A new approach to the side chain formation of 24-alkyl-22-hydroxy steroids: Application to the preparation of early brassinolide biosynthetic precursors

Article abstract of DOI:10.1016/j.steroids.2012.03.010

A new synthetic route to 22S-hydroxy-24R-methyl steroids has been developed and applied for the preparation of cathasterone, (22S)-hydroxycampesterol, and 6-deoxocathasterone, which are precursors in the early stages of the biosynthesis of brassinolide. The construction of the steroid side chain with the correct stereochemistry at C-24 is based on the use of Claisen rearrangement. The introduction of the 22-hydroxyl group has been achieved by epoxidation of the Δ²²-double bond, nucleophilic opening of the intermediate mesyl epoxide with sodium sulfide, and desulfurization of the formed tetrahydrothiophenes with Raney nickel.

Full text of DOI:10.1016/j.steroids.2012.03.010

Steroids 77 (2012) 780–790
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
A new approach to the side chain formation of 24-alkyl-22-hydroxy steroids:
Application to the preparation of early brassinolide biosynthetic precursors
⇑
Alaksiej L. Hurski, Vladimir N. Zhabinskii, Vladimir A. Khripach
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Kuprevich Str., 5/2, 220141 Minsk, Belarus
a r t i c l e i n f o
a b s t r a c t
Article history:
A new synthetic route to 22S-hydroxy-24R-methyl steroids has been developed and applied for the prep-
aration of cathasterone, (22S)-hydroxycampesterol, and 6-deoxocathasterone, which are precursors in
the early stages of the biosynthesis of brassinolide. The construction of the steroid side chain with the
correct stereochemistry at C-24 is based on the use of Claisen rearrangement. The introduction of the
Received 18 January 2012
Received in revised form 19 March 2012
Accepted 21 March 2012
Available online 2 April 2012
22-hydroxyl group has been achieved by epoxidation of the
D
22-double bond, nucleophilic opening of
the intermediate mesyl epoxide with sodium sulfide, and desulfurization of the formed tetrahydrothi-
ophenes with Raney nickel.
Keywords:
Brassinosteroids
Cathasterone
Ó 2012 Elsevier Inc. All rights reserved.
(22S)-22-Hydroxycampesterol
6-Deoxocathasterone
Deuterium labeling
1. Introduction
However, the subsequent transformation into the desired
24-alkyl-22-hydroxy derivatives (involving
D
22-bond epoxidation
The 22-hydroxy steroids 1–3 are known biosynthetic precursors
of the steroidal plant hormone brassinolide [1] (Fig. 1). These
compounds and their deuterated analogs are useful tools for
studies of the initial steps of the biosynthetic transformations of
campesterol into brassinolide. A number of synthetic routes have
been developed for the preparation of 24-alkyl-22-hydroxy ste-
roids. One of the first was based on the coupling of the steroidal
moiety with an appropriate C23–C28 fragment [2]. The use of race-
mic 2,3-dimethylbutylmagnesium bromide is an evident drawback
of this method. Somewhat better results were obtained with a
chiral C23–C28 fragment, prepared from commercial (R)-Roche
ester [3]. The major problems of this approach were connected
with the use of relatively expensive reagents and a rather lengthy
synthetic sequence.
followed by epoxide reduction) suffered from a low overall
efficiency owing to the low regio- and stereoselectivity of these
reactions [6]. The main task of the present work was to develop
an improved synthetic route to 24-alkyl-22-hydroxy steroids,
using products of the Claisen rearrangement as key intermediates.
2. Experimental
2.1. General
Melting points were recorded on a Boetius micro-melting point
apparatus and are uncorrected. 1H and 13C NMR spectra were
obtained using a Bruker AVANCE 500 (Bruker Biospin, Rheinstet-
ten, Germany) spectrometer in CDCl3 operating at 500 MHz for
1H and 125 MHz for 13C. Chemical shifts were determined relative
to the residual solvent peaks (CHCl3, d = 7.26 for hydrogen atoms,
d = 77.00 for carbon atoms). Mass spectra were performed on a
LCQ Fleet mass spectrometer (Thermo Electron Corporation, USA)
with an APCI source. Spectra were collected in a positive ion mode
and analyzed by Xcalibur software. Chemicals were purchased
from Aldrich and Fluka and used as received. Reactions were
monitored by TLC using aluminum or plastic sheets, silica gel 60
F254 precoated (Merck Art. 5715). Column chromatography was
carried out on Kieselgel 60 (Merck Art. 7734).
Among the large number of steroidal side chain syntheses,
those making use of the Claisen rearrangement proved to be very
effective and were repeatedly used for the preparation of
D
22-24-
alkyl steroids with a predefined stereochemistry at C-24 [4,5].
Abbreviations: DMM, dimethoxymethane; Na2EDTA, disodium ethylenediamine
tetraacetate; MOM, methoxy methyl; (DHQD)2AQN, hydroquinidine (anthraqui-
none-1,4-diyl) diether; brsm, based on recovered starting material; DMP, dime-
thoxy propane; DIPA, diisopropanolamine; PPTS, pyridinium p-toluenesulfonate;
MCPBA, 3-chloroperoxybenzoic acid.
⇑
Corresponding author. Tel./fax: +375 172 678 647.
E-mail address: khripach@iboch.bas-net.by (V.A. Khripach).
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.03.010

A.L. Hurski et al. / Steroids 77 (2012) 780–790
781
Fig. 1. Structures of early brassinolide biosynthetic precursors: cathasterone 1, (22S)-22-hydroxy campesterol 2, and 6-deoxocathasterone 3.
2.2. Synthesis of compounds
69.58, 109.87, 124.99, 133.13. MS (APCI+) m/z (%): 415 ([M+H]+,
24), 397 ([MꢀH2O+H]+, 100).
2.2.1. Addition of lithium methyl acetylide to (20S)-6-(1,3-dioxolan-2-
yl)-3a,5-cyclo-20-formyl-5a-pregnane (5)
2.2.3. (22E,24R,25R)- and (22E,24R,25S)-6-(1,3-Dioxolan-2-yl)-24-
Methylacetylene (20 mL, 0.32 mol) was condensed into THF
(75 mL), which was a cooled to ꢀ65 °C. Then 1.6 M BuLi (57 mL,
91 mmol) was added at ꢀ65 °C. After keeping at this temperature
methyl-3a,5-cyclo-5a-cholest-22-en-26-oic acid ethyl esters [(25R)-
8a and (25S)-8a]
A solution of allylic alcohol 7 (10.0 g, 20.1 mmol), triethyl ortho-
propionate (80 mL, 0.4 mol) and propionic acid (2 mL, 27 mmol) in
benzene (250 mL) was refluxed under argon for 2 h. Then the solu-
tion was cooled to room temperature, diluted with water (100 mL)
and extracted with EtOAc (3 ꢁ 30 mL). The organic layer was dried
(Na2SO4) and evaporated. The residue was chromatographed on
SiO2 (cyclohexane–EtOAc = 30:1)10:1) to give the ester 8a
(8.5 g, 75%, an oil) as a mixture of 25R- and 25S-isomers (ratio
5.4:1 based on the ratio of integral intensity peaks with d 129.96
and 130.40 or 137.71 and 136.89). 1H NMR: d 0.31 (t, J = 4.2 Hz,
1H, C4–H), 0.60 (dd, J = 8.2, 4.6 Hz, 1H, C4–H), 0.71 (s, 3H, C18–
H), 0.95 (d, J = 6.8 Hz, 3H), 0.99 (s, 3H, C19–H), 1.06 (d, J = 6.9 Hz,
3H), 1.24 (d, J = 7.1 Hz, 3H, C27–H), 3.73 (q, J = 6.6 Hz, 1H, –OCH2-
CH2O–), 3.83 (q, J = 6.4 Hz, 1H, –OCH2CH2O–), 3.86–3.94 (m, 1H,
–OCH2CH2O–), 4.01 (dt, J = 8.0, 6.0 Hz, 1H, –OCH2CH2O–), 4.04–
4.17 (m, 2H, –OCH2CH3), 5.08 (dd, J = 15.1, 8.5 Hz, 1H, C22– or
C23–H), 5.18–5.29 (m, 1H, C23– or C22–H). 13C NMR d: 7.25,
12.32, 13.85, 14.26, 15.04, 17.23, 18.95, 19.30, 20.65, 20.77,
22.55, 23.02, 24.12, 24.18, 24.88, 28.53, 28.73, 29.67, 33.21,
34.08, 39.23, 39.39, 39.98, 40.16, 40.26, 42.73, 45.14, 45.52,
45.58, 47.42, 55.83, 56.31, 59.95, 60.00, 64.59, 64.83, 77.25,
109.85, 109.93, 129.96, 130.40, 136.89, 137.71, 175.84, 176.30.
MS (APCI+) m/z (%): 499 ([M+H]+, 100).
for 10 min, a solution of (20S)-6-(1,3-dioxolan-2-yl)-3a,5-cyclo-20-
formyl-5a-pregnane 5 (11.0 g, 29.6 mmol, prepared according to
[7]) was added. The mixture was stirred at ꢀ65 °C for 3 h, and then
NH4Cl (10 g) was added. After warming to room temperature, the
mixture was diluted with water (150 mL) and extracted with CHCl3
(3 ꢁ 75 mL). The organic layer was dried (Na2SO4) and evaporated.
The residue was chromatographed on SiO2 (petroleum ether–
EtOAc = 30:1)10:1) to give:
(22R)-6-(1,3-Dioxolan-2-yl)-3a,5-cyclo-26,27-bisnor-5a-cholest-
23-yn-22-ol 6 (7.7 g, 63%) as an oil. 1H NMR d: 0.32 (t, J = 4.2 Hz, 1H,
C4–H), 0.60 (dd, J = 8.1, 4.6 Hz, 1H, C4–H), 0.72 (s, 3H, C18–H), 1.00
(s, 3H, C19–H), 1.09 (d, J = 6.6 Hz, 3H, C21–H), 1.85 (d, J = 2.2 Hz,
3H, C25–H), 3.74 (q, J = 6.6 Hz, 1H, –OCH2CH2O–), 3.80–3.86 (m,
1H, –OCH2CH2O–), 3.89 (dt, J = 7.9, 6.3 Hz, 1H, –OCH2CH2O–),
4.02 (dt, J = 7.9, 6.0 Hz, 1H, –OCH2CH2O–), 4.43 (s, 1H, C22–H).
13C NMR d: 3.55, 7.24, 12.11, 13.14, 18.95, 22.60, 23.01, 24.15,
24.88, 27.57, 33.20, 34.14, 39.21, 39.84, 40.13, 42.29, 42.70,
45.57, 47.32, 51.79, 56.02, 64.59, 64.84, 65.63, 80.37, 81.11,
109.85. MS (APCI+) m/z (%): 413 ([M+H]+, 35), 395 ([MꢀH2O+H]+,
100).
(22S)-6-(1,3-Dioxolan-2-yl)-3a,5-cyclo-26,27-bisnor-5a-cholest-
23-yn-22-ol (3.8 g, 31%) as an oil. 1H NMR d: 0.32 (t, J = 4.2 Hz, 1H,
C4–H), 0.61 (ddd, J = 8.3, 4.6, 1.8 Hz, 1H, C4–H), 0.72 (s, 3H, C18–H),
1.00 (s, 3H, C19–H), 1.03 (d, J = 6.5 Hz, 1H, C21–H), 1.85 (d,
J = 2.2 Hz, 3H, C25–H), 3.74 (q, J = 6.6 Hz, 1H, –OCH2CH2O–),
3.80–3.86 (m, 1H, –OCH2CH2O–), 3.89 (dt, J = 7.9, 6.3 Hz, 1H,
–OCH2CH2O–), 4.02 (dt, J = 7.9, 6.0 Hz, 1H, –OCH2CH2O–), 4.42 (d,
J = 16.9 Hz, 1H, C22–H).
2.2.4. (22R,23R,24S,25R)-6-(1,3-Dioxolan-2-yl)-22-hydroxy-24-
methyl-3a,5-cyclo-5a-cholestano-26,23-lactone (9)
To a stirred solution of isomeric mixture of 8a (25R:25S = 5.4:1)
in t-BuOH (18 mL) and water (18 mL), K2CO3 (0.79 g, 5.72 mmol),
MsNH2 (0.27 g, 2.84 mmol), (DHQD)2AQN (81 mg, 0.189 mmol),
K3Fe(CN)6 (1.87 g, 5.68 mmol) and K2OsO4ꢂ2H2O (7 mg,
0.019 mmol) were added. The reaction mixture was stirred at room
temperature for 48 h, diluted with water (40 mL) and acidified to
pH 6 with tartaric acid. The water layer was separated and ex-
tracted with EtOAc (3 ꢁ 20 mL). The combined organic layers were
dried (Na2SO4), concentrated in vacuo and the residue was purified
by column chromatography on silica gel (petroleum ether–
EtOAc = 9:1)1:1) to give lactone 9 (0.26 g, 49%) as white crystals.
Mp 236–238 °C (MeCN). 1H NMR d: 0.33 (t, J = 4.2 Hz, 1H, C4–H),
0.61 (dd, J = 8.1, 4.6 Hz, 1H, C4–H), 0.74 (s, 3H, C18–H), 0.90 (d,
J = 7.1 Hz, 3H, C28–H), 1.00 (d, J = 6.9 Hz, 3H, C21–H), 1.01 (s, 3H,
C19–H), 1.17 (d, J = 7.2 Hz, 3H, C27–H), 2.50 (dp, J = 6.9, 4.2 Hz,
1H, C24–H), 2.86 (p, J = 7.1 Hz, 1H, C25–H), 3.75 (dd, J = 13.4,
6.5 Hz, 1H, –OCH2CH2O–), 3.81–3.87 (m, 2H, C22-H, –OCH2CH2O–
), 3.90 (td, J = 7.9, 6.2 Hz, 1H, –OCH2CH2O–), 4.03 (td, J = 7.7,
6.2 Hz, 1H, –OCH2CH2O–), 4.34 (dd, J = 8.9, 4.3 Hz, 1H, C23–H).
13C NMR d: 7.25, 8.55, 9.56, 12.14, 12.56, 18.95, 22.58, 23.05,
23.91, 24.86, 27.43, 33.20, 34.19, 35.91, 36.17, 39.19, 40.06,
40.11, 41.59, 42.55, 45.54, 47.26, 52.03, 56.01, 64.62, 64.86,
2.2.2. (22S,23Z)-6-(1,3-Dioxolan-2-yl)-3a,5-cyclo-26,27-bisnor-5a-
cholest-23-en-22-ol (7)
A mixture of propargyl alcohol 6 (6.0 g, 14.5 mmol), 5% Pd on
BaSO4 (0.9 g), quinoline (1.0 mL), ethanol (70 mL) and THF
(10 mL) was stirred under H2 until the gas adsorption had ceased.
The catalyst was removed by filtration, the solvent was evaporated
and the residue was chromatographed on SiO2 (hexane–
EtOAc = 40:1)10:1) to give allylic alcohol 7 (4.2 g, 70%) as an oil.
1H NMR d: 0.32 (t, J = 4.2 Hz, 1H, C4–H), 0.60 (dd, J = 8.2, 4.6 Hz,
1H, C4–H), 0.71 (s, 3H, C18–H), 0.95 (d, J = 6.3 Hz, 3H, C21–H),
1.00 (s, 3H, C19–H), 1.65 (dd, J = 6.6, 1.3 Hz, 1H, C25–H), 3.74 (q,
J = 6.5 Hz, 1H, –OCH2CH2O–), 3.80–3.86 (m, 1H, –OCH2CH2O–),
3.90 (dt, J = 8.0, 6.3 Hz, 1H, –OCH2CH2O–), 4.02 (dt, J = 8.0, 6.0 Hz,
1H, –OCH2CH2O–), 4.52–4.61 (m, 1H, C22–H), 5.45–5.59 (m, 1H,
C22– and C23–H). 13C NMR d: 7.25, 12.03, 12.31, 13.41, 18.95,
22.58, 23.04, 24.16, 24.88, 27.81, 33.19, 34.15, 39.21, 40.00,
40.17, 41.90, 42.75, 45.56, 47.34, 52.50, 56.11, 64.60, 64.84,

782
A.L. Hurski et al. / Steroids 77 (2012) 780–790
71.37, 83.71, 109.83, 178.21. MS (APCI+) m/z (%): 487 ([M+H]+,
100), 469 ([MꢀH2O+H]+, 15).
(Na2SO4), concentrated and the residue was purified by column
chromatography on silica gel (petroleum ether–EtOAc = 9:1)1:1)
to give mesylate 12 (23 mg, 79%) as an oil. 1H NMR d: 0.32 (t,
J = 4.2 Hz, 1H, C4–H), 0.60 (dd, J = 8.1, 4.6 Hz, 1H, C4–H), 0.73 (s,
3H, C18–H), 0.88 (d, J = 6.8 Hz, 1H, >CHCH3), 0.89 (d, J = 6.4 Hz,
3H, >CHCH3), 1.00 (s, 1H, C19–H), 1.11 (d, J = 6.8 Hz, 3H, >CHCH3),
3.00 (s, 3H, OMs), 3.34 (d, J = 8.2 Hz, 1H, C23–H), 3.43 (s, 3H, CH3O-
CH2O–), 3.71 (d, J = 8.2 Hz, 1H, C22–H), 3.74 (dd, J = 13.5, 6.6 Hz,
1H, –OCH2CH2O–), 3.82–3.86 (m, 1H, –OCH2CH2O–), 3.90 (dt,
J = 7.9, 6.3 Hz, 1H, –OCH2CH2O–), 3.99–4.05 (m, 1H, –OCH2CH2O–
), 4.18 (dd, J = 9.6, 6.4 Hz, 1H, C26–H), 4.39 (dd, J = 9.6, 3.9 Hz,
1H, C26–H), 4.64 (d, J = 6.5 Hz, 1H, CH3OCH2O–), 4.76 (d,
J = 6.5 Hz, 1H, CH3OCH2O–). 13C NMR d: 7.24, 9.72, 12.13, 12.69,
15.50, 18.93, 22.51, 23.02, 24.11, 24.86, 28.40, 33.19, 34.09,
35.60, 36.27, 36.99, 37.50, 39.17, 40.12, 42.65, 45.54, 47.33,
52.03, 55.81, 56.25, 64.61, 64.87, 68.05, 71.81, 73.76, 88.49,
99.31, 109.79. MS (APCI+) m/z (%): 613 ([M+H]+, 9), 581
([MꢀMeOH+H]+, 100), 551 [MꢀMeOHꢀCH2O+H]+, 21).
2.2.5. (22R,23R,24S,25R)-6-(1,3-Dioxolan-2-yl)-22-methoxymethyl-
24-methyl-3a,5-cyclo-5a-cholestano-26,23-lactone (10)
A 2 M solution of MOMCl in toluene (0.6 mL, 1.2 mmol) was
added to a mixture of alcohol 9 (99 mg, 0.203 mmol), N,N-diisopro-
pylethylamine (0.32 mL, 1.84 mmol) and Bu4NI (15 mg,
0.041 mmol) in THF (1.5 mL). The reaction mixture was stirred at
room temperature overnight and quenched with saturated
NaHCO3 (5 mL). The water layer was separated and extracted with
EtOAc (3 ꢁ 5 mL). The combined organic layers were dried
(Na2SO4), concentrated and the residue was purified by column
chromatography on silica gel (petroleum ether–EtOAc = 9:1)3:1)
to give ether 10 (91 mg, 85%) as an oil. 1H NMR d: 0.32 (t,
J = 4.0 Hz, 1H, C4–H), 0.61 (dd, J = 8.2, 4.5 Hz, 1H, C4–H), 0.74 (s,
3H, C18–H), 0.89 (d, J = 6.9 Hz, 3H, C28–H), 0.98–1.02 (m, 6H,
C19– and C21–H), 1.15 (d, J = 7.1 Hz, 3H, C27–H), 2.43 (td, J = 7.0,
4.3 Hz, 1H, C24–H), 2.78 (p, J = 7.0 Hz, 1H, C25–H), 3.43 (s, 3H,
CH3OCH2O–), 3.66 (d, J = 9.5 Hz, 1H, C22–H), 3.74 (q, J = 6.6 Hz,
1H, –OCH2CH2O–), 3.84 (q, J = 6.3 Hz, 1H, –OCH2CH2O–), 3.87–
3.93 (m, 1H, –OCH2CH2O–), 4.02 (ddd, J = 11.7, 6.6, 0.9 Hz, 1H,
–OCH2CH2O–), 4.41 (dd, J = 9.4, 3.9 Hz, 1H, C23–H), 4.67 (d,
J = 6.8 Hz, 1H, CH3OCH2O–), 4.87 (d, J = 6.8 Hz, 1H, CH3OCH2O–).
13C NMR d: 7.25, 8.24, 9.53, 12.05, 13.17, 18.93, 22.55, 23.05,
24.05, 24.86, 27.59, 33.22, 34.16, 36.36, 36.67, 39.20, 40.06,
40.26, 41.35, 42.65, 45.54, 47.29, 52.11, 56.12, 56.34, 64.61,
64.86, 77.97, 83.94, 97.89, 1i09.80, 178.51. MS (APCI+) m/z (%):
2.2.8. (22E,24R)-6-(1,3-Dioxolan-2-yl)-26-methanesulfonyloxy-24-
methyl-3a,5-cyclo-5a-cholest-22-ene (13a)
A solution of ester 8a (0.39 g, 0.78 mmol) in Et2O (5 mL) was
added to an ice cooled stirred suspension of LiAlH4 (30 mg,
0.78 mmol) in Et2O (2 mL). The reaction mixture was stirred at
room temperature for 30 min, quenched at 0 °C with water
(0.11 mL) and filtered. The filter cake was washed thoroughly with
EtOAc (3 ꢁ 5 mL). The filtrate was concentrated under reduced
pressure and the residue was chromatographed on silica gel
(petroleum ether–EtOAc = 9:1)7:3) to give the 26-alcohol as an
oil (0.34 g). It was dissolved, without further purification, in CH2Cl2
(7 mL), and Et3N (0.5 mL, 3.6 mmol) and MsCl (0.1 mL, 1.3 mmol)
were added at 0 °C. The reaction mixture was stirred at room tem-
perature for 10 min and quenched with saturated aqueous NaH-
CO3. The water layer was separated and extracted with EtOAc.
The combined organic layers were dried (Na2SO4) and concen-
trated. The residue was purified by column chromatography on sil-
ica gel (petroleum ether–EtOAc = 9:1)7:3) to give mesylate 13a
(0.39 g, 94%) as an oil. 1H NMR d: 0.32 (t, J = 4.1 Hz, 1H, C4–H),
0.61 (dd, J = 8.1, 4.6 Hz, 1H, C4–H), 0.72 (s, 3H, C18–H), 0.90 (t,
J = 6.7 Hz, 3H, >CHCH3), 0.99 (d, J = 5.3 Hz, 3H, >CHCH3), 1.00 (s,
1H, C19–H), 2.98, 2.99 (s, 3H, OMs), 3.74 (q, J = 6.6 Hz, 1H, –OCH2-
CH2O–), 3.84 (dd, J = 12.5, 6.4 Hz, 1H, –OCH2CH2O–), 3.94–3.86 (m,
1H, –OCH2CH2O–), 4.06–3.96 (m, 2H, C26–H), 4.11 (dd, J = 9.4,
6.3 Hz, 1H, –OCH2CH2O–), 5.19–5.10 (m, 1H, C22– or C23–H),
5.28–5.20 (m, 1H, C23– or C22–H). 13C NMR d: 7.24, 12.34, 12.50,
13.91, 17.30, 18.57, 18.96, 20.76, 22.57, 23.01, 24.19, 24.88,
28.93, 29.68, 33.20, 34.07, 37.21, 37.66, 38.08, 38.56, 39.21,
39.95, 40.13, 40.34, 42.73, 45.58, 47.37, 55.70, 55.82, 56.27,
64.60, 64.84, 73.46, 73.57, 109.85, 128.67, 130.50, 137.50, 138.29.
MS (APCI+) m/z (%): 535 ([M+H]+, 100).
531
([M+H]+,
25),
499
([MꢀMeOH+H]+,
100),
487
(MꢀCH2CH2O+H]+, 26), 469 ([MꢀMeOHꢀCH2O+H]+ 39), 456 (15).
2.2.6. (22R,23R,24S,25R)-6-(1,3-Dioxolan-2-yl)-22-methoxymethyl-
24-methyl-3a,5-cyclo-5a-cholestan-23,26-diol (11)
LiAlH4 (8 mg, 0.21 mmol) was added to a stirred ice cooled solu-
tion of lactone 10 (35 mg, 0.066 mmol) in Et2O (2 mL). The reaction
mixture was stirred at room temperature for 30 min, quenched
with water (30lL), filtered and the filter cake was washed thor-
oughly with EtOAc (3 ꢁ 5 mL). The filtrate was concentrated under
reduced pressure and the residue was chromatographed on silica
gel (petroleum ether–EtOAc = 9:1)1:1) to give diol 11 (30 mg,
88%) as an oil. 1H NMR d: 0.32 (t, J = 4.2 Hz, 1H, C4–H), 0.60 (dd,
J = 8.1, 4.6 Hz, 1H, C4–H), 0.73 (s, 3H, C18–H), 0.82 (d, J = 7.1 Hz,
3H, C28–H), 0.90 (d, J = 7.0 Hz, 3H, C21– or C27–H), 0.92 (d,
J = 7.4 Hz, 3H, C27– or C21–H), 0.99 (s, 3H, C19–H), 3.30–3.37 (m,
2H, C23– and C26–H), 3.43 (s, 3H, CH3OCH2O), 3.53 (dd, J = 11.3,
9.0 Hz, 1H, C26–H), 3.67 (dd, J = 8.3, 0.8 Hz, 1H, C22–H), 3.74 (dd,
J = 13.4, 6.4 Hz, 1H, –OCH2CH2O–), 3.83 (dd, J = 12.5, 6.5 Hz, 1H,
–OCH2CH2O–), 3.90 (td, J = 7.9, 6.3 Hz, 1H, –OCH2CH2O–), 4.02
(td, J = 7.8, 6.2 Hz, 1H, –OCH2CH2O–), 4.63 (d, J = 6.6 Hz, 1H, CH3O-
CH2O–), 4.76 (d, J = 6.5 Hz, 1H, CH3OCH2O–). 13C NMR d: 6.39, 7.24,
12.12, 12.68, 17.93, 18.93, 22.51, 23.01, 24.11, 24.86, 28.41, 33.18,
34.08, 37.75, 38.03, 39.16, 40.08, 40.12, 40.89, 42.63, 45.52, 47.30,
52.01, 55.89, 56.22, 63.83, 64.60, 64.87, 74.92, 88.24, 99.29, 109.79.
MS (APCI+) m/z (%): 535 ([M+H]+, 13), 503 ([MꢀMeOH+H]+, 64),
485 [MꢀH2O+H]+, 84), 473 ([MꢀMeOHꢀCH2O+H]+ 100), 455
([MꢀMeOH–CH2O–H2O+H]+ 91).
2.2.9. (24S)-6-(1,3-Dioxolan-2-yl)-26-methanesulfonyloxy-22,23-
epoxy-24-methyl-3a,5-cyclo-5a-cholestane (14a)
Variant A. NaHCO3 (0.45 g, 5.5 mmol) and MCPBA (0.22 g,
0.982 mmol) were added sequentially to a stirred solution of olefin
13a (99 mg, 0.185 mmol) in a mixture of CH2Cl2 (2 mL) and i-PrOH
(2 mL). The resulting suspension was stirred at room temperature
for 48 h, diluted with EtOAc (5 mL) and quenched with a 10% aque-
ous solution of Na2CO3 (5 mL). The water layer was separated and
extracted with EtOAc (3 ꢁ 5 mL). The combined organic layers
were dried (Na2SO4) and concentrated. The residue was purified
by column chromatography on silica gel (petroleum ether–
EtOAc = 9:1)3:2) to give unreacted 8a (8 mg, 8%) and a mixture
(RR:SS = 70:30, based on relative intensity of signals at d 2.75 and
d 2.78 attributed to epoxide proton of (22R,23R,25R/S) isomer,
and signals at d 2.39–2.54 attributed to epoxide proton of
2.2.7. (22R,23R,24S,25R)-6-(1,3-Dioxolan-2-yl)-26-
methanesulfonyloxy-22-methoxymethyl-24-methyl-3a,5-cyclo-5a-
cholestan-23-ol (12)
MsCl (0.015 mL, 0.19 mmol) was added to a stirred ice cooled
solution of diol 11 (25 mg, 0.048 mmol) and Et3N (0.054 mL,
0.39 mmol) in THF (2 mL). The reaction mixture was stirred at
room temperature overnight and quenched with saturated aque-
ous NaHCO3 (2 mL). The water layer was separated and extracted
with EtOAc (3 ꢁ 5 mL). The combined organic layers were dried

A.L. Hurski et al. / Steroids 77 (2012) 780–790
783
(22R,23R,25R/S) isomer and two epoxide protons of (22S,23S,25R/S)
isomer) of isomeric epoxides 14a (85 mg, 84%, the yield was 91%
based on recovered starting material). 1H NMR d: 0.31 (t,
J = 4.2 Hz, 1H, C4–H), 0.60 (dd, J = 8.0, 4.6 Hz, 1H, C4–H), 0.69 (s,
3H, C18–H), 0.99 (s, 3H, C19–H), 2.39–2.54 (m, 1.3H, epoxide),
2.75 (dd, J = 5.6, 1.8 Hz, 0.14H, epoxide), 2.78 (dd, J = 5.0, 2.0 Hz,
0.56H, epoxide), 3.00 (s, 1H, OMs), 3.71–3.76 (m, 1H, –OCH2-
CH2O–), 3.80–3.85 (m, 1H, –OCH2CH2O–), 3.86–3.91 (m, 1H,
–OCH2CH2O–), 3.98–4.03 (m, 1H, –OCH2CH2O–), 4.07–4.14 (m,
1H, C26–H), 4.22–4.28 (m, 1H, C26–H). MS (APCI+) m/z (%): 551
([M+H]+, 100), 533 ([MꢀH2O+H]+, 8), 455 ([MꢀMsOH+H]+, 9).
Variant B. Olefin 13a (30 mg, 0.056 mmol) and 1,1,1-trifluoroac-
etone (63 mg, 0.56 mmol) were dissolved in an ice cooled mixture
of MeCN (0.2 mL), dimethoxymethane (DMM) (0.4 mL) and an
aqueous 4 ꢁ 10ꢀ4M solution of Na2EDTA (0.2 mL). A mixture of Ox-
one (0.35 g, 0.56 mmol) and NaHCO3 (0.15 g, 1.79 mmol) was
added in portions to the vigorously stirred reaction mixture at
0 °C over 2 h. After stirring for 15 min, EtOAc (5 mL) and water
(5 mL) were added, and the water layer was separated and ex-
tracted with EtOAc (2 ꢁ 3 mL). The combined organic layers were
dried (Na2SO4) and concentrated. The residue was purified by
column chromatography on silica gel (petroleum ether–
EtOAc = 9:1)3:2) to give isomeric mixture (RR:SS = 1:4, based on
relative intensity of signals at d 2.75 and d 2.78 attributed to epox-
ide proton of (22R,23R,25R/S) isomer, and signals at d 2.39–2.54
attributed to epoxide proton of (22R,23R,25R/S) isomer and two
epoxide protons of (22S,23S,25R/S) isomer) of epoxides 14a
(24 mg, 78%). 1H NMR d: 0.31 (t, J = 4.2 Hz, 1H, C4–H), 0.60 (dd,
J = 8.0, 4.6 Hz, 1H, C4–H), 0.69 (s, 3H, C18–H), 0.96 (d, J = 6.5 Hz,
1H, >CHCH3), 0.99 (s, 3H, C19–H), 1.00 (d, J = 6.2 Hz, 1H, >CHCH3),
1.08 (d, J = 7.0 Hz, 1H, >CHCH3), 2.39–2.54 (m, 1.8H, epoxide), 2.75
(dd, J = 5.6, 1.8 Hz, 0.04H, epoxide), 3.01 (s, 3H, OMs), 2.78 (dd,
J = 5.0, 2.0 Hz, 0.16H, epoxide), 3.69–3.77 (m, 1H, OCH2CH2O),
3.79–3.86 (m, 1H, OCH2CH2O), 3.86–3.92 (m, 1H, OCH2CH2O),
3.98–4.05 (m, 1H, OCH2CH2O), 4.13 (dd, J = 9.6, 6.9 Hz, 0.8H, H-
26), 4.08–4.13 (m, 1H, 0.2H, H-26), 4.25 (dd, J = 9.6, 6.5 Hz, 0.8H,
H-26), 4.25–4.29 (m, 1H, 0.2H, H-26).
3H, >CHCH3), 2.48 (t, J = 10.0 Hz, 1H, C26–H), 2.78 (dd, J = 10.0,
6.3 Hz, 1H, C26–H), 2.93 (dd, J = 10.2, 1.9 Hz, 1H, C23–H), 3.47 (d,
J = 10.2 Hz, 1H, C22–H), 3.70–3.78 (m, 1H, –OCH2CH2O–), 3.80–
3.86 (m, 1H, –OCH2CH2O–), 3.86–3.92 (m, 1H, –OCH2CH2O–),
3.98–4.04 (m, 1H, –OCH2CH2O–). MS (APCI+) m/z (%): 489
([M+H]+, 22), 471 ([MꢀH2O+H]+, 100).
2.2.12. (24S)-6b-Methoxy-23,26-thio-24-methyl-3a,5-cyclo-5a-
cholestan-22-ol (15b)
The title compound (165 mg, an isomeric mixture at C-22, C-23,
and C-25 positions) was prepared in 97% yield as an oil from epox-
ide 14b as described above for the preparation of tetrahydrothio-
phene 15a. 1H NMR signals of major isomer d: 0.42 (dd, J = 7.9,
5.0 Hz, 1H, C4–H), 0.60–0.66 (t, J = 4.4 Hz, 1H, C4–H), 0.74 (s, 3H,
C18–H), 0.83 (d, J = 6.84 Hz, 1H, >CHCH3), 0.96 (d, J = 7.02 Hz, 1H,
>CHCH3), 1.01 (s, 3H, C19–H), 1.02 (d, J = 6.8 Hz, 3H, >CHCH3),
2.47 (t, J = 10.1 Hz, 1H, C26–H), 2.74–2.80 (m, 2H, C6– and C26–
H), 2.92 (dd, J = 10.3, 2.1 Hz, 1H, C23–H), 3.31 (s, 3H, OMe), 3.48
(d, J = 10.2 Hz, 1H, C22–H). MS (APCI+) m/z (%): 443 ([MꢀH2O+H]+,
100), 429 ([MꢀMeOH+H]+, 20), 411 ([MꢀH2OꢀMeOH+H]+, 43).
2.2.13. Reduction of tetrahydrothiophene (15a) with Raney nickel
A solution of tetrahydrothiophene 15a (82.6 mg, 0.169 mmol) in
EtOH (6 mL) was added via syringe to a stirred suspension of a large
excess of freshly prepared Raney-Ni (W-7) in EtOH (3 mL) under a
hydrogen atmosphere. The reaction mixture was stirred for 12 h
under hydrogen atmosphere and then the liquid was decanted.
The catalyst was washed with EtOH (3 ꢁ 5 mL) and the combined
organic phases were evaporated under reduced pressure. The resi-
due was purified by column chromatography on silica gel (petro-
leum ether–EtOAc = 20:1)4:1) to give an inseparable mixture of
16a and 17a (16a:17a = 2:1, 66.1 mg, 85%) as an oil. 1H NMR d:
0.32 (t, J = 4.2 Hz, 1H, C4–H), 0.60 (dd, J = 8.0, 4.6 Hz, 1H, C4–H),
0.72 and 0.73 (s, 1H, C18–H), 0.80 (d, J = 6.8 Hz, 3H, >CHCH3), 0.82
(d, J = 6.5 Hz, 3H, >CHCH3), 0.86 (d, J = 6.6 Hz, 3H, >CHCH3), 0.88
(d, J = 6.3 Hz, 3H, >CHCH3), 1.00 (s, 3H, C19–H), 3.69–3.78 (m, 2H,
–OCH2CH2O– and C22–H), 3.81–3.86 (m, 1H, –OCH2CH2O–), 3.87–
3.92 (m, 1H, OCH2CH2O–), 3.99–4.04 (m, 1H, –OCH2CH2O–). 13C
NMR d: 7.24, 11.20, 12.09, 12.17, 12.24, 14.89, 15.75, 17.80, 18.30,
18.95, 19.90, 19.97, 22.53, 22.57, 23.05, 24.09, 24.24, 24.87, 27.29,
27.75, 27.80, 29.67, 31.98, 33.19, 33.39, 34.04, 34.11, 34.14, 34.50,
35.28, 39.20, 39.27, 39.35, 40.05, 40.08, 40.16, 42.33, 42.72, 43.08,
45.56, 47.35, 47.41, 52.60, 53.11, 55.79, 56.10, 56.15, 64.60, 64.84,
70.89, 71.60, 109.83, 109.88. MS (APCI+) m/z (%): 459 ([M+H]+,
26), 441 ([MꢀH2O+H]+, 100), 397 ([MꢀOHCH2CH2OH+H]+, 8).
2.2.10. (24S)-6b-Methoxy-26-methanesulfonyloxy-22,23-epoxy-24-
methyl-3a,5-cyclo-5a-cholestane (14b)
Compound 14b (75 mg, an isomeric mixture at C-22, C-23, and
C-25 positions) was prepared in 73% yield (91% based on recovered
starting material) as an oil from (22E,24R)-6-(1,3-dioxolan-2-yl)-26-
methanesulfonyloxy-24-methyl-3
a
,5-cyclo-5
a
-cholest-22-ene
13b
[8] as described above for the preparation of epoxide 14a (variant
A). 1H NMR d: 0.42 (dd, J = 7.51, 5.10 Hz, 1H, C4–H), 0.59–0.65 (m,
1H, C4–H), 0.69 (s, 1H, C18–H), 1.00 (s, 1H, C19–H), 2.39–2.54 (m,
1.3H, epoxide), 2.73–2.80 (m, 1.7H, epoxide and C6–H), 3.00 (s, 3H,
OMs), 3.31 (s, 3H, OMe), 4.05–4.15 (m, 1H, C26–H), 4.28–4.22 (m,
1H, C26–H). MS (APCI+) m/z (%): 523 ([M+H]+, 17), 491
([MꢀMeOH+H]+, 100), 473 ([MꢀMeOHꢀH2O+H]+, 82), 395
([MꢀMeOHꢀMsOH+H]+, 18), 371 (19).
2.2.14. Reduction of tetrahydrothiophene (15b) with Raney nickel
The reaction was carried out as described above for the reduc-
tion of tetrahydrothiophene 15a. The obtained mixture of isomeric
alcohols 16b and 17b was separated by column chromatography
on silica gel (petroleum ether–EtOAc = 20:1)3:1) to afford:
(22S,24R)-6b-Methoxy-24-methyl-3a,5-cyclo-5a-cholestan-22-ol
(16b) as an oil (60 mg, 52%). 1H NMR d: 0.43 (dd, J = 8.1, 5.1 Hz,
1H, C4–H), 0.64 (t, J = 4.3 Hz, 1H, C4–H), 0.72 (s, 3H, C18–H), 0.80
(d, J = 6.8 Hz, 3H, >CHCH3), 0.82 (d, J = 6.7 Hz, 3H, >CHCH3), 0.86
(d, J = 6.1 Hz, 3H, >CHCH3), 0.88 (d, J = 4.9 Hz, 3H, >CHCH3), 1.02
(s, 3H, C19–H), 2.75–2.78 (m, 1H, C6–H), 3.32 (s, 3H, OMe), 3.77
(t, J = 6.9 Hz, 1H, C22–H). 13C NMR d: 11.19, 12.16, 13.05, 15.75,
17.80, 19.25, 19.96, 21.41, 22.76, 24.08, 24.92, 27.86, 30.52,
31.99, 33.31, 35.06, 35.16, 35.27, 39.28, 39.32, 40.24, 42.66,
43.33, 47.93, 52.64, 56.39, 56.55, 71.65, 82.36. MS (APCI+) m/z
(%): 399 ([MꢀMeOH+H]+, 7), 381 ([MꢀH2OꢀMeOH+H]+, 100).
2.2.11. (24S)-6-(1,3-Dioxolan-2-yl)-23,26-thio-24-methyl-3
-cholestan-22-ol (15a)
Na2Sꢂ9H2O (0.66 g, 2.75 mmol) was added to a solution of 14a
a,5-cyclo-
5
a
(0.19 g, 0.35 mmol) in DMF (11 mL). The reaction mixture was stir-
red at 85 °C for 5 h and evaporated under reduced pressure. The
residue was diluted with water (20 mL) and extracted with EtOAc
(3 ꢁ 10 mL). The combined organic layers were dried (Na2SO4), and
concentrated. The residue was purified by column chromatography
on silica gel (petroleum ether–EtOAc = 20:1)4:1) to give com-
pound 15a (0.16 g, 94%). 1H NMR signals of major isomer d: 0.32
(t, J = 4.0 Hz, 1H, C4–H), 0.60 (dd, J = 7.9, 4.6 Hz, 1H, C4–H), 0.74
(s, 1H, C18–H), 0.83 (d, J = 6.83 Hz, 3H, >CHCH3), 0.97 (d,
J = 7.0 Hz, 3H, >CHCH3), 0.99 (s, 1H, C19–H), 1.03 (d, J = 6.7 Hz,
(22R,24R)-6b-Methoxy-24-methyl-3a,5-cyclo-5a-cholestan-22-ol
(17b) as an oil (39 mg, 27%). 1H NMR d: 0.43 (dd, J = 8.1, 5.1 Hz, 1H,
C4–H), 0.65 (t, J = 4.5 Hz, 1H, C4–H), 0.74 (s, 3H, C18–H), 0.82 (d,
J = 6.7 Hz, 3H, >CHCH3), 0.83 (d, J = 6.4 Hz, 3H, >CHCH3), 0.87 (d,

784
A.L. Hurski et al. / Steroids 77 (2012) 780–790
J = 6.4 Hz, 3H, >CHCH3), 0.91 (d, J = 6.8 Hz, 3H, >CHCH3), 1.02 (s, 3H,
C19–H), 2.77 (m, 1H, C6–H), 3.32 (s, 3H, OMe), 3.74 (dd, J = 10.6,
2.6 Hz, 1H, C22–H). 13C NMR d: 12.26 (ꢁ2), 13.05, 14.92, 18.32,
19.27, 19.90, 21.46, 22.78, 24.27, 24.95, 27.37, 30.50, 33.33,
33.41, 34.07, 34.53, 35.03, 35.24, 40.24, 42.36, 43.04, 43.36,
48.03, 53.21, 56.07, 56.55, 70.97, 82.35. MS (APCI+) m/z (%): 399
([MꢀMeOH+H]+, 8), 381 ([MꢀH2OꢀMeOH+H]+, 100).
12.05, 12.27, 14.90, 18.31, 19.65, 19.88, 22.83, 24.12, 25.86,
27.18, 33.38, 33.42, 34.04, 34.48, 34.74, 35.37, 39.64, 42.20,
42.96, 44.73, 46.04, 46.27, 46.68, 52.96, 56.49, 70.81, 209.67. MS
(APCI+) m/z (%): 415 ([M+H]+, 100), 397([MꢀH2O]+, 17).
2.2.18. (22S,24R)-22-Acetoxy-24-methyl-3a,5-cyclo-5a-cholestan-6-
one (20)
2.2.15. (24R)-6b-Methoxy-24-methyl-3
one (18)
a
,5-cyclo-5
a
-cholestan-22-
Ac2O (0.024 mL, 0.25 mmol) was added to a stirred solution of
alcohol 19 (54 mg, 0.13 mmol) and DMAP (3 mg, 0.03 mmol) in
pyridine (1.3 mL). The reaction mixture was stirred at room tem-
perature overnight, and then evaporated under reduced pressure.
The residue was distributed between EtOAc (5 mL) and H2O
(5 mL). The aqueous layer was separated and extracted with EtOAc
(3 ꢁ 3 mL). The combined organic layers were dried (Na2SO4) and
concentrated. The residue was purified by column chromatography
on silica gel (petroleum ether–EtOAc = 20:1)9:1) to give acetate
20 (59 mg, 99%) as an oil. 1H NMR d: 0.71 (s, 3H, C18–H), 0.79 (d,
J = 6.9 Hz, 3H, >CHCH3), 0.82 (d, J = 6.6 Hz, 3H, >CHCH3), 0.84 (d,
J = 6.6 Hz, 3H, >CHCH3), 0.95 (d, J = 6.9 Hz, 3H, >CHCH3), 0.99 (s,
3H, C19–H), 2.02 (s, 3H, OAc), 5.04 (t, J = 7.1 Hz, 1H, C22–H). 13C
NMR d: 11.59, 11.80, 12.56, 15.44, 17.91, 19.64, 19.83, 21.28,
22.83, 23.95, 25.85, 28.03, 32.07, 33.41, 34.75, 35.00, 35.26,
35.63, 38.10, 39.61, 42.57, 44.66, 45.98, 46.24, 46.68, 52.40,
56.80, 74.80, 170.72, 209.59. MS (APCI+) m/z (%): 457 ([M+H]+,
100), 397 ([MꢀAcOH+H]+, 21).
A solution of Dess-Martin periodinane in CH2Cl2 (15%, 0.94 mL,
140 mg, 0.33 mmol) was added to a stirred solution of alcohol 17b
(115 mg, 0.267 mmol) in CH2Cl2 (5 mL) at 0 °C. The reaction mix-
ture was stirred at room temperature for 1 h and quenched with
a mixture of saturated aqueous solutions of NaHCO3 (2.5 mL) and
Na2S2O3 (2.5 mL). The water layer was separated and extracted
with CH2Cl2 (3 ꢁ 5 mL). The combined organic layers were dried
(Na2SO4) and concentrated. The residue was purified by column
chromatography on silica gel (ethyl acetate-petrol ether) to give
ketone 18 as an oil (85 mg, 74%). 1H NMR d: 0.42 (dd, J = 8.1,
5.0 Hz, 1H, C4–H), 0.64 (t, J = 4.4 Hz, 1H, C4–H), 0.72 (s, 3H, C18–
H), 0.78 (d, J = 6.8 Hz, 3H, >CHCH3), 0.81 (d, J = 6.8 Hz, 3H, >CHCH3),
0.85 (d, J = 6.8 Hz, 3H, >CHCH3), 1.01 (s, 3H, C19–H), 1.06 (d,
J = 6.9 Hz, 3H, >CHCH3), 2.23 (dd, J = 16.9, 9.3 Hz, 1H), 2.35 (dd,
J = 17.0, 4.1 Hz, 1H), 2.49 (dq, J = 10.4, 6.9 Hz, 1H), 2.76 (t,
J = 2.9 Hz, 1H, C6–H), 3.31 (s, 3H, OMe). 13C NMR d: 12.43, 13.06,
15.85, 16.27, 18.33, 19.25, 19.83, 21.41, 22.68, 24.39, 24.91,
27.68, 30.45, 32.06, 33.32, 33.49, 35.04, 35.16, 40.07, 42.85,
43.34, 46.59, 47.96, 49.70, 51.86, 55.81, 56.54, 82.30, 214.39. MS
(APCI+) m/z (%): 429 ([M+H]+, 70), 397 ([MꢀMeOH+H]+, 100), 379
([MꢀMeOHꢀH2O+H]+, 23), 282 (22), 255 (13).
2.2.19. (22S,24R)-3b,22-Diacetoxy-24-methyl-5a-cholestan-6-one
(21)
An aqueous solution of H2SO4 (15%, 0.015 mL) was added to a
solution of cyclopropyl ketone 20 (58 mg, 0.126 mmol) in AcOH
(4 mL). The reaction mixture was stirred at 110 °C for 3 h and the
volatiles were evaporated under reduced pressure. The residue
was diluted with EtOAc (5 mL) and H2O (5 mL). The water layer
was separated and extracted with EtOAc (3 ꢁ 5 mL). The combined
organic layers were dried (Na2SO4) and concentrated. The residue
was purified by column chromatography on silica gel (petroleum
ether–EtOAc = 9:1)7:3) to give diacetate 21 (47 mg, 72%) as white
crystals. Mp 213–215 °C (MeCN). 1H NMR d: 0.65 (s, 3H, C18–H),
0.75 (s, 3H, C19–H), 0.78 (d, J = 6.9 Hz, 3H, >CHCH3), 0.82 (d,
J = 6.7 Hz, 3H, >CHCH3), 0.83 (d, J = 6.9 Hz, 3H, >CHCH3), 0.94 (d,
J = 6.7 Hz, 3H, >CHCH3), 2.01 (s, 3H, OAc), 2.02 (s, 3H, OAc), 2.29
(dd, J = 13.2, 4.5 Hz, 1H, C5–H), 4.65 (tt, J = 11.7, 4.7 Hz, 1H,
C3–H), 5.03 (t, J = 7.0 Hz, 1H, C22–H). 13C NMR d: 11.78, 12.51,
12.99, 15.43, 17.89, 19.82, 21.27, 21.30, 21.41, 23.85, 26.05,
26.76, 27.90, 32.06, 35.00, 35.63, 36.36, 37.87, 38.08, 39.33,
40.85, 42.83, 46.51, 52.42, 53.74, 56.40, 56.48, 72.75, 74.74,
170.55, 170.70, 210.21. MS (APCI+) m/z (%): 517 ([M+H]+, 7), 457
([MꢀAcOH+H]+, 65), 397 ([Mꢀ2AcOH+H]+, 100).
2.2.16. (22S,24R)-6b-Methoxy-24-methyl-3
22-ol (16b)
a,5-cyclo-5a-cholestan-
A solution of ketone 18 (95 mg, 0.22 mmol) in Et2O (1 mL) was
added to an ice cooled stirred suspension of LiAlH4 (17 mg,
0.45 mmol) in Et2O (2 mL). The reaction mixture was stirred at
room temperature for 30 min, quenched with water (0.07 mL),
and filtered. The filter cake was washed thoroughly with EtOAc
(3 ꢁ 5 mL). The filtrate was concentrated under reduced pressure
and the residue was chromatographed on silica gel (petroleum
ether/EtOAc) to give an oily product (78 mg, 82%). The NMR data
were identical with those described above for alcohol 16b.
2.2.17. Acid catalyzed hydrolysis of a mixture of dioxolanes 16a and
17a
PPTS (16 mg) was added to a solution of the dioxolanes 16a and
17a (159 mg, 0.347 mmol) in 80% aqueous acetone (6 mL). After
3 h the solvents were evaporated under reduced pressure and the
remaining residue was subjected to column chromatography on
silica gel (petroleum ether–EtOAc = 9:1)3:2) to give:
(22S,24R)-24-Methyl-3a,5-cyclo-5a-cholestan-6-on-22-ol (22S-
19) (89 mg, 62%) as white crystals. Mp 179–182 °C (MeCN). 1H
NMR d: 0.72 (s, 3H, C18–H), 0.80 (d, J = 6.9 Hz, 3H, >CHCH3), 0.82
(d, J = 6.7 Hz, 3H, >CHCH3), 0.86 (d, J = 6.9 Hz, 3H, >, >CHCH3),
0.89 (d, J = 6.7 Hz, 3H, >CHCH3), 1.00 (s, 3H, C19–H), 3.76 (td,
J = 6.8, 1.7 Hz, 1H, C22–H). 13C NMR d: 11.21, 11.62, 11.94, 15.75,
17.79, 19.66, 19.95, 22.84, 23.96, 25.86, 27.72, 31.99, 33.42,
34.79, 35.29 (ꢁ2), 39.31, 39.35, 39.67, 42.59, 44.73, 45.99, 46.28,
46.71, 52.40, 56.84, 71.52, 209.67. MS (APCI+) m/z (%): 415
([M+H]+, 100), 397([MꢀH2O]+, 17).
2.2.20. (22S,24R)-24-Methyl-5
cathasterone/
a-cholestan-6-one-3b,22-diol (1)/
A solution of diacetate 21 (47 mg, 0.09 mmol) in methanolic
KOH (5%, 7 mL) was stirred at 45 °C for 10 h. The solvent was evap-
orated under reduced pressure and the residue was distributed be-
tween EtOAc (5 mL) and H2O (5 mL). The water layer was
separated and extracted with EtOAc (3 ꢁ 5 mL). The combined or-
ganic layers were dried (Na2SO4) and concentrated. The residue
was purified by column chromatography on silica gel (petroleum
ether–EtOAc = 4:1)1:2) to give cathasterone 1 (37 mg, 76%). Mp
175–177 °C (EtOAc). Lit. [9] mp 176–177.5 °C, lit. [6] mp 188–
190 °C. The 1H and 13C NMR data agreed with the literature values
[6,9]. MS (APCI+) m/z (%): 433 ([M+H]+, 31), 415 ([MꢀH2O+H]+, 82),
397 ([Mꢀ2H2O+H]+, 100), 379 ([Mꢀ3H2O+H]+, 20).
(22R,24R)-24-Methyl-3a,5-cyclo-5a-cholestan-6-on-22-ol (22R-
19) (47 mg, 33%) as an oil. 1H NMR d: 0.73 (s, 3H, C18–H), 0.81
(d, J = 6.6 Hz, 3H, >CHCH3), 0.83 (d, J = 6.7 Hz, 3H, >CHCH3), 0.87
(d, J = 6.5 Hz, 3H, >CHCH3), 0.92 (d, J = 6.7 Hz, 3H, >CHCH3), 1.00
(s, 3H, C19–H), 3.67–3.78 (m, 1H, C22–H). 13C NMR d: 11.65,

A.L. Hurski et al. / Steroids 77 (2012) 780–790
785
2.2.21. (22S,24R)-24-Methylcholest-5-en-22-ol (2)/(22S)-22-
hydroxycampesterol/
1H, C4–H), 0.60 (dd, J = 8.1, 4.6 Hz, 1H, C4–H), 0.69 (s, 3H, C18–
H), 0.99 (s, 3H, C19–H), 2.54–2.39 (m, 1.3H, epoxide), 2.75 (dd,
J = 5.6, 2.2 Hz, 0.14H, epoxide), 2.78 (dd, J = 5.1, 2.2 Hz, 0.56H,
epoxide), 3.01 (s, 3H, OMs), 3.76–3.71 (m, 1H, –OCH2CH2O–),
3.85–3.81 (m, 1H, –OCH2CH2O–), 3.92–3.86 (m, 1H, –OCH2CH2O–),
4.04–3.98 (m, 1H, –OCH2CH2O–). MS (APCI+) m/z (%): 553 ([M+H]+,
18), 509 ([MꢀCH2CH2O+H]+, 100).
A mixture of ether 16b (90 mg, 0.209 mmol), TsOHꢂH2O (9 mg),
dioxane (9 mL) and H2O (3 mL) was stirred at 75 °C for 3 h. The sol-
vents were evaporated under reduced pressure. The residue was
diluted with water (10 mL) and extracted with EtOAc (3 ꢁ 5 mL).
The combined organic layers were dried (Na2SO4) and concen-
trated. The residue was purified by column chromatography on
silica gel (petroleum ether–EtOAc = 4:1)1:1) to give (22S)-22-
hydroxycampesterol 2 (70 mg, 80%). Mp 187–190 °C (EtOAc). Lit.
[6] mp 193–195 °C. 1H NMR d: 0.71 (s, 3H, C18–H), 0.82 (d,
J = 6.8 Hz, 3H, >CHCH3), 0.85 (d, J = 6.7 Hz, 3H, >CHCH3), 0.89 (d,
J = 6.9 Hz, 3H, >CHCH3), 0.91 (d, J = 6.4 Hz, 3H, >CHCH3), 1.02 (s,
3H, 19-H), 3.52 (tt, J = 11.0, 4.5 Hz, 1H, C3–H), 3.77 (t, J = 6.7 Hz,
1H, C22–H), 5.35 (dd, J = 5.1, 2.5 Hz, 1H, C5–H). 13C NMR d:
11.36, 11.83, 15.92, 17.89, 19.41, 19.99, 21.21, 24.28, 27.86,
31.81, 31.94, 32.12, 32.16, 35.66, 36.60, 37.38, 39.65 (ꢁ2), 39.96,
42.39, 42.44, 50.31, 52.80, 56.83, 71.84, 71.93, 121.61, 140.93.
MS (APCI+) m/z (%): 399 ([MꢀH2O+H]+, 64), 381 ([Mꢀ2H2O+H]+,
100).
2.2.24. (24S)-[26,26-2H2]-6-(1,3-Dioxolan-2-yl)-23,26-thio-24-
methyl-3a,5-cyclo-5a-cholestan-22-ol (23)
The title compound was prepared in 97% (141 mg) yield as an
oil from epoxide 22 as described above for the preparation of tet-
rahydrothiophene 15a. 1H NMR signals of the major isomer d:
0.32 (t, J = 3.9 Hz, 1H, C4–H), 0.60 (dd, J = 8.0, 4.7 Hz, 1H, C4–H),
0.74 (s, 3H, C18–H), 0.83 (d, J = 6.8 Hz, 3H, >CHCH3), 0.97 (d,
J = 7.03 Hz, 3H, >CHCH3), 1.00 (s, 3H, C19–H), 1.03 (d, J = 6.7 Hz,
3H, >CHCH3), 2.93 (dd, J = 10.2, 1.9 Hz, 1H, C23–H), 3.48 (d,
J = 10.1 Hz, 1H, C22–H), 3.77–3.71 (m, 1H, –OCH2CH2O), 3.86–
3.81 (m, 1H, –OCH2CH2O–), 3.93–3.87 (m, 1H, –OCH2CH2O–),
4.05–3.99 (m, 1H, –OCH2CH2O–). MS (APCI+) m/z (%): 491
([M+H]+, 25), 473 ([MꢀH2O+H]+, 100).
2.2.22. (22S,24R)-24-Methyl-5a-cholestan-22-ol (3)/6-
deoxocathasterone/
2.2.25. (24R)-[26,26-2H2]-6-(1,3-Dioxolan-2-yl)-24-methyl-3
cyclo-5 -cholestan-22-ol (24)
a,5-
A mixture of 10% Pd/C (10 mg), alkene 2 (37 mg, 0.089 mmol)
and EtOH (4 mL) was stirred under hydrogen atmosphere over-
night and then filtered. The filter cake was washed thoroughly with
EtOH. The filtrate was concentrated under reduced pressure and
the residue was chromatographed on silica gel (petroleum ether–
EtOAc = 4:1)1:1) to give 6-deoxocathasterone 3 (35 mg, 95%).
Mp 188–191 °C (EtOAc). Lit. [6] mp 200–202 °C. 1H NMR d: 0.66
(s, 3H, C18–H), 0.79 (s, 3H, C19–H), 0.80 (d, J = 6.8 Hz, 3H, >CHCH3),
0.81 (d, J = 7.8 Hz, 3H, >CHCH3), 0.86 (d, J = 6.5 Hz, 3H, >CHCH3),
3.58 (tt, J = 10.7, 4.8 Hz, 1H, C3–H), 3.75 (t, J = 6.8 Hz, 1H, C22–H).
13C NMR d: 11.16, 11.98, 12.30, 15.74, 17.80, 19.96, 21.24, 24.11,
27.80, 28.66, 31.46, 31.99 (ꢁ2), 35.25, 35.41, 35.50, 36.95, 38.14,
39.26, 39.31, 39.99, 42.49, 44.78, 52.60, 54.27, 56.39, 71.29,
71.63. MS (APCI+) m/z (%): 401 ([MꢀH2O+H]+, 23), 383
([Mꢀ2H2O+H]+, 100).
a
The title compound was prepared from tetrahydrothiophene 23
as described above for the preparation of alcohols 16a and 17a. An
inseparable mixture of C-22 epimers was obtained in 78% (65 mg)
yield. 1H NMR signals of the major isomer d: 0.31 (t, J = 4.0 Hz, 1H,
C4–H), 0.60 (dd, J = 7.7, 4.7 Hz, 1H, C4–H), 0.72 (s, 1H, C18–H), 0.81
(d, J = 6.5 Hz, 3H, >CHCH3), 0.86 (d, J = 6.7 Hz, 3H, >CHCH3), 0.87 (d,
J = 6.2 Hz, 3H, >CHCH3), 0.99 (s, 3H, C19–H), 3.77–3.70 (m, 2H,
–OCH2CH2O– and C22–H), 3.85–3.81 (m, 1H, –OCH2CH2O–),
3.92–3.87 (m, 1H, –OCH2CH2O–), 4.04–3.99 (m, 1H, –OCH2CH2O–
). 13C NMR d: 7.24, 11.20, 12.08, 12.16, 12.23, 14.89, 15.75, 17.19
(p, J = 19 Hz), 17.75, 18.94, 19.85, 19.92, 22.53, 22.56, 23.04,
24.08, 24.24, 24.87, 27.28, 27.74, 27.79, 29.66, 31.82, 33.19,
33.23, 34.04, 34.11, 34.14, 34.46, 35.25, 39.19, 39.27, 39.35,
40.05, 40.08, 40.15, 42.32, 42.71, 43.07, 45.55, 47.34, 47.40,
52.59, 53.10, 55.79, 56.10, 56.15, 64.59, 64.84, 70.88, 71.59,
109.83, 109.87. MS (APCI+) m/z (%): 461 ([M+H]+, 22), 443
([MꢀH2O+H]+, 100).
2.2.23. (24S)-[26,26-2H2]-6-(1,3-Dioxolan-2-yl)-26-
methanesulfonyloxy-22,23-epoxy-24-methyl-3a,5-cyclo-5a-
cholestane (22)
A solution of ester 8a (0.82 g, 1.64 mmol) in Et2O (6 mL) was
added to an ice cooled stirred suspension of LiAlD4 (138 mg,
3.29 mmol) in Et2O (4 mL). The reaction mixture was stirred at
room temperature for 30 min, quenched at 0 °C with water
(0.47 mL) and filtered. The filter cake was washed thoroughly with
EtOAc (3 ꢁ 10 mL). The filtrate was concentrated under reduced
pressure and the residue was chromatographed on silica gel
(petroleum ether–EtOAc = 4:1)3:1) to give (24R)-[26,26-2H2]-6-
2.2.26. (24R)-1-2H2]-6-(1,3-Dioxolan-2-yl)-24-methyl-3
a,5-cyclo-5a-
cholestan-22-one (25)
The title compound was prepared in 94% (60 mg) yield as an oil
from alcohols 16a and 17a as described above for the preparation
of ketone 18. 1H NMR d: 0.32 (t, J = 4.2 Hz, 1H, C4–H), 0.60 (dd,
J = 8.1, 4.5 Hz, 1H, C4–H), 0.73 (s, 3H, C18–H), 0.78 (d, J = 6.7 Hz,
3H, >CHCH3), 0.85 (d, J = 6.9 Hz, 3H, >CHCH3), 1.00 (s, 3H, C19–
H), 1.07 (d, J = 7.0 Hz, 3H, >CHCH3), 2.23 (dd, J = 17.0, 9.3 Hz, 1H),
2.35 (dd, J = 17.0, 4.0 Hz, 1H), 2.44–2.53 (m, 1H), 3.73 (q,
J = 6.6 Hz, 1H, –OCH2CH2O–), 3.83 (q, J = 6.4 Hz, 1H, –OCH2CH2O–
), 3.86–3.92 (m, 1H, –OCH2CH2O–), 4.01 (dt, J = 7.7, 6.0 Hz, 1H,
–OCH2CH2O–). 13C NMR d: 7.26, 12.36, 15.87, 16.28, 18.94, 19.80,
22.56, 22.98, 24.40, 24.87, 27.61, 31.92, 33.23, 33.47, 34.07,
39.21, 39.93, 40.16, 42.91, 45.57, 46.47, 47.38, 49.74, 51.84,
55.59, 64.61, 64.85, 109.78, 214.24. MS (APCI+) m/z (%): 459
([M+H]+, 100).
(1,3-dioxolan-2-yl)-24-methyl-3a,5-cyclo-5a-cholest-22-en-26-ol
(0.69 g, 92%) as an oil.
This compound was mesylated as described above for the prep-
aration of compound 13a, providing [26,26-2H2]-6-(1,3-dioxolan-2-
yl)-26-methanesulfonyloxy-24-methyl-3a,5-cyclo-5a-cholest-22-ene
in 99% yield as an oil.
This compound was dissolved in CH2Cl2 (10 mL), then NaHCO3
(1.23 g, 14.7 mmol) and MCPBA (0.58 g, 2.61 mmol) were added
sequentially to a stirred solution. The resulting suspension was
stirred at room temperature for 3 h and quenched with 10%
aqueous solution of Na2CO3 (20 mL). The water layer was
separated and extracted with EtOAc (3 ꢁ 10 mL). The combined
organic layers were dried (Na2SO4) and concentrated. The residue
was purified by column chromatography on silica gel (petroleum
ether–EtOAc = 9:1)3:2) to give an isomeric mixture (RR:SS =
70:30) of epoxides 22 (0.57 g, 70%). 1H NMR d: 0.32 (t, J = 4.1 Hz,
2.2.27. (24R)-[22,26,26-2H3]-6-(1,3-Dioxolan-2-yl)-24-methyl-3
cyclo-5 -cholestan-22-ol (26)
a,5-
a
A solution of ketone 25 (123 mg, 0.269 mmol) in Et2O (1.5 mL)
was added to an ice cooled stirred suspension of LiAlD4 (11 mg,
0.26 mmol) in Et2O (0.5 mL). The reaction mixture was stirred at
room temperature for 30 min, then quenched with water
(0.04 mL), and filtered. The filter cake was washed thoroughly with

786
A.L. Hurski et al. / Steroids 77 (2012) 780–790
EtOAc (3 ꢁ 5 mL). The filtrate was concentrated under reduced
pressure and the residue was chromatographed on silica gel
(petroleum ether–EtOAc = 9:1)3:1) to give an inseparable mix-
ture of alcohols 26 (115 mg, 93%) as an oil. 1H NMR signals of major
isomer d: 0.32 (t, J = 4.3 Hz, 1H, C4–H), 0.61 (dd, J = 8.2, 4.6 Hz, 1H,
C4–H), 0.73 (s, 3H, C18–H), 0.82 (d, J = 6.6 Hz, 3H, >CHCH3), 0.87 (d,
J = 6.6 Hz, 3H, >CHCH3), 0.88 (d, J = 5.3 Hz, 3H, >CHCH3), 1.00 (s, 3H,
C19–H), 3.74 (q, J = 6.6 Hz, 1H, –OCH2CH2O–), 3.84 (q, J = 6.3 Hz,
1H, –OCH2CH2O–), 3.90 (dt, J = 8.1, 6.3 Hz, 1H, –OCH2CH2O–),
4.02 (dt, J = 7.9, 6.0 Hz, 1H, –OCH2CH2O–). 13C NMR d: 7.25,
11.19, 12.10, 15.76, 17.20 (p, J = 18.9 Hz), 17.76, 18.95, 19.93,
22.58, 23.05, 24.09, 24.88, 27.80, 29.68, 31.84, 33.20, 34.15,
35.24, 39.20, 39.23, 40.09, 40.17, 42.72, 45.56, 47.36, 52.60,
56.16, 64.61, 64.85, 71.18 (t, J = 21 Hz), 109.88. MS (APCI+) m/z
(%): 462 ([M+H]+, 29), 444 ([MꢀH2O]+, 100).
1. Mp 179–182 °C (white crystals from MeOH). 1H NMR d: 067 (s,
3H, C18–H), 0.74 (s, 3H, C19–H), 0.81 (d, J = 6.7 Hz, 3H, >CHCH3),
0.86 (d, J = 7.0 Hz, 3H, >CHCH3), 0.87 (d, J = 6.9 Hz, 3H, >CHCH3),
2.30 (dd, J = 13.2, 4.5 Hz, 1H, C5–H), 3.56 (tt, J = 11.1, 4.7 Hz, 1H,
C3–H). 13C NMR d: 11.15, 11.93, 13.12, 15.76, 17.20 (p,
J = 19.0 Hz), 17.76, 19.90, 21.51, 23.88, 27.60, 29.99, 30.65, 31.84,
35.26, 36.63, 37.93, 39.15, 39.32, 39.47, 40.91, 42.85, 46.64,
52.45, 53.84, 56.63, 56.74, 70.59, 71.13 (t, J = 21 Hz), 210.84. MS
186 (APCI+) m/z (%): 436 ([M+H]+, 30), 418 [MꢀH2O+H]+, 55), 400
([Mꢀ2H2O+H]+, 100), 382 ([Mꢀ3H2O+H]+, 15).
2.2.32. Oxidation of (22E,24R)-6-(1,3-dioxolan-2-yl)-26-
methanesulfonyloxy-24-methyl-3
with methyl(trifluoromethyl)dioxirane
(22E,24R)-6-(1,3-Dioxolan-2-yl)-26-methanesulfonyloxy-24-
methyl-3 ,5-cyclo-5 -cholest-22-ene 13b [8] (30 mg, 0.059 mmol)
a,5-cyclo-5a-cholest-22-ene (13b)
2.2.28. (22S,24R)-[22,26,26-2H3]-24-methyl-3
a,5-cyclo-5a-cholestan-
a
a
6-on-22-ol (27)
and 1,1,1-trifluoroacetone (63 mg, 0.56 mmol) were dissolved in
an ice cooled mixture of MeCN (0.2 mL), DMM (0.4 mL) and an
aqueous 4 ꢁ 10ꢀ4M solution of Na2EDTA (0.2 mL). A mixture of
Oxone (0.35 g, 0.56 mmol) and NaHCO3 (0.15 g, 1.79 mmol) was
added in portions to this vigorously stirred solution at 0 °C over
2 h. After stirring for another 15 min, EtOAc (5 mL) and water
(5 mL) were added, and the water layer was separated and ex-
tracted with EtOAc (2 ꢁ 5 mL). The combined organic layers were
dried (Na2SO4) and concentrated. The residue was purified by
column chromatography on silica gel (petroleum ether–EtOAc =
9:1)1:1) to give epoxides 14b (9 mg, 30%) as a mixture of isomers
(22R,23R:22S,23S = 1:4). 1H NMR d: 0.43 (dd, J = 7.7, 5.1 Hz, 1H,
C4–H), 0.67–0.60 (m, 1H, C4–H), 0.70 (s, 3H, C18–H), 0.97 (d,
J = 6.3 Hz, 3H, >CHCH3), 1.00 (d, J = 7.0 Hz, 3H, >CHCH3), 1.02 (s,
3H, C19–H), 1.08 (d, J = 7.0 Hz, 3H, >CHCH3), 2.39–2.55 (m, 1.8H,
epoxide), 2.74–2.80 (m, 1.2H, C6–H and epoxide), 3.01 (s, 3H,
OMs), 3.32 (s, 3H, OMe), 4.09–4.16 (m, 1H, C26–H), 4.23–4.31
(m, 1H, C26–H).
PPTS (10 mg) was added to a solution of compound 26 (95 mg,
0.207 mmol) in 90% aqueous acetone (10 mL). After 3 h the sol-
vents were evaporated under reduced pressure and residue was
purified by column chromatography on silica gel (petroleum
ether–EtOAc = 9:1)2:1) to give compound 27 (72 mg, 83%) as
white crystals. Mp178–181 °C (MeCN). 1H NMR d: 0.72 (s, 3H,
C18–H), 0.81 (d, J = 6.7 Hz, 3H, >CHCH3), 0.86 (d, J = 6.8 Hz, 3H,
>CHCH3), 0.89 (d, J = 6.7 Hz, 3H, >CHCH3), 0.99 (s, 3H, C19–H),
2.42 (d, J = 12.9 Hz, 1H). 13C NMR d:11.18, 11.61, 11.93, 15.73,
17.19 (t, J = 19 Hz), 17.75, 19.65, 19.89, 22.83, 23.95, 25.85, 27.70,
29.65, 31.82, 33.41, 34.78, 35.21, 35.29, 39.16, 39.25, 39.66,
42.57, 44.72, 45.98, 46.26, 46.70, 52.38, 56.84, 71.07 (t, J = 21 Hz),
209.65. MS (APCI+) m/z (%): 418 ([M+H]+, 100), 400 ([MꢀH2O+H]+,
8).
2.2.29. (22S,24R)-[22,26,26-2H3]-22-Acetoxy-24-methyl-3
-cholestan-6-one (28)
The title compound was prepared in 99% (59 mg) yield as an oil
a,5-cyclo-
5a
Further elution gave (24S)-26-methanesulfonyloxy-22,23-epoxy-
24-methyl-3a,5-cyclo-5a-cholestan-6-one (31) (13 mg, 43%) as an
from alcohol 27 as described above for the preparation of acetate
20. 1H NMR d: 0.71 (s, 1H, C18–H), 0.82 (d, J = 6.3 Hz, 3H, >CHCH3),
0.83 (d, J = 6.4 Hz, 3H, >CHCH3), 0.95 (d, J = 6.7 Hz, 3H, >CHCH3),
0.98 (s, 1H, C19–H), 2.01 (s, 1H, OAc). 13C NMR d: 11.56, 11.79,
12.51, 15.43, 17.29 (p, J = 18.9 Hz), 17.85, 19.62, 19.77, 21.25,
22.82, 23.93, 25.84, 28.01, 31.91, 33.40, 34.74, 34.94, 35.22,
35.54, 37.98, 39.60, 42.55, 44.64, 45.97, 46.22, 46.66, 52.38,
56.79, 74.44 (t, J = 21 Hz), 170.68, 209.53. MS (APCI+) m/z (%):
460 ([M+H]+, 100), 400 ([MꢀAcOH+H]+, 13).
oil. 13C NMR signals of major isomer d: 0.70 (s, 3H, C18–H),
0.98 (d, J = 6.6 Hz, 3H, >CHCH3), 1.00 (s, 3H, C19–H), 1.01 (d,
J = 6.0 Hz, 3H, >CHCH3), 1.08 (d, J = 7.0 Hz, 3H, >CHCH3), 2.43
(dd, J = 7.1, 2.1 Hz, 1H, epoxide), 2.49 (dd, J = 8.8, 2.1 Hz, 1H,
epoxide), 3.01 (s, 3H, OMs), 4.14 (dd, J = 9.6, 6.8 Hz, 1H, C26–H),
4.25 (dd, J = 9.6, 6.5 Hz, 1H, C26–H). 13C NMR d: 11.65, 11.75,
12.12, 12.19, 12.55, 12.98, 14.61, 16.13, 19.65, 22.81, 24.24,
25.87, 26.93, 33.45, 34.73, 35.30, 36.30, 37.05, 37.34, 37.50,
38.15, 39.53, 43.03, 44.71, 46.03, 46.09, 46.28, 46.70, 53.50,
55.92, 56.49, 57.90, 59.46, 61.84, 62.58, 73.42, 209.48. MS (APCI+)
m/z (%): 507 ([M+H]+, 100), 489 ([MꢀH2O+H]+, 9), 411
([MꢀMsOH+H]+, 67).
2.2.30. (22S,24R)-[22,26,26-2H3]-3b,22-Diacetoxy-24-methyl-5
cholestan-6-one (29)
a-
The title compound was prepared in 72% (47 mg) yield from
cyclopropyl ketone 28 as described above for the preparation of
acetate 21. Mp 214–216 °C (white crystals from MeCN). 1H NMR
d: 0.65 (s, 3H, C18–H), 0.75 (s, 3H, C19–H), 0.82 (d, J = 6.7 Hz, 3H,
>CHCH3), 0.83 (d, J = 6.7 Hz, 3H, >CHCH3), 0.94 (d, J = 6.7 Hz, 3H,
>CHCH3), 2.005 (s, 3H, OAc), 2.013 (s, 3H, OAc), 2.24 (dd, J = 12.7,
2.9 Hz, 1H), 2.29 (dd, J = 13.3, 4.5 Hz, 1H, C5–H), 4.65 (tt, J = 11.7,
4.7 Hz, 1H, C3–H). 13C NMR d: 11.77, 12.48, 12.98, 15.42, 17.29
(p, J = 19.0 Hz), 17.85, 19.77, 21.26, 21.30, 21.40, 23.84, 26.04,
26.76, 27.89, 31.90, 34.94, 35.54, 36.35, 37.86, 37.95, 39.32,
40.84, 42.81, 46.50, 52.40, 53.73, 56.38, 56.47, 72.74, 74.38 (t,
J = 21 Hz), 170.54, 170.68, 210.19. MS (APCI+) m/z (%): 520
([M+H]+, 11), 460 ([MꢀAcOH+H]+, 83), 400 ([Mꢀ2AcOH+H]+, 100).
2.2.33. Oxidation of the ether (16b) with
methyl(trifluoromethyl)dioxirane
A mixture of Oxone (0.92 g, 1.5 mmol) and NaHCO3 (0.4 g,
4.8 mmol) was added portionwise over 4 h to an ice cooled
vigorously stirred solution of ether 16b (60 mg, 0.139 mmol) and
1,1,1-trifluoroacetone (0.17 g, 1.04 mmol) in a mixture of DMM
(1 mL), MeCN (0.5 mL) and 4 ꢁ 10ꢀ4
M aqueous Na2EDTA
(0.6 mL). The reaction mixture was stirred at 0 °C for 1 h, diluted
with water (10 mL) and extracted with EtOAc (3 ꢁ 5 mL). The com-
bined organic layers were dried (Na2SO4) and concentrated. The
residue was purified by column chromatography on silica gel
(petroleum ether–EtOAc = 9:1)3:2) to give starting ether 16b
(10 mg, 17%) and ketone 19 (37 mg, 64%, 77% based on recovered
starting material).
2.2.31. (22S,24R)-[22,26,26-2H3]-24-Methyl-5
a-cholestan-6-one-
3b,22-diol (30)/[22,26,26-2H3]-cathasterone/
The title compound was prepared in 79% (37 mg) yield from
diacetate 29 as described above for the preparation of cathasterone

A.L. Hurski et al. / Steroids 77 (2012) 780–790
787
Scheme 1.
It should be noted that the hydroxy lactone 9 was isolated as a
single isomer starting from the inseparable C-25 diastereomeric
mixture 8a (for the meaning of a and b see Scheme 2). The forma-
tion of a 4:1 diastereomeric mixture of such esters was earlier
shown by us [8]. At that time the preferred (R)-configuration at
C-25 was assumed, based on analysis of the possibility of only
one chair like transition state for the Claisen rearrangement. The
experimental results obtained in this study are in agreement with
the previous suggestions [8]. The ratio of 25R-8a (precursor of the
lactone 9) to its 25S-8a epimer was evaluated to be 5.4:1 based on
the ratio of integral intensity peaks with d 129.96 and 130.40 or
137.71 and 136.89. The assignment of the stereochemistry at
C-24 became apparent from the NOESY spectrum of MOM ether
10, which clearly showed a NOE correlation between the 27- and
28-methyl groups. This indicates a syn orientation of both methyl
groups, confirming the stereochemistry at C-25 as being (R).
3. Results and discussion
The ester 8 was easily synthesized from stigmasterol 4 via the
known aldehyde 5 [10] according to described procedures [8,11],
in 8 steps and in 20% overall yield. Compound 8 already contains
a methyl group at C-24, and the stereochemistry of C-24 is defined
by the Claisen rearrangement of the propionate of allyl alcohol 7
(Scheme 1). The C-22 and C-23 hydroxyl groups were introduced
by Sharpless asymmetric dihydroxylation [12]. Under the reaction
conditions the C-23 hydroxyl group reacted with the ester group
[13,14], which led to the formation of hydroxy lactone 9, thus
allowing selective manipulations at the 22- and 23-position. Pro-
tection of the free hydroxyl group in 9 was the most obvious
way to continue. The use of the TBS protecting group failed because
of its instability in a later stage under reduction conditions. Finally
the 22-hydroxyl group was protected as a MOM ether.
Scheme 2.

788
A.L. Hurski et al. / Steroids 77 (2012) 780–790
The lactone 10 was reduced with LiAlH4 to give diol 11. It was
transformed into cathasterone 1 by standard methods, involving
acid-catalyzed cyclopropane ring opening [22] of the acetate pro-
tected derivative 20.
Regeneration of the AB-cycles by treatment of 16b with acid led
in one step to (22S)-hydroxycampesterol 2 (Scheme 4). Hydroge-
nation of 2 over Pd catalyst gave 6-deoxocathasterone 3, which is
another biosynthetic precursor of brassinolide [23,24].
treated with an excess of MsCl, but unexpectedly the secondary
hydroxyl group at C-23 was left intact. The search for an appropri-
ate protocol for the preparation of the 23,26-dimesylate, using var-
ious solvents and catalysts [15], gave no results. Either the starting
compound 11 was isolated or, under more severe conditions, cycli-
zation took place with formation of the corresponding tetrahydro-
furan. These results prohibited the simultaneous removal of both
hydroxyl groups via dimesylate reduction. A successful implemen-
tation of this approach would require additional chemical steps
thus making the whole synthetic sequence less attractive.
However this approach deserves consideration as a starting
point for the preparation of brassinosteroid metabolites with a chi-
ral center at C-25 [16].
Modern techniques in detection, identification and quantifica-
tion of brassinolide precursors are based on the use of the corre-
sponding labeled derivatives as internal standards [25,26]. These
compounds have to meet some requirements [27]. At least three
deuterium atoms should be present in the molecule to avoid pos-
sible interferences with the peaks [M+1] and [M+2] belonging to
natural 13C and 18O containing species. We have already reported
the synthesis of labeled brassinosteroids containing three [28] or
six [29] deuterium atoms at the end of the side chain. The present
synthetic route to 22-hydroxysteroids provides an obvious method
for the introduction of two deuterium atoms at C-26 via the reduc-
tion of the ester 8a with LiAlD4 (Scheme 5). The third deuterium
atom can in principle be introduced at C-26 by hydrogenation of
tetrahydrothiophene 23 over Raney nickel that is prepared in deu-
terated water. The main concern with such an approach is that it
could not guarantee a sufficient isotopic purity level for the final
compound. Labeling at C-22 by reduction of the ketone 25 with
LiAlD4 seems to be a more reliable and predictable alternative.
The remaining steps of the synthesis of triple-deuterated cathas-
terone 30 were performed as described above for the preparation
of its non-labeled congener 1.
Better results for the preparation of 22-hydroxy steroids were
obtained by using the tetrahydrothiophene derivatives 15a,b as
intermediates (Scheme 2). The synthesis of cathasterone 1 started
from ester 8a, and the 6-deoxo- and
D
5-derivatives 3 and 2 were
obtained starting from ester 8b [8]. A lithium aluminum hydride
reduction of the esters 8a,b followed by mesylation of the interme-
diate alcohols gave the bishomoallylic mesylates 13a,b. The search
for optimal epoxidation conditions of the
D
22-double bond was
carried out with compound 13a. Its epoxidation with MCPBA in
the presence of NaHCO3 in methylene chloride led to a 2:1 mixture
of isomers in favor of the 22R,23R-epimer 14a in 70% yield. The ste-
reochemical course of this epoxidation was assessed by chemical
correlation with the alcohols 22R-16a and 22S-16a (vide infra). At-
tempts to increase the stereoselectivity by using different epoxida-
tion conditions were unsuccessful. A solution of dimethyldioxirane
in acetone [17] and Shi’s catalyst [18,19] gave no reaction at all.
Epoxidation with in situ generated methyl(trifluoromethyl)dioxira-
ne [20] gave the undesired 22S,23S-epoxide 14a as the main iso-
mer. Ultimately it was found that epoxidation with MCPBA with
isopropanol as a co-solvent gave a better yield of the epoxides
14a (up to 91% based on the recovered starting material 13a),
but the 2:1 (22R,23R):(22S,23S)-isomeric ratio remained
unchanged.
Compounds 14a and 14b produced a mixture of isomeric tetra-
hydrothiophenes 15a,b with good regioselectivity when reacted
with sodium sulfide. The desulfurization [21] of 15a,b was
smoothly achieved with Raney nickel to give mixtures of the 22S
and 22R alcohols 16a and 17a. Their ratio depended on the epoxi-
dation method used for the preparation of mesyl epoxides 13a,b.
The proportion of the desired 16a,b could be increased by oxida-
tion of the 22R-alcohols 17a,b into corresponding 22-ketones fol-
lowed by hydride reduction. The alcohols 16b and 17b could be
separated easily by column chromatography, but we failed to do
the same with the dioxolane derivatives 16a and 16b.
While optimizing the preparation of 22,23-epoxides, we had
observed that the epoxidation of the
D
22-olefin 13b with
methyl(trifluoromethyl)dioxirane yielded not only the expected
product 14b, but also the 6-ketone 31 (Scheme 6). Oxidation of ste-
roidal ethers is known [30], but this direct reaction has never been
used for the transformation of 3a,5-cyclo-6b-methoxy derivatives
into the corresponding 6-ketones. Such a reaction would be highly
profitable for the chemistry of brassinosteroids instead of the four-
step reaction sequence that is in use for that purpose now
[28,31,32].
To check the synthetic utility of this method, we studied the
oxidation of the less functionalized ether 16b (Scheme 7). Its reac-
tion with in situ generated methyl(trifluoromethyl)dioxirane pro-
ceeded smoothly to give 6-ketone 32 in a reasonable 64% yield
(77% based on recovered starting material).
4. Conclusion
In summary, we have successfully developed a new method for
the synthesis of 22S-hydroxy-22R-methyl steroids. Construction of
the side chain skeleton with the correct stereochemistry at C-24
was based on the use of a Claisen rearrangement. After Sharpless
dihydroxylation of the obtained C-25 isomeric mixture of
Fortunately, this problem was solved by separation of the
isomeric alcohols after removal of the dioxolane protecting group.
The pure 22S-alcohol 19 with the desired side chain could be
isolated in 62% yield from the isomeric mixture of 16a and 17a
by chromatography (Scheme 3). Compound 19 was further
D
22-26-esters, a (25R)-26,23-lactone was isolated as the major
product. This is
a possible synthetic intermediate for the
Scheme 3.

A.L. Hurski et al. / Steroids 77 (2012) 780–790
789
Scheme 4.
Scheme 5.
Scheme 6.
are biosynthetic precursors in the early stages of the brassinolide
biosynthesis. A possibility to prepare the corresponding labeled
22-hydroxy steroids was demonstrated by the synthesis of
[22,26,26-2H3]-cathasterone 30 containing three deuterium atoms
in positions that did not allow isotopic exchange. In addition, a
new method for the synthesis of steroidal 6-ketones was
developed based on the oxidation of the 6-methyl ether with
methyl(trifluoromethyl)dioxirane.
Scheme 7.
Acknowledgment
preparation of brassinosteroid metabolites with a chiral center at
C-25, although attempts to use it for the introduction of the
required 22-hydroxyl group did not give the expected results.
We thank Prof. Ae de Groot (Wageningen) for helpful discus-
sions and linguistic support in the preparation of this manuscript.
The successful functionalization at C-22 was achieved by
D
22-dou-
ble bond epoxidation, nucleophilic epoxide ring opening with
sodium sulfide of the intermediate mesyl epoxide, and desulfuriza-
tion of the formed tetrahydrothiophenes with Raney nickel. The
new methodology was applied to the preparation of cathasterone
1, (22S)-hydroxycampesterol 2, and 6-deoxocathasterone 3 which
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.steroids.2012.03.
010.

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A.L. Hurski et al. / Steroids 77 (2012) 780–790
[16] Hai T, Schneider B, Porzel A, Adam G. Metabolism of 24-epi-castasterone in cell
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