Δ5-7-Ketosterols with modified side chain: The synthesis and the effects on viability and cholesterol biosynthesis in Hep G2 cells

Article abstract of DOI:10.1134/S1068162006050141

(22E)-3β-Hydroxysitosta-5,22-dien-7-one, (22R,23R)-3β,22,23- trihydroxysitost-5-en-7-one, and (22R,23R)-3β-hydroxy-22,23- isopropylidenedioxysitost-5-en-7-one were synthesized. The cytotoxicity and effects on cholesterol biosynthesis of the resulting 7-ketosterols, 7-ketocholesterol, and (22S,23S)-3β-hydroxy-22,23-oxidositost-5-en-7-one were studied in hepatoblastoma Hep G2 cells. Pleiades Publishing, Inc., 2006.

Full text of DOI:10.1134/S1068162006050141

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2006, Vol. 32, No. 5, pp. 497–503. © Pleiades Publishing, Inc., 2006.
Original Russian Text © E.A. Piir, G.E. Morozevich, F.V. Drozdov, V.P. Timofeev, A.Yu. Misharin, 2006, published in Bioorganicheskaya Khimiya, 2006, Vol. 32, No. 5,
pp. 551−558.
D5-7-Ketosterols with Modified Side Chain:
The Synthesis and the Effects on Viability and Cholesterol
Biosynthesis in Hep G2 Cells
E. A. Piira, G. E. Morozevicha, F. V. Drozdova, V. P. Timofeevb, and A. Yu. Misharina, 1
a Orechovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Pogodinskaya ul. 10,
Moscow, 119992 Russia
b Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 117984 Russia
Received August 1, 2005; in final form, October 21, 2005
Abstract—(22E)-3β-Hydroxysitosta-5,22-dien-7-one, (22R,23R)-3β,22,23-trihydroxysitost-5-en-7-one, and
(22R,23R)-3β-hydroxy-22,23-isopropylidenedioxysitost-5-en-7-one were synthesized. The cytotoxicity and
effects on cholesterol biosynthesis of the resulting 7-ketosterols, 7-ketocholesterol, and (22S,23S)-3β-hydroxy-
22,23-oxidositost-5-en-7-one were studied in hepatoblastoma Hep G2 cells.
Key words: oxysterols, synthesis, Hep G2 cells, cholesterol biosynthesis, cytotoxicity
DOI: 10.1134/S1068162006050141
INTRODUCTION
7-one (XV), and 7-ketocholesterol (XVI) in human
hepatoma Hep G2 cell line.
7-Ketocholesterol is one of the major oxysterols of
mammalian organisms.2 In a cell culture, 7-ketocholes-
terol affects the biosynthesis, metabolism, and trans-
port of lipids and oxidative processes in mitochondria,
promotes inflammation, activates AHR, and is involved
in the regulation of proliferation and apoptosis [1–6].
The side chain of 7-ketocholesterol is rapidly degraded
by the 27-hydroxylase route in hepatocytes [7, 8]. Like
7-ketocholesterol, 7-ketositosterol, its close structural
analogue, affects the growth and viability of C57BL/6
macrophages and the activity of mitochondrial dehy-
drogenases in the same cells [9].
RESULTS AND DISCUSSION
The synthesis of 7-ketosterols (XI)–(XIII) from
stigmasterol (I) is given in the scheme. Various meth-
ods of stereoselective hydroxylation of 22,23-double
bond in stigmasterol derivatives have currently been
developed [14–18]; however, high prices of the neces-
sary reagents and long reaction times are shortcomings
of these methods. We decided to use the classic Wood-
ward procedure for the hydroxylation of 22,23-double
bond.
We continue our studies of new analogues of biolog-
ically active oxysterols containing modified side chains
[10–13], and present here (1) the synthesis of ∆5-7-
ketosterols, namely, (22Ö)-3β-hydroxysitosta-5,22-
dien-7-one (XI), (22R,23R)-3β,22,23-trihydroxysitost-
5-en-7-one (XII), and (22R,23R)-3β-hydroxy-22,23-
isopropylidenedioxysitost-5-en-7-one (XIII) (scheme)
and (2) a comparative examination of cytotoxicities and
the effects on cholesterol biosynthesis of the synthe-
sized 7-ketosterols (XI)–(XIII) and the earlier obtained
(22S,23S)-3β-hydroxy-22,23-oxidositost-5-en-7-one
(XIV), (22R,23R)-3β-hydroxy-22,23-oxidositost-5-en-
Stigmasterol (I) was transformed into 3α,5α-cyclo-
6β-methoxystigmastane (II) via the corresponding
tosylate with the aim to protect 5,6-double bond [19].
The treatment of cyclosterol (II) by iodine in 95%
AcOH in the presence of AcOAg at room temperature
for 1 h resulted in a mixture, from which iodoacetate
(III) was isolated as the major product in 65% yield.
The presence of iodine atom in (III) was confirmed by
a molecular ion of m/z 612 in its mass spectrum. The
treatment of (III) with K2CO3 in aqueous MeOH
accompanied with deacetylation led to the only prod-
uct, the earlier described (22R,23R)-22,23-oxido-
3α,5α-cyclo-6β-methoxysitostane [13], which unequi-
vocally confirmed the 22S,23R-configuration of (III).
Note that the 3α,5α-cyclo-6β-methoxy fragment of (II)
was sufficiently stable under the conditions; it was
shown in a separate experiment that the half-time of
1
Corresponding author; phone: +7 (495) 246-3375;
e-mail: alexander.misharin@ibmc.msk.ru.
2
Abbreviations: AHR, aromatic hydrocarbon receptor; CPBA,
meta-chloroperbenzoic acid; FCS, fetal calf serum; and MTT,
[3-(4,5-dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide.
497

498
PIIR et al.
CH3
CH3
CH3
CH3
CH3
I
CH3
CH3
OAc CH3
H3C
H3C
H3C
H3C
H3C
H3C
CH3
H3C
H3C
H3C
‡, b
c
HO
OCH3
OCH3
d
(I)
(II)
(III)
CH3
CH3
OH CH3
AcO
H3C
H3C
CH3
CH3
OH CH3
OH
H3C
H3C
H3C
f
CH3
CH3
e
+
OH
AcO
H3C
H3C
H3C
OAc CH3
(VI)
HO
H3C
AcO
f
(IV) + (V)
X
X
X
H3C
H3C
H3C
H3C
H3C
H3C
h
g
O
O
AcO
H3C
AcO
H3C
HO
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H3C
CH3
CH3
CH3
CH3
CH3
, X =
(VII)
(XI), X =
OAc
OH
OAc
H3C
CH3
OAc CH3
H3C
CH3
H3C
(VIII), X =
(X), X =
(XII), X =
OH CH3
OAc CH3
h
H3C
O
CH3
(22R,23R)-
H3C
H3C
O
CH3
H3C
CH3
CH3
HO
O
(XIII)
O
O
H3C
H3C
H3C
H3C
H3C
H3C
(22S,23S)-
H3C
(22R,23R)-
CH3
CH3
CH3
CH3
H3C
H3C
CH3
CH3
CH3
CH3
AcO
O
HO
O
HO
O
(XVI)
(XIV)
(XV)
Reagents: a TosCl + Py; b AcONa/MeOH, heating; c I , AcOAg/95% AcOH; d AcOAg/AcOH, heating; e K CO /MeOH–H O;
2
2
3
2
f Ac O/Py; g K Cr O /AcOH–Ac O, heating; h (CH ) C(OCH ) , TosOH;
2
2
2
7
2
3 2
3 2
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 5 2006

∆5-7-KETOSTEROLS WITH MODIFIED SIDE CHAIN
499
MTT test, % of control
150
cyclosterol (II) conversion into acetate (VII) in 95%
AcOH at room temperature was 78 h.
Iodoacetate (III) was transformed into a 1 : 1 mix-
ture of diacetylated triols (IV) and (V) in 62% yield
under a reflux in glacial acetic acid in the presence of
AcOAg. The presence of 5,6-double bond and 3β-ace-
toxy group in products (IV) and (V) was proved by the
1H NMR spectrum of the mixture (see the Experimental
section). Evidently, compounds (IV) and (V) are iso-
meric 22- and 23-monoacetates of 22,23-diol. Since the
transformation of (22S,23R)-iodoacetate (III) into
products (IV) and (V) is realized under these conditions
[20] through the cyclic intermediate and requires the
retention of the C23 configuration and the inversion of
configuration at C22, compounds (IV) and (V) have the
(22R,23R)-configuration. When the mixture of (IV) and
(V) was treated with K2CO3 in aqueous methanol, the
crystalline (22R,23R)-sitost-5-ene-3β,22,23-triol (VI)
was obtained in 95% yield. Based on the above-
described experiments, a simple preparative procedure
was developed that allowed the preparation of the target
triol (VI) from stigmasterol (I) in 28%yield without
isolation of intermediates (see the Experimental sec-
tion).
140
130
120
110
1
2
3
100
90
80
70
60
0
10
20
30
40
50
60
[7-Ketocholesterol], µM
Stigmasterol (I) and triol (VI) were acetylated
(Ac2O/Py), and the corresponding acetates (VII) and
(VIII) were transformed into acetylated 7-ketosterols
(IX) and (X) by the treatment with K2Cr2O7 in a mix-
ture of AcOH and Ac2O at 50°ë using the method [21].
The yields of ketosterol triacetate (X) and ketosterol
acetate (IX) were 65 and 31%, respectively. The allyl
oxidation [21] in the presence of 22,23-double bond is
known to lead to side reactions. More effective methods
of keto group introduction in position 7 of stigmasterol
derivatives have been proposed [22, 23]; however, we
used the method [21] because of its simplicity and a
short time. Saponification of acetates (IX) and (X)
resulted in high yields of target ketosterols (XI) and
(XII). The treatment of ((XII) with 2,2-dimethoxypro-
pane in the presence of TosOH gave acetonide (XIII) in
79% yield.
Fig. 1. The effect of 7-ketocholesterol (XVI) on the viabil-
ity of Hep G2 cells after (1) 24, (2) 48, and (3) 72 h of incu-
bation.
the results of this test were practically indistinguishable
from control, whereas, after 72-h incubation, 7-keto-
sterol manifested a moderate toxicity (viability of
~75%) at a concentration of 60 µM. Alterations in cell
morphology were also observed including a decreased
spreading and the presence of rounded cells.
The effects of 7-ketosterols (XI)–(XVI) on the viabil-
ity of Hep G2 cells after 48-h incubation are shown in
Fig. 2. Among the compounds of this series, (22R,23R)-
3β,22,23-trihydroxysitost-5-en-7-one (XII) was most
toxic. Epimeric 22,23-epoxy-7-ketosterols differently
affected the cell viability: (22S,23S)-isomer (XIV) dis-
played a rather high toxicity, whereas (22R,23R)-isomer
showed no noticeable effect. Under the conditions of our
experiments, we revealed no toxicity to Hep G2 cells for
compounds (XI), (XIII), (XV), and (XVI).
The next stage was a comparative study of biologi-
cal activities of new 7-ketosterols (XI)–(XIII) and the
earlier synthesized (22S,23S)-3β-hydroxy-22,23-oxi-
dositost-5-en-7-one (XIV), (22R,23R)-3β-hydroxy-
22,23-oxidositost-5-en-7-one (XV), and 7-ketocholes-
terol (XVI) [13] in human hepatoma Hep G2 cells.
The effects of (XI)–(XVI) on the cholesterol biosynthe-
It is known that 7-ketocholesterol (XVI) manifests a sis in Hep G2 cells after incubation for a short time (3 h) in
high toxicity toward numerous cell lines; however, it is the serumless medium are shown in Fig. 3. (22S,23S)-3β-
limitedly toxic for hepatocytes (particularly, for Hep Hydroxy-22,23-oxidositost-5-en-7-one (XIV) effectively
G2 cells) [2, 3]. We studied cytotoxic effects of 7-keto- inhibited cholesterol biosynthesis; the calculated IC50
cholesterol (XVI) for Hep G2 cells by the method [24] value was 4 0.3 µM. (22R,23R)-3β,22,23-Trihydroxysi-
using a tetrazolium dye MMT after 24-, 48-, and 72-h tost-5-ene-7-one (XII) selectively inhibited cholesterol
incubation (Fig. 1). A considerable increase in the biosynthesis at concentrations up to 20 µM without affect-
results of MMT assay, which indicates an increased ing fatty acid and triglyceride biosynthesis monitored in
activity of mitochondrial dehydrogenases, with the 7- this case and thereafter by the incorporation of [14C]ace-
ketosterol maximal concentration of 30 µM was tate into the corresponding fractions. At a concentration of
observed after 24-h incubation. After 48-h incubation, 30 µM, it inhibited biosynthesis of not only cholesterol,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 5 2006

500
Viability of cells, % of control
PIIR et al.
Cholesterol biosynthesis level, % of control
140
120
100
80
(XI)
(XII)
(XIII)
(XIV)
(XV)
(XVI)
120
100
80
60
60
(XI)
(XII)
(XIII)
(XIV)
(XV)
(XVI)
40
20
40
20
0
5
10
15
20
25
30
[7-Ketocholesterol], µM
0
10
20
30
40
50
60
Fig. 3. The effects of (XI)–(XVI) on the cholesterol biosyn-
thesis level in Hep G2 cells after 3-h incubation in the
serumless medium (the curve numbers correspond to the
compound numbers).
[7-Ketocholesterol], µM
Fig. 2. The effects of (XI)–(XVI) on the viability of Hep G2
cells after 48-h incubation (the curve numbers correspond
to the compound numbers).
EXPERIMENTAL
but also of fatty acids and triglycerides. The inhibitory
effects of (XI), (XIII), and (XV) were small.
(22R,23R)-3β-Hydroxy-22,23-isopropylidenedioxysi-
tost-5-en-7-one (XIII) stimulated fatty acid and triglyc-
eride biosynthesis by 40% at a concentration of 30 µM.
Our results demonstrate that side chain modifications
essentially affect 7-cholesterol cytotoxicity and its abil-
ity to regulate cholesterol biosynthesis in Hep G2 cells.
(22R,23R)-3β,22,23-Trihydroxysitost-5-en-7-one (XII)
efficiently decreased the cell viability and markedly
inhibited cholesterol biosynthesis. In comparison with
ketosterol (XII) (22S,23S)-3β-hydroxy-22,23-oxidosi-
tost-5-en-7-one (XIV) was a more potent inhibitor of
cholesterol biosynthesis, but was less toxic.
Stigmasterol was obtained from ICN; reagents and
solvents were from Aldrich, Merck, and MedKhimLab
(Russia); media, sera, and plastic utensil for cell cul-
tures were from Gibco BRL and Costar; PBS and MTT,
from Sigma; and [1-14C]AcONa, from Amersham.
Stigmasteryl acetate (VII) was obtained by treatment of
stigmasterol (I) with excess ÄÒ2é in pyridine; 6β-
methoxy-3α,5α-cyclostigmastane (II) was synthesized
by the method [19]; (22S,23S)-3β-hydroxy-22,23-oxi-
dositost-5-ene-7-one (XIV) and (22R,23R)-3β-
hydroxy-22,23-oxidositost-5-ene-7-one (XV) were
prepared as in [13], and 7-ketocholesterol was obtained
according to [21]. Mps of crystalline compounds were
determined in a glass capillaries.
Column chromatography was carried out on silica
gel G (70–200 µm (Merck), andTLC, on precoated
HPTLC Kieselgel plates (Merck). Substance spots on
plates were detected with 3% ammonium molybdate in
5% H2SO4 and/or by 5% SbCl3 in dry CHCl3 followed
by heating.
One may presume that further studies of the afore-
mentioned compounds in various cell cultures can be of
interest for both the study of the role of oxysterols in
regulatory processes and the evaluation of pharmaco-
logical potential of these compounds.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 5 2006

∆5-7-KETOSTEROLS WITH MODIFIED SIDE CHAIN
501
1ç- and 13C NMR spectra were registered on an (1 g), methanol (5 ml), and water (3 ml) was refluxed
AMX-III-400 (Bruker) in CDCl3; chemical shifts are
given in ppm; coupling constants, in Hz; chemical
shifts of CHCl3 in 1ç- and 13C NMR spectra were 7.25
and 77.16 ppm, respectively. Mass spectrum of com-
pound (III) was registered on a Kratos MS-890 mass
spectrometer at electron impact ionization with energy
of 70 eV.
Radioactivity was measured in a toluene scintillator
on an LKB counter. The cell viability based on the
MTT assay [24] was determined on a Microtiter plate
reader (LKB).
The human cell line Hep G2 obtained from the
European collection of cell cultures (ECACC) was cul-
tured at 37°C in the atmosphere containing 5% ëé2 in
the 1 : 1 OptiMEM : F12 medium containing 10% FCS
in 24- or 96-well plates. The cells were kept for 24 h in
the serumless medium before the experiments.
for 30 min, and chloroform (20 ml) and water (10 ml)
were added. The chloroform layer was separated, the
aqueous layer was extracted with a 2 : 1 ëHCl3 –MeOH
(10 ml); the combined extract was dried with Na2SO4;
and the residue was recrystallized from acetonitrile to
give (VI); yield 167 mg (0.38 mmol, 95%); mp 182–
184°C; 1H NMR: 0.71 (3 H, s), 0.86 (3 H, d, J 6.8), 0.93
(3 H, d, J 6.8), 0.95 (3 H, t, J 7.5), 1.00 (3 H, s), 1.02 (3
H, d, J 6.8), 3.51 (1 H, m), 3.55–3.68 (2 H, m), 5.34 (1
H, m); 13C NMR: 11.70, 13.96, 14.10, 14.45, 17.72,
18.54, and 19.30 (C18, C19, C21, C26, C27, C28, and
C29), 21.03, 21.67, 24.48, 26.87, 27.96 and 29.61 (C2,
C7, C11, C15, C16, and C25), 31.62, 31.79, 31.88,
37.21, and 39.71 (C1, C8, C10, C12, and C20), 42.25,
42.35, 49.60, 50.06, 52.64, and 56.36 (C4, C9, C13,
C14, C17, and C24); 70.65, 71.72, 72.30 (C3, C22, and
C23), 121.48, and 140.76 (C5 and C6).
(22S,23R)-3a,5a-Cyclo-6b-methoxy-22-iodo-23-
acetoxysitostane (III). Water (0.5 ml) and AcOAg
(220 mg, 1.3 mmol) were added to a solution of cyclos-
terol (II) (240 mg, 0.5 mmol) in AcOH (10 ml). Finely
ground iodine (130 mg, 1.1 mmol) was then added in
portions for 20 min under vigorous stirring. The mix-
ture was stirred for another 40 min, and the precipitate
was separated and washed with toluene (3 × 15 ml).
The filtrate was combined with the toluene extract,
water (10 ml) was added, and the toluene layer was suc-
cessively washed with saturated NaHCO3 and water,
dried with Na2SO4, and evaporated. The residue was
chromatographed on a column eluted with 3 : 1 hex-
ane–EtOAc. Compound (III) (260 mg, 0.35 mmol,
Preparative synthesis of (22R,23R)-3b,22,23-tri-
hydroxysitost-5-ene (VI) from stigmasterol (I). Tosyl
chloride (8.64 g, 40 mmol) was added to a solution of
stigmasterol (8.24 g, 20 mmol) in anhydrous pyridine
(50 ml). The mixture was stirred for 14 h, poured into a
mixture of saturated NaHCO3 (500 ml) and ice (100 g),
stirred for 2 h, and the precipitate was separated. The
aqueous solution was extracted with toluene (2 ×
100 ml). The combined toluene extract was washed
with saturated Na2SO4, dried with Na2SO4, and evapo-
rated to a volume of 40 ml. The resulting solution was
slowly poured into a solution of AcONa (20 g) in meth-
anol (250 ml) under reflux, and the mixture was
refluxed for another 40 min and evaporated. Toluene
(200 ml) and water (50 ml) were added to the residue;
the toluene solution was washed with water (2 × 50 ml),
dried with Na2SO4, and evaporated to dryness. The res-
idue was dissolved in AcOH (40 ml); water (2 ml) and
AcOAg (6.72 g, 40 mmol) were added followed by the
addition of finely ground I2 (5.04 g, 40 mmol) in por-
tions for 20 min. The mixture was stirred at room tem-
perature for 40 min, ÄÒ2O (15 ml) and AcOAg (3.36 g,
20 mmol) were added, and the mixture was refluxed
under stirring for 1 h and filtered. The filtrate was evap-
orated, the residue was dissolved in 1 : 1 toluene–ethyl
acetate (50 ml), and the resulting solution was washed
with saturated NaHCO3 and evaporated. Potassium car-
bonate (20 g), methanol (100 ml), and water (40 ml)
were added to the residue, and the mixture was refluxed
under stirring for 40 min. After cooling, chloroform
(200 ml) and water (20 ml) were added; the organic
layer was separated; and the aqueous layer was
extracted with ëçël3 containing 10% MeOH (3 ×
50ml). The combined chloroform extract was washed
with Na2SO4 and evaporated. The residue was treated
with boiling light petroleum (40 ml) and kept for 14 h
at room temperature. The resulting precipitate was
twice recrystallized from a minimal volume of acetone
to give 2.45 g (5.6 mmol, 28%) of (VI). Mp and 1H- and
1
65%) was isolated as a colorless glass-like film; H
NMR: 0.43 (1 H, m), 0.64 (1 H, m), 0.75 (3 H, s), 0.76
(3 H, d, J 6.8), 0.88 (3 H, d, J 6.8), 0.89 (3 H, t, 7.5),
1.01 (3 H, s), 2.05 (3 H, s), 2.78 (1 H, s), 3.32 (3 H, s),
4.36 (1 H, d, J 7.5), and 5.43 (1 H, d, J 10.5); MS, m/z
(I, %): 612 (4) [M+]; 597 (15), 580 (18), 557 (34), 443
(15), 393 (100).
(22R,23R)-3b,22-Diacetoxysitost-5-en-23-ol (IV)
and (22R,23R)-3b,23-diacetoxysitost-5-en-23-ol (V).
A mixture of (III) (185 mg, 0.3 mmol), AcOAg (86 mg,
0.5 mmol) and glacial AcOH (10 ml) was refluxed for 1
h, and the precipitate was separated and washed with
toluene (3 × 10 ml). The filtrate was combined with the
toluene extract, combined with water (10 ml); the tolu-
ene layer was washed with saturated NaHCO3 and
water, dried with Na2SO4 and evaporated. The residue
was chromatographed on a silica gel column in 3 : 1
hexane–ethyl acetate to give a mixture of (IV) and (V)
1
as a clear film; yield 105 mg (0.2 mmol, 66%); H
NMR: 0.68 (3 H, s), 0.81 (d, J 6.6) and 0.85 (3 H total,
d, J 6.6), 0.87 (3 H, t, J 7.5), 0.92 (3 H, d, J 6.8), 1.00
(3 H, s), 1.08 (3 H, d, J 6.8), 2.02 (3 H, s), 2.05 (s) and
2.10 (3 H total, s), 3.66 (m) and 4.91 (1 H total, m), 3.72
(m) and 5.06 (1 H total, m), 4.59 (1 H, m), 5.36 (1 H, m).
(22R,23R)-3b,22,23-Trihydroxysitost-5-ene (VI).
A mixture of (IV) and (V) (210 mg, 0.4 mmol), K2CO3 13C NMR spectra coincided with those given above.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 5 2006

502
PIIR et al.
(22R,23R)-3b,22,23-Triacetoxysitost-5-ene (VIII). and 138.21 (C5, C22, and C23), 165.09 (C6), and 202.22
A mixture of 3β,22,23-trihydroxystigmast-5-ene (VI) (C7).
(446 mg, 1 mmol), Ac2O (2 ml), and anhydrous pyridine
(22R,23R)-3b,22,23-Trihydroxystigmast-5-ene-
7-one (XII) was recrystallized from 1 : 2 EtOAc–hex-
ane; mp136°ë; quantitative yield; 1H NMR: 0.72 (3 H,
s), 0.93 (3 H, d, J 6.8), 0.95 (3 H, t, J 7.5), 0.95 (3 H, d,
J 6.8), 1.03 (3 H, d, J 6.8), 1.20 (3 H, s), 3.55–3.72 (3 H,
m), 5.68 (1 H, d, J 1.2); 13C NMR: 12.05, 14.25, 14.37,
14.60, 17.44, 17.93, and 18.73 (C18, C19, C21, C26,
C27, C28, and C29), 21.37, 21.92, 26.66, 27.09, and
28.50 (C11, C15, C16, C24, and C25), 31.50, 36.54,
37.44, and 38.83 (C1, C2, C12, and C20), 42.00, 42.59,
43.75, 45.57, 49.78, 50.70, and 51.56 (C4, C8, C9, C10,
C13, C14, and C17); 70.66, 70.76, and 72.29 (C3, C22,
and C23); 126.15 (C5), 165.39 (C6), and 202.28 (C7).
Found, %: C 76.05; H 10.39. ë29ç48é4. Calc., %: C
75.61; H 10.50.
(5 ml) was refluxed for 1 h, cooled, diluted with pyridine
(10 ml) and methanol (5 ml), kept for 10 min, twice
reevaporated with water, and the resulting glasslike film
was dried in a vacuum to give (VIII); yield 620 mg (1
mmol, quantitative); 1H NMR: 0.66 (3 H, s), 0.82 (3 H,
d, J 6.8), 0.91 (6 H, d, J 6.8), 0.92 (3 H, t, J 7.5), 0.99 (3
H, s); 2.02 (3 H, s), 2.03 (3 H, s), 2.08 (3 H, s), 4.58 (1 H,
m), 5.03 (1 H, m), 5.24 (1 H, m), and 5.36 (1 H, m).
(22Ö)-3b-Acetoxystigmasta-5,22-dien-7-one (IX)
and (22R,23R)-3b,22,23-triacetoxystigmast-5-en-7-
one (X). Acetic anhydride (10 ml) and finely ground
ä2ër2O7 (0.36 g, 1.20 mmol) were added to a solution
of (VII) or (VIII) (1 mmol of each) in AcOH (10 ml),
and the mixture was stirred at 50°C for 50 min with
TLC monitoring in 5 : 1 hexane–EtOAc. The resulting
mixtures were poured into toluene (150 ml), shaken for
5 min, and, after 15 min, the toluene solution was fil-
tered through a paper filter into stirred saturated
NaHCO3 solution (100 ml). The toluene solution was
separated, aqueous layer was extracted with toluene
(100 ml), and combined toluene extracts were washed
with water, dried with Na2SO4, and evaporated.
(22E)-3β-Acetoxystigmasta-5,22-dien-7-one (IX) was
isolated by chromatography on a silica gel column in
5 : 1 hexane–EtOAc; yield 146 mg (0.31 mmol, 31%);
colorless clear film; 1H NMR: 0.69 (3 H, s), 0.79 (3 H,
t, J 7.5), 0.83 (3 H, d, J 6.8), 0.85 (3 H, d, J 6.8), 1.01 (3
H, d, J 6.8), 1.20 (3 H, s), 2.04 (3 H, s), 4.70 (1 H, m),
5.04 (1 H, m), 5.24 (1 H, m), 5.69 (1 H, d, J 1.2).
(22R,23R)-3β,22,23-Triacetoxystigmast-5-en-7-one (X)
was isolated by crystallization from light petroleum;
yield 303 mg (0.65 mmol, 65%); 1H NMR: 0.69 (3 H,
s), 0.83 (3 H, d, J 6.8), 0.92 (3 H, t, J 7.5), 0.92 (3 H, d,
J 6.8), 0.94 (3 H, d, J 6.8), 1.19 (3 H, s); 2.02 (3 H, s),
2.03 (3 H, s); 2.07 (3 H, s), 4.70 (1 H, m); 5.01 (1 H, m),
5.16 (1 H, m), and 5.69 (1 H, d, J 1.2).
(22E)-3b-Hydroxystigmasta-5,22-dien-7-one (XI)
and (22R,23R)-3b,22,23-trihydroxystigmast-5-en-7-
one (XII). Compounds (IX) and (X) were refluxed with
a tenfold excess of K2CO3 in 2 : 1 MeOH–H2O for 30
min. Products (VIII) and (XI) were extracted with
CHCl3, and chloroform extracts were dried with Na2SO4
and evaporated. (22E)-3β-Hydroxystigmasta-5,22-
diene-7-one (XI) was recrystallized from 7 : 3 Meéç–
ç2é mixture; mp 124–126°ë; Found, %: C 81.35; H
10.70. C29H46O2. Calc., %: C 81.63; H 10.87. quantita-
tive yield; 1H NMR: 0.69 (3 H, s), 0.78 (3 H, d, J 6.8);
0.79 (3 H, t, J 7.5); 0.84 (3 H, d, J 6.8); 1.01 (3 H, d, J
6.8); 1.20 (3 H, s); 3.67 (1 H, m); 5.01 (1 H, m), 5.16 (1
H, m), and 5.68 (1 H, d, J 1.2). 13C NMR: 12.36, 14.37,
17.48, 19.16, 21.17, 21.39, and 21.57 (C18, C19, C21,
C26, C27, C28, and C29), 25.50, 26.56, 29.16, and 29.85
(C11, C15, C16, and C25), 31.40, 32.03, 36.55, and
38.78 (C1, C2, C12, and C20), 40.35, 42.00, 43.15,
(22R,23R)-3b-Hydroxy-22,23-isopropylidene-
dioxystigmast-5-en-7-one (XIII). A mixture of keto-
sterol (XII) (92 mg, 0.2 mmol), 2,2-dimethoxypropane
(3 ml), and TosOH (5 mg) was stirred for 10 min,
diluted with ëHCl3 (20 ml), washed with saturated
NaHCO3 (2 × 5 ml), dried with Na2SO4, and evapo-
rated. Acetonide (XIII) was isolated by chromatogra-
phy on a silica gel column eluted with 2 : 1 hexane–ace-
tone; colorless gel-like film; Found, %: C 76.30; H
10.32. ë32ç52é4. Calc., %: C 76.75; H 10.47. yield
80 mg (0.16 mmol, 79%); 1H NMR: 0.70 (3 H, s), 0.93
(3 H, d, J 6.8), 0.93 (3 H, t, J 7.5), 0.94 (3 H, d, J 6.8),
1.03 (3 H, d, J 6.8), 1.19 (3 H, s), 1.36 (6 H, s), 3.56 (1
H, m), 3.86–4.00 (2 H, m), and 5.68 (1 H, s); 13C NMR:
11.54, 13.27, 14.12, 17.28, 18.64, and 19.67 (C18, C19,
C21, C26, C27, and C29), 21.22, 23.31, 26.50, 26.82,
27.31, 27.56, 28.61, and 29.68 (C11, C15, C16, C24,
C25, C28, and (ëH3)2C(O)2); 31.21, 36.39, 38.39, and
38.71 (C1, C2, C10, and C12), 41.82, 43.80, 45.44,
46.50, 49.70, 49.91, and 51.46 (C4, C8, C9, C13,
NC14, C17, and C20), 70.52, 72.29. and 79.72 (C3,
C22, and C23), 106.66 106.66 (ëH3)2C(O)2), 126.06
(C5), 165.10 (C6), and 202.00 (C7).
Cytotoxicity of compounds (XI)–(XVI) to Hep
G2 cells [24]. Hep G2 cells were incubated in 96-well
plates in a serumless medium containing the tested
compounds at varied concentrations for 48 h [for 7-
ketocholesterol (XVI), the incubation was also carried
out for 24 and 72 h], the cultural medium was removed,
PBS (125 µl) containing MTT (1 mg/ml) was added
into each well, and the cells were incubated for 4 h at
37°ë. The reaction was terminated by addition into
each well of a solution of 0.1 M HCl in isopropanol
containing 10% Triton X-100 (125 µl). Optical density
was measured every 2 h at 540 and 690 nm in each well.
The results of the MMT assay (the difference in absorp-
tion at 540 and 690 nm) were averaged for six indepen-
dent determinations. The result of the MMT assay in
45.58, 50.02, 50.17, 51.39, and 54.93 (C4, C8, C9, C10, the absence of the tested compounds was taken as
C13, C14, C17, and C24), 70.71 (C3), 126.30, 129.71, 100%.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 5 2006

∆5-7-KETOSTEROLS WITH MODIFIED SIDE CHAIN
503
7. Norlin, M., von Bahr, S., Bjorkhem, I., and Wikvall, K.,
The effect of compounds (XI)–(XVI) on choles-
terol biosynthesis in Hep G2 cells [25]. Hep G2 cells
were incubated in 24-well plates in a serumless
medium at varied concentrations for 3 h, the medium
was replaced by a fresh one containing 0.1 mM [1-
14ë]ÄÒéNa (1 µCi per ml of water) and the tested com-
pounds, and the incubation was carried out for 3 h. The
cells were washed thrice with PBS, the lipids were
extracted with a 3 : 2 mixture of hexane–isopropanol,
the extracts were separated by TLC in 70 : 29 : 1 hex-
ane–Et2O–AcOH in the presence of internal standards
(cholesterol oleate, trioleine, oleic acid, and choles-
terol). The chromatograms were treated with iodine
vapors, the areas corresponding to the standards were
collected in vials for radioactivity counting. The level
of cholesterol biosynthesis (cpm/mg of cell protein for
3 h) was calculated by incorporation of the radioactive
label into cholesterol. The incorporation of the radioac-
tive label in the absence of tested compounds was taken
as 100%. Each determination was repeated thrice in
three independent experiments.
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ACKNOWLEDGMENTS
We are grateful to A.P. Pleshkova (Nesmeyanov
Institute of Organoelement Compounds, RussianAcad-
emy of Sciences) for registration of mass-spectra.
The work was supported by the Russian Foundation
for Basic Research, project no. 03-04-48700, and the
program Molecular and Cell Biology of the Presidium
of Russian Academy of Sciences.
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