Synthesis of secasterol and 24-episecasterol and their toxicity for MCF-7 cells

Article abstract of DOI:10.1134/S1068162010060117

The convergent synthesis of biosynthetic precursors of brassinosteroids with a Δ₂-bond in cycle A-secasterol and 24-episecasterol-was performed. The key stages in the construction of the side chain in these compounds were the Julia olefination of the steroid 22-aldehyde followed by the Sharpless asymmetric dihydroxylation of the intermediate Δ₂₂- olefin. The cytotoxicity of the synthesized compounds for breast carcinoma MCF-7 cells was assessed.

Full text of DOI:10.1134/S1068162010060117

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2010, Vol. 36, No. 6, pp. 746–754. © Pleiades Publishing, Ltd., 2010.
Original Russian Text © V.A. Khripach, V.N. Zhabinskii, O.V. Gulyakevich, O.V. Konstantinova, A.Yu. Misharin, A.R. Mekhtiev, V.P. Timofeev, Ya.V. Tkachev, 2010, published in
Bioorganicheskaya Khimiya, 2010, Vol. 36, No. 6, pp. 815–824.
Synthesis of Secasterol and 24ꢀEpisecasterol and Their Toxicity
for MCFꢀ7 Cells
1
V. A. Khripacha, , V. N. Zhabinskiia, O. V. Gulyakevicha, O. V. Konstantinovaa, A. Yu. Misharinb,
A. R. Mekhtievb, V. P. Timofeevc, and Ya. V. Tkachevc
a Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, ul. Kuprevicha 5/2, Minsk, 220141 Belarus
b Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Moscow, Russia
c Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
Received March 2, 2010; in final form, April 28, 2010
Abstract—The convergent synthesis of biosynthetic precursors of brassinosteroids with a
A—secasterol and 24ꢀepisecasterol—was performed. The key stages in the construction of the side chain in
these compounds were the Julia olefination of the steroid 22ꢀaldehyde followed by the Sharpless asymmetric
dihydroxylation of the intermediate
22ꢀolefin. The cytotoxicity of the synthesized compounds for breast carꢀ
cinoma MCFꢀ7 cells was assessed.
Δ
2ꢀbond in cycle
Δ
Keywords: brassinosteroids, secasterol, brassinolide, sulfones, olefination
DOI: 10.1134/S1068162010060117
1
INTRODUCTION
est to assess their cytotoxic activity [20]. Among such
compounds is secasterol (IIа), discovered in Secale
cereale seedlings [21]. As has been shown, it is a preꢀ
cursor of brassinolide (IIIа) in the synthesis of the latꢀ
Integrated studies of brassinosteroids have led to
the discovery of several properties of scientific and
applied interest not only for plant research, but also for
studies of other organisms beyond the plant kingdom
ter in natural objects from campesterol
(Iа)
(Scheme 1). Similar to other brassinosteroids, which
are contained in plant material in vanishing amounts,
the detection of secasterol (IIа) requires a specimen
with a known structure obtained by chemical syntheꢀ
sis. A chemically synthesized specimen with a known
structure is also necessary to search for isomeric
24ꢀepisecasterol (IIb), which is undoubtedly present
in plants as an intermediate in the biosynthesis of
24ꢀepibrassinolide (IIIb) from 22,23ꢀdihydrobrassiꢀ
casterol (Ib).
Thus, the goal of this work was to synthesize secasꢀ
terol (IIa) and 24ꢀepisecasterol (IIb) as specimens
with a known structure necessary in the search for and
isolation of brassinosteroids from plant extracts, as
well as in the study of the cytotoxic activity of comꢀ
pounds (II) and their analogs towards breast carciꢀ
noma MCFꢀ7 cells.
2
[1–12]. Note separately the cytotoxic effect of brassiꢀ
nosteroids on tumor cells [11, 13–16]. A distinctive
specific feature of the brassinosteroid structure is the
presence of the (22R,23R)ꢀ22,23ꢀdihydroxylated side
chain [1], which plays an important role in the cytoꢀ
toxicity displayed by steroids [17, 18]. It has been
found that the (22
tane derivatives with an equatorial
R
,23
R
)ꢀ22,23ꢀdihydroxystigmasꢀ
3βꢀhydroxy group
are the most toxic towards breast carcinoma MCFꢀ7,
ovary carcinoma CaOV, and prostate carcinoma
LnCaP; the cytotoxic effect of the mentioned comꢀ
pounds is correlated with their affinity for the phosꢀ
pholipid bilayer membranes and the ability to inhibit
Hh signaling. It has also been demonstrated that an
increase in the hydrophobicity of the steroid molecule
elevates its antiproliferative activity [17, 18].
It is known that brassinosteroid biosynthesis in
plants is a multistage process, which in several cases
commences from the introduction of functional
groups into the steroid side chain [19]. Such intermeꢀ
diate compounds are more hydrophobic as compared
with the final brassinosteroids; therefore, it is of interꢀ
RESULTS AND DISCUSSION
We planned to accomplish the most complex part
in the synthesis of compounds (II)—side chain conꢀ
struction—by a Julia olefination via the extension of
the side chain in aldehyde (IV) followed by the Sharpꢀ
less dihydroxylation of the Δ22ꢀolefin fragment of
compound (VI) with the introduction of (22
diol groups (Scheme 2). The target compounds, (IIa
and (IIb), differed only in the stereochemistry of the
1
Corresponding author; phone: +375 172 678 647; eꢀmail: khriꢀ
pach@iboch.basꢀnet.by.
R,23R)ꢀ
2
Abbreviations: (DHQD) ꢀPHAL, hydroquinidine 1,4ꢀphthalaꢀ
2
)
zinediyl diether; MTT, 3ꢀ(4,5ꢀdimethylthiazolylꢀ2)ꢀ2,5 dipheꢀ
nyltetrazolium bromide.
746

SYNTHESIS OF SECASTEROL AND 24ꢀEPISECASTEROL
747
OH
R1
OH
R1
R2
R2
R1
R2
HO
HO
HO
O
HO
H
H
HO
O
O
(Iа, b
)
(IIа, b
)
(IIIа, b)
a: R1 = Me, R2 = H
b: R1 = H, R2 = Me
Scheme 1.
OH
HO
SO2Ph
CHO
(V)
(VIIа
)
OH
(IV
)
(VI)
HO
VIIb
(
)
Scheme 2.
methyl grout at the C24 atom; therefore, we decided to described protocols used for synthesizing brassinolide
use racemic sulfone (V). Such an approach allows both
[22]. The interaction between aldehyde (XII) and
isomers to be concurrently produced using a more
accessible reagent as compared with the correspondꢀ
ing optically pure sulfones. This method could be of
practical value only in the case of the successful sepaꢀ
ration of isomeric products (VIIa) and (VIIb). Further
study demonstrated that the risk paid off.
organolithium derivative (XIII), produced by treating
sulfone (V) with butyllithium, gave a mixture of
hydroxy sulfones (XIV), which was acetylated with
acetic anhydride without separation (Scheme 4). The
resulting acetoxy sulfones (XV) were treated with magꢀ
nesium amalgam to obtain a mixture of compounds
(
XVI) epimeric at C24; then, the protective ketone
Commercially available 2,3ꢀdimethylbutꢀ1ꢀene
group was eliminated via acid hydrolysis in the presꢀ
ence of pyridinium tosylate (Scheme 5).
(
VIII) was chosen as the initial compound for producꢀ
ing a side chain synthon for the target compounds; this
compound allowed sulfone ( ) with the necessary
V
The further synthesis of target compounds (II
implied the construction of 22,23ꢀdiol groups. It is
known that the conventional dihydroxylation of Δ22
steroids with osmium tetroxide leads to the predomiꢀ
nant formation of nonnatural (22 ,23 )ꢀdiols. The
)
structure of the carbon backbone to be obtained
(Scheme 3). The hydroboration—oxidation of olefin
ꢀ
(
VIII) gave alcohol (IX), which was converted into
tosylate ( ) by treatment with TsCl. The nucleophilic
X
S
S
substitution of the tosyl group with the help of a phenyl
sulfide anion gave sulfide (XI). The oxidation of
the last compound with hydrogen peroxide gave sulꢀ
required (22R,23R)ꢀisomers are usually formed by the
catalytic oxidation of Δ22ꢀolefins with osmium tetroxꢀ
ide in the presence of dihydroquinidine derivatives
[23]. Note that it is impossible to completely avoid the
fone (
The carbon backbone of the side chain for comꢀ
pounds (II) was constructed according to the earlier alytic variant of the reaction. For a Sharpless dihyꢀ
V).
formation of side (22S,23S)ꢀdiols even in a chiral catꢀ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
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2010

748
KHRIPACH et al.
O
S
(
1. BH
3
2. H O
S
–
TsCl/Py
PhS
H O
2 2
2
2
O
HO
TsO
(VIII
)
(
IX
)
(X)
(XI
)
V)
Scheme 3.
CHO
Li
O
OH
S
O
(XIII)
SO2Ph
XIV
O
O
(XII)
(
)
OAc
Ac O
2
Mg/Hg
SO2Ph
XV
O
O
(XVI)
(
)
Scheme 4.
droxylation of olefin (XVII), we used hydroquinidine
1,4ꢀphthalazinediyl diether ((DHQD)2ꢀPHAL) as a
catalyst; this compound provides the highest yield of
Indeed, in the reaction conducted under condiꢀ
tions analogous to those described above, derivative
(
XX) with a protected diol group was transformed into
the product of (XXI) rearrangement with a yield of
86%, and its saponification gave the target secasterol
(22R,23R)ꢀisomers [24]. This reaction gave a complex
mixture of products, from which we succeeded in isoꢀ
lating all four isomeric diols (XVIII) (Scheme 5) by
chromatography on silica gel.
(
XIXd) (Scheme 7).
Then we set the goal of clarifying the interaction
between the stereochemical configuration of C22,
C23, and C24 atoms, the conformation of the side
chain, and the cytotoxicity of compounds (XVIIIa),
A convenient singleꢀstage method for synthesizing
Δ2ꢀ6ꢀketones giving a yield of the target products
exceeding 80% was the heating of steroid 3α,5ꢀcycloꢀ
(
XVIIIb), (XVIIId), and (XIXc). Energetically favorꢀ
6ꢀketones in dimethylformamide [25] or dimethylacꢀ
etamide [26] in the presence of pyridinium salts.
The boiling of compounds (XVIIIa), (XVIIIc), and
XVIIId) in dimethylacetamide in the presence of
pyridinium bromide actually led to the products of
XIXa), (XIXc), and (XIXd) rearrangement (Scheme
able conformations for the side chain of the comꢀ
pounds in question were computed with the help of
conformational search methods [27–29]; the cytotoxꢀ
icity was assessed in the breast carcinoma MCFꢀ7 cell
line using the MTT test [30]. The results are consoliꢀ
dated in the table.
(
(
6); however, their yields (20–51%) were considerably
lower as compared with the expected values. After the
chromatographic separation of the reaction mixtures,
the low polarity fractions with NMR spectra displayꢀ
ing intensive signals in the region of olefin protons
were isolated in addition to the expected compounds
The comparison of the sets of low energy conformꢀ
ers for compounds (XVIIIa), (XVIIIb), (XVIIId), and
(
XIXc) suggests that the stereochemical configuration
of C22 and C23 determines the side chain flexibility.
For compounds (XIXc) and (XVIIId), which have a
(22R,23R)ꢀconfiguration, at least 90% of all comꢀ
(
XIXa),
diol group in the side chain of compounds (XVIIIa),
XVIIIc), and (XVIIId) was insufficiently stable under
the reaction conditions.
(XIXc), and (XIXd). Presumably, the
puted conformers differing in the value of the potential
energy by 1 kcal/mol clustered in the region of one
energy minimum; in addition, the terminal methyl
≤
(
groups C26–C28 were located in the βꢀregion. On the
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
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2010

SYNTHESIS OF SECASTEROL AND 24ꢀEPISECASTEROL
749
Py ⋅ TsOH
(XVI)
(
XVII)
O
Sharpless oxidation
OH
OH
(R)
(S)
(S)
(R)
(S)
(S)
OH
OH
HO
HO
(R)
(
S
)
+
+
+
(R)
(S)
(
R)
(R)
(
XVIIIа), 14%
(XVIIId), 34%
HO
HO
(
XVIIIb), 0.83%
(XVIIIc), 29%
Scheme 5.
contrary, characteristic of compounds (XVIIIa) and derivatives of 22,23ꢀdihydroxycampestane and 22,23ꢀ
XVIIIb), which have a (22 ,23 ) conformation, is a dihydroxyergostane: (22
,23 )ꢀisomers (XIXc) and
wide range of low energy side chain conformers. Conꢀ
XVIIId) displayed a high cytotoxicity for MCFꢀ7
cells, whereas the (22 ,23 )ꢀisomers either had a low
(
S
S
R
R
(
S
S
sequently, the side chain of (22 ,23 )ꢀ22,23ꢀdihyꢀ
R
R
toxicity (compound (XVIIIb)) or were nontoxic (comꢀ
pound (XVIIIa)).
droxyergostane and (22 ,23 )ꢀ22,23ꢀdihydroxyꢀ
campestane is more rigid as compared with the side
chain of the corresponding (22 ,23 )ꢀisomers. The
R
R
S
S
stereochemical configuration of the C24 atom has an
insignificant effect on the distribution of low energy
conformers, as is evident from the table.
EXPERIMENTAL
Used in the work were 2,3ꢀdimethylbutꢀ1ꢀene, tolꢀ
uenesulfonyl chloride, pyridinium bromide,
roperoxybenzoic acid, thiophenol, potassium osmate,
and butyllithium produced by Aldrich (United States).
In our previous paper [31], we demonstrated that
mꢀchloꢀ
the (22R,23R)ꢀ22,23ꢀdihydroxystigmastane derivaꢀ
tives with a conformationally rigid side chain displayed
considerable toxicity towards tumor cells. The data NMR spectra (
δ, ppm, and J,Hz) were recorded in a
listed in table demonstrate that this is also true for the Bruker Avance DRXꢀ500 (Germany) device
OH
OH
HO
HO
Py ⋅ HBr
20–51%
H
O
O
(
(
(
XVIIIа): (22
XVIIIc): (22
XVIIId): (22
S
R
R
,23
,23
,23R,24
S
R
,24
,24
S
R
S
)
)
)
(
(
(
XIXа): (22
XIXc): (22
XIXd): (22
S,23S,24S)
,23R,24
R
R
)
R
,23R,24
S
)
Scheme 6.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
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2010

750
KHRIPACH et al.
OAc
OAc
AcO
AcO
Ac O, Py
2
Py
⋅
HBr
KOH
(
XVIIId
)
(XIXd)
H
(XX
)
(XXI)
O
O
Scheme 7.
(500 MHz for 1H NMR and 125 MHz for 13C NMR) 0.85 (3 H, d,
for solutions in CDCl3 (unless another solvent is specꢀ (3 H, d, 6.9, Me), 1.62 (1 H, m, H3), 1.77 (1 H, m,
ified). Chemical shifts were determined relative to the H2), 2.70 (1 H, dd, 12, 8.5, H1), 3.01 (1 H, dd, 12,
J 6.8, Me), 0.90 (3 H, d, J 6.9, Me), 0.96
J
J
J
residual chloroform signals. The melting temperatures 5.2, H1), 7.15 (1 H, m, Ph), 7.25 (2 H, m, Ph), 7.32 (2
13
were measured on a Koffler hot stage. TLC was perꢀ H, m, Ph). C NMR spectrum: 15.07, 17.68, 20.25,
formed on Kieselgel 60 F254 chromatography plates 31.38, 38.80, 53.39, 125.46, 128.66, 128.74, 137.48.
in the following solvent systems: (a) petroleum ether–
ethyl acetate (6 : 1); (b) petroleum ether–ethyl acetate
(3 : 1); (c) petroleum ether–ethyl acetate (2 : 1); and
(d) CHCl3–MeOH (9 : 1). Column chromatography
was performed on Kieselgel 60 (VWR, Art. 7734)
silica gel.
2,3ꢀDimethylbutylphenyl sulfone (V). A solution
containing 2.26 g (11.6 mmol) of sulfide (XI) in 56 ml
of acetic acid was dropwise supplemented with 56 ml
of 30% H2O2. The mixture was stirred at room temperꢀ
ature for 24 h and diluted with water to extract with
petroleum ether (3 × 25 ml). The organic layer was
2,3ꢀMethylbutanol tosylate (X). 2,3ꢀDimethylbutꢀ washed with a saturated NaHCO3 solution (15 ml) and
1ꢀene (VIII) solution (0.37 ml, 3 mmol) in THF (3 ml) dried with Na2SO4. The solvent was evaporated in a
was dropwise supplemented with the complex vacuum. The yield of sulfone (
V
) as a colorless oilꢀlike
THF (1 M, 3.2 ml, 3.2 mmol) over 40 min. The product was 2.5 g (95%). H NMR spectrum: 0.75 (3
reaction mixture was left overnight at room temperaꢀ H, d, 6.8, Me), 0.80 (3 H, d, 6.8, Me), 1.00 (3 H, d,
ture and then treated with 30% H2O2 (1.5 ml) under 6.9, Me), 1.66 (1 H, m, H3), 2.00 (1 H, m, H2), 2.86
stirring. After stirring for 40 min at room temperature, (1 H, dd, 14, 8.8, H1), 3.08 (1 H, dd, 14, 3.3, H1),
1
BH3×
J
J
J
J
J
the reaction mixture was diluted with water and 7.56 (2 H, t, J 7.6, Ph), 7.64 (1 H, m, Ph), 7.90 (2 H,
extracted with CH2Cl2. The extract was dried with m, Ph). 13C NMR spectrum: 15.85, 17.78, 19.07,
Na2SO4 and evaporated. The mixture was dissolved in 32.33, 33.69, 60.39, 127.79, 129.20, 133.48, 139.96.
pyridine (3 ml) and supplemented with TsCl (3 g,
16 mmol). The mixture was left overnight at room
temperature, diluted with water, and extracted with
CHCl3. The extract was dried with Na2SO4, the solvent
was evaporated in a vacuum, and the residue was
6,6ꢀEthylenedioxyꢀ24ꢀmethylꢀ3
22ꢀene (XVI). A solution containing 0.88 g (3.9 mmol) of
sulfone ( ) in 35 ml of THF cooled to –76 was supꢀ
plemented in an argon flow with 4.5 ml of a 1.2ꢀN
butyllithium solution in hexane. The mixture was
stirred at the same temperature for 30 min and suppleꢀ
mented with 1.21 g (3.25 mmol) of aldehyde (XII) in
15 ml of THF. Stirring of the mixture was continued at
α,5ꢀcycloꢀ5αꢀcholestꢀ
V
°С
coevaporated with toluene. The yield of tosylate (
a colorless oil was 630 mg (82%). 1H NMR spectrum:
0.77 (3 H, d, 6.5, Me), 0.83 (3 H, d, 6.5, Me), 0.84
(3 H, d, 6.3, Me), 1.66 (2 H, m, H2 and H3), 2.46
(3 H, s, Me), 3.85 (1 H, dd, 9.4, 6.4, H1), 3.95 (1 H,
dd, 9.4, 5.6, H1), 7.35 (2 H, d, 8.0, Ts), 7.79 (2 H,
d,
8.2, Ts). 13C NMR spectrum: 12.47, 17.85, 20.16,
28.65, 38.27, 73.77, 127.84, 129.75, 133.06, 144.59.
Х) as
J
J
J
a temperature of –76°С. After 2 h, the mixture was
J
supplemented with 0.9 ml of acetic anhydride; the
cooling bath was removed, and the reaction mixture
was left overnight. A saturated NH4Cl solution (20 ml)
was added, and the aqueous phase was separated from
J
J
J
2,3ꢀDimethylbutylphenyl sulfide (XI). The solution the organic phase to extract it with petroleum ether
of sodium methylate prepared from 185 mg 30 ml). The pooled organic phases were dried with
(8.0 mmol) of sodium and 12 ml of methanol was Na2SO4 and evaporated. The residue was dissolved in
gradually supplemented with 375 mg (3.4 mmol) of 250 ml of absolute methanol, cooled to , and supꢀ
thiophenol and 869 mg (3.4 mmol) of tosylate ( ) in plemented with 2.5 g of activated magnesium and 1.0 g
(3 ×
0°С
Х
5 ml of methanol. The mixture was kept at room temꢀ of HgCl2 under stirring. The reaction mixture was
perature for 2 days and then diluted with 10 ml of a 5% stirred for 6 h and filtered through the layer of silica
KOH aqueous solution and extracted with petroleum gel. The filtrate was acidified with diluted acetic acid
ether (3 × 15 ml). The extract was dried with Na2SO4 to pH 6 and extracted with petroleum ether (3 ×
and evaporated in vacuum. The yield of sulfide (XI) as 30 ml). The extract was dried with Na2SO4 and evapoꢀ
a colorless oil was 579 mg (88%). 1H NMR spectrum: rated in a vacuum; the residue was separated by chroꢀ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
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SYNTHESIS OF SECASTEROL AND 24ꢀEPISECASTEROL
751
Set of lowꢀenergy conformations of compounds (XVIIIa), (XVIIIb), (XVIIId), and (XIXc) and their cytotoxicity for the breast
carcinoma MCFꢀ7 cell line
Configuration of C22,
C23, and C24 atoms
Toxicity for MCFꢀ7 cells
Compound
Lowꢀenergy conformations
(IC50, µM)
(
XIXc
)
(R
,
R,
R
)
1.6
(
XVIIId
)
(R
,
R
,
S
)
1.8
(
XVIIIb
)
(
S
,
S
,
R)
49
(
XVIIIa
)
(S
,
S
,
S
)
Nontoxic
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752
KHRIPACH et al.
matography on a silica gel column using a mixture of trum: 0.742 (3 H, d,
petroleum ether and ethyl acetate (10 : 1) as an eluent. 0.80 (3 H, d, 6.8, Me), 0.93 (3 H, d,
Totally, 0.64 g of compound (XVI) as an oil (Rf 0.28 (3 H, s, H19), 1.05 (3 H, d, 6.9, Me), 3.45 (1 H, dd,
J
6.9, Me), 0.745 (3 H, s, H18),
J
J
6.9, Me), 1.00
J
(a)) and 0.62 g of initial aldehyde (XII) were obtained
J 8.6, 6.3, H23 or H22), 3.68 (1 H, br. s, H22 or H23).
(the yield calculated per reacted aldehyde (XII) was 13C NMR spectrum: 10.07, 11.67, 11.92, 14.09, 15.67,
1
91%). H NMR spectrum: 0.74 (3 H, s, H18), 0.79 19.65, 21.63, 22.82, 24.25, 25.87, 26.40, 27.72, 33.42,
(3 H, d,
(3 H, d,
J
J
6.8, H21 or H24), 1.01 (3 H, s, H18), 1.08 34.71, 35.42, 39.61, 41.64, 42.23, 43.20, 44.70, 45.97,
6.8, H24 or H21), 3.70–4.03 (4 H, m, dioxꢀ 46.29, 46.68, 52.68, 56.52, 71.03, 71.51, 209.72.
olane). 13C NMR spectrum: 7.36, 12.43, 15.99, 16.34,
18.97, 22.61, 23.08, 24.48, 24.95, 27.71, 33.35, 34.19,
39.35, 40.09, 40.29, 43.03, 45.69, 46.58, 47.56, 49.80,
52.03, 55.75, 64.68, 64.92, 109.86, 214.10.
(22
cycloꢀ
Melting temperature, 172–173
EtOAc);
f 0.23 (c). 1H NMR spectrum: 0.74 (3 H, s,
H18), 0.87 (3 H, d, 6.8, Me), 0.89 (3 H, d, 6.9, Me),
0.96 (3 H, d, 6.7, Me), 0.99 (3 H, s, H19), 1.02 (3 H,
d,
S
,23
ꢀcholestanꢀ6ꢀone (XVIIIb) (9.7 mg, 0.83%).
(petroleum ether–
S,24R)ꢀ22,23ꢀDihydroxyꢀ24ꢀmethylꢀ3α,5ꢀ
5α
°С
R
J
J
24ꢀMethylꢀ3α,5ꢀcycloꢀ5αꢀcholestꢀ22ꢀenꢀ6ꢀone
J
(XVII). The solution containing 1.43 g (3.25 mmol) of
steroid (XVI) in the mixture of methanol (120 ml) and
water (2.5 ml) was supplemented with 0.155 g
(0.61 mmol) of pyridinium tosylate. The reaction
mixture was kept at room temperature for 24 h and
evaporated in a vacuum; the residue was diluted with
J
6.8, Me), 3.59 (1 H, m, H22), 3.71 (1 H, m, H23).
13CꢀNMR spectrum: 9.91, 11.66, 11.91, 13.95, 18.89,
19.62, 21.45, 22.82, 24.23, 25.86, 27.97, 29.81, 33.43,
34.73, 35.37, 39.66, 41.97, 43.19, 43.88, 44.66, 45.98,
46.29, 46.68, 52.51, 56.58, 70.19, 73.20, 209.66.
water (120 ml) and extracted with chloroform (3 ×
(22
cycloꢀ
Melting temperature, 192–193
temperature according to published data, 187–190°C
[33] and 189.4–190.3°C [32]; Rf 0.17 (c). H NMR
,23
ꢀcholestanꢀ6ꢀone (XVIIIc) (344 mg, 29%).
(EtOAc); melting
R,24R)ꢀ22,23ꢀDihydroxyꢀ24ꢀmethylꢀ3α,5ꢀ
40 ml). The extract was dried with Na2SO4 and evapoꢀ
rated in a vacuum. The residue was separated by chroꢀ
matography on a column filled with silica gel using a
mixture of petroleum ether and ethyl acetate (10 : 1) as
°С
1
an eluent. The yield of cycloketone (XVII) as an oil
spectrum: 0.72 (3 H, s, H18), 0.84 (3 H, d,
0.86 (3 H, d, 6.8, Me), 0.91 (3 H, d, 6.9, Me), 0.98
(3 H, d, 6.7, Me), 1.00 (3 H, s, H19), 3.40 (1 H, t,
5.2, H23), 3.70 (1 H, d,
3.6, H22). 13C NMR specꢀ
J 7.0, Me),
1
(
Rf 0.32 (a)) was 0.96 g (74%). H NMR spectrum:
J
J
0.72 (3 H, s, H18), 0.81 (3 H, d,
d, 6.8, Me), 0.90 (3 H, d, 6.8, Me), 1.00 (3 H, s,
H19), 1.01 (3 H, d, 6.8, Me), 5.10–5.22 (2 H, m,
J 6.8, Me), 0.83 (3 H,
J
J
J
J
J
J
trum: 10.81, 11.66, 11.85, 12.46, 17.26, 19.68, 22.12,
22.84, 23.97, 25.88, 26.96, 27.85, 33.43, 34.80, 35.38,
39.65, 40.23, 41.38, 42.54, 44.73, 45.95, 46.30, 46.72,
52.57, 56.77, 72.64, 76.34, 209.75.
H22 and H23). 13C NMR spectrum: 11.62, 12.20,
18.07, 19.61, 19.68, 19.94, 20.16, 20.96, 22.83, 24.06,
25.88, 28.75, 33.04, 33.18, 33.43, 34.74, 35.29, 39.58,
40.23, 42.57, 42.76, 43.06, 44.78, 46.05, 46.29, 46.74,
55.83, 55.89, 57.07, 131.92, 132.02, 135.54, 135.78,
209.75.
(22
cycloꢀ
Melting temperature, 167–168
1H NMR spectrum: 0.73 (3 H, s, H18), 0.84 (3 H, d,
6.8, Me), 0.91 (3 H, d, 6.6, Me), 0.94 (3 H, d, 7.1
Me), 0.96 (3 H, d, 6.6, Me), 1.00 (3 H, s, H19), 3.56
(1 H, d, 8.1, H22), 3.71 (1 H, d,
8.1, H23). 13C
R
,23
ꢀcholestanꢀ6ꢀone (XVIIId) (397 mg, 34%).
С (EtOAc); f 0.11(c).
R,24S)ꢀ22,23ꢀDihydroxyꢀ24ꢀmethylꢀ3α,5ꢀ
5α
°
R
Hydroxylation
cholestꢀ22ꢀenꢀ6ꢀone (XVII). A suspension of olefin
XVII) (1.185 g, 2.99 mmol) in a mixture of tert
of
24ꢀmethylꢀ3α,5ꢀcycloꢀ5αꢀ
J
J
J
,
(
ꢀ
J
butanol and water (72 ml, 1 : 1) was supplemented
with K3Fe(CN)6 (2.95 g, 8.97 mmol), K2CO3 (1.24 g,
8.97 mmol), methanesulfonamide (0.85 g,
J
J
NMR spectrum: 10.10, 11.66, 11.92, 19.68, 20.74,
20.86, 22.84, 23.89, 25.87, 27.72, 30.71, 33.42, 34.79,
35.39, 36.77, 39.68, 40.00, 42.50, 44.71, 45.95, 46.29,
46.71, 52.23, 56.81, 73.45, 74.69, 209.74.
8.97 mmol), K2OsO4 ⋅ 2H2O (22 mg, 0.06 mmol), and
(DHQD)2ꢀPHAL (23 mg, 0.03 mmol). The reaction
mixture was stirred at room temperature for 14 days,
supplemented with sodium sulfite (2 g, 16 mmol), and
stirred for 1 day. The aqueous phase was separated
from the organic phase and extracted with chloroform
(3 × 50 ml). The pooled organic phases were evapoꢀ
rated; the residue was separated by chromatography
on a column with SiO2 using a mixture of petroleum
(22
cholestꢀ2ꢀenꢀ6ꢀone (XIXa). A solution of cycloketone
XVIIIа) (115 mg, 0.27 mmol) in dimethylacetamide
S,23S,24S)ꢀ22,23ꢀDihydroxyꢀ24ꢀmethylꢀ5αꢀ
(
(4 ml) was supplemented with pyridinium bromide
(133 mg, 0.84 mmol). The mixture was boiled under
reflux in an atmosphere of argon for 3 h. The solvent
was evaporated in a vacuum; the residue was diluted in
water (10 ml) and extracted with chloroform
ether and EtOAc (20 :
1
4 : 1) as an eluent. The folꢀ
(3 × 5 ml). The organic layer was dried with Na2SO4
and evaporated. The residue was chromatographed on
lowing compounds were distributed according to the
order of elution.
a column with SiO2 using petroleum ether–EtOAc
(22
cycloꢀ
Melting temperature, 183–185
EtOAc); melting temperature according to published
S
,23
ꢀcholestanꢀ6ꢀone (XVIIIa) (173 mg, 14%).
(petroleum ether–
S,24S)ꢀ22,23ꢀDihydroxyꢀ24ꢀmethylꢀ3α,5ꢀ
(20 :
XIXа) was 60 mg (51%). Melting temperature, 192–
194 (petroleum ether–EtOAc); Rf 0.20 (b).
data, 171.1–171.4°С [32]); R
f 0.26 (c). H NMR specꢀ 1H NMR spectrum: 0.70 (6 H, s, H18 and H19), 0.74
1
8
: 1) as an eluent. The yield of Δ2ꢀ6ꢀketone
5α
(
°С
°С
1
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
No. 6
2010

SYNTHESIS OF SECASTEROL AND 24ꢀEPISECASTEROL
753
(3 H, d,
J
6.9, Me), 0.80 (3 H, d,
J
6.8, Me), 0.92 (3 H,
(22R,23R,24S)ꢀ22,23ꢀDiacetoxyꢀ24ꢀmethylꢀ5αꢀ
d, 6.9, Me), 1.04 (3 H, d,
J
J
6.9, Me), 3.44 (1 H, m, cholestꢀ2ꢀenꢀ6ꢀone (XXI). A solution of cycloketone
H23), 3.67 (1 H, br. s, H22), 5.56 (1 H, m, H2 or H3),
5.68 (1 H, m, H3 or H2). 13C NMR spectrum: 10.05,
11.81, 13.48, 14.07, 15.65, 21.06, 21.62, 21.67, 24.15,
26.38, 27.58, 37.63, 39.30, 39.38, 39.98, 41.61, 42.18,
43.30, 46.92, 52.71, 53.31, 53.81, 56.28, 71.02, 71.52,
124.48, 124.88, 212.03.
(XX) (71 mg) in dimethylacetamide (2 ml) was suppleꢀ
mented with pyridinium bromide (73 mg, 0.46 mmol).
The reaction mixture was heated at 160 in an atmoꢀ
°С
sphere of argon for 2 h, supplemented with an addiꢀ
tional 20 mg (0.12 mmol) of pyridinium bromide, and
heated for 2 h. The solvents were evaporated in a vacꢀ
uum, the residue was supplemented with water, and
the mixture was extracted with CHCl3. The organic
layer was dried over Na2SO4 and evaporated. The resiꢀ
due was chromatographed on a column with SiO2
(22
cholestꢀ2ꢀenꢀ6ꢀone (XIXc) was obtained according to
the analogous protocol from cycloketone (XVIIIc
with a yield of 38%. Melting temperature, 166–168
(petroleum ether–EtOAc); melting temperature
according to published data, 136–138 С [33]; Rf 0.25
(c). 1H NMR spectrum: 0.68 (3 H, s, H18), 0.71 (3 H,
s, H19), 0.84 (3 H, d, 7.0, Me), 0.86 (3 H, d, 6.8
Me), 0.91 (3 H, d, 6.9, Me), 0.98 (3 H, d, 6.7, Me), H18), 0.71 (3 H, d,
3.41 (1 H, t, 4.9, H23), 3.70 (1 H, d, 6.8, H22), 5.56 0.94 (3 H, d,
6.7, Me), 0.95 (3 H, d,
(1 H, m, H2 or H3), 5.68 (1 H, m, H3 or H2). 13C (3 H, d,
6.8, Me), 1.99 (3 H, s, OAc), 2.02 (3 H, s,
NMR spectrum: 10.84, 11.76, 12.43, 13.49, 17.29, OAc), 5.16 (1 H, dd,
9.0, 0.9, H22), 5.32 (1 H, dd,
9.0, 1.7, H23), 5.56 (1 H, m, H2 or H3), 5.68 (1 H,
R,23R,24R)ꢀ22,23ꢀDihydroxyꢀ24ꢀmethylꢀ5αꢀ
)
С
°
using petroleum ether–EtOAc (15 :
eluent. The yield of enediacetate
1
6
: 1) as an
(XXI) was 61 mg
°
(86%); Rf 0.37 (b). Melting temperature, 204–206°С
1
J
J
,
(petroleum ether). H NMR spectrum: 0.70 (3 H, s,
6.7, Me), 0.90 (3 H, d, 6.7, Me),
6.7, Me), 1.02
J
J
J
J
J
J
J
J
J
J
21.11, 21.68, 22.13, 23.87, 26.98, 27.72, 37.75, 39.31,
39.45, 40.03, 40.16, 41.37, 42.66, 46.93, 52.62, 53.29,
53.81, 56.56, 72.68, 76.38, 124.49, 124.91, 212.05.
J
m, H3 or H2). 13C NMR spectrum: 11.03, 11.74,
12.82, 13.50, 20.29, 20.86, 20.93, 21.09, 21.69, 23.82,
28.06, 30.41, 36.95, 37.68, 39.36, 39.44, 39.83, 39.98,
42.66, 46.85, 52.38, 53.34, 53.81, 56.65, 74.15, 75.75,
124.43, 124.98, 170.50, 170.53, 211.76.
(22
cholestꢀ2ꢀenꢀ6ꢀone (XIXd) was obtained according to
the analogous protocol from cycloketone (XVIIId
with a yield of 20%. Melting temperature, 172–174
(petroleum ether–EtOAc); melting temperature
according to published data, 164 С [33]); Rf 0.50 (d).
1H NMR spectrum: 0.69 (3 H, s, H18), 0.71 (3 H, s,
H19), 0.84 (3 H, d, 6.9, Me), 0.91 (3 H, d, 6.6, Me),
0.94 (3 H, d, 6.7, Me), 0.96 (3 H, d, 6.7, Me), 3.56
(1 H, dd, 8.2, 2.6, H23), 3.72 (1 H, dd, 8.2, 2.4,
R,23R,24S)ꢀ22,23ꢀDihydroxyꢀ24ꢀmethylꢀ5αꢀ
)
С
°
Saponification of diacetate (XXI). Diacetate (XXI
)
(45 mg, 0.087 mmol) was dissolved in a 5% KOH soluꢀ
tion in methanol (2 ml). The reaction mixture was
boiled with reflux for 1 h, cooled, and acidified to neuꢀ
tral pH with a 20% acetic acid solution in methanol.
The solvents were evaporated in a vacuum, the residue
was chromatographed on a column with SiO2 using
CHCl3 (30 : 1), and Rf 0.50 (d) was the eluent. The
yield of the compound with spectral characteristics
matching the properties described above for enediol
°
J
J
J
J
J
J
H23), 5.56 (1 H, m, H2 or H3), 5.68 (1 H, m, H3 or
13
H2). C NMR spectrum: 10.08, 11.84, 11.90, 13.50,
20.72, 20.86, 21.12, 21.68, 23.80, 27.61, 30.75, 36.78,
37.75, 39.31, 39.49, 40.03, 42.63, 46.92, 52.31, 53.30,
53.80, 56.61, 73.52, 74.71, 124.48, 124.92, 212.02.
(
XIXd) was 35 mg (92%).
Computation of side chain conformation. The conꢀ
(22
cycloꢀ
XVIIId) (69 mg, 0.16 mmol) in pyridine (1 ml) was
supplemented with acetic anhydride (0.1 ml,
0.11 mmol). The reaction mixture was kept at 60 for
R
,23
R,24S)ꢀ22,23ꢀDiacetoxyꢀ24ꢀmethylꢀ3α,5ꢀ
former structures were computed by a usage directed
approach to the stochastic conformational search [27]
of the HyperChem molecular modeling program [28].
The energies were computed using the MM+ force
field parameters. Six dihedral angles formed by the
side chain carbon atoms were used as the structural
parameters of the conformational search space. At
least 10000 minimizations of the total energy were
performed during each search procedure, and no more
than 2000 conformations with an energy within
16 kcal of the minimal value were stored. The VMD
Molecular Graphics program [29] was used for the
structural analysis of the conformers. The population
of local energy levels was estimated based on the comꢀ
puted total energies of the conformers on the assumpꢀ
tion of the Boltzmann distribution at room temperaꢀ
ture (293 K).
5αꢀcholestanꢀ6ꢀone (XX). A solution of diol
(
°С
6 h. Then, the solvents were evaporated to dryness, and
the residue was loaded onto a column with SiO2. A
petroleum ether–EtOAc mixture (10 :
used for elution. The yield of diacetate was 79 mg
(92%). Melting temperature, 173–175 (petroleum
f 0.42 (b). H NMR spectrum: 0.74 (3 H, s,
H18), 0.90 (3 H, d, 6.7, Me), 0.94 (3 H, d, 6.9, Me),
0.95 (3 H, d, 6.9, Me), 1.00 (3 H, s, H19), 1.02 (3 H,
d, 6.8, Me), 1.99 (3 H, s, OAc), 2.01 (3 H, s, OAc),
5.16 (1 H, d,
1
8 : 1) was
°С
1
ether);
R
J
J
J
J
J
9.0, H23), 5.32 (1 H, dd, J 9.0, 1.6,
H23). 13C NMR spectrum: 11.04, 11.61, 11.83, 12.83,
19.65, 20.30, 20.86, 20.94, 22.83, 23.91, 25.87, 28.16,
30.39, 33.45, 34.77, 35.27, 36.94, 39.64, 39.81, 42.55,
44.63, 45.98, 46.27, 46.70, 52.31, 56.86, 74.15, 75.73, (XIXc) for MCFꢀ7 cells [30]. The breast carcinoma
170.53, 209.46.
Cytotoxicity of compounds (XVIIIa)–(XVIIId) and
MCFꢀ7 cell line, obtained from the American
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
No. 6
2010

754
KHRIPACH et al.
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Tissue Culture Collection (ATCC), were cultivated in
96ꢀwell plates at 37 in an atmosphere of 5% СО2 in
a RPMIꢀ1640 medium containing ꢀglutamine
(2 mM), antibiotics (100 U/ml penicillin and
100 g/ml streptomycin), and 10% fetal bovine serum
⎯
°C
L
⎯
μ
inactivated by heating. Before the experiment, the
cells were kept for 24 h in a serumꢀfree medium and
then incubated for 48 h in a serumꢀfree medium with
the tested compounds at various concentrations. The
culture medium was removed, each well was suppleꢀ
13. Swaczynová J., Šíša M., Hnilicková J., Kohout L.,
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⎯
⎯
mented with 125
(1 mg/ml), and cells were incubated for 4 h at 37
The reaction was stopped by adding 125 l of 0.1 M
μl of PBS containing MTT
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Phytochemistry, 2008, vol. 69, pp. 418 426.
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°С.
⎯
μ
HCl in PrOH containing 10% Triton Xꢀ100; after 2 h,
optical absorption at 540 nm was measured in each
well in a Multiskan Spectrum Microplate Reader
(Thermo Scientific, United States). The MTT test
values were averaged over six independent measureꢀ
ments. The MTT test values in the absence of tested
compounds were regarded as 100%. The IC50 values
were calculated from the plots of the cell viability rate
versus the concentration of the tested compounds.
⎯
,
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⎯
⎯
ACKNOWLEDGMENTS
20. Hamdy, A.ꢀH.A., Aboutabl, E.A., Sameer, S., Hussein, A.A.,
The work was supported by the Russian Foundaꢀ
tion for Basic Research (project nos. 08ꢀ04ꢀ90023ꢀ
Bel_a and 07ꢀ04ꢀ01155ꢀa) and the Belarusian Repubꢀ
lican Foundation for Basic Research (project no.
Kh08Rꢀ031).
DíazꢀMarrero, A.R., Darias, J., and Cueto, M., Steꢀ
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