Palmitates of isomeric 15-oxygenated Δ8(14)-sterols

Article abstract of DOI:10.1023/B:RUBI.0000023106.52513.6a

3β-Hexadecanoyloxy-5α-cholest-8(14)-en-15-one, 3α-hexadecanoyloxy-5α-cholest-8(14)-en-15-one, 15β- hexadecanoyloxy-5α-cholest-8(14)-en-3β-ol, 15α-hexadecanoyloxy- 5α-cholest-8(14)-en-3β-ol, 15β-hexadecanoyloxy-5α-cholest- 8(14)-en-3-one, and 15α-hexadecan

Full text of DOI:10.1023/B:RUBI.0000023106.52513.6a

Russian Journal of Bioorganic Chemistry, Vol. 30, No. 2, 2004, pp. 184–190. Translated from Bioorganicheskaya Khimiya, Vol. 30, No. 2, 2004, pp. 208–214.
Original Russian Text Copyright © 2004 by Ignatov, Prokof’ev, Timofeev, Misharin.
Palmitates of Isomeric 15-Oxygenated D8(14)-Sterols
, 1
D. V. Ignatov*, Yu. I. Prokof’ev*, V. P. Timofeev**, and A. Yu. Misharin*
*Orekhovich Research Institute of Biomedical Chemistry, Russian Academy of Medical Sciences,
Pogodinskaya ul. 10, Moscow, 119992 Russia
**Engelhardt Institute of Molecular Biology, Russian Academy of Sciences,
ul. Vavilova 32, GSP Moscow, 117984 Russia
Received October 28, 2002; in final form, February 10, 2003
Abstract—3β-Hexadecanoyloxy-5α-cholest-8(14)-en-15-one, 3α-hexadecanoyloxy-5α-cholest-8(14)-en-15-one,
15β-hexadecanoyloxy-5α-cholest-8(14)-en-3β-ol, 15α-hexadecanoyloxy-5α-cholest-8(14)-en-3β-ol, 15β-hexa-
decanoyloxy-5α-cholest-8(14)-en-3-one, and 15α-hexadecanoyloxy-5α-cholest-8(14)-en-3-one were synthe-
sized and their chromatographic and 1H NMR characteristics were determined.
Key words: oxysterol acyl derivatives, oxysterols
1
INTRODUCTION
cal synthesis and characterization of palmitoyl deriva-
tives of isomeric 15-oxygenated ∆8(14)-sterols (II),
(IV), (VIII), (IX), (XV), and (XVI) (Schemes 1, 2).
15-Oxygenated derivatives of cholesterol and lanos-
terol exhibit a wide spectrum of biological activities in
cultured cells and have hypocholesterolemic and anti-
2
atherogenic properties in mammals [1]. 3β-Hydroxy-
RESULTS AND DISCUSSION
5α-cholest-8(14)-en-15-one [15-ketocholesterol (I)] is
known to form acyl derivatives in blood plasma and
mammal cells [2–6]. Acyl derivatives of (I) were iden-
tified in chylomicrons at the dietary administration of
(I) to experimental animals. Along with polar metabo-
lites, they are formed at the incubation of ∆8(14),15-
ketosterols with cell cultures [7–12]. 15-Ketosteryl
esters containing residues of various carboxylic acids
were obtained by (I) acylation [13, 14]. It was
described in [4, 15] that the toxic effect of 15-ketosteryl
oleate and its ability to inhibit activity of HMG-CoA
reductase in CHO K1 cells are determined by the rate
of intracellular hydrolysis of the ester and the formation
of free ketosterol (I).
Acylation of 15-ketosterol (I) with palmitic acid in
the presence of TPS and DMAP or N-methylimidazole
by the method [14] resulted in palmitate (II) (Scheme 1)
in yields 75–85%. To obtain 3α-palmitoyloxy-5α-
cholest-8(14)-en-15-one (IV), (I) was treated with
MsCl in pyridine and, then, 3β-mesylate (III) was con-
verted into 3α-palmitate (IV) by the reaction with
cesium palmitate [16, 17]. The reaction in DMF [16]
resulted in a better yield (72% after boiling for 40 min)
in comparison with the modified procedure [17] [after
the 12-h boiling with a cesium palmitate excess in tol-
uene in the presence of 0.5 equiv of DMAP reacted
only 15% of (III)]. An alkaline hydrolysis of palmitate
(IV) led to the previously described [18] 3α-hydroxy-
5α-cholest-8(14)-en-15-one, which unequivocally con-
firmed 3α-configuration of (IV).
One can presume that the acylation of free hydroxyl
group of biologically active oxysterol with a fatty acid
residue, which substantially increases the molecule
hydrophobicity, should contribute to binding of the
oxysterol derivative to cell and affect the processes of
its internalization and subsequent intracellular metabo-
lism. Therefore, the obtaining of acyl derivatives of 15-
oxygenated ∆8(14)-sterols that differ in the position
and configuration of substituents and the study of their
biological activities and metabolism in liver cells is of
interest. The goal of this communication is the chemi-
The next problem was the introduction of palmitoyl
residue in position 15β. To this end, hydroxyl group of
(I) was primarily protected by tritylation: tritylated
ketosterol (V) was obtained in 75% yield after the treat-
ment of (I) with TrCl excess in boiling pyridine. We
showed previously [19] that the reduction of keto group
in tritylated (V) with LiAlH4 proceeded stereospecifi-
cally and in quantitative yield led to the trityl derivative
of ∆8(14),15β-hydroxysterol (VI). Acylation of (VI)
with palmitic acid in the presence of TPS and N-meth-
ylimidazole [14] gave the corresponding palmitate
(VII) in 74% yield. The removal of trityl group from
(VII) led to 15β-palmitate of 3β-sterol (VIII) in 66%
yield from the starting (VI).
1
Corresponding author; e-mail: misharin@ibmh.msk.su
Abbreviations: CPBA, m-chloroperbenzoic acid; DMAP, N,N-
2
dimethlaminopyridine; HMG-CoA, hydroxymethylglutarylcoen-
zyme A; HMPTA, hexamethylphosphotriamide; Ms, mesyl
(methanesulfonyl); Palm, palmitoyl (hexadecanoyl); TPS, 2,4,6-
triisopropylbenzenesulfonyl chloride; and Tr, trityl (triphenylme-
thyl).
1068-1620/04/3002-0184 © 2004 MAIK “Nauka /Interperiodica”

d
‡
O
O
O
HO
MsO
HO
TrO
TrO
TrO
PalmO
H
H
H
H
H
(II)
(I)
(V)
b
e
c
O
O
OH
PalmO
H
H
(IV)
(III)
(VI)
‡
g
f
OPalm
OPalm
OPalm
O
H
H
(IX)
(VIII)
(VII)
Scheme 1. (‡) ëH (CH ) COOH, TPS, N-methylimidazole/toluene; (b) MsCl/Py; (c) ëH (CH ) COOës/DMF; (d) TrCl/Py; (e) LiAlH /Et O; (f) 90% HCOOH; (g) CrO –
3
2 14
3
2 14
4
2
3
Py/CH Cl
2
2

186
IGNATOV et al.
‡
b
O
HO
TrO
TrO
H
H
H
(X)
(XI)
(XII)
c
e
d
OPalm
OPalm
OH
HO
TrO
TrO
H
H
H
(XV)
(XIV)
(XVI)
(XIII)
f
OPalm
O
H
Scheme 2. (‡) TrCl/Py; (b) ëPBA, NaHCO /Et O; (c) LiAlH /Et O; (d) ëH (CH ) COOH, TPS, N-methylimidazole/toluene;
3
2
4
2
3
2 14
(e) 90% HCOOH; (f) CrO –Py/CH Cl .
3
2
2
We initially planned to use SN2 substitution of palm- epoxide (XII) was without isolation reduced with
itoyloxy group for 15β-mesyloxy group in 3β-trity-
loxy-15β-mesyloxy-5α-cholest-8(14)-ene. 3β-Trity-
loxy-15β-mesyloxy-5α-cholest-8(14)-ene was actually
obtained from (VI) in 79% yield [1H NMR spectrum:
0.695 (3 H, s, H18), 0.795 (6 H, d, J6.6, H26, H27),
0.927 (3 H, d, J 6.6, H21), 0.965 (3 H, s, H19), 3.341 (1
H, m, H3), 3.350 (3 H, s, CH3S), 6.397 (1 H, m, H15),
LiAlH4 in ether. After separation of the reduction prod-
ucts, 3β-trityloxy-5α-cholest-8(14)-ene-15α-ol (XIII)
was isolated in 39% yield from (XI). The acylation of
15α-hydroxysterol (XIII) with palmitic acid and the
removal of trityl group in (XIV) were carried out under
the conditions described above for the obtaining of
7.180–7.550 (15 H, m, trityl)]. However, its heating in 15β-isomer (VIII). The yield of 15α-palmitate (XV)
DMF or HMPTA rapidly led to the product of mesy-
loxy group elimination and the formation of 3β-trity-
loxy-5α-cholesta-8(14),15-diene. The treatment of this
compound with 90% HCOOH resulted in 3β-hydroxy-
5α-cholesta-8(14),15-diene [1H NMR spectrum: 0.759
(3 H, s, H18), 0.862 (6 H, d, J 6.6, H26, H27), 0.942
(3 H, d, J 6.6, H21), 0.963 (3 H, s, H19), 3.627 (1 H, m,
H3), 5.894 (1 H, d, J15,16 8.0, H15), 6.326 (1 H, dd, J15,16
8.0, J16,17 6.5, H16)].
was 63% from starting (XIII).
The configuration of the substituent at C15 in (VIII)
and (XV) was confirmed as follows. The compounds
were subjected to alkaline hydrolysis, and the resulting
sterols were compared with the known 3β,15α-dihy-
droxy-5α-cholest-8(14)-ene and 3β,15β-dihydroxy-
5α-cholest-8(14)-ene [21] in respect of 1H NMR spec-
tra and chromatographic mobility. The oxidation of 3β-
hydroxyl group in 15-palmitates (VIII) and (XV) by
the CrO3–Py complex led to 3-ketosteryl-15-palmitates
(IX) and (XVI), respectively.
The alternative (successful) scheme (Scheme 2) of
the preparation of 15α-palmitoyloxy-5α-cholest-
8(14)-en-3β-ol was based on the known LiAlH4 reduc-
tion of sterol ∆7,14α,15α-epoxides to ∆8(14)-15α-
hydroxysterols
[20].
3β-Triphenylmethoxy-5α-
The chromatographic characteristics of (II), (IV),
(VIII), (IX), (XV), and (XVI) are given in Table 1 and
the 1H NMR spectra, in Table 2.
cholesta-7,14-diene (XI), prepared by tritylation of
diene (X) in 82% yield, was treated with CPBA in ether
solution in the presence of dry NaHCO3. The resulting
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PALMITATES OF ISOMERIC 15-OXYGENATED ∆8(14)-STEROLS
187
Table 1. HPTLC characteristics of (II), (IV), (VIII), (IX), (XV), and (XVI)
Rf values in systems:
Compound
hexane–EtOAc hexane–EtOAc hexane–acetone hexane–acetone ëHCl3–acetone toluene–EtOAc
(10 : 1)
(4 : 1)
(10 :1)
(5 : 1)
(10 :1)
(10 : 1)
(II)
(IV)
(VIII)
(IX)
(XV)
(XVI)
0.38
0.44
0.09
0.24
0.06
0.21
0.71
0.75
0.30
0.53
0.26
0.51
0.64
0.66
0.15
0.31
0.14
0.26
0.76
0.77
0.29
0.51
0.23
0.42
0.99
0.99
0.79
0.98
0.72
0.95
0.77
0.81
0.31
0.80
0.30
0.74
Table 2. Chemical shifts in the 1H NMR spectra of (II), (IV), (VIII), (IX), (XV), and (XVI)
Com-
18H 26H; 27H 19H
21H
15ç
–
3H
7ç
Protons of acyl residue
pound
(II)
0.721 s 0.852 0.960 s 0.987
(d, J 6.6) (d, J 6.6)
0.694 s 0.857 0.948 s 0.968
(d, J 6.6) (d, J 6.6)
(VIII) 0.705 s 0.849 0.975 s 0.920 5.544 m 3.638 m 2.40–2.55 m 0.870 (t, J 7.0), 1.242 (br. s); 2.252 (t, J 7.0)
(d, J 6.6) (d, J 6.6)
0.831 s 0.860 0.998 s 0.949 5.540 m
(d, J 6.6) (d, J 6.6)
(XV) 0.688 s 0.838; 0.867 s 0.940 5.771 m 3.649 m 2.15–2.40 m 0.846 (t, J 7.0), 1.242 (br. s); 2.275 (t, J 7.0)
4.730 m 4.126 m 0.870 (t, J 7.0), 1.242 (br. s); 2.249 (t, J 7.0)
(IV)
–
5.046 m 4.137 m 0.872 (t, J 7.0), 1.242 (br. s); 2.302 (t, J 7.0)
(IX)
–
2.45–2.60 m 0.866 (t, J 7.0), 1.242 (br. s); 2.252 (t, J 7.0)
0.841
(d, J 6.6)
(d, J 6.6)
(XVI) 0.850 s 0.851; 0.869 s 0.904 5.789 m
–
2.45–2.60 m 0.866 (t, J 7.0), 1.242 (br. s); 2.270 (t, J 7.0)
0.858
(d, J 6.6)
(d, J 6.6)
* The signal form and spin-coupling constant (Hz) are given in parentheses.
EXPERIMENTAL
was performed by a modified procedure [23] and the
removal of benzoyl groups in 3β-benzoyloxy-5α-
cholesta-7,14-diene and 3β-benzoyloxy-5α-cholest-
8(14)-ene-15-one by alkaline hydrolysis. 3β,15α-
Dihydroxy-5α-cholest-8(14)-ene and 3β,15β-dihy-
droxy-5α-cholest-8(14)-ene-15-one were obtained by
1H NMR spectra were registered on an AMX-III-
400 instrument (Bruker, Germany) in deuterochloro-
form. Analytical TLC was carried out on precoated
HPTLC Kieselgel F254 plates (Merck, Germany). Sub-
stance spots were visualized in the light of a UV-lamp the reduction of 3β-benzoyloxy-5α-cholest-8(14)-ene-
(254-nm filter) and by spraying with 3% solution of 15-one by the method [21].
ammonium molybdate in 5% aqueous H2SO4, followed
3b-Hexadecanoyloxy-5a-cholest-8(14)-ene-15-one
by heating. Preparative TLC was carried out on PSC
(II). A mixture of (I) (400 mg, 1 mmol) and palmitic
Kieselgel F254 plates (Merck, Germany), and column
acid (512 mg, 2.0 mmol) was dried by coevaporation
chromatography on Silica gel L (40–100 µm, Che-
mapol).
with anhydrous pyridine and toluene. DMAP (218 mg,
5 mmol) was added, and the mixture was cooled to 0°C,
treated with TPS (758 mg, 2.5 mmol), and stirred for
15 min at room temperature. After dilution with toluene
(50 ml), a saturated solution of NaHCO3 (20 ml) was
added, and the toluene solution was washed with a sat-
urated solution of Na2SO4 (2 × 15 ml), dried with
Na2SO4, and evaporated. The residue was chromato-
Chemical reagents were purchased from Aldrich
and Merck, and solvents were purified by standard pro-
cedures. 3β-hydroxy-5α-cholest-8(14)-ene-15-one (I)
and 3β-hydroxy-5α-cholesta-7,14-diene (X) were syn-
thesized by a common scheme [22] with the following
changes: the isomerization of 3β-benzoyloxycholesta-
5,7-diene into 3β-benzoyloxy-5α-cholesta-7,14-diene graphed on a silica gel column eluted with 19 : 1 ben-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 2 2004

188
IGNATOV et al.
zene–ethyl acetate mixture. The product (II) was drous ether (5 ml) was dropwise added to a suspension
recrystallized from acetone; it had no clear mp; yield of LiAlH4 (150 mg) in anhydrous ether (15 ml) at 0°C.
490 mg (75%); chromatographic characteristics: Table
The mixture was stirred for 30 min at 20°C, LiAlH4
excess was quenched with water at cooling, and the
aqueous layer was extracted with ether (4 × 20 ml). The
combined ether extract was washed with water, dried
with NaOH, and evaporated to dryness. A glassy (VI)
(640 mg) was obtained, which was crystallized upon
1; 1H NMR spectrum: Table 2.
The use of N-methylimidazole as an acylation cata-
lyst raised yield of (II) to 85%.
3b-Methansulfonyloxy-5a-cholest-8(14)-ene-15-
one (III). Ketosterol (I) (400 mg, 1 mmol) was dis-
solved in dry pyridine (5 ml) and treated with MsCl
(0.2 ml) upon cooling. The reaction mixture was stirred
for 4 h at room temperature, a saturated NaHCO3 solu-
tion (20 ml) was added, the mixture was stirred for 2 h
and extracted with chloroform (3 × 15 ml). The chloro-
form extract was washed with water (2 × 5 ml), dried
with Na2SO4, evaporated, and the residue was dissolved
in boiled chloroform and treated with boiled methanol
until emergence of turbidity. After 24 h at 0°C, color-
less crystals were obtained; mp 146°C; yield 440 mg
1
storing; yield quantitative; H NMR spectrum: 0.663
(3 H, s, H18), 0.834 and 0.839 (6 H, two d, H26 and
H27), 0.895 (3 H, d, J 6.6, H21), 0.992 (3 H, s, H19),
2.625 (1 H, m, H7), 3.412 (1 H, m, H7), 3.412 (1 H, m,
H3), 4.572 (1 H, m, H15), 7.180–7.550 (15 H, m, tri-
tyl).
3b-Triphenylmethoxy-15b-hexadecanoyloxy-5a-
cholest-8(14)-ene (VII) and 15b-hexadecanoyloxy-
5a-cholest-8(14)-ene-3b-ol (VIII). A mixture of (VI)
(129 mg, 0.2 mmol) and palmitic acid (128 mg,
0.5 mmol) was dried by coevaporation with anhydrous
pyridine and toluene, dissolved in anhydrous toluene,
successively treated with N-methylimidazole (0.5 ml,
6.1 mmol) and TPS (182 mg, 0.6 mmol), and stirred for
30 min at room temperature. The reaction mixture was
diluted with toluene (25 ml), a saturated solution of
NaHCO3 (10 ml) was added, the toluene solution was
washed with a saturated solution of Na2SO4 (2 × 5 ml),
dried with Na2SO4, evaporated, and the residue was dis-
solved in chloroform (10 ml).
1
(0.92 mmol, 92%); H NMR spectrum: 0.730 (3 H, s,
H18), 0.854 (6 H, d, J 6.6, 26-CH3, H27), 0.962 (3 H,
s, H19), 0.988 (3 H, d, J 6.6, H21), 2.996 (3 H, s,
CH3S), 4.136 (1 H, m, H7), 4.656 (1 H, m, H3).
3a-Hexadecanoyloxy-5a-cholest-8(14)-ene-15-one
(IV). Ketosterol mesylate (III) (230 mg, 0.5 mmol) was
dried by coevaporation with dry toluene, dissolved in
anhydrous DMF (10 ml), treated with a dried salt
CH3(CH2)14COOës (1165 mg, 3 mmol) and the reac-
tion mixture was refluxed for 40 min, with monitoring
the reaction by TLC in 10 : 1 hexane–ethyl acetate sys-
tem. The mixture was then diluted with toluene (30 ml),
washed with water (3 × 10 ml), dried by Na2SO4, and
evaporated. The residue was chromatographed on a sil-
ica gel column (2.5 × 30 cm). Elution with 10 : 1 hex-
ane–ethyl acetate gave the product (IV), which was
recrystallized from acetonitrile; it has no definite mp;
yield 470 mg (72%); chromatographic characteristics:
Table 1; 1H NMR: Table 2.
An aliquot of the chloroform solution (2 ml) was
separated by preparative TLC in 10 : 1 hexane–EtOAc.
Compound (VII) gave a positive reaction to trityl
group. It was isolated as a transparent film; yield 23 mg
1
(29 µmol, 74%); H NMR spectrum: 0.660 (3 H, s,
H18), 0.835 and 0.840 (6 H, two d, H26 and H27),
0.871 (3 H, t, J 7.0, CH3, palmitoyl), 0.895 (3 H, d, J
6.6, H21), 0.932 (3 H, s, H19), 1.190–1.290 (br. s, CH2
of palmitoyl), 3.412 (1 H, m, H3), 5.482 (1 H, m, H15),
7.180–7.550 (15 H, m, trityl).
3b-Triphenylmethoxy-5a-cholest-8(14)-ene-15-
one (V). A mixture of ketosterol (I) (800 mg, 2 mmol),
TrCl (840 mg, 3 mmol), and dry pyridine (30 ml) was
refluxed for 1 h, treated with an additional quantity of
TrCl (280 mg, 1 mmol), and refluxed for other 2 h. The
mixture was then cooled, poured into the stirred satu-
rated solution of NaHCO3 (50 ml), stirred for 30 min,
and extracted with toluene (3 × 30 ml). The extract was
washed with a saturated solution of Na2SO4 (2 × 25 ml),
dried with Na2SO4, and evaporated. The residue was
chromatographed on a silica gel column (2.5 × 30 cm).
Elution with 15 : 1 hexane–ethyl acetate gave the prod-
uct (V), which was recrystallized from an 1 : 2 acetone–
methanol mixture; yield 480 mg (75%); mp 168°C; 1H
NMR spectrum: 0.633 (3 H, s, H18), 0.834 (6 H, d,
J 6.6, H26, H27), 0.911 (3 H, s, H19), 0.949 (3 H, d, J
6.6, H21), 3.410 (1 H, m, H3), 4.018 (1 H, m, H7),
7.18–7.50 (15 H, m, trityl).
The remaining chloroform solution (8 ml) was evap-
orated to dryness, 90% HCOOH (10 ml) and dropwise
ether (until dissolution) were added to the residue at
stirring. The mixture was stirred for 30 min, treated
with water, and again evaporated to dryness. The resi-
due was chromatographed on a silica gel column (2.5 ×
30 cm). Elution with 4 : 1 hexane–ethyl acetate gave
(VIII) with an admixture of palmitic acid. It was
rechromatographed on a plate in a 5 : 1 hexane–acetone
system to give (VIII); yield 76 mg [106 µmol, 66%
from starting (VI)]; chromatographic characteristics:
Table 1; 1H NMR: Table 2.
3b-Triphenylmethoxy-5a-cholesta-7,14-diene (XI).
A mixture of (X) (384 mg, 1.0 mmol), TrCl (420 mg
1.5 mmol), and dry pyridine (20 ml) was refluxed for
1 h, treated with additional TrCl (140 mg, 0.5 mmol),
again refluxed for 1 h, poured into a saturated solution
of NaHCO3 (30 ml), stirred for 30 min, and extracted
3b-Triphenylmethoxy-5a-cholest-8(14)-ene-15b-
ol (VI). A solution of (V) (640 mg, 1 mmol) in anhy- with toluene (3 × 30 ml). The toluene solution was
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PALMITATES OF ISOMERIC 15-OXYGENATED ∆8(14)-STEROLS
189
washed with a saturated solution of Na2SO4 (2 × 25 ml), H3), 5.772 (1 H, m, H15), 7.180–7.550 (15 H, m, tri-
tyl).
dried with Na2SO4, evaporated, and the residue was
chromatographed on a silica gel column (2.5 × 30 cm).
Elution with a 15 : 1 hexane–EtOAc mixture gave (XI);
a colorless glassy mass; yield 513 mg (0.82 mmol,
82%). After recrystallization from a 3 : 1 acetone–
methanol mixture, colorless crystals of (XI) were
15a-Hexadecanoyloxy-5a-cholest-8(14)-ene-3b-ol
(XV). A mixture of (XIV) (85 mg, 100 µmol) and 90%
HCOOH (5 ml) was dropwise diluted with ether until
the dissolution of precipitate. The solution was stirred
for 30 min and evaporated to dryness. The residue was
separated by preparative TLC in a 5 : 1 hexane–acetone
system to give (XV) as a white waxy film; yield 51 mg
(85 µmol, 85%). Chromatographic characteristics:
Table 1; 1H NMR: Table 2.
1
obtained; mp 114–116°C; H NMR spectrum: 0.720
(3 H, s, H18), 0.780 (3 H, s, H19), 0.858 (6 H, d, J 6.6,
H26 and H27), 0.894 (3 H, d, J 6.6, H21), 3.366 (1 H,
m, H3), 5.422 (1 H, CH2 of palmitoyl), 5.651 (1 H, m,
H7), 7.18–7.55 (15 H, m, trityl).
15b-Hexadecanoyloxy-5a-cholest-8(14)-ene-3-one
(IX) and 15a-hexadecanoyloxy-5a-cholest-8(14)-
ene-3-one (XVI). A solution of (VIII) or (XV) in
CH2Cl2 (5 ml) was added to CrO3–Py complex pre-
pared from CrO3 (60 mg) in CH2Cl2 (5 ml) [24]. The
mixture was stirred for 2 h, monitoring the oxidation
course by TLC. Methanol (1 ml) was then added, and,
after 15 min, silica gel (1.0 g) was added to the reaction
mixture, and it was evaporated to dryness. The reaction
products adsorbed on silica gel were applied onto a col-
umn with dry silica gel (2 × 10 cm), and ketosterol
palmitate (IX) or (XVI) was eluted with a 4 : 1 toluene–
EtOAc mixture. The solvent was removed, and the
product was additionally purified by preparative TLC in
a 5 : 1 hexane–acetone system. Compounds (IX)
(20 mg, 36 µmol, 72%) and (XVI) (19 mg, 34 µmol,
68%) were isolated as white waxy films. Chromato-
graphic characteristics: Table 1; 1H NMR: Table 2.
3b-Triphenylmethoxy-14a,15a-oxido-5a-cholest-
7-ene (XII) and 3b-triphenylmethoxy-5a-cholest-
8(14)-en-15a-ol (XIII). Dry NaHCO3 (0.50 g) and,
then, CPBA (170 mg, 1.0 mmol) were added to a solu-
tion of (XI) (375 mg, 0.6 mmol) in dry ether (20 ml).
The mixture was stirred for 20 min at 0°C, ether solu-
tion was decanted, and the precipitate was washed with
ether (2 × 20 ml). The combined ether extract was
washed at 0°C with a saturated solution of Na2SO3,
dried with MgSO4, and evaporated to a final volume of
10 ml. The resonances at 5.69 (m, H7) and 3.69 (br. s,
1
H15) were seen in H NMR of unpurified product
(XII). The ether solution containing epoxide (XII) was
dropwise added to a stirred suspension of LiAlH4
(100 mg) in dry ether (15 ml). The mixture was stirred
for 30 min, cooled to 0°C, LiAlH4 excess was decom-
posed with ice water, the ether layer was decanted, and
the aqueous layer was extracted with ether (4 × 15 ml).
The combined ether extract was dried with MgSO4,
evaporated, and the residue was chromatographed on a
silica gel column (3.5 × 15 cm), eluting product (XIII)
with 4 : 1 hexane–EtOAc as a transparent film; yield
ACKNOWLEDGMENTS
This work was supported by Russian Foundation for
Basic Research, project no. 00-04-48643.
1
143 mg (0.23 mmol, 39%); H NMR spectrum: 0.648
(3 H, s, H18), 0.778 (3 H, s, H19), 0.855 (6 H, d, J 6.6,
H26 and H27), 0.927 (3 H, d, J 6.6, H21), 3.446 (1 H,
m, H3), 4.836 (1 H, m, H15), 7.180–7.550 (15 H, m, tri-
tyl).
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