Efficient, stereoselective synthesis of 24(s), 25-epoxycholesterol

Article abstract of DOI:10.1021/jo981753v

Efficient, stereoselective syntheses of 24(S), 25-epoxycholesterol (1) have been developed starting from cholenic acid (4) or stigmasterol (8), both featuring as the key step Sharpless asymmetric dihydroxylation of desmosterol acetate (2). This work permits preparation of gram quantities of 1 for further evaluation as a natural regulator of cholesterol metabolism, specifically, e.g., as a ligand for the LXRα. nuclear receptor.

Full text of DOI:10.1021/jo981753v

J . Org. Chem. 1998, 63, 9919-9923
9919
Efficien t, Ster eoselective Syn th esis of 24(S),25-Ep oxych olester ol
Nicholas C. O. Tomkinson and Timothy M. Willson
Department of Medicinal Chemistry, Glaxo Wellcome Research and Development,
Research Triangle Park, North Carolina 27709
J onathon S. Russel and Thomas A. Spencer*
Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755
Received August 26, 1998
Efficient, stereoselective syntheses of 24(S),25-epoxycholesterol (1) have been developed starting
from cholenic acid (4) or stigmasterol (8), both featuring as the key step Sharpless asymmetric
dihydroxylation of desmosterol acetate (2). This work permits preparation of gram quantities of 1
for further evaluation as a natural regulator of cholesterol metabolism, specifically, e.g., as a ligand
for the LXRR nuclear receptor.
Since 24(S),25-epoxycholesterol (1) was shown to be
formed enzymatically from squalene 2,3(S);22(S),23-
dioxide1 and identified as a mammalian natural product,2
evidence has slowly accumulated consistent with par-
ticipation of that oxysterol in the natural regulation of
cholesterol metabolism.3-6 An important recent develop-
ment in this regard was the discovery that the previously
orphan nuclear LXR receptors are activated by oxys-
terols,7,8 including 1 at physiologic concentration. In
response to 1, the LXRR receptor subtype regulates
transcription of the gene encoding cholesterol 7R-hy-
droxylase,8 the enzyme that catalyzes the rate-limiting
step in cholesterol degradation. The fact that 24(S),25-
epoxycholesterol has a unique biosynthetic origin among
putatively regulatory oxysterols, being formed via squalene
dioxide rather than by oxidation of cholesterol,5 has been
used to explain the effectiveness of inhibitors of oxido-
squalene cyclase as cholesterol-lowering agents, since
cyclase inhibition leads to accumulation of squalene
dioxide and, consequently, 1.9-11
In view of this mounting evidence of the biochemical
importance of 1, it was desired to have access to sub-
stantial quantities of this epoxide, free of the C24 epimer
(epi-1), which is not known to be naturally occurring. To
date, only a few milligrams of 1 have ever been prepared
at one time, usually by painstaking HPLC separation of
the mixture of C24 epimeric epoxides formed by peracid
treatment of desmosterol.12 Development of an efficient,
highly stereoselective, and reasonably economical syn-
thesis of 1 is described herein.
The fundamental strategy adopted for steroselective
generation of the 24(S),25-epoxide function was to apply
Sharpless asymmetric dihydroxylation13 to an appropri-
ate derivative of desmosterol, such as acetate 2, to
produce 24(R),25-diol 3. Mesylation of the secondary
hydroxyl group of 3, followed by treatment with base,
would then lead to 1.
However, desmosterol is extremely expensive and
available only in limited supply, so it was also essential
to develop an efficient and economical synthesis of key
prospective intermediate 2. A number of syntheses of
desmosterol from a variety of steroidal precursors have
been reported;14 we decided to attempt to seek improve-
ment of two of these previous approaches. The first
approach utilized as starting material cholenic acid (4),
which has been converted, as different derivatives of the
3â-hydroxyl group, to C24 aldehydes,15,16 which in turn
(1) Nelson, J . A.; Steckbeck, S. R.; Spencer, T. A. J . Biol. Chem. 1981,
256, 1067-1068.
(2) Nelson, J . A.; Steckbeck, S. R.; Spencer, T. A. J . Am. Chem. Soc.
1981, 103, 6974-6975.
(3) Spencer, T. A.; Gayen, A. K.; Phirwa, S.; Nelson, J . A.; Taylor,
F. R.; Kandutsch, A. A.; Erickson, S. K. J . Biol. Chem. 1985, 260,
13391-13394.
(4) Saucier, S. E.; Kandutsch, A. A.; Taylor, F. R.; Spencer, T. A.;
Phirwa, S.; Gayen, A. K. J . Biol. Chem. 1985, 260, 14571-14579.
(5) Spencer, T. A. Acc. Chem. Res. 1994, 27, 83-90.
(6) Sinensky, M.; Logel, J .; Phirwa, S.; Gayen, A. K.; Spencer, T. A.
Exp. Cell Res. 1987, 171, 492-497.
(7) J anowski, B. A.; Willy, P. J .; Rama Devi, T.; Falck, J . R.;
Mangelsdorf, D. A. Nature 1996, 383, 728-731.
(8) (a) Lehmann, J . M.; Kliewer, S. A.; Moore, L. B.; Smith-Oliver,
T. A.; Oliver, B. B.; Su, J .-L.; Sundseth, S. S.; Winegar, D. A.;
Blanchard, D. E.; Spencer, T. A.; Willson, T. M. J . Biol. Chem. 1997,
272, 3137-3140. (b) Peet, D. J .; Turley, S. D.; Ma, W.; J anowski, B.
A.; Lobaccaro, J . A.; Hammer, R. E.; Mangelsdorf, D. J . Cell 1998, 93,
693-704.
(12) Saucier, S. E.; Kandutsch, A. A.; Gayen, A. K.; Nelson, J . A.;
Spencer, T. A. J . Lipid Res. 1990, 31, 2179-2185.
(13) Kolb, H. C.; VanNieuwenhze, M. S.; Sharpless, K. B. Chem.
Rev. 1994, 94, 2483-2547.
(14) Kircher, H. W.; Rosentein, F. U. J . Org. Chem. 1987, 52, 2586-
2588. References to other syntheses of desmosterol are provided in this
paper.
(9) Mark, M.; Mu¨ller, P.; Maier, R.; Eisele, B. J . Lipid Res. 1996,
37, 148-158.
(10) Morand, O. H.; Aebi, J . D.; Dehmlow, H.; J i, Y.-H.; Gains, N.;
Lengsfeld, H.; Himber, J . J . Lipid Res. 1997, 38, 373-390.
(11) Eisele, B.; Budzinski, R.; Mu¨ller, P.; Maier, R.; Mark, M. J .
Lipid Res. 1997, 38, 564-575.
10.1021/jo981753v CCC: $15.00 © 1998 American Chemical Society
Published on Web 12/05/1998

9920 J . Org. Chem., Vol. 63, No. 26, 1998
Tomkinson et al.
Sch em e 1a
Sch em e 2a
a
Reagents: (a) CH2N2; (b) (CH3)2AlCl, CH3O(CH3)NH2Cl; (c)
(iBu)2AlH; (d) AcCl, py; (e) (CH3)2CPPh3.
can be transformed to the desmosterol skeleton by Wittig
isopropylidenation.3,17 Our variation on this theme pro-
ceeded, as outlined in Scheme 1, via esterification of 4
to 5 (98%), conversion to Weinreb amide 6 by the Nakata
procedure (94%),18 and reduction to aldehyde 7 (89%),
followed by acetylation and isopropylidenation to afford
2 (82%).
The second approach utilized the less costly stigmas-
terol (8) as starting material and, as shown in Scheme
2, followed the route developed by Partridge et al.,19
through tosylation to 9, methanolysis to i-steroid 10,
reductive ozonolysis to 11, and conversion, via tosylate
12,20 to iodide 13 in 42% overall yield, compared to the
originally reported 41%.19 Coupling of 13 with π-(di-
methylallyl)nickel bromide in 65% yield has been used
in a previous synthesis of desmosterol,21 but we wished
to avoid large-scale use of Ni(CO)4. Apfel22 effected direct
alkylation of a C22 bromide analogous to 13 with pren-
yllithium in 39% yield, and we hoped to be able to
improve this type of coupling by, for example, use of
organocuprate methodology. However, extensive efforts,
with or without involvement of a variety of cuprates, to
couple 13 or tosylate 12 with prenyl organometallic
reagents,23 or to couple metalated 13 with prenyl bro-
mide,24 all resulted in either very low yield or in a high
proportion of product of coupling at the undesired, more
a
Reagents: (a) TsCl, py; (b) CH3OH, ∆; (c) (1) O3, CH2Cl2, (2)
NaBH4; (d) TsCl, py; (e) KI, acetone, ∆; (f) PhSO2Na, DMF, ∆; (g)
(1) BuLi, (2) 4-bromo-2-methyl-2-butene; (h) Li, NH3; (i) HOAc,
∆.
substituted, terminus of the allylic system.25 The best
yield of desired alkylation product 14 was obtained by
reaction of 13 with prenyllithium by the procedure of
Apfel,22 which afforded a mixture judged by H NMR to
1
contain 51% 14, 7% of the alternate alkylation isomer,
and 17% of reduction product 16.26
Attention was then turned to known C22 sulfone 15,
which was formed in 92% yield from 13 upon treatment
with sodium phenyl sulfinate in DMF at a temperature
intermediate between those employed by Ourisson27 and
Moriarty.28 Alkylation of 15 with prenyl bromide gave
81% of 17 as a separable mixture of diastereomers. The
phenylsulfonyl group was removed with Li/NH3 to pro-
duce 74% of 14, which was heated in acetic acid19 to afford
87% of 2.
With adequate quantities of key intermediate 2 avail-
able by either route, its asymmetric dihydroxylation by
the Sharpless procedure13 was explored. Gratifyingly,
dihydroxylation was found to give the desired 24(R),25-
diol 3 in 92% yield using in situ generated AD-mix-â
reagent with addition of CH3SO2NH2 to improve catalyst
turnover13 (Scheme 3). This reaction was regio- and
(15) Kris Prasad, V. V.; Ponticorvo, L.; Lieberman, S. J . Steroid
Biochem. 1984, 21, 733-736.
(16) Okamoto, M.; Tabe, M.; Fujii, T.; Tanaka, T. Tetrahedron:
Asymmetry 1995, 6, 767-778.
(17) Fagerlund, U. H. M.; Idler, D. R. J . Am. Chem. Soc. 1957, 79,
6473-6475.
(18) Shimizu, T.; Osaka, K.; Nakata, T. Tetrahedron Lett. 1997, 38,
2685-2688.
(19) Partridge, J . J .; Faber, S.; Uskokovic, M. R. Helv. Chim. Acta
1974, 57, 764-770.
(20) Direct conversion of 11 to 13, as reported by Schmittberger and
Uguen (Schmittberger, T.; Uguen, D. Tetrahedron Lett. 1996, 37, 29-
32) gave poor results in our hands.
(24) Lithiated 13 + prenyl bromide + Cu: Gorlier, J .-P.; Hamon,
L.; Levisalles, J .; Wagnon, J . J . Chem. Soc. Chem. Commun. 1973,
88.
(25) Morisaki, M.; Shibata, M.; Duque, C.; Imamura, N.; Ikekawa,
N. Chem. Pharm. Bull. 1980, 28, 606-611. This work describes an
analogous “backwards” coupling product from the same type of reaction
of a C22 tosylate.
(26) Dasgupta, S. K.; Gut, M. J . Org. Chem. 1975, 40, 1475-1477.
This paper reports that 12 gives only 16 or dimeric product upon
reaction with prenyl Grignard reagent. Ikekawa, ref 25, also reports
a reduction product analogous to 16.
(27) Koch, P.; Nakatani, Y.; Luu, B.; Ourisson, G. Bull. Soc. Chim.
Fr. 1983, 189-194.
(28) Moriarty, R. M.; Enache, L. A.; Kinney, W. A.; Allen, C. S.;
Canary, J . W.; Tuladhar, S. M.; Guo, L. Tetrahedron Lett. 1995, 36,
5139-5142.
(21) Dasgupta, S. K.; Crump, D. R.; Gut, M. J . Org. Chem. 1974,
39, 1658-1661.
(22) Apfel, M. A. J . Org. Chem. 1978, 43, 2284-2285.
(23) Prenyl Grignard reagent (PGR) + 12 or 13: (a) Lipshutz, B.
H.; Hackmann, C. J . Org. Chem. 1994, 59, 7437-7444. PGR + 12 +
various copper reagents (Cu): (b) Tamura, M.; Kochi, J . Synthesis 1971,
303-305. (c) Fouquet, G.; Schlosser, M. Angew. Chem., Int. Ed. Engl.
1974, 13, 82-83. (d) Schlosser, M. Angew. Chem., Int. Ed. Engl. 1974,
13, 701-706. (e) Reference 25. Prenyllithium (PL): (f) Seyferth, D.;
Weiner, M. A. J . Org. Chem. 1961, 26, 4797-4800. PL + 12 or 13: (g)
Reference 22. PL + 12 or 13 + Cu: See ref 23b-d. (h) Lipshutz, B. H.;
Elworthy, T. R. J . Org. Chem. 1990, 55, 1695-1696. (i) Lipshutz, B.
H.; Ellsworth, E. L.; Dimock, S. H.; Smith, R. A. J . J . Am. Chem. Soc.
1990, 112, 4404-4410.

Synthesis of 24(S),25-Epoxycholesterol
J . Org. Chem., Vol. 63, No. 26, 1998 9921
Sch em e 3a
with silica gel 60 F-245. Visualization was obtained by
exposure to 5% phosphomolybdic acid in ethanol. MgSO4 was
used to dry all solvents and organic layers from reaction
mixtures. Cholenic acid was purchased from Steraloids Inc.,
Wilton, NH. All other reagents, unless noted, were obtained
from Aldrich Chemical Co., Milwaukee, WI.
5-Ch olen ic Acid -3â-ol N-Meth oxy-N-m eth yla m id e (6).
To a stirred suspension of 2.54 g (26 mmol) of CH3ONHCH3‚HCl
in 100 mL of CH2Cl2 under N2 at 0 °C was added 26 mL (26
mmol, 1 M in hexane) of (CH3)2AlCl over 5 min, and the
mixture was stirred for 1 h while the temperature rose to rt.
Then a solution of 2.0 g (5.15 mmol) of 5, prepared in 98%
yield by diazomethane esterification of 4, in 80 mL of CH2Cl2
was added dropwise via cannula, stirring was continued at rt
for 4 h, and 80 mL of phosphate buffer (pH 8.0) was added.
The resulting mixture was stirred for 20 min, diluted with 200
mL of CHCl3, and filtered through Celite, which was washed
with 200 mL of CHCl3. The aqueous layer was extracted with
3 × 100 mL of CHCl3, and the combined organic layers were
washed with brine, dried, and evaporated to give 2.02 g (94%)
of 6, which was recrystallized from EtOAc/hexanes to give 6:
mp 138-139 °C; [R]D -7 (c 0.03, CHCl3); 1H NMR 5.28 (t, J )
2.6 Hz, 1 H), 3.62 (s, 3 H), 3.46 (m, 1 H), 3.11 (s, 3 H), 2.38-
2.16 (m, 4 H), 1.95-0.82 (m, 26 H), 0.94, (s, 3 H), 0.88 (d, J )
6.5 Hz, 3 H), 0.62 (s, 3 H); 13C NMR 140.8, 121.7, 71.8, 61.2,
56.7, 55.9, 50.1, 42.4, 42.3, 39.7, 37.2, 36.5, 35.6, 31.9, 31.6,
30.7, 28.9, 28.1, 24.3, 21.1, 19.4, 18.5, 11.9; MS m/z 418.3
(MH+). Anal. Calcd for C26H43N1O3: C, 74.77; H, 10.38; N, 3.35.
Found: C, 74.72; H, 10.50; N, 3.41.
3â-Hyd r oxych olest-5-en -24-a l (7). To a stirred solution
of 2.0 g (4.54 mmol) of 6 in 70 mL of CH2Cl2 under N2 at -78
°C was added a solution of 9.08 mL (1.0 M in toluene, 9.08
mmol) of DIBAH in 10 mL of CH2Cl2 over 1 h, and stirring
was continued at -78 °C for 30 min. This mixture was added
via cannula to a cooled (0 °C) solution of 12 g of sodium
potassium tartrate in 40 mL of H2O, and the resulting mixture
was stirred for 3 h during which time the biphasic mixture
became clear. The aqueous phase was extracted with 3 × 50
mL of ether, and the combined organic layers were washed
with brine, dried, and evaporated to give crude 7, which was
purified by chromatography (3:17 EtOAc/hexanes) to give 1.53
g (89%) of 7, which was recrystallized from CH2Cl2/CH3OH to
give 7: mp 132-133 °C; [R]D -14 (c 0.03, CHCl3); 1H NMR
9.7 (t, J ) 1.9 Hz, 1 H), 5.28 (t, J ) 2.5 Hz, 1 H), 3.49 (m, 1
H), 2.49-0.82 (m, 26 H), 0.94 (s, 3 H), 0.86 (d, J ) 6.5 Hz, 3
H), 0.61 (s, 3 H); 13C NMR 203.3, 140.8, 121.7, 121.6, 71.8,
56.7, 55.8, 50.9, 50.0, 42.4, 42.3, 40.9, 39.7, 37.2, 36.5, 35.3,
31.9, 31.6, 28.2, 27.9, 24.2, 21.0, 19.4, 18.4, 11.9; MS m/z 381.2
(M + Na). Anal. Calcd for C24H38O‚H2O: C, 76.55; H, 10.71.
Found: C, 76.62; H, 10.65.
Desm oster ol Aceta te (2) fr om 7. A solution of 2.0 g (5.6
mmol) of 7, 10 mL of CH2Cl2, and 5 mL of pyridine was cooled
to 0 °C and treated with 0.794 mL (11.2 mmol) of acetyl
chloride. After 30 min, 5 mL of H2O was added, and the
reaction mixture was diluted with 100 mL of EtOAc. The
organic layer was washed with 3 × 50 mL of 2 M hydrochloric
acid, 50 mL of H2O, and 50 mL of brine, dried, and evaporated
to give the acetate of 7, mp 145-147 °C (lit.15 mp 146-148
°C), which was dissolved in 30 mL of THF. A suspension of
4.82 g (11.2 mmol) of isopropyltriphenylphosphonium iodide
in 50 mL of THF was cooled to 0 °C and treated with 4.46 mL
(11.2 mmol, 2.5 M in hexanes) of n-BuLi, and stirring was
continued at 0 °C for 30 min, after which time the THF
solution of acetate was added via cannula. The reaction
mixture was allowed to warm to rt, stirred for 18 h, and
quenched with 50 mL of saturated NH4Cl solution, and the
aqueous phase was extracted with 3 × 50 mL of EtOAc. The
combined organic layers were washed with 150 mL of H2O and
150 mL of brine, dried, and evaporated to give crude 2, which
was chromatographed (1:19 EtOAc/hexanes) to give 1.95 g
(82%) of 2: mp 91-92 °C (lit.14 mp 94-95 °C): 1H NMR 5.38-
5.33 (m, 1H), 5.07 (t, J ) 9 Hz, 1H), 4.65-4.53 (m, 1H), 2.33-
2.27 (m, 2H), 2.01 (s, 3H), 1.66 (s, 3H), 1.58 (s, 3H), 1.01 (s,
3H), 0.93-0.91 (d, J ) 6 Hz, 3H), 0.66 (s, 3H); 13C NMR 170.8,
139.8, 131.1, 125.4, 122.8, 74.2, 56.9, 56.2, 50.2, 42.5, 39.9, 38.3,
a
Reagents: (a) AD-mix-â, CH3SO2NH2; (b) AD-mix-R,
CH3SO2NH2; (c) CH3SO2Cl, py; (d) K2CO3, CH3OH, H2O.
stereospecific; no dihydroxylation of the ∆5,6 double bond
or formation of 24(S)-diol (epi-3) could be detected by 13
C
NMR. Diol 3 was converted to its C24 mesylate, which
was treated with K2CO3 in methanol to generate the
epoxide function and, more slowly, to free the C3 hydroxyl
group. Dilution of the reaction mixture with H2O pro-
vided the hemihydrate of 24(S),25-epoxycholesterol (1)
as a white powder in 87% yield from 3 (80% from 2, with
one purified intermediate). 24(R),25-Epoxycholesterol
(epi-1) was also prepared from intermediate 2 by a
parallel route by use of the AD-mix-R reagent13 in the
asymmetric dihydroxylation step. The C24 configuration
of 1 was initially confirmed by HPLC comparison with
samples of 1 and epi-1 generated by HPLC separation of
the diastereomeric mixture.12 To provide final confirma-
tion of the relative configurations, single-crystal X-ray
analysis was performed on both 1 and epi-1. The resulting
structures showed unequivocally that the assignment of
C24 stereochemistry to 1 and epi-1 was correct.29
The synthesis of 1 from cholenic acid (4) requires 8
steps and affords 54% overall yield. From stigmasterol
(8), which costs approximately 1% as much as 4, prepa-
ration of 1 requires 12 steps and affords 16% overall yield.
All steps in either synthesis can readily be conducted on
a multigram scale.
Exp er im en ta l Section
NMR spectra were taken in CDCl3 on either a 300 or 400
MHz spectrophotometer. The 1H and 13C chemical shifts are
reported in units of δ from TMS. Tetrahydrofuran (THF) and
ether were distilled from sodium/benzophenone. Acetone was
distilled from calcium carbonate onto 3 Å molecular sieves.
Pyridine was distilled from calcium hydride onto 4 Å molecular
sieves. Methanol was distilled from magnesium and iodine
onto 3 Å molecular sieves. Acetic acid was distilled from acetic
anhydride. All reactions were magnetically stirred. Melting
points are uncorrected.
Flash column chromatography was carried out on EM
Reagent silica gel 60 (230-400 mesh). Thin-layer chromatog-
raphy (TLC) was conducted on EM plastic sheets precoated
(29) Atomic coordinates, bond lengths and angles, and thermal
parameters for 1 and epi-1 have been deposited at the Cambridge
Crystallographic Data Centre, with registration nos. 102766 for 1 and
102765 for epi-1.

9922 J . Org. Chem., Vol. 63, No. 26, 1998
Tomkinson et al.
37.2, 36.8, 36.3, 35.8, 32.1, 32.0, 28.4, 28.0, 26.0, 24.9, 24.5,
21.7, 21.2, 19.5, 18.8, 17.9, 12.1.
22.8, 21.6, 19.5, 18.0, 15.0, 13.3, 12.2. Anal. Calcd for
C34H50O3S: C, 75.79; H,9.36. Found: C, 75.77; H. 9.30.
3r,5-Cyclo-22-iod o-5r-23,24-bisn or ch ola n -6â-ol 6-Meth -
yl Eth er (13). The sequence of Partridge et al.19 was used to
convert stigmasterol (8) to 13 with the following results and
modifications in procedure, if any, for each step. Tosylate 9,
mp 146-147.5 °C (lit.19 mp 148-149 °C), 95% from 8, was
converted to 10 in 71% yield, but consumption of 8 required
12 h at reflux. Similar results were obtained with the
procedure of Steele and Mosettig.30 Ozonolysis of 10 was
conducted as described by Salmond and Sobala,31 followed by
NaBH4 reduction and workup as described by Boger and
Coleman,32 to afford 11 (81%), which was carried on to tosylate
12 (88%), mp 144-146 °C (lit.19 mp 144-145 °C), using a
workup described by Gut et al.,21 and then to iodide 13 (87%),
mp 103.5-104.5 °C (lit.19 mp 103-104 °C).
3r,5-Cyclo-22-p h en ylsu lfon yl-5r-23,24-b isn or ch ola n -
6â-ol 6-Meth yl Eth er (15). According to a modification of a
procedure by Ourisson,27 a mixture of 500 mg (1.1 mmol) of
13 and 542 mg (3.3 mmol) of sodium benzenesulfinate in 10
mL of DMF was heated (oil bath temp 95-100 °C) for 3 h with
stirring under argon. The reaction mixture was treated with
10 mL of water and extracted with 3 × 10 mL of ether. The
combined ether layers were washed with 3 × 10 mL of water
and 2 × 10 mL of brine, dried, filtered, and evaporated to
afford 570 mg of colorless residue that was recrystallized from
ether to yield 475 mg (92%) of colorless 15: mp 142-143 °C
(lit.27 mp 143-144 °C); 1H NMR 7.91-7.87 (m, 2H), 7.65-7.52
(m, 3H), 3.29 (s, 3H), 3.15-3.10 (dd, J ) 3, 12 Hz, 1H), 2.87-
2.79 (dd, J ) 12, 15 Hz, 1H), 2.73 (t, J ) 3 Hz, 1H), 1.18-1.16
(d, J ) 6 Hz, 3H), 0.98 (s, 3H), 0.66 (s, 3H).
3r,5-Cyclo-5r-ch olest-24-en -6â-ol 6-Meth yl Eth er (14).
A solution of 152 mg (22.0 mmol) of lithium in 8 mL of 3:1
ammonia/THF at -78 °C was treated dropwise with 296 mg
(0.55 mmol) of 17 dissolved in 3 mL of THF and 0.5 mL of
absolute ethanol. The blue solution was stirred for 30 min at
-78 °C, quenched by dropwise addition of acetone until the
solution became colorless, and kept at rt for 2 h to allow the
ammonia to evaporate. The residue was dissolved in 10 mL of
EtOAc and washed with 10 mL of water. The aqueous layer
was washed with 2 × 10 mL of EtOAc, and the combined
organic layers were washed with 25 mL of saturated NH4Cl
solution and 25 mL of brine, dried, filtered, and evaporated to
afford 418 mg of residue that was chromatographed on AgNO3-
impregnated silica gel33 (2:98 EtOAc/hexanes) to yield 162 mg
(74%) of colorless oily 14: mp 60-62 °C after crystallization
from methanol (lit.21 mp 64-66 °C); 1H NMR 5.06 (t, J ) 9
Hz, 1H), 3.30 (s, 3H), 2.74, (t, J ) 3 Hz, 1H), 1.66 (s, 3H), 1.58
(s, 3H), 1.01 (s, 3H), 0.93-0.91 (d, J ) 6 Hz, 3H), 0.66 (s, 3H);
13C NMR 131.1, 125.5, 82.6, 56.8, 56.7, 56.5, 48.2, 43.6, 43.0,
40.5, 36.3, 35.8, 35.5, 35.3, 33.6, 30.7, 28.5, 26.0, 25.2, 25.0,
24.4, 23.0, 21.7, 19.5, 18.8, 17.8, 13.3, 12.5.
Desm oster ol Aceta te (2) fr om 14. According to a modi-
fication of a procedure by Partridge,19 a solution of 335 mg
(0.84 mmol) of 14 in 5 mL of glacial HOAc was heated (oil
bath temp 70 °C) with stirring for 6 h. The mixture was treated
with 5 mL of water and extracted with 3 × 5 mL of EtOAc.
The organic layers were washed with 3 × 5 mL of water and
3 × 5 mL of saturated NaHCO3 solution, dried, filtered, and
evaporated to afford 351 mg of residue that was chromato-
graphed (2:98 EtOAc/hexanes) to yield 313 mg (87%) of 2: mp
91-93 °C.
3r,5-Cyclo-5r-ch olest-24-en -22-p h en ylsu lfon yl-6â-ol 6-
Meth yl Eth er (17). To a solution of 854 mg (1.82 mmol) of
15 in 10 mL of THF was added dropwise 1.1 mL (2.00 mmol)
of 1.8 M butyllithium in pentane at -78 °C with stirring under
argon. After being stirred for 15 min, the yellow solution was
treated dropwise with 1.36 mg (9.10 mmol) of neat 4-bromo-
2-methyl-2-butene. The mixture was allowed to warm to room
temperature over 2 h, and stirring was continued for 12 h.
The solvent was evaporated, and the residue was dissolved in
15 mL of EtOAc and washed with 15 mL of saturated NH4Cl
solution. The aqueous layer was extracted with 2 × 15 mL of
EtOAc, and the combined organic layers were washed with
15 mL of 5% NaHCO3 solution and 15 mL of brine, dried,
filtered, and evaporated to afford 827 mg of residue that was
recrystallized twice from acetone to yield 793 mg (81%) of
colorless 17 (mp 163-167 °C). Chromatography (1:9 EtOAc/
hexanes) of 205 mg of crude 17 afforded 25 mg of a less polar
C22 epimer and 64 mg of a more polar C22 epimer. Less polar
24(R),25-Dih yd r oxych olester yl-3â-a ceta te (3). To a mix-
ture of 10 mg (0.023 mmol) of K2Os2(OH)4, 73 mg (0.094 mmol)
of (DHQD)2PHAL, 970 mg (7.03 mmol) of K2CO3, and 2.32 g
(7.03 mmol) of potassium ferricyanate was added 20 mL of
H2O and 20 mL of t-BuOH. The resulting mixture was stirred
vigorously at rt until it was clear, and then 223 mg (2.34 mmol)
of CH3SO2NH2 was added. After 15 min, 1.0 g (2.3 mmol) of 2
was added in one portion, and stirring was continued for 4 d.
The reaction mixture was cooled to 0 °C, treated with 4 g of
Na2SO3, and stirred at 0 °C for 1 h. The aqueous layer was
extracted with 4 × 50 mL of ether, and the combined organic
layers were washed with 2 × 100 mL of 1 N potassium
hydroxide solution and 100 mL of brine, dried, and evaporated
to give crude 3, which was chromatographed (3:7 EtOAc/
hexanes) to give 0.993 g (92%) of 3, which was recrystallized
from methanol to give 3: mp 158-159 °C (lit.34 mp 164-165
1
isomer: mp 166-168 °C; H NMR 7.88-7.87 (m, 2H), 7.85-
1
°C); [R]D -36 (c 0.01, CHCl3); H NMR 5.31 (d, J ) 4.8 Hz, 1
7.84 (m, 3H), 4.76 (t, J ) 6 Hz, 1H), 3.31 (s, 3H), 3.11-3.03
(m, 1H), 2.76 (t, J ) 3 Hz, 1H), 2.47-2.33 (m, 1H), 2.24-2.11
(m, 2H), 1.59 (s, 3H), 1.38 (s, 3H), 1.33-1.30 (d, J ) 9 Hz,
3H), 1.01 (s, 3H), 0.63 (s, 3H), 0.63 (s, 3H); 13C NMR 141.1,
135.8, 133.4, 129.2, 128.4, 119.8, 82.6, 67.1, 56.8, 56.5, 53.2,
48.1, 43.6, 43.2, 40.2, 37.6, 35.4, 35.2, 33.5, 30.8, 28.9, 27.0,
25.9, 25.2, 24.3, 23.0, 21.7, 19.5, 17.9, 14.7, 13.3, 11.8; FAB-
HRMS (MH+) calcd for C34H51O3S 539.3553, found 539.3559.
H), 4.54 (m, 1 H), 3.27 (dd, J ) 6.2, 6.0 Hz, 1 H), 2.25 (m, 2
H), 1.97 (s, 3 H), 1.93-0.89 (m, 25 H), 1.15 (s, 3 H), 1.09 (s, 3
H), 0.95 (s, 3 H), 0.87 (d, J ) 6.5 Hz, 3 H), 0.62 (s, 3 H); 13C
NMR 170.6, 139.7, 122.6, 78.8, 77.2, 74.0, 73.2, 56.6, 56.0, 50.0,
42.3, 39.7, 38.1, 37.0, 36.6, 35.6, 32.8, 31.9, 28.3, 28.1, 27.8,
26.6, 24.3, 23.2, 21.5, 21.0, 19.3, 18.6, 11.9; FAB-HRMS (MH+)
calcd for C29H49O4 461.3628, found 461.3628. Anal. Calcd for
C
29H48O4: C, 75.61; H, 10.50. Found: C, 75.09; H, 10.48.
1
More polar isomer: mp 163-165 °C; H NMR 7.83-7.80 (m,
24(S),25-Ep oxych olester ol (1). To a stirred solution of 1.0
2H), 7.61-7.48 (m, 3H), 4.79 (t, J ) 6 Hz, 1H), 3.29 (s, 3H),
3.07-3.02 (m, 1H), 2.73 (t, J ) 3 Hz, 1H), 2.58-2.50 (m, 1H),
2.43-2.34 (m, 2H), 1.51 (s, 3H), 1.49 (s, 3H), 1.11-1.08 (d, J
) 9 Hz, 3H), 0.98 (s, 3H), 0.64 (s, 3H); 13C NMR 140.1, 133.6,
133.4, 129.1, 128.7, 121.4, 82.5, 67.3, 56.8, 54.2, 48.1, 43.6, 43.3,
40.5, 35.4, 35.2, 34.5, 33.5, 30.7, 28.5, 25.8, 25.1, 24.2, 22.9,
g (2.17 mmol) of 3 in 20 mL of CH2Cl2 was added 1 mL of
pyridine. The resulting solution was cooled to 0 °C, and 0.34
mL (4.34 mmol) of CH3SO2Cl was added. The mixture was
allowed to warm to rt, stirred overnight, diluted with 100 mL
of EtOAc, washed with 3 × 50 mL of 2 N hydrochloric acid
and 50 mL of brine, dried, and evaporated to give a crude
mesylate, which was dissolved in 20 mL of CH3OH and 2 mL
of H2O and treated with 1.5 g (11 mmol) of K2CO3. The mixture
was stirred at room temperature for 18 h and diluted with 50
mL of H2O. The resulting precipitate was filtered, washed with
water, and dried under vacuum to give 0.756 g (87%) of a
hemihydrate of 1 as a white powder: mp 156-157 °C;35 [R]D
(30) Steele, J . A.; Mosettig, E. J . Org. Chem. 1963, 28, 571-572 used
KOAc in place of pyridine in the methanolysis of 9 for 3 h at reflux in
the conversion of a tosylate analogous to 9 to an i-steroid methyl ether.
For the use of KOAc in CH3OH at reflux for 24 h, see: Catalan, C. A.
N.; Kokke, W. C. M. C.; Duque, C.; Djerassi, C. J . Org. Chem. 1983,
48, 5207-5214.
(31) Salmond, W. G.; Sobala, M. C. Tetrahedron Lett. 1977, 1695-
1698.
(32) Boger, D. L.; Coleman, R. S. J . Am. Chem. Soc. 1988, 110,
4796-4807.
(33) Gupta, A. S.; Dev, S. J . Chromatogr. 1963, 12, 189-195.
(34) Partridge, J . J .; Uskokovic, M. R. U. S. Patent 4,038,272, 1977.

Synthesis of 24(S),25-Epoxycholesterol
J . Org. Chem., Vol. 63, No. 26, 1998 9923
1
-44 (c 0.01, CHCl3); H NMR 5.35 (d, J ) 5.3 Hz, 1 H), 3.51
24(R),25-E p oxych olest er ol (ep i-1). 24(R),25-Epoxy-
cholesterol (epi-1) was synthesized by the procedure described
above for 1 except that (DHQ)2PHAL was used as the chiral
ligand in the asymmetric dihydroxylation protocol. Thus, 47
mg (0.11 mmol) of 2 yielded 31 mg (63%) of epi-1 as a white
solid: mp 154-155 °C (lit.2 mp 166.5-168 °C); [R]D -26 (c 0.4,
CHCl3); 1H NMR 5.29 (d, J ) 5.3 Hz, 1 H), 3.46 (m, 1 H), 2.62
(t, J ) 6.1 Hz, 1 H), 2.25-2.16 (m, 2 H), 1.98-1.75 (m, 5 H),
1.56-0.83 (m, 19 H), 1.24 (s, 3 H), 1.19 (s, 3 H), 0.94 (s, 3 H),
0.87 (d, J ) 6.5 Hz, 3 H), 0.62 (s, 3 H); 13C NMR 140.8, 121.7,
71.8, 64.8, 58.4, 56.7, 55.8, 50.1, 42.3, 42.3, 39.7, 37.2, 36.5,
35.6, 32.4, 31.9, 31.6, 28.2, 25.4, 24.9, 24.3, 21.0, 19.4, 18.7,
18.6,. 11.8 ppm; MS m/z 423.3 (M + Na). X-ray analysis29 was
performed on a crystal grown from Et2O.
(m, 1 H), 2.68 (m, 1 H), 2.31 (m, 2 H), 2.28-1.95 (m, 5 H),
1.86-0.89 (m, 26 H), 1.3 (s, 3 H), 1.26 (s, 3 H), 1.00 (s, 3 H),
0.94 (d, J ) 6.5 Hz, 3 H), 0.68 (s, 3 H); 13C NMR 140.8, 121.7,
77.2, 71.8, 65.0, 58.2, 56.7, 56.0, 50.1, 42.3, 42.3, 39.8, 37.3,
36.5, 35.7, 32.5, 31.9, 31.7, 28.2, 25.7, 25.0, 24.3, 21.1, 19.4,
18.7, 18.6, 11.9; MS m/z 423.3 (M + Na). Anal. Calcd for
27H44O2‚0.5 H2O: C, 79.16; H, 11.07. Found: C, 79.07; H,
10.92.
Comparison of the HPLC retention time of this 1 with that
C
of 1 and epi-1 previously prepared12 was conducted under
conditions previously described for analysis of 24(S)- vs 24-
(R)-hydroxycholesterol,36 except that 98:2 hexane/2-propanol
was used as solvent at a flow rate of 1 mL/min. Typical
retention times were as follows: 1, 14.92 min; epi-1, 15.34 min.
X-ray analysis29 was performed on a crystal grown from
EtOAc.
Ack n ow led gm en t. The research at Dartmouth was
supported by NIH grant HL52069. The HPLC analyses
of 1 and epi-1 were kindly performed by Dansu Li. The
X-ray structures of 1 and epi-1 were determined by
Peter White, University of North Carolina, Chapel Hill.
(35) Anhydrous 1, mp 160-162 °C, has previously been fully
characterized: Reference 2. See also: Steckbeck, S. R. PhD Disserta-
tion, Dartmouth College, 1981.
(36) Saucier, S. E.; Kandutsch, A. A.; Gayen, A. K.; Swahn, D. K.;
Spencer, T. A. J . Biol. Chem. 1989, 264, 6863-6869.
J O981753V