1H, 3-H), 0.64 (t,
J = 4.5 Hz, 1H, 4-H), 0.71 (s, 3H, 18-H), 1.04 (s, 3H, 19-H),
2.78 (brs, 1H, 6-H), 3.08–3.28 (m, 1H, 26-H), 3.33 (s, 3H,
OMe), 3.44–3.68 (m, 3H, OTHP and 26-H), 3.80–3.94 (m,
1H, OTHP), 4.58 (m, 1H, 3-H). HRMS calc. for C33H56O3:
500.4245; found: 500.4229. EIMS m/z: 500 ([M]+, 41), 485
([M − CH3]+, 30), 468 ([M − CH3OH]+, 50), 445 (43), 416
([M − THP + H]+, 6), 384 ([M − THP CH3O]+, 15), 366
(7), 255 ([M − C13H25O2 (fission C-17/C-20)-CH3OH]+,
12), 213 ([C13H25O2 (fission C-17/C-20)]+, 8), 95 (14), 85
([THP H]+, 100).
2.23. (25R)-Cholest-5-en-3β,26-diol
((25R)-26-hydroxycholesterol) (34)
HCl (37%, 0.4 ml) was added to a stirred solution of
tetrahydropyranyl ether 32 (200 mg, 0.41 mmol) in THF
(1.5 ml) and MeOH (1.5 ml), and stirring was continued
for 15 min. After addition of pyridine (0.4 ml), the reac-
tion mixture was evaporated. The residue was dissolved
in CHCl3, washed with saturated NaHCO3 solution, dried
(Na2SO4), and evaporated. The obtained oily product was pu-
rified by column chromatography on SiO2 (toluene–EtOAc,
20:1 ⇒ 2:1) to give 34 (90 mg, 54%). Mp 172–174 ◦C
(hexane–EtOAc) (lit. mp 169–170 ◦C [41], 177–178 ◦C [44],
175–177 ◦C [42]). IR (cm−1): 2945, 2880, 1755, 1695, 1480,
2.20. (25S)-24-Phenylsulfonylcholest-5-en-3β,26-diol
26-(2ꢀ-tetrahydropyranyl) ether (31)
The title compound was prepared in 79% yield
as an oil from iodide 23 and 2-[(2S)-2-methyl-3-
phenylsulfonylpropyloxy]tetrahydro-2H-pyran 30 as de-
scribedaboveforthesynthesisof 26from 23and25. 1HNMR
δ: 3.09–3.82 (m, 3H, 26-H and OTHP), 4.35–4.68 (m, 1H,
OTHP), 5.36 (d, J = 4.5 Hz, 1H, 6-H), 7.58 (m, 3H, arom.),
7.90 (m, 2H, arom.).
1
1390, 1285, 1065. H NMR δ: 0.68 (s, 3H, 18-H), 0.93 (d,
J = 6.5 Hz, 3H, 21-H), 1.02 (s, 3H, 19-H), 3.36–3.63 (m, 3H,
3- and 26-H), 5.36 (d, J = 5.0 Hz, 1H, 6-H). 13C NMR δ: 11.9,
16.5, 18.7, 19.4, 21.1, 23.4, 24.3, 28.3, 31.7, 31.9, 33.6, 35.7,
35.8, 36.2, 36.5, 37.3, 39.8, 42.3, 50.1, 56.2, 56.8, 68.5, 71.8,
121.7, 140.8. HRMS calc. for C27H46O2: 402.34978; found:
402.34974. EIMS m/z: 402 ([M]+, 100), 387 ([M − CH3]+,
20), 384 ([M − H2O]+, 50), 369 ([M − H2O CH3]+, 19), 317
(19), 291 (25), 273 ([M − C8H17O (fission C-17/C-20)]+,
10), 255 ([M − C8H17O (fission C-17/C-20)-H2O]+, 12), 213
(12), 145 (14), 107 (13).
2.21. (25R)-Cholest-5-en-3β,26-diol
26-(2ꢀ-tetrahydropyranyl) ether (32)
The title compound was prepared in 68% yield as an oil
from sulfone 31 according to the procedure described above
for the preparation of 28 from 26. 1H NMR δ: 0.69 (s, 3H, 18-
H), 1.02 (s, 3H, 19-H), 3.08–3.28 (m, 1H, 26-H), 3.42–3.66
(m, 3H, OTHP and 26-H), 3.78–3.94 (m, 1H, OTHP), 4.58
(m, 1H, 3-H), 5.35 (d, J = 4.5 Hz, 1H, 6-H). HRMS calc.
for C32H54O3: 486.4073; found: 486.4080. EIMS m/z: 486
([M]+, 6), 468 ([M − H2O]+, 38), 402 ([M − THP + H]+, 14),
384 ([M − THPOH]+, 38), 366 ([M − THPOH H2O]+, 18),
351 (6), 271 ([M − C13H25O2 (fission C-17/C-27)-2H]+, 18),
255 (13), 213 ([C13H25O2 (fission C-17/C-27)]+, 15), 159
(17), 145 (21), 107 (22), 85 ([THP H]+, 100).
2.24. (25S)-Cholest-5-en-3β,26-diol((25S)-26-
hydroxycholesterol) (41)
The title compound was isolated in 62% yield from
tetrahydropyranyl ether 28 according to the procedure de-
scribed above for the preparation of 34 from 32. Mp
160–162 ◦C (MeOH) (lit. mp 171–174 ◦C [43]). [α]D = −50
(c 0.003, MeOH). IR (cm−1): 2945, 2875, 1640, 1475,
1385, 1060. 1H NMR (CDCl3:CD3OD) δ: 0.68 (s, 3H,
18-H), 0.92 (d, J = 7.0 Hz, 3H, 21-H), 1.03 (s, 3H, 19-H),
3.27–3.61 (m, 3H, 3- and 26-H), 5.34 (d, J = 4.0 Hz, 1H, 6-
H). 13C NMR (CDCl3:CD3OD) δ: 11.9, 16.7, 18.8, 19.4,
21.1, 23.5, 24.3, 28.3, 31.3, 32.0, 33.8, 35.8, 35.9, 36.3,
36.6, 37.3, 39.9, 42.0, 42.4, 50.2, 56.0, 56.2, 56.9, 67.9,
2.22. (25S)-3β-Acetoxy-cholest-5-en-26-ol (33)
The mixture of compound 29 (3.2 g, 6.4 mmol) and acetic
acid (30 ml) was refluxed for 1.5 h. After cooling down to
room temperature, 37% HCl (2 ml) and water (1 ml) were
added to the reaction mixture, and the resulting mixture was
stirred at room temperature for 1 h. Then, the reaction mixture

V.A. Khripach et al. / Steroids 70 (2005) 551–562
557
71.4, 121.6, 140.9. HRMS calc. for C27H46O2: 402.3495;
found: 402.3498. EIMS m/z: 402 ([M]+, 100), 388 (34), 384
([M − H2O]+, 48), 369 ([M − H2O CH3]+, 22), 317 (22),
291 (32), 273 ([M − C8H17O (fission C-17/C-20)]+, 17), 255
([M − C8H17O (fission C-17/C-20)-H2O]+, 18), 213 (18),
145 (14), 107 (13.5).
solved in THF (25 ml), and MeOH (25 ml) and HCl (37%,
3 ml) were added. The mixture was stirred at room tempera-
ture for 30 min. After addition of pyridine (3.5 ml), the sol-
vents were evaporated. The residue was dissolved in CHCl3,
washed with saturated NaHCO3 solution, dried (Na2SO4),
and evaporated. The obtained product was purified by col-
umn chromatography on SiO2 to give acetate 37 (1.6 g, 89%)
as an oil. 1H NMR δ: 0.62 (s, 3H, 18-H), 0.94 (s, 3H, 19-H),
2.00 (s, 3H, OAc), 2.26 (d, J = 8.0 Hz, 2H, 4-H), 3.90 (m,
2H, 26-H), 4.54 (m, 1H, 3-H), 5.32 (d, J = 4.5 Hz, 1H, 6-H).
13C NMR δ: 12.3, 17.0, 19.1, 19.7, 21.8, 21.4, 23.7, 24.7,
28.2, 32.3, 33.1, 34.0, 36.1, 37.0, 37.4, 38.5, 40.1, 42.7, 50.5,
56.5, 57.1, 68.9, 73.4, 74.4, 123.0, 140.1, 170.9. HRMS calc.
for C29H46O2 ([M − H2O]+): 426.3484; found: 426.3498.
EIMS m/z: 426 ([M − H2O]+), 384 ([M − AcOH]+, 100), 370
([M − H2O AcOH]+, 9), 281 (7), 255 ([C19H28 (fission C-
17/C-20)-AcOH]+, 8), 231 (8), 181 (14), 147 (10), 131 (17),
119 (15), 107 (7.5), 81 (9.5).
2.25. (25R)-Cholest-4-en-3-on-26-ol (38)
A mixture of alcohol 32 (0.4 g, 0.82 mmol), cyclohex-
anone (0.4 ml), and toluene (50 ml) was heated under ni-
trogen until about 10 ml of the solvents as an azeotropic
mixture were distilled off to remove traces of water. Then,
Al(OiPr)3 (168 mg, 0.82 mmol) was added, and the reaction
mixture was refluxed for 2 h. After cooling to room tem-
perature, methanol (2 ml) was added to the reaction mix-
ture, and the solvents were evaporated in vacuo. The ob-
tained tetrahydropyranyl ether 36 was dissolved in methanol
(5 ml), and HCl (37%, 1.2 ml) was added dropwise to this
solution. After 1 h, pyridine (0.6 ml) was added to the reac-
tion mixture, and the solvents were concentrated in vacuo.
The residue was mixed with a saturated solution of NaHCO3
and extracted with CHCl3. The combined organic fractions
were dried (Na2SO4) and evaporated in vacuo. The residue
was purified by column chromatography on SiO2 to give
enone 38 (214 mg, 65%). Mp 124–126 ◦C (acetone) (lit.
mp 129–131 ◦C [46]). IR (cm−1): 2940, 1675, 1475, 1385,
2.28. (25S)-3β-Acetoxy-cholest-5-en-26-ol (33)
The title compound was prepared as an oil in 75% yield
from 28 according to the procedure described above for the
preparation of 37 from 32.
2.29. (25R)-3β-Hydroxy-cholest-5-en-26-oic acid (39)
1
1340, 1240, 1200, 1050. H NMR δ: 0.71 (s, 3H, 18-H),
TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl, 50 mg,
0.32 mmol) and phosphate buffer (1.3 ml, pH 6.86) were
added to a solution of alcohol 37 (1.6 g, 3.6 mmol) in a mix-
ture of THF (20 ml) and CH3CN (10 ml). Then, solutions
of NaClO2 (650 mg in 5 ml H2O) and NaClO (0.2 ml in 2 ml
H2O)wereaddedsimultaneouslyat35 ◦Ctothismixtureover
0.5 h. The reaction mixture was stirred at this temperature for
5 h, then cooled to room temperature, and mixed with a cold
saturated solution of Na2SO3. After 15 min, an additional
amount of phosphate buffer was added, and the reaction mix-
ture was extracted with EtOAc. The combined organic frac-
tions were dried (Na2SO4) and evaporated in vacuo. The ob-
tained crude acetate was dissolved in THF (10 ml) and treated
with a 3% solution of KOH in MeOH (13 ml) for 15 min at
room temperature. Then, acetic acid (0.4 ml) and pyridine
(five drops) were added, and the reaction mixture was fil-
tered through a short pad of SiO2. The filtrate was concen-
trated in vacuo and purified by column chromatography on
SiO2 togiveacid 39(820 mg, 55%). Mp175–177 ◦C(MeOH)
(lit. mp 168–170 ◦C [46]). [α]D = −28 (c 0.002, MeOH). IR
(cm−1): 1065, 1230, 1390, 1480, 1720, 2875, 2945. 1H NMR
(500 MHz) δ: 0.67 (s, 3H, 18-H), 0.90 (d, J = 6.4 Hz, 3H,
21-H), 1.00 (s, 3H, 19-H), 1.17 (d, J = 7.2 Hz, 3H, 27-H),
2.38–2.50 (m, 1H, 25-H), 3.53 (m, 1H, 3-H), 5.34 (brs, 1H, 6-
H). 13C NMR δ: 11.5, 16.4, 18.3, 19.0, 20.8, 23.3, 24.0, 27.9,
31.3, 31.6, 33.6, 35.3, 35.4, 36.2, 36.9, 38.9, 39.5, 41.9, 42.0,
49.8, 55.8, 56.4, 71.5, 121.4, 140.4, 181.7. HRMS calc. for
C26H44O ([M − CO2]+): 372.3392; found: 372.3398. EIMS
m/z: 416 ([M]+, 100), 398 (59), 331 (23), 305 (50), 273 (14),
0.92 (d, J = 6.5 Hz, 6H, 21- and 27-H), 1.18 (s, 3H, 19-H),
3.46 (m, 1H, 26-H), 5.74 (s, 1H, 4-H). 13C NMR δ: 11.9,
16.5, 17.3, 18.6, 21.0, 23.4, 24.1, 28.2, 32.0, 32.9, 33.5,
33.9, 35.56, 35.64, 35.8, 36.1, 38.6, 39.6, 42.4, 53.8, 55.8,
56.0, 68.4, 123.7, 171.8, 199.8. HRMS calc. for C27H44O2:
400.3341; found: 400.3338. EIMS m/z: 400 ([M]+, 100), 385
([M − CH3]+, 5), 358 ([M − CH3 CO + H]+, 16), 277 (13),
229 (19), 124 (35).
2.26. (25S)-Cholest-4-en-3-on-26-ol (43)
The title compound was obtained from 28 in 63% yield
as an oil according to the procedure described above for the
synthesis of 38 from 32. IR (cm−1): 2940, 1670, 1470, 1385,
1
1340, 1245, 1210. H NMR δ: 0.70 (s, 3H, 18-H), 1.19 (s,
3H, 19-H), 3.46 (m, 2H, 26-H), 5.73 (s, 1H, 4-H). 13C NMR
(125 MHz) δ: 17.3, 18.7, 21.0, 23.5, 24.2, 28.2, 32.1, 32.9,
33.7, 34.0, 35.6, 35.70, 35.74, 35.8, 36.2, 38.6, 39.6, 42.4,
53.8, 55.9, 56.1, 68.3, 123.7, 171.8, 199.6.
2.27. (25R)-3β-Acetoxy-cholest-5-en-26-ol (37)
A mixture of alcohol 32 (2 g, 4.1 mol), pyridine (3 ml),
Ac2O (0.8 ml, 8.2 mmol), and DMAP (2 mg) was stirred at
room temperature for 2 h. Then, the reaction mixture was
treated with saturated NaHCO3 solution and extracted with
CHCl3. The organic fractions were dried (Na2SO4) and evap-
orated in vacuo to give crude 35. The crude product was dis-

558
V.A. Khripach et al. / Steroids 70 (2005) 551–562
Scheme 2.
255 ([C19H28 (fission C-17/C-20)-H2O]+, 19), 213 (23), 159
(16), 145 (21), 107 (24), 81 (21).
17.4, 19.0, 19.7, 21.4, 24.1, 24.6, 28.6, 32.3, 32.0, 34.4, 36.0,
36.9, 37.6, 39.6, 40.1, 42.7, 50.5, 56.5, 57.1, 72.2, 122.1,
141.1, 181.8. HRMS calc. for C25H41O ([M − C2H3O2]+):
357.3157; found: 357.3161. EIMS m/z: 416 ([M]+, 100), 398
(60), 383 (28), 331 (25), 305 (48), 291 (11), 273 (14), 255
([C19H28 (fission C-17/C-20)-H2O]+, 20), 213 (24), 159 (15),
145 (25), 140 (34), 107 (23), 81 (20).
2.30. (25S)-3β-Hydroxy-cholest-5-en-26-oic acid (42)
The title compound was obtained from 33 in 48% yield by
the procedure described above for the synthesis of compound
39. Mp 157–160 ◦C (MeOH). IR (cm−1): 1030, 1060, 1200,
1245, 1390, 1480, 1725, 2875, 2950. 1H NMR δ: 0.66 (s, 3H,
18-H), 0.92 (d, J = 6.5 Hz, 3H, 21-H), 1.02 (s, 3H, 19-H),
1.19 (d, J = 7.2 Hz, 3H, 27-H), 2.46 (m, 1H, 25-H), 3.51 (m,
1H, 3-H), 5.36 (d, J = 5.0 Hz, 1H, 6-H). 13C NMR δ: 12.2,
2.31. (25R)-Cholest-4-en-3-on-26-oic acid (40)
TEMPO (6 mg, 0.04 mmol) and phosphate buffer (2 ml,
pH 6.86) were added to a solution of alcohol 38 (0.16 g,
Scheme 3.

V.A. Khripach et al. / Steroids 70 (2005) 551–562
559
Scheme 4.
0.40 mmol) in a mixture of THF (2 ml) and CH3CN (1 ml).
2.32. (25S)-Cholest-4-en-3-on-26-oic acid (44)
Then, solutions of NaClO2 (70 mg in 0.5 ml H2O) and Na-
ClO (1.3 ml of water solution with 2–3% of active Cl) were
added simultaneously to this mixture at 35 ◦C. The reaction
mixture was stirred at this temperature for 5 h, then cooled
to room temperature, and mixed with a cold saturated so-
lution of Na2SO3. After 15 min, an additional amount of
phosphate buffer was added, and the reaction mixture was
extracted with CHCl3. The combined organic fractions were
dried (Na2SO4) and evaporated in vacuo. The residue was
purified by column chromatography on SiO2 to give acid 40
The title compound was obtained from alcohol 43 in
51% yield by the procedure described above for the syn-
thesis of compound 40. Mp 172–175 ◦C (EtOAc). 1H NMR
(500 MHz) δ: 0.70 (s, 3H, 18-H), 0.90 (d, J = 6.4 Hz, 3H, 21-
H), 1.20 (s, 3H, 19-H), 2.22–2.50 (m, 3H, 2- and 25-H), 5.73
(s, 1H, 4-H). 13C NMR (125 MHz) δ: 18.6, 21.0, 23.7, 24.2,
28.2, 32.1, 33.0, 33.9, 34.0, 35.61, 35.64, 35.71, 35.73, 38.6,
39.3, 39.6, 42.4, 53.8, 55.9, 56.1, 123.8, 171.8, 181.9, 199.7.
(80 mg, 48%). Mp 147–148 ◦C (hexane–EtOAc). H NMR
1
δ: 0.70 (s, 3H, 18-H), 0.90 (d, J = 6.4 Hz, 3H, 21-H), 1.20
(s, 3H, 19-H), 1.20 (d, J = 7.2 Hz, 3H, 27-H), 5.73 (s, 1H,
4-H). HRMS calc. for C26H41O ([M − COOH]+): 369.3156;
found: 369.3157. EIMS m/z: 414 ([M]+, 100), 400 (15), 369
([M − COOH]+), 291 (16), 271 ([C19H27O (fission C-17/C-
20)]+, 11), 229 (43), 147 (16), 124 (67), 107 (15), 95 (19).
3. Results and discussion
Syntheticstudieson26-functionalizedsteroidshavealong
history, and early attempts of their stereoselective preparation
were made in the 1950’s [44]. There are several common ap-
proaches to the 26-functionalized steroids with a chiral center
Scheme 5.

560
V.A. Khripach et al. / Steroids 70 (2005) 551–562
at C-25. The simplest variant is the use of the appropriate
starting material where this stereocenter is already present. A
few steps are necessary to get the desired functionality, but in
practice, only one epimer can be obtained in such a way (e.g.,
(25R)-26-hydroxy cholesterol from diosgenin [45]). Access
to both diastereomers is possible via the corresponding
achiral precursors (e.g., 25-enes [46]). Most syntheses of
26-functionalized steroids are based on transformation of
derivatives with a preformed carbon side chain skeleton. In
our opinion, better results can be achieved by using a conver-
gent scheme when a low-molecular building block is attached
to a steroidal part (Scheme 2). Similar methodology was used
for the preparation of related deuterium labeled compounds
[47].
Preparation of the desired compounds requires formation
ofboththesidechainandthecyclicpart. Thereareatleasttwo
possibilities to achieve the final goal with respect to formation
of the cyclic part, and we decided to study both of them
(Scheme 3). The first one makes use of the commercially
available acid 7, and the second one relies on the acid 8 [48]
available in a few steps from stigmasterol. First stages in both
cases were essentially the same and were directed to the one-
carbon homologation of the side chain and introduction of the
23-iodine. Reduction of acids 7 and 8 with borane proceeded
smoothly. Even in the case of 7, which had a ꢀ5-double bond,
the desired reduction product 9 was isolated in a reasonable
(64%) yield. Tosylation of the primary alcohol 9 by treatment
with TsCl in pyridine gave poor results because of formation
Scheme 6.

V.A. Khripach et al. / Steroids 70 (2005) 551–562
561
of a considerable amount (up to 22%) of 22-chloride 13 along
with the desired tosylate 11 (57%). Both 11 and 13 could be
used for preparation of nitrile 14 at the next stage, but for
practical considerations, formation of 13 should be avoided.
In this respect, tosylation of alcohols 9 and 10 in CHCl3 in
the presence of pyridine proved to be more efficient. The
hydride reductions of nitriles 14 and 15 with DIBAL-H and
then, aldehydes 16 and 17 with LiAlH4 proceeded smoothly
and gave the expected 23-alcohols 18 and 19.
Synthesis of 23-iodides 23 and 24 was carried out by tosy-
lation of 18 and 19 followed by nucleophilic substitution of
the corresponding tosylates with KI. There was no problem
with 19 which contained only one hydroxyl group. Tosylation
of 18 proceeded with the formation of di- and monotosylates
20 and 21, but the primary hydroxyl group was more reactive,
and the desired monotosylate 21 could be obtained in up to
58% yield.
mass measurements, and Emily Wheeler (Jena) for linguistic
support in the preparation of this manuscript.
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