Synthesis of (24S)-hydroxy-and (24S)-24,25-epoxycholesterol analogues, potential agonists of nuclear LXR receptors

Article abstract of DOI:10.1134/S1068162006060124

A new approach to the synthesis of a series of isomeric 24-hydroxy-and 24,25-epoxysteroids starting from lithocholic acid was proposed. Sharpless asymmetric hydroxylation of intermediate Δ²⁴-olefines was used as a reaction determining the stereochemistry of target compounds. The resulting derivatives are potential agonists of nuclear receptors LXR_α and LXR_β and are potentially useful in the structure-function studies.

Full text of DOI:10.1134/S1068162006060124

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2006, Vol. 32, No. 6, pp. 586–594. © Pleiades Publishing, Inc., 2006.
Original Russian Text © V.A. Khripach, V.N. Zhabinskii, A.V. Antonchik, A.P. Antonchik, 2006, published in Bioorganicheskaya Khimiya, 2006, Vol. 32, No. 6, pp. 651–659.
Synthesis of (24S)-Hydroxy-
and (24S)-24,25-Epoxycholesterol Analogues,
Potential Agonists of Nuclear LXR Receptors
1
V. A. Khripach, V. N. Zhabinskii, A. V. Antonchick, and A. P. Antonchick
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, ul. Kuprevicha 5/2, Minsk,
220141 Belarus
Received March 29, 2006; in final form, April 17, 2006
Abstract—A new approach to the synthesis of a series of isomeric 24-hydroxy- and 24,25-epoxysteroids
starting from lithocholic acid was proposed. Sharpless asymmetric hydroxylation of intermediate ∆24-ole-
fines was used as a reaction determining the stereochemistry of target compounds. The resulting deriva-
tives are potential agonists of nuclear receptors LXRα and LXRβ and are potentially useful in the struc-
ture–function studies.
DOI: 10.1134/S1068162006060124
Key words: asymmetric hydroxylation, cholesterol, oxysterols, steroids
INTRODUCTION
OH
The identification of oxysterols as ligands of recep-
tors LXRα and LXRβ [1–3] suggests that these com-
pounds and their analogues may serve as tools for
studying the metabolism of lipids in human and animal
organisms and also offers a possibility of design of new
HO
HO
(24S)-24-Hydroxycholesterol
2
drugs. We can first mention the following compounds
as endogenous oxysterols: (24S)-hydroxycholesterol,
which is of great importance in the maintenance of cho-
lesterol level in human and mammalian brain [4], and
(24S)-24,25-epoxycholesterol, which is involved in the
regulation of transcription of the gene encoding 7α-
hydroxylase [5] controlling the rate of cholesterol
transformation into bile acids in liver. Other natural and
synthetic oxygenated cholesterol derivatives that are
capable of LXR binding are also known [6, 7]. The
study of biological properties of such compounds and
their analogues is of great interest for the investigation
O
(24S)-24,25-Epoxycholesterol
The highest level of LXR receptors is observed in
liver where the biosynthesis of bile acids from choles-
terol occurs. Therefore, it is of interest to obtain deriv-
of metabolic processes proceeding in organism with atives, the structure of which would be a combination
of the structural moieties of both oxysterols and bile
acids. To this end, we undertook the synthesis of new
oxysterol analogues with substituents at C24 and/or
C25 and the cyclic moiety of lithocholic acid. The syn-
thesis of such compounds is of additional interest,
because lithocholic acid itself is an agonist of another
nuclear receptor, PXR, and participates in the regula-
participation of cholesterol, since it is directly related to
the problems of atherosclerosis, ischemic heart disease,
hypertension, and stenocardia (angina pectoris) [8, 9].
1
Corresponding author; phone: (+375) 172-678-647;
e-mail: khripach@iboch.bas-net.by.
2
Abbreviations: DHP, dihydropyran; Ms, methanesulfonyl; and
Thp, tetrahydropyranyl.
586

SYNTHESIS OF (24S)-HYDROXY- AND (24S)-24,25-EPOXYCHOLESTEROL ANALOGUES
587
O
OH
(V)
OH
OH
CO2H
(I)
(III)
(VI)
O
(VII)
OH
(II)
OH
OH
(IV)
(VIII)
Scheme 1.
tion of biosynthesis, transport, and metabolism of bile
acids [10].
RESULTS AND DISCUSSION
Lithocholic acid (IX), a relatively inexpensive and
available steroid, was used as starting compound. In
this work, the methyl ester of (IX) was obtained by both
the treatment with diazomethane and the alternative
procedure consisting in keeping acid (IX) in methanol
at room temperature in the presence of conc. H2SO4.
The target product (X) was obtained by both methods
in quantitative yield. At the next stage, the hydroxyl
group at C3 was protected through conversion into
ether (XI). The presence of Thp protecting group at C3
resulted in that (XI) and some other intermediate prod-
ucts at subsequent stages represented inseparable mix-
tures of diastereomers. To experimentally confirm the
assigned structures, we obtained 3α-hydroxy deriva-
tives (XIIa), (XIVa), (XVa), and (XVIIIa)–(XXIVa).
A detailed study of the relationship between the
structure and function of oxysterols implies that as
many as possible of appropriate derivatives are avail-
able for it. The formation of side chain in an oxysterol
molecule is the main and most difficult problem of the
chemical synthesis, which determines the range of
compounds available for the study. The method of
choice should enable the preparation of individual com-
pounds with predictable stereochemistry of substitu-
ents in the side chain and should also provide a possi-
bility of introduction of additional functional groups in
both the side chain and the cyclic moiety of steroid
molecule.
The further sequence of reactions implied the prep-
aration of aldehyde (XIII) and its conversion by the
Wittig reaction into ∆24-olefin (XIV). Aldehyde (XIII)
could be obtained from (X) in one step; however, the
reaction sequence we previously have tested in the syn-
thesis of related compounds [16] proved to be more
efficient. Consequently, the key intermediate com-
pound, olefin (XIV), was prepared from lithocholic
acid (IX) in five stages in total yield of 70%
(Scheme 2).
The main stages of formation of the side chain in
oxysterols are described in literature [11–16]. The most
effective solution of the formulated task may be the use
of derivatives of bile acids (I) as starting compounds
with their subsequent transformation into ∆24-olefins
(II). The stereosselective hydroxylation of these olefins
in the presence of catalysts of quinine or quinidine
series (the conditions of Sharpless reaction [17]) under
the action of a commercial mixture AD-mixα or AD-
mixβ would ensure obtaining any of isomeric 24,25-
diols. Taking into consideration that the ∆24-bond is
rather remote from the chiral tetracyclic backbone of
steroid, we guess that the result of the hydroxylation
would mainly depend on the choice of catalyst. Diols
(III) and (IV) could subsequently be converted into
both 24-alcohols (VI) and (VIII) and 24,25-epoxides
(V) and (VII) (Scheme 1).
Hydroxylation of olefin (XIV) using the commer-
cial mixture AD-mixβ, which contained chiral catalyst
on the basis of quinidine, led to the formation of diol
(XV) in 90% yield (Scheme 3). A similar reaction with
AD-mixα, which contained chiral catalyst on the basis
of quinine, led to the formation of isomeric diol (XX).
An analysis of the 13C NMR spectra of derivatives
(XVa) and (XXa) unambiguously showed that they are
individual compounds exhibiting very similar sets of
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 6 2006

588
KHRIPACH et al.
CO2Me
MeOH, H+
100%
(COCl)2
CH2OH
DMSO
CO2H
LiAlH4
CH2N2
St
St
St
(IX) St = M
(X) St = M
(XII) St = N
DHP
93%
H+
(XI) St = N
(XII‡) St = M
CHO
Ph3P=CMe2
St
St
(XIII) St = N
(XIV) St = N
(XIV‡) St = M
H+
M =
N =
ThpO
HO
H
H
Scheme 2.
NMR signals. The stereochemistry of hydroxyl group (XIX) and (XXII) and subsequent reduction of double
at C24 in diols (XV) and (XX) was assigned by analogy bond with diitmide yielded saturated (XXIV) and
with the literature data [18, 19], which indicated the (XXIII) [18, 19]. This approach is promising for the
predominant formation of (24R)-24,25-diols on introduction of additional functional groups in posi-
hydroxylation of ∆24-steroids in the presence of cata- tions 25 and 26 due to the presence of double bond in
lysts of quinidine series and (24S)-24,25-diols, in the (XIX) and (XXII).
presence of quinine derivatives.
Thus, we have synthesized a series of new com-
The treatment of diols (XV) and (XX) with MsCl in pounds, which are potential agonists of receptors LXR
pyridine yielded corresponding 24-mesylates without and combine in their structures the structural moieties
affecting the tertiary hydroxyl group at C25. The incu- of oxysterols and bile acids.
bation of intermediate mesylates in aqueous methanol
in the presence of K2CO3 led to epoxides (XVI) and
EXPERIMENTAL
(XVII). The configuration of chiral center C24 was
inverted in this reaction.
The following chemicals were used in this work:
The following problem consisted in the transforma- 3α-hydroxy-5β-cholan-24-oic acid from Sigma (United
tion of 24,25-diols (XV) and (XX) into the correspond- States); N-methyl-N-nitroso-p-toluenelsulfonamid, iso-
ing 24-hydroxyderivatives (XXVI) and (XXV). Previ- propyltriphenylphosphonium iodide, AD−mixα, AD-mixβ,
ously, we have solved a similar problem by the method methanesulfonamide, and sodium cyanoborohydride
involving the opening of epoxide cycle with HBr with from Aldrich (United States). NMR spectra (δ, ppm)
subsequent debromination of intermediate bromohy- were measured in CDCl3 (unless another solvent is indi-
drin [16]. The drawback of this method is the formation cated) on a BrukerAvance 500 spectrometer (Germany)
of a mixture of regioisomeric bromohydrins at the stage at 500 MHz for 1ç and 125 MHz for 13C. Tetramethyl-
of epoxide opening; moreover, the target 24-alcohol is silane was used as internal standard. Melting points
a minor product. Here, we used two new approaches. were measured using a Koeffler block. The reactions
First of all, we studied a possibility of anti-Markowni- were monitored by TLC on Kieselgel 60 F254 precoated
koff reduction of epoxides (XVI) and (XVII) by aluminum sheets (VWR Art. 5715, Merck, Germany).
NaBH3CN in the presence of trifluoroboron etherate Column chromatography was carried out on Kieselgel
[20]. After the removal of Thp protection, 24-alcohols 60 (VWR, Art. 7734).
(XXV) and (XXVI) were obtained in 66–72% yields
(Scheme 4).
General procedure for removal of Thp protecting
group; the synthesis of (XIIa), (XIVa), (XVa), and
An alternative method of the synthesis of (XVIIIa)–(XXIVa). p-Toluenesulfonic acid monohy-
24−hydroxyderivatives consisted in the primary elimi- drate (1 mg, 5.26 µmol) was added to a solution of
nation of hydroxy group at C25 under the action of 3α-ThpO-steroid (XII), (XIV), (XV), or (XVIII)–
MsCl in the presence of Et3N [21] with preliminary (XXIV) (0.1 mmol) in methanol (5 ml). The mixture
protection of the secondary hydroxyl group in diols was stirred for 24 h at room temperature, a saturated
(XV) and (XX) by acetylation. The elimination of inter- aqueous solution (2 ml) of NaHCO3 was then added,
mediate 25-mesylates with the formation of ∆25-olefins and the mixture was evaporated in a vacuum to dryness.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 6 2006

SYNTHESIS OF (24S)-HYDROXY- AND (24S)-24,25-EPOXYCHOLESTEROL ANALOGUES
589
O
O
St
St
(XVI) St = N
1. MsCl
(XVII) St = N
1. MsCl
2.K2CO3
2.K2CO3
OH
OH
AD-mixβ
AD-mixα
OH
OH
St
St
St
(XV) St = N
(XV‡) St = M
(XIV) St = N
(XX) St = N
(XX‡) St = M
H+
H+
Ac2O
Ac2O
OAc
OAc
OAc
OAc
MsCl
MsCl
OH
OH
St
St
St
St
(XVIII) St = N
(XVIII‡) St = M
(XIX) St = N
(XIX‡) St = M
(XXII) St = N
(XXII‡) St = M
(XXI) St = N
(XXI‡) St = M
H+
H+
H+
H+
M =
N =
HO
ThpO
H
H
Scheme 3.
The resulting residue was mixed with water (10 ml) and
extracted with ethyl acetate (3 × 10 ml). The combined
organic extracts were dried with MgSO4 and evapo-
rated in a vacuum. The residue was chromatographed
on a column eluted with a petroleum ether–ethyl ace-
tate mixture to yield the corresponding 3α-hydroxys-
teroids.
Methyl ester of 3a-hydroxy-5b-cholan-24-oic
acid (X) (variant B). Conc. H2SO4 (0.1 ml, 1.87 mmol)
was added to a solution of acid (IX) (1.00 g,
2.65 mmol) in methanol (50 ml). After 16 h, the reac-
tion mixture was mixed with a saturated aqueous
NaHCO3 solution (10 ml) and evaporated in a vacuum
to dryness. The residue was mixed with water (20 ml)
and extracted with ethyl acetate (3 × 20 ml). The com-
bined organic extracts were dried with MgSO4 and
evaporated in a vacuum to get ether (X); yield 1.04 g
(100%); it was identical to that obtained by variant A.
Methyl ester of 3a-hydroxy5b-cholan-24-oic
acid (X) (variant A). Acid (IX) (9.7 g, 25.8 mmol) was
added in portions to a solution of diazomethane (from
20 g of N-methyl-N-nitroso-p-toluenesulfonamide
[22]) in diethyl ether. The reaction mixture was kept for
60 min at room temperature and evaporated in a vac-
uum to get ester (X); yield 10.0 g (100%); mp 126–
128°ë (hexane); 1H NMR (δ, ppm, J, Hz): 3.66 (3 H, s,
ëéëç3), 3.62 (1 H, m, H3), 0.92 (3 H, s, H19), 0.91
Methyl ester of 3a-tetrahydropyranyloxy-5b-
cholan-24-oic acid (XI). p-Toluenesulfonic acid
monohydrate (100 mg, 0.53 mmol) was added to a
stirred at 0°ë mixture of (X) (10.37 g, 26.5 mmol),
DHP (4.0 ml, 44.2 mmol), and CH2Cl2 (100 ml). The
reaction mixture was stirred for 1 h and kept at room
temperature for 19 h. The solution was then diluted
with saturated aqueous NaHCO3 solution (50 ml) and
extracted with CHCl3 (3 × 50 ml). The combined
organic extracts were dried with MgSO4 and evapo-
13
(3 H, d, J 6.8, H21), 0.64 (3 H, s, H18). C NMR (δ,
ppm): 174.81, 71.86, 56.49, 55.94, 51.49, 42.73, 42.09,
40.43, 40.17, 36.45, 35.84, 35.37, 35.35, 34.57, 31.06,
31.00, 30.55, 28.19, 27.19, 26.42, 24.21, 23.38, 20.82,
18.27, 12.04.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 6 2006

590
KHRIPACH et al.
OH
OAc
OAc
O
1. NaBH3CN
2. H+
OH–
N2H4
‰Îfl St = M
St
St
(XXII) St = N
St
St
(XVI) St = N
(XXV) St = M
(XXIII) St = N
H+
(XXIII‡) St = M
OAc
OAc
OH
O
1. NaBH3CN
2. H+
OH–
‰Îfl St = M
N2H4
St
(XVII) St = N
St
(XXIV) St = N
(XXIV‡) St = M
St
St
(XXVI) St = M
(XIX) St = N
H+
M =
HO
N =
ThpO
H
H
Scheme 4.
rated in a vacuum. The residue was chromatographed
3a-Tetrahydropyranyloxy-5b-cholan-24-al (XIII).
on a column eluted with 40 : 1
10 : 1 petroleum A solution of DMSO (5.7 ml, 80.3 mmol) in CH2Cl2
ether–ethyl acetate gradient to get ester (XI) as an oil;
(15 ml) was dropwise added in argon atmosphere to a
cooled to –80°C mixture of CH2Cl2 (50 ml) and
(ëéël)2 (3.3 ml, 38.9 mmol).After stirring the reaction
mixture for 20 min at –60°C, a solution of (XII)
(5.64 g, 12.6 mmol) in CH2Cl2 (35 ml) was dropwise
added. After 20 min at –60°ë, Et3N (21 ml, 153,7
mmol) was dropwise added. The reaction mixture was
heated to room temperature, diluted with water (50 ml),
and extracted with CH2Cl2 (3 × 50 ml). The combined
organic extracts were dried with MgSO4 and evapo-
rated in a vacuum. The residue was chromatographed
1
yield 11.7 g (93%); H NMR (δ, ppm): 4.72 (1 H, m,
Thp), 3.92 (1 H, m, H3), 3.65 (1 H, m, ëç2é of Thp),
3.66 (3 H, s, ëéëç3), 3.48 (1 H, m, ëç2é of Thp),
0.90 (3 H, s, H19), 0.63 (3 H, s, H18).
3a-Tetrahydropyranyloxy-5b-cholan-24-ol (XII).
A solution of (XI) (11.72 g, 24.7 mmol) in THF (90 ml)
was dropwise added to a suspension of LiAlH4 (3.0 g,
79 mmol) in THF (150 ml). The reaction mixture was
stirred for 2 h at room temperature, then successively
treated with water (3 ml), 15% NaOH (3 ml), and again
water (9 ml). The resulting suspension was filtered, and
the residue was additionally washed with ethyl acetate
(50 ml). The filtrate was evaporated in a vacuum and the
residue was chromatographed on a column eluted with
on a column eluted with 40 : 1
10 : 1 petroleum
ether–ethyl acetate gradient to get aldehyde (XIII) as
1
an oil; yield 5.00 g (89%); H NMR (δ, ppm): 9.76
(1 H, s, H24), 4.72 (1 H, m, CH from Thp), 3.92 (1 H,
m, H3), 3.63 (1 H, m, ëç2é of Thp), 3.49 (H, m,
ëç2é of Thp), 0.90 (3 H, s, H19), 0.62 (3 H, s, H18).
20 : 1
3 : 1 petroleum ether–ethyl acetate gradient
1
to get (XII); yield 10.63 g (96%); H NMR (δ, ppm):
4.72 (1 H, m, CH of Thp), 3.92 (1 H, m, H3), 3.58–3.66
(3 H, m, ëç2é of Thp and H24), 3.49 (1 H, m, ëç2é
of Thp), 0.90 (3 H, s, H19), 0.63 (3 H, s, H18).
3a-Tetrahydropyranyloxy-5b-cholest-24-ene (XIV).
tert-BuOK (4 g, 35.7 mmol) was added to a stirred sus-
pension of isopropyltriphenylphosphonium iodide
(13.1 g, 30.3 mmol) in THF (150 ml). After 5 min, the
reaction mixture was treated with a solution of alde-
hyde (XIII) (4.20 g, 9.46 mmol) in THF (50 ml), stirred
for 20 min, then mixed with MeIi (2 ml), and stirred
again for 16 h. The resulting suspension was filtered,
the precipitate was washed with THF (50 ml), and the
filtrate was evaporated in a vacuum. The residue was
chromatographed on a column eluted with 50: 1
20 : 1 petroleum ether–ethyl acetate gradient to get ole-
5b-Cholan-3a,24-diol (XIIa) was obtained
according to the standard procedure described above
for the removal of Thp protecting group in 85% yield;
mp 179–182°ë (EtOAc–CHCl3) (lit. [23]: mp 177–
178°ë); 1H NMR (CDCl3–CD3OD) (δ, ppm): 3.55 (3 H,
m, H3 and H24), 0.90 (3 H, s, H19), 0.63 (3 H, s,
H18).13C NMR (CDCl3–CD3OD) (δ, ppm): 71.63,
63.15, 56.74, 56.46, 42.87, 42.32, 40.70, 40.41, 36.19,
36.06, 35.84, 35.53, 34.75, 32.07, 30.29, 29.36, 28.44,
27.40, 26.63, 24.38, 23.53, 21.01, 18.71, 12.14.
1
fin (XIV) as an oil; yield 3.52 g (79%); H NMR (δ,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 6 2006

SYNTHESIS OF (24S)-HYDROXY- AND (24S)-24,25-EPOXYCHOLESTEROL ANALOGUES
591
ppm): 5.09 (1 H, t, J 7.0 Hz, H24), 4.72 (1 H, m, CH of of K2CO3 (0.3 g, 2.1 mmol) in water (0.7 ml). The reac-
Thp), 3.92 (1 H, m, H3), 3.63 (1 H, m, ëç2é of Thp),
tion mixture was stirred for 5 h, diluted with water
3.49 (1 H, m, ëç2é of Thp), 1.68 (3 H, s, H27 or H26), (10 ml), and extracted with CHCl3 (3 × 20 ml). The
1.60 (3 H, s, H26 or H27), 0.90 (3 H, s, H19), 0.63 (3 H, combined organic extracts were dried with MgSO4 and
s, H18).
evaporated in a vacuum. The residue was chromato-
graphed on a column eluted with 30 : 1
8 : 1 petro-
5b-Cholest-24-en-3a-ol (XIVa) was obtained from
leum ether–ethyl acetate gradient to get epoxide
olefin (XIV) according to the procedure described
above in 95% yield; mp 114–116°ë (hexane) (lit. [24]
mp 117–118°ë). 1H NMR (δ, ppm): 5.09 (1 H, t, J 7.0
Hz, H24), 3.62 (1 H, m, H3), 1.68 (3 H, s, H27 or H26),
1.60 (3 H, s, H26 or H27), 0.92 (3 H, s, H19), 0.92 (3 H,
d, J 6.3 Hz, H21), 0.64 (3 H, s, H18). 13C NMR (δ,
ppm): 130.86, 125.23, 71.88, 56.52, 56.26, 42.71,
42.12, 40.47, 40.20, 36.47, 36.06, 35.86, 35.59, 35.35,
34.57, 30.56, 28.27, 27.21, 26.43, 25.69, 24.72, 24.24,
23.37, 20.83, 18.57, 17.60, 12.02.
1
(XVII) as an oil; yield 138 mg (77%); H NMR (δ,
ppm, J, Hz): 4.72 (1 H, m, CH from Thp), 3.92 (1 H, m,
H3), 3.63 (1 H, m, ëç2é of Thp), 3.49 (1 H, m, ëç2é
of Thp), 2.68 (1 H, t, J 6.0, H24), 1.30 (3 H, s, H27 or
H26), 1.27 (3 H, s, H26 or H27), 0.90 (3 H, s, H19),
0.64 (3 H, s, H18). 13C NMR (δ, ppm): 96.92, 96.67,
76.15, 75.98, 65.01, 62.87, 58.16, 56.55, 56.26, 42.78,
42.38, 42.14, 40.31, 40.26, 36.76, 35.91, 35.64, 35.39,
34.84, 34.43, 32.82, 32.59, 31.39, 31.36, 28.65, 28.38,
27.42, 27.27, 26.82, 26.43, 25.78, 25.58, 25.00, 24.29,
23.43, 20.86, 20.10, 18.72, 18.57, 12.08.
(24R)-3a-Tetrahydropyranyloxy-5b-cholestane-
24,25-diol (XV). A mixture of olefin (XIV) (192 mg,
0.408 mmol), AD-mixβ (400 mg), MeSO2NH2 (30 mg,
0.315 mmol), tert-BuOH (5 ml), and water (4 ml) was
stirred for 64 h, treated with Na2SO3 (0.5 g, 4 mmol),
and stirred for additional 30 min. The reaction mixture
was evaporated in a vacuum; the resulting residue was
mixed with water (30 ml) and extracted with ethyl ace-
tate (3 × 20 ml). The combined organic extracts were
dried with MgSO4 and evaporated in a vacuum. The
residue was chromatographed on a column eluted with
(24S)-3a-Tetrahydropyranyloxy-24,25-epoxy-
5b-cholestane (XVI) was obtained in 84% yield
according to the procedure described above for (XVII)
1H NMR (δ, ppm, J, Hz): 4.72 (1 H, m, CH of Thp),
3.92 (1 H, m, H3), 3.63 (1 H, m, ëç2é of Thp), 3.49
(1 H, m, ëç2é of Thp), 2.68 (1 H, t, J 6.0, H24), 1.30
(3 H, s, H27 or H26), 1.27 (3 H, s, H26 or H27), 0.90
13
(3 H, s, H19), 0.64 (3 H, s, H18). C NMR (δ, ppm):
96.87, 96.62, 76.10, 75.93, 64.96, 62.83, 56.50, 56.22,
42.73, 42.33, 42.09, 40.26, 40.21, 35.86, 35.72, 35.60,
35.34, 34.79, 34.38, 32.54, 31.34, 31.31, 28.33, 27.37,
27.22, 26.77, 26.38, 25.73, 25.53, 24.95, 24.24, 23.38,
20.82, 20.05, 18.67, 18.52, 12.03.
20 : 1
5 : 1 petroleum ether–ethyl acetate gradient
to get diol (XV) as an oil; yield 186 mg (90%); 1H NMR
(δ, ppm): 4.72 (1 H, m, CH from Thp), 3.92 (1 H, m,
H3), 3.63 (1 H, m, ëç2é of Thp), 3.49 (1 H, m, ëç2é
of Thp), 3.32 (1 H, m, H24), 1.21 (3 H, s, H27 or H26),
1.16 (3 H, s, H26 or H27), 0.90 (3 H, s, H19), 0.64 (3 H,
s, H18).
(24R)-3a-Tetrahydropyranyloxy-24-acetoxy-5b-
cholestan-25-ol (XVIII). A mixture of diol (XV)
(242 mg, 0.480 mmol) in pyridine (1.1 ml) and Ac2O
(1.1 ml) was kept at room temperature for 9 h, diluted
with water (20 ml), and extracted with ethyl acetate
(3 × 20 ml). The combined organic extracts were dried
with MgSO4 and evaporated in a vacuum. The residue
was chromatographed on a column eluted with a 30 : 1
5 : 1 petroleum ether–ethyl acetate gradient to get
(24R)-5b-Cholestan-3a,24,25-triol (XVa) was
obtained in 94% yield from diol (XV) according to the
procedure described above; mp 150–152°ë (ethyl ace-
tate) (lit. [25] mp 148–150°ë); H NMR (δ, ppm, J,
1
Hz): 3.62 (1 H, m, H3), 3.33 (1 H, m, H24), 1.18 (3 H,
s, H27 or H26), 1.14 (3 H, s, H26 or H27), 0.92 (3 H, s,
H19), 0.92 (3 H, d, J 6.3, H21), 0.66 (3 H, s, H18).
13C NMR (CDCl3–CD3OD) (δ, ppm): 78.54, 72.85,
71.25, 56.32, 56.15, 42.49, 41.89, 40.27, 40.00, 37.74,
35.79, 35.64, 35.47, 35.10, 34.34, 29.89, 28.08, 27.86,
26.98, 26.22, 25.72, 23.98, 23.13, 22.74, 20.60, 18.21,
11.76.
1
(XVIII) as an oil; yield 246 mg (94%); H NMR (δ,
ppm, J, Hz): 4.77 (1 H, dd, J 2.3 and 10.5 Hz, H24),
4.72 (1 H, m, CH of Thp), 3.92 (1 H, m, H3), 3.63 (1 H,
m, ëç2é of Thp), 3.49 (1 H, m, ëç2é of Thp), 2.11
(3 H, s, OAc), 1.20 (3 H, s, H27 or H26), 1.19 (3 H, s,
H26 or H27), 0.90 (3 H, s, H19), 0.63 (3 H, s, H18).
(24R)-24-Acetoxy5b-cholestane-3a,25-diol (XVIIIa)
was obtained in 92% yield from alcohol (XVI) accord-
ing to the procedure described above; mp 129–132°ë
(hexane–ethyl acetate); 1H NMR (δ, ppm, J, Hz): 4.77
(1 H, dd, J 2.2 and 10.4, H24), 3.62 (1 H, m, H3), 2.11
(3 H, s, OAc), 1.20 (3 H, s, H27 or H26), 1.19 (3 H, s,
H26 or H27), 0.91 (3 H, s, H19), 0.90 (3 H, d, J 6.6 Hz,
(24R)-3a-Tetrahydropyranyloxy-24,25-epoxy-
5b-cholestane (XVII). Pyridine (0.6 ml, 7.4 mmol)
and MsCl (dropwise, 0.2 ml, 2.6 mmol) were added to
a stirred at 0°ë solution of diol (XX) (186 mg,
0.369 mmol) in ëH2Cl2 (10 ml). The reaction mixture
was stirred at room temperature for 5 h, diluted with
saturated aqueous NaHCO3 solution (10 ml), and
extracted with CHCl3 (3 × 15 ml). The combined
organic extracts were dried with MgSO4 and evapo-
13
H21), 0.63 (3 H, s, H18). C NMR (δ, ppm): 171.27,
80.05, 72.50, 71.83, 56.48, 55.99, 42.68, 42.08, 40.42,
40.18, 36.43, 35.83, 35.32, 35.21, 34.54, 32.01, 30.51,
rated in a vacuum. The residue was dissolved in meth- 28.12, 27.17, 26.78, 26.40, 25.69, 24.84, 24.16, 23.35,
anol (10 ml) and treated under stirring with a solution 21.04, 20.80, 18.40, 12.00.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 6 2006

592
KHRIPACH et al.
(24R)-3a-Tetrahydropyrnyloxy-24-acetoxy-5b-
0.65 (3 H, s, H18); 13C NMR (CDCl3–CD3OD) (δ,
cholest-25-ene (XIX). Triethylamine (0.9 ml, 6.48 ppm): 79.21, 72.84, 71.19, 56.26, 55.89, 42.42, 41.84,
mmol) and then dropwise MsCl (0.28 ml, 3.61 mmol) 40.24, 39.95, 35.74, 35.72, 35.60, 35.05, 34.30, 33.03,
were added to a stirred solution of acetoxy alcohol 29.82, 27.95, 27.83, 26.94, 26.17, 25.57, 23.94, 23.09,
(XVIII) (194 mg, 0.355 mmol) in CH2Cl2 (10 ml) at 22.68, 20.56, 18.41, 11.69.
0°ë. The reaction mixture was stirred for 16 h, diluted
(24S)-3a-Tetrhydropyranyloxy-24-acetoxy-5b-
with water (20 ml), and extracted with CHCl3 (3 ×
cholestan-25-ol (XXI) was obtained from diol (XX) in
20 ml). The combined organic extracts were dried with
MgSO4 and evaporated in a vacuum. The residue was
93% yield according to the procedure described above
for (XVIII); 1H NMR (δ, ppm, J, Hz): 4.73 (2 H, m, CH
chromatographed on a column eluted 40 : 1
5 : 1
of Thp and H24), 3.92 (1 H, m, H3), 3.63 (1 H, m,
ëç2é of Thp), 3.49 (1 H, m, ëç2é of Thp), 2.10 (3 H,
s, OAc), 1.20 (3 H, s, H27 or H26), 1.19 (3 H, s, H26 or
H27), 0.90 (3 H, s, H19), 0.62 (3 H, s, H18).
petroleum ether–ethyl acetate gradient to get olefin
(XIX) (yield 100 mg (53%), total yield of 78% when
taking into account 50 mg (25%) of the recovered start-
ing alcohol (XVIII)); 1H NMR of (XIX) (δ, ppm): 5.11
(1 H, m, H24), 4.93 (1 H, s, H26), 4.87 (1 H, m, H26),
4.72 (1 H, m, CH of Thp), 3.92 (1 H, m, H3), 3.63 (1 H,
m, ëç2é of Thp), 3.49 (1 H, m, ëç2é of Thp), 2.06
(3 H, s, OAc), 1.71 (3 H, s, H27), 0.90 (3 H, s, H19),
0.62 (3 H, s, H18).
(24S)-24-Acetoxy-5b-cholestane-3a,25-diol (XXIa)
was obtained in 95% yield from alcohol (XXI) accord-
ing to the standard procedure for removal Thp protect-
ing group; mp 140–142°ë (hexane-ethyl acetate);
1H NMR (δ, ppm, J, Hz): 4.73 (1 H, dd, J 2.4 and 10.2,
H24), 3.62 (1 H, m, H3), 2.10 (3 H, s, OAc), 1.20 (3 H,
s, H27 or H26), 1.19 (3 H, s, H26 or H27), 0.91 (3 H, s,
H19), 0.91 (3 H, d, J 6.8, H21), 0.63 (3 H, s, H18);
13C NMR (δ, ppm): 171.28, 80.77, 72.45, 71.78, 56.40,
55.86, 42.63, 42.05, 40.38, 40.09, 36.39, 35.79, 35.78,
35.30, 34.52, 32.36, 30.47, 28.13, 27.15, 26.68, 26.37,
25.97, 24.89, 24.15, 23.33, 21.02, 20.76, 18.72, 11.97.
(24R)-24-Acetoxy-5b-cholest-25-en-3a-ol (XIXa)
was obtained from olefin (XIX) in 97% yield according
to the procedure described above; mp 101–103°ë (hex-
ane–ethyl acetate); 1H NMR (δ, ppm, J, Hz): 5.11 (1 H,
m, H24), 4.93 (1 H, s, H26), 4.87 (1 H, m, H26), 3.62
(1 H, m, H3), 2.06 (3 H, s, OAc), 1.71 (3 H, s, H27),
0.92 (3 H, s, H19), 0.91 (3 H, d, J 6.6, H21), 0.63 (3 H,
s, H18); 13C NMR (δ, ppm): 170.41, 143.45, 112.47,
77.64, 71.89, 56.53, 55.99, 42.71, 42.13, 40.48, 40.20,
36.49, 35.88, 35.38, 34.59, 31.30, 30.57, 29.11, 28.21,
27.22, 26.45, 24.22, 23.39, 21.23, 20.84, 18.62, 18.19,
12.03.
(24S)-3a-Tetrhydropyranyloxy-24-acetoxy-5b-
cholest-25-ene (XXII) was obtained from alcohol
(XXI) in 86% yield according to the procedure
1
described above for (XIX); H NMR (δ, ppm, J, Hz):
5.11 (1 H, t, J 6.8, H24), 4.93 (1 H, s, H26), 4.88 (1 H,
m, H26), 4.72 (1 H, m, CH of Thp), 3.92 (1 H, m, H3),
3.63 (1 H, m, ëç2é of Thp), 3.49 (1 H, m, ëç2é of
Thp), 2.05 (3 H, s, OAc), 1.71 (3 H, s, H27), 0.90 (3 H,
s, H19), 0.62 (3 H, s, H18).
(24S)-3a-Tetrahydropyranyloxy-5b-cholestan-
24,25-diol (XX). A mixture of olefin (XIV) (2.19 g,
4.66 mmol), AD-mixα (5.25 g), MeSO2NH2 (300 mg,
3.15 mmol), tert-BuOH (50 ml), and water (40 ml) was
stirred for 64 h, then mixed with Na2SO3 (5.0 g,
40 mmol), and stirred again for 30 min. The reaction
mixture was evaporated to dryness in a vacuum. The
resulting residue was mixed with water (100 ml) and
extracted with ethyl acetate (3 × 70 ml). The combined
organic extracts were dried with MgSO4 and evapo-
rated in a vacuum. The residue was chromatographed
(24S)-24-Acetoxy-5b-cholest-25-en-3a-ol (XXIIa)
was obtained in 88% yield from olefin (XXII) accord-
ing to the standard procedure for removal Thp protect-
1
ing group; mp 95–97°ë (hexane); H NMR (δ, ppm):
5.11 (1 H, t, J 6.8 Hz, H24), 4.94 (1 H, s, H26), 4.88
(1 H, m, H26), 3.62 (1 H, m, H3), 2.05 (3 H, s, OAc),
1.71 (3 H, s, H27), 0.92 (3 H, s, H19), 0.91 (3 H, d, J 6.8
Hz, H21), 0.63 (3 H, s, H18); 13C NMR (δ, ppm):
170.41, 143.03, 113.11, 78.11, 71.87, 56.50, 56.00,
42.69, 42.09, 40.43, 40.17, 36.44, 35.85, 35.44, 35.34,
34.57, 31.29, 30.54, 28.84, 28.20, 27.20, 26.43, 24.20,
23.38, 21.28, 20.82, 18.60, 17.88, 12.02.
on a column eluted with 20 : 1
5 : 1 petroleum
ether–ethyl acetate gradient to get diol (XX) as an oil;
yield 2.10 g (89%); 1H NMR (δ, ppm, J, Hz): 4.72 (1 H,
m, CH of Thp), 3.92 (1 H, m, H3), 3.63 (1 H, m, ëç2é
of Thp), 3.49 (1 H, m, ëç2é of Thp), 3.28 (1 H, dd,
J 1.7 and 10.0, H24), 1.22 (3 H, s, H27 or H26), 1.16
(3 H, s, H26 or H27), 0.90 (3 H, s, H19), 0.63 (3 H, s,
H18).
(24S)-24-Acetoxy-5b-cholestan-3a-ol (XXIIIa)
was obtained in 90% yield from acetate (XXIII)
according to the standard procedure for the removal of
Thp protecting group; mp 100–102°ë (hexane);
(24S)-5b-Cholestan-3a,24,25-triol (XXa) was 1H NMR (δ, ppm): 4.68 (1 H, m, H24), 3.62 (1 H, m,
obtained in 92% yield from diol (XX) according to the H3), 2.05 (3 H, s, OAc), 0.93–0.87 (12 H, m, H19, H21,
standard procedure described above; mp 159–161°ë H26, and H27), 0.63 (3 H, s, H18); 13C NMR (δ, ppm):
1
(ethyl acetate) (lit. [25] mp 162–164°ë); H NMR (δ, 171.07, 79.10, 71.81, 56.43, 55.91, 42.63, 42.05, 40.38,
ppm, J, Hz): 3.59 (1 H, m, H3), 3.22 (1 H, dd, J 1.3 and 40.11, 36.39, 35.79, 35.72, 35.30, 34.52, 31.44, 30.96,
10.0, H24), 1.19 (3 H, s, H27 or H26), 1.14 (3 H, s, H26 30.49, 28.16, 27.52, 27.16, 26.38, 24.16, 23.34, 21.14,
or H27), 0.93 (3 H, d, J 7.7, H21), 0.92 (3 H, s, H19), 20.77, 18.70, 18.63, 17.27, 11.98.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 6 2006

SYNTHESIS OF (24S)-HYDROXY- AND (24S)-24,25-EPOXYCHOLESTEROL ANALOGUES
593
(24R)-3a-Tetrhydropyranyloxy-24-acetoxy-5b- extracts were dried with MgSO4 and evaporated in a
cholestane (XXIV). A mixture of NH2OH · HCl
(8.47 g, 0.121 mmol), KOH (6.80 g, 0.121 mmol), umn eluted a 20 : 1
vacuum. The residue was chromatographed on a col-
5 : 1 petroleum ether–ethyl ace-
DMF (23 ml), and water (1.2 g) was vigorously stirred tate gradient to get diol (XXVI); yield 13 mg (90%);
for 2 h at 0°ë, then filtered, and the precipitate was mp 131–133 and 145–146°ë (hexane–ethyl acetate);
1H NMR (δ, ppm): 3.62 (1 H, m, H3), 3.31 (1 H, m,
washed with DMF (7 ml). Ethyl acetate (5.2 ml,
53.3 mmol) was added at 0°ë under stirring to the fil-
trate. The reaction mixture was stirred for 30 min at
0°ë, the resulting solution was added in portions under
stirring during 5 h to olefin (XIX) (78 mg, 0.147 mmol)
at 90°ë. After cooling, the reaction mixture was diluted
with saturated NaCl (50 ml) and extracted with ethyl
acetate (3 × 50 ml). The combined organic extracts
were dried with MgSO4 and evaporated in a vacuum.
The residue was chromatographed on a column eluted
H24), 0.93–0.90 (12 H, m, H19, H21, H26, and H27),
0.64 (3 H, s, H18). 13C NMR (δ, ppm): 77.05, 71.87,
56.49, 56.20, 42.69, 42.09, 40.43, 40.18, 36.44, 35.84,
35.68, 35.33, 34.56, 33.52, 31.99, 30.58, 30.53, 28.34,
27.19, 26.41, 24.21, 23.36, 20.81, 18.89, 18.59, 17.22,
12.03.
(24R)-5b-Cholestane-3a,24-diol (XXVI) (vari-
ant B). Trifluoroboron etherate was dropwise added
until appearance of yellow color to a stirred solution of
epoxide (XVII) (96 mg, 0.197 mmol), NaBH3CN
(50 mg, 0.793 mmol), and bromocresol green indicator
(2–3 mg) in THF (5 ml). The reaction mixture was
stirred for 48 h, diluted with saturated aqueous
NaHCO3 solution (10 ml), and extracted with ethyl ace-
tate (3 × 10 ml). The combined organic extracts were
dried with Na2SO4 and evaporated in a vacuum. The
residue was dissolved in methanol (25 ml) and mixed
with TosOH · H2O (100 mg, 0.53 mmol). The reaction
mixture was kept at room temperature for 12 h, mixed
with pyridine (1 ml), and evaporated in a vacuum. The
residue was chromatographed on a column eluted with
with 40 : 1
10 : 1 petroleum ether–ethyl acetate
gradient to get acetate (XXIV) as an oil; yield 65 mg
(83%); 1H NMR (δ, ppm): 4.71 (2 H, m, CH of Thp and
H24), 3.92 (1 H, m, H3), 3.63 (1 H, m, ëç2é of Thp),
3.49 (1 H, m, ëç2é of Thp), 2.05 (3 H, s, OAc), 0.90
(3 H, s, H19), 0.62 (3 H, s, H18).
(24S)-3a-Tetrhydropyranyloxy-24-acetoxy-5b-
cholestan (XXIII) was similarly obtained from olefin
(XXII) in 76% yield; 1H NMR (δ, ppm): 4.71 (2 H, m,
CH of Thp and H24), 3.92 (1 H, m, H3), 3.63 (1 H, m,
ëç2é of Thp), 3.49 (1 H, m, ëç2é of Thp), 2.05 (3 H,
s, OAc), 0.90 (3 H, s, H19), 0.62 (3 H, s, H18).
(24R)-24-Acetoxy-5b-cholestan-3a-ol (XXIVa)
was obtained in 83% yield from acetate (XXIV)
according to the standard procedure for the removal of
Thp protecting group; mp 112–114°ë (hexane–ethyl
20 : 1
5 : 1 petroleum ether–ethyl acetate gradient
to yield 53 mg (66%) of diol (XXVI), mp and spectral
data of which corresponded to those of the sample
obtained by variant A.
1
acetate); H NMR (δ, ppm): 4.69 (1 H, m, H24), 3.62
(1 H, m, H3), 2.05 (3 H, s, OAc), 0.91 (3 H, s, H19),
0.91–0.87 (9 H, m, H21, H26, and H27), 0.63 (3 H, s,
H18); 13C NMR (δ, ppm): 171.00, 78.73, 71.85, 56.50,
56.02, 42.68, 42.10, 40.44, 40.18, 36.46, 35.84, 35.38,
35.34, 34.56, 31.43, 31.37, 30.54, 28.14, 27.39, 27.19,
26.41, 24.18, 23.36, 21.13, 20.81, 18.51, 17.73, 12.01.
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