The introduction of a double bond on the steroid skeleton - The preparation of enol silyl ether derivatives from vicinal diols

Article abstract of DOI:10.1135/cccc2010145

The ways of converting steroid vicinal diol into an unsaturated derivative were studied with the intention of preparing suitable precursors for the introduction of deuterium or tritium into the molecules of brassinosteroids. The model vicinal diol compound, 2α,3α-dihydroxy-5α-pregnane-6, 20-dione (3), was converted with high regioselectivity to the α-hydroxy ketone derivative, 2α-hydroxy-5α-pregnane-3,6,20-trione (8), in a 75% yield. The attempts to convert α-hydroxy ketone 8 to the dioxolene derivative, 2α,3α-(isopropylidenedioxy)-5α-pregn-2-ene-6,20- dione (6), failed. The conditions for the conversion of ketone to the corresponding enol trialkylsilyl ether were optimized using cyclohexanone as a model compound. The best and reproducible results were obtained by using tert-butyldimethylsilyl trifluoromethansulfonate (TBDMSiOTf) as the silylating reagent and triethylamine as the base. Under these conditions, the 3,6,20-trione (8) was converted to 2,3-bis(tert-butyldimethylsilyloxy)-5α-pregn-2-ene- 6,20-dione (14) with a 66% yield.

Full text of DOI:10.1135/cccc2010145

Double Bond on the Steroid Skeleton
443
THE INTRODUCTION OF A DOUBLE BOND ON THE
STEROID SKELETON – THE PREPARATION OF
ENOL SILYL ETHER DERIVATIVES FROM VICINAL DIOLS
1
2
3,
Aleš MAREK , Blanka KLEPETÁŘOVÁ and Tomáš ELBERT *
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i.,
1
Flemingovo nám. 2, 166 10 Prague 6, Czech Republic; e-mail: marek@uochb.cas.cz,
2
3
klepetarova@uochb.cas.cz, elbert@uochb.cas.cz
Received December 13, 2010
Accepted March 8, 2011
Published online April 14, 2011
The ways of converting steroid vicinal diol into an unsaturated derivative were studied with
the intention of preparing suitable precursors for the introduction of deuterium or tritium
into the molecules of brassinosteroids. The model vicinal diol compound, 2α,3α-dihydroxy-
5α-pregnane-6,20-dione (3), was converted with high regioselectivity to the α-hydroxy
ketone derivative, 2α-hydroxy-5α-pregnane-3,6,20-trione (8), in a 75% yield. The attempts
to convert α-hydroxy ketone 8 to the dioxolene derivative, 2α,3α-(isopropylidenedioxy)-
5α-pregn-2-ene-6,20-dione (6), failed. The conditions for the conversion of ketone to the
corresponding enol trialkylsilyl ether were optimized using cyclohexanone as a model com-
pound. The best and reproducible results were obtained by using tert-butyldimethylsilyl
trifluoromethansulfonate (TBDMSiOTf) as the silylating reagent and triethylamine as the
base. Under these conditions, the 3,6,20-trione (8) was converted to 2,3-bis(tert-butyl-
dimethylsilyloxy)-5α-pregn-2-ene-6,20-dione (14) with a 66% yield.
Keywords: Enol silyl ether; Vicinal diol; Steroids; Brassinosteroids; X-ray diffraction.
Compounds containing one or more double bonds are ideal precursors
for preparing tritium-labeled tracers with specific activities higher than
60 Ci/mmol. Tritium is simply introduced by catalytic tritiation with
carrier-free tritium gas. Such tracers are indispensable for the mechanism of
action studies of highly biologically active compounds with low or medium
molecular weight, most often referred to as hormones. The vicinal diol
grouping on Ring A is typical for brassinosteroids – plant hormones1 dis-
covered thirty years ago. In Fig. 1, the formulas of two typical brassino-
steroids – 24-epibrassinolide (1) and castasterone (2) – are given. These
compounds have been intensively studied and exhibit not only growth
regulation functions in plants but also some promising antiviral activities2.
However, no synthesis leading to a tritium-labeled brassinosteroid with a
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 5, pp. 443–456
© 2011 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2010145

444
Marek, Klepetářová, Elbert:
specific activity higher than 60 Ci/mmol has yet been reported. We decided
to exploit the vicinal diol grouping for the introduction of a double bond
to the molecule of brassinosteroids. The 2α,3α-dihydroxy-5α-pregnane-
6,20-dione (3) was chosen as a readily accessible3 model compound for
the elucidation of a transformation pathway leading from the particular
brassinosteroid via its enol derivative as a precursor to a tritium-labeled
compound.
OH
OH
O
H
H
H
OH
OH
HO
HO
HO
HO
HO
HO
O
H
H
H
1
2
3
O
O
O
FIG. 1
Formulas of 24-epibrassinolide (1), castasterone (2) and 2α,3α-dihydroxy-5α-pregnane-
6,20-dione (3)
The ready formation of the dioxolene ring (Scheme 1) during the oxida-
tion of methyl 9,10,12-trihydroxyoctadecanoate (4) by the Jones reagent
has been described in the literature4. The authors explain the formation
of methyl 9,10-(isopropylidenedioxy)-12-oxo-9-octadecenoate (5) as the re-
sult of the acid-catalyzed reaction of the enol form of the primarily formed
α-hydroxy ketone with acetone.
OH
O
i
O CH3
O CH3
HO
O
OH
O
O
O
4
5
i = CrO3 , H2SO4 , acetone , water, -15 °C
SCHEME 1
RESULTS AND DISCUSSION
The oxidation of dihydroxy pregnane dione 3 by the Jones reagent yielded
a complicated mixture of products as revealed by TLC (Scheme 2). Only
2α,3α-(isopropylidenedioxy)-5α-pregnane-6,20-dione (7) in a 22% yield
and the starting compound 3 in a 44% yield were isolated by chromatogra-
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 5, pp. 443–456

Double Bond on the Steroid Skeleton
445
phy on a silica gel, the other compounds decomposed during the workup.
We decided to try a two-step conversion of dihydroxy pregnane dione 3 to
2,3-(isopropylidenedioxy)-5α-pregn-2-ene-6,20-dione (6). As the first step,
we prepared 2α-hydroxy-5α-pregnane-3,6,20-trione (8) through the oxida-
tion of dihydroxy pregnane dione 3 by dimethyldioxirane (DMD) (Scheme 2).
O
O
H
H
O
O
O
O
i
3
7
6
H
H
O
O
ii
22% isolated yield
not observed
O
O
H
O
O
H
H
H
HO
O
O
O
HO
O
H
H
H
O
H
O
O
9
8
O
i = CrO3 , dil. H2SO4 , acetone, -15 °C
ii = Dimethyldioxirane, acetone, 4 °C
SCHEME 2
When one equivalent of DMD was used for the oxidation, some unreacted
starting compound 3 and the regioisomeric 3α-hydroxy-5α-pregnane-
2,6,20-trione (9) were isolated in addition to 3,6,20-trione 8. The yield of
3,6,20-trione 8 after chromatographic separation was 54%. The ratio of
major 3,6,20-trione 8 to minor 2,6,20-trione 9 was 10:1. The observed high
regioselectivity of the oxidation of dihydroxy pregnane dione 3 to 3,6,20-
trione 8 is in accordance with the published data on the oxidation of 22,23-
(isopropylidenedioxy)-24-epicastasterone with methyl(trifluoromethyl)-
dioxirane5. When a fourfold excess of DMD was used, the yield of 3,6,20-
trione 8 increased to 80% and regioisomer 9 was not observed. Still some
unreacted starting compound 3 was present (TLC, about 20% according
to NMR). However, no overoxidation to the 2,3-diketo derivative was ob-
served.
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446
Marek, Klepetářová, Elbert:
Keeping the reaction mixture at 4 °C proved to be important; at r.t.,
a mixture of products was observed (TLC). At r.t. the rate of disintegration
of excess DMD to acetic acid is much more higher and the 3,6,20-trione 8
in acidic solution isomerizes to 2,6,20-trione 9. It is very likely that both 8
and 9 undergo further acid-catalyzed elimination of the vicinal hydroxy
group giving conjugated enones (see the structures in brackets in Scheme 2).
The instability of both α-hydroxy ketones 8 and 9 under acidic condi-
tions is the most probable reason for the unsuccessful attempts to convert
3,6,20-trione 8 to 2,3-(isopropylidenedioxy)-5α-pregn-2-ene-6,20-dione (6) by
a reaction with acetone under catalysis with 4-toluensulfonic acid (TsOH).
We tried to trap the enol form of α-hydroxy ketone 8 also by other meth-
ods normally used for the conversion of vicinal diols to the corresponding
isopropylidene derivatives6,7 but without success. These included trans-
isopropylidenation with 2,2-dimethoxypropane catalyzed with a TsOH,
reaction with acetone catalyzed by FeCl3 and a reaction with acetone cata-
lyzed by Ce(IV)(NH4)2(NO3)6. The elimination of condensation water from
the reaction mixtures using an azeotropic distillation with benzene or tolu-
ene or adding a molecular sieve failed to ameliorate the conditions of the
conversion.
We therefore decided to try to convert 3,6,20-trione 8 to another enol
derivative – 2,3-bis(trialkylsilyloxy)-5α-pregn-2-ene-6,20-dione – as an alter-
native possible precursor for catalytic tritiation. Owing to the limited
amount of 3,6,20-trione 8 available, we decided to investigate the optimal
combination of the trialkylsilyl reagent, base and solvent on cyclohexan-
one (10) as a model compound (Scheme 3).
OSiR1R2R3
O
O
i
+
13
11 R1 = R2 = R3 = methyl
12 R1 = t-butyl R2 = R3 = methyl
10
i = R1R2R3SiX, base, solvent, argone atmosphere
TMSiCl
R1 = R2 = R3 = methyl
R1 = t-butyl R2 = R3 = methyl X = Cl
X = Cl
TBDMSiCl
TBDMSiOTf R1 = t-butyl R2 = R3 = methyl X = trifluoromethansulfonyl
SCHEME 3
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Double Bond on the Steroid Skeleton
447
The results are summarized in Table I. The only reasonable yield was
obtained by the use of tert-butyldimethylsilyl trifluoromethanesulfonate
(TBDMSiOTf) as the silylating reagent, triethylamine as the base and di-
chloromethane (DCM) as the solvent8. Thus, these conditions were selected
as the starting point for the development of 2,3-bis(tert-butyldimethyl-
silyloxy)-5α-pregn-2-ene-6,2-dione (14) synthesis.
The reaction of 3,6,20-trione 8 with TBDMSiOTf turned out to be
strongly dependent on the molar ratio of the reagent to the starting com-
pound and on the mode of mixing all the reaction components together
(Scheme 4). In Table II, the results of the pilot experiments are summarized.
The conversion of the starting compound to the products was better than
80% in all cases (with one exception). The attempts for the stepwise con-
TABLE I
The results of the reaction of cyclohexanone (10) with the silylating reagents under various
reaction conditions
Product yield, %
Silylation
reagent
Reaction
conditions
enol 11 or 12 13
TMSiCl (2 eq.)
Et3N (8 eq.), DCM,
r.t., 2 days
<10a
0
Et3N (8 eq.), CH3CN,
r.t., 2 days
TMSiCl (2 eq.)
20a
0b
0
0
Et3N (24 eq.), CH3CN,
reflux, 3 days
TMSiCl (1.2 eq.)
TBDMSiCl (3 eq.)
TBDMSiCl (1.2 eq.)
TBDMSiCl (1.5 eq.)
TBDMSiCl (1.5 eq.)
imidazole (6 eq.), CH3CN,
reflux, 3 day
0b
0
Et3N (2.4 eq.), CH3CN,
r.t., 6 days
1a
0
imidazole (2 eq.), Et3N (3 eq.),
DCM, sonication, 4 h
<10b
11c
0
Et3N (3 eq.), after 1 h LDA (2 eq.),
THF, –78 °C, 3 h
50
Et3N (3 eq.) and LDA (2 eq.), THF,
from –78 °C to –5 °C in 3 h
TBDMSiCl (1.5 eq.)
TBDMSiOTf (1.5 eq.)
8c
0
0
Et3N (1.5 eq.), DCM,
r.t., 10 min
83c
a
b
c
According to 1H NMR; according to TLC; isolated yield.
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448
Marek, Klepetářová, Elbert:
version of 3,6,20-trione 8 to the enol 14 via 2α-tert-butyldimethylsilyloxy-
5α-pregnane-3,6,20-trione (15) failed, only 2α,20-bis(tert-butyldimethyl-
silyloxy)-5α-pregn-20-ene-3,6-dione (16) was isolated. When larger and
larger excesses of TBDMSiOTf to 3,6,20-trione 8 were used, derivatives with
enol functions in Rings A and B 17, 18 and 19 gradually started to appear
in the reaction mixtures. The enol 14 was eventually obtained in a satisfac-
tory 66% yield when the molar ratio of TBDMSiOTf to 3,6,20-trione 8 was
40:1, and TBDMSiOTf was added to the reaction mixture in two equal por-
tions. Even though forty equivalents of triethylamine were present, the re-
action mixture became slightly acidic at the end. The most plausible expla-
nation of this unexpected result is that 2,3,6,20-tetrakis(tert-butyldimethyl-
silyloxy)-5α-pregna-2,6,20-triene (17) (or a mixture of dienes) is formed as
a transitional product. When the reaction mixture becomes acidic at the
end of the reaction time, the enol groups at C-6 and C-20 are hydrolyzed
back to the parent keto groups. The 2,3-bis(tert-butyldimethylsilyloxy) enol
O
O
H
H
i
TBDMSiO
O
TBDMSiO
TBDMSiO
8
H
H
O
O
14
15
OSiTBDM
OSiTBDM
H
H
TBDMSiO
TBDMSiO
O
TBDMSiO
H
H
OSiTBDM
O
16
17
O
OSiTBDM
O
H
H
H
TBDMSiO
TBDMSiO
HO
O
TBDMSiO
TBDMSiO
H
H
OSiTBDM
19
O
OSiTBDM
18
20
i = TBDMSiOTf, base, solvent, argone atmosphere
SCHEME 4
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Double Bond on the Steroid Skeleton
449
group in 14 is sterically hindered and the double bond is symmetrically
substituted with electronegative substituents. This could explain the higher
stability of this group in comparison with the tert-butyldimethylsilyloxy
enol groups on C-20 and C-6.
TABLE II
The results of the reaction of the 3,6,20-trione 8 with TBDMSiOTf
Yield, %a
TBDMSiOTf
Starting
compd.
to steroid
ratio
Conditions
mixture of
14 15
16
20
17, 18, 19
8
15
15
15
8
2.2:1
1.2:1
3.2:1
10:1
40:1
Et3N (4.4 eq.), 30 min
Et3N (2.2 eq.), 30 min
Et3N (4.4 eq.), 30 min
Et3N (20 eq.), 30 min
0
0
0
0
0
28
45
18
4
37
47
76
91
0
0
0
15
0
0
0
0
88b
0
Et3N (80 eq.), 1 ml of DCM,
2 h
0
0
8
8
40:1
40:1
Et3N (80 eq.), 5 ml of DCM,
2 h
0
0
0
94
0
0
0
0
0
Et3N (2 × 20 eq.), 1 ml of
DCM, TBDMSiOTf 2 × 20 eq.
(see Experimental)
66
a
b
Isolated yield calculated on starting compound; that the mixture of regioisomers had
been formed followed from the 1D and 2D NMR.
EXPERIMENTAL
NMR spectra were recorded with a Bruker Avance II 300 MHz spectrometer (1H at 300 MHz,
13C at 75.5 MHz) in CDCl3 unless stated otherwise. Chemical shifts are reported in ppm
(δ-scale) relative to TMS. The residual solvent signals in the 1H and 13C NMR spectra were
used as an internal reference (CDCl3: δ 7.26 for 1H and δ 77.23 for 13C). The coupling con-
stants (J) are given in Hz. The apparent resonance multiplicity is described as s (singlet), br s
(broad singlet), d (doublet), dd (doublet of doublets), t (triplet), AB system (A-B quartet) and
m (multiplet). Additional NMR techniques such as C-APT, H,H-COSY, H,C-HMBC and
H,C-HMQC were used for regular signal assignment. Occasionally, the 1H and 13C NMR spec-
tra showed broad signals or two sets of signals without estimated spin–spin interactions as
a result of the hindered rotation of the silyl function. The mass spectra were obtained in the
ESI mode either on Q-Tof micro from Waters or on LTQ Orbitrap XL from Thermo Fisher
Scientific for the HRMS spectra.
The TLC was performed on Merck Silica gel 60 F254 on aluminium sheets or on Merck
Aluminium oxide 60 F254 neutral on aluminium sheets in a solvent mixture indicated sepa-
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450
Marek, Klepetářová, Elbert:
rately for each experiment. The detection of the compounds on the TLC plates was first
done under a UV254 lamp and then the plates were developed by spraying with a 5% solu-
tion of phosphomolybdic acid in methanol (w/w) and heating with heat gun set to 350 °C.
Flash chromatography was performed on Silica gel 60 from Fluka or on Aluminium oxide
Type WN-6, Neutral, Activity Grade Super 1 from Sigma–Aldrich. DCM was dried by MgSO4,
distilled and stored over molecular sieve under argon. Tetrahydrofuran (THF) was purified
by distillation in a sodium/benzophenone still. Acetonitrile “Gradient grade for HPLC” was
purchased from Merck. All of the other reagents were reagent grade purchased from Sigma–
Aldrich and used as received. The evaporation of the solvents was done on a rotary evapora-
tor under reduced pressure. The melting points were determined on a Boetius micro-heating
block (VEB Analytik, Dresden, Germany) and are uncorrected. The optical rotation values
were measured on the AUTOPOL IV (Rudolph Research Analytical, USA) and are given in
10–1 deg cm2/g, the concentrations c are given in g/100 ml.
Reaction of 2α,3α-Dihydroxy-5α-pregnane-6,20-dione (3) with the Jones Reagent
First, 36 µl of 8 N Jones reagent (prepared from 2.67 g of CrO3, 2.3 ml of conc. sulfuric acid
and diluted with water to the volume of 10 ml) were added to a solution of 100 mg of
dihydroxy pregnane dione 3 (provided by Dr. Kohout, prepared according to ref.3) in 8 ml
of acetone vigorously stirred and cooled to –15 °C under argon atmosphere. The reaction
mixture was left for 20 min to warm up to r.t. while being continuously stirred under argon
atmosphere. Subsequently, 20 ml of water were added and the reaction mixture was ex-
tracted thrice with 5 ml of chloroform. The combined chloroform extracts were washed with
5 ml of a 10% water solution of sodium bicarbonate and dried over MgSO4. TLC (silica gel,
chloroform–diethyl ether 1:1) revealed a mixture of products (0.35 < RF < 0.7) besides the re-
sidual starting compound 3 (RF 0.2). After flash chromatography on a silica gel (chloroform–
diethyl ether 5:1), the least polar compound – 2α,3α-(isopropylidenedioxy)-5α-pregnane-
20
6,20-dione (7) – was isolated (24 mg, 22%), m.p. 238–240 °C, [α]D + 93 (c 0.32, CHCl3),
RF 0.7 (silica gel; chloroform–diethyl ether 1:1). MS (ESI): 411.2 (100, M + Na), 799.5 (15,
2 × M + Na). HRMS (ESI): for C24H36O4Na calculated 411.2506, found 411.2506. 1H NMR:
0.61 (3 H, s, CH3-18); 0.68 (3 H, s, CH3-19); 1.34 and 1.50 (2 × 3 H, 2 × s, 2 × CH3 of the
isopropylidene); 1.49 and 1.98 (2 × 1 H, 2 × m, CH2-15); 1.35 and 2.15 (2 × 1 H, 2 × m,
CH2-16); 1.35 and 1.53 (2 × 1 H, 2 × m, CH2-11); 1.43 (1 H, m, CH-14); 1.45 (1 H, m, CH-9);
1.50 and 2.10 (2 × 1 H, 2 × m, CH2-12); 1.32 and 2.02 (2 × 1 H, 2 × m, CH2-1); 2.00 and
2.15 (2 × 1 H, 2 × m, CH2-4); 1.77 (1 H, m, CH-8); 2.03 and 2.33 (2 × 1 H, m and dd, J =
13.2, 4.5, CH2-7); 2.13 (3 H, CH3-21); 2.52–2.58 (2 H, m, CH-5α and CH-17α); 4.07–4.14
(1 H, m, CH-2β); 4.27 (1 H, m, CH-3β). 13C NMR: 12.92 (CH3-19); 13.54 (CH3-18); 21.33
(CH2-11); 22.79 (CH2-16); 22.99 (CH2-15); 24.39 (CH2-4); 26.74 and 28.85 (2 × CH3 of the
isopropylidene); 31.65 (CH3-21); 37.57 (CH-8); 38.66 (CH2-12); 41.41 (CH2-1); 42.59 (C-10);
44.46 (C-13); 46.92 (CH2-7); 51.73 (CH-5); 53.49 (CH-9); 56.98 (CH-14); 63.59 (CH-17);
72.33 (CH-3); 72.49 (CH-2); 108.82 (C of the isopropylidene); 209.15 (CO-20); 211.05
(CO-6).
The starting dihydroxy pregnane dione 3 was isolated back (44 mg, 44%). HRMS (ESI): for
C
21H32O4Na calculated 371.2193, found 371.2193. 1H NMR: 0.62 (3 H, s, CH3-18); 0.76
(3 H, s, CH3-19); 1.33 and 1.71 (2 × 1 H, 2 × m, CH2-15); 1.35 and 1.80 (2 × 1 H, 2 × m,
CH2-16); 1.35 and 1.7 (2 × 1 H, 2 × m, CH2-11); 1.39 (1 H, m, CH-14); 1.40 (1 H, m, CH-9);
1.49 and 2.05 (2 × 1 H, 2 × m, CH2-12); 1.55 and 1.73 (2 × 1 H, 2 × m, CH2-1); 1.70 and
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Double Bond on the Steroid Skeleton
451
1.89 (2 × 1 H, 2 × m, CH2-4); 1.75 (1 H, m, CH-8); 2.00 and 2.32 (2 × 1 H, m and dd, J =
13.2, 4.5, CH2-7); 2.14 (3 H, CH3-21); 2.60 (1 H, t, J = 9.0, CH-17α); 2.70 (1 H, dd, J = 12.6,
3.0, CH-5α); 3.76 (1 H, m, CH-2β); 4.05 (1 H, m, CH-3β). 13C NMR: 13.55 (CH3-18); 13.73
(CH3-19); 21.29 (CH2-11); 22.87 (CH2-16); 24.26 (CH2-15); 26.43 (CH2-4); 31.65 (CH3-21);
37.62 (CH-8); 38.54 (CH2-12); 40.24 (CH2-1); 42.60 (C-10); 44.51 (C-13); 46.67 (CH2-7);
50.88 (CH-5); 53.66 (CH-9); 56.82 (CH-14); 63.45 (CH-17); 68.30 (CH-2); 68.44 (CH-3);
209.65 (CO-20); 212.02 (CO-6).
2α-Hydroxy-5α-pregnane-3,6,20-trione (8)
First, 15 ml of 0.08 M solution of dimethyldioxirane (1.2 mmol, prepared according to ref.9)
were added to a solution of dihydroxy pregnane dione 3 (415 mg, 1.2 mmol) in 5 ml of
DCM, and the reaction mixture was kept in the refrigerator at 4 °C overnight. It was left for
1 h to warm up to r.t. and the solvents were evaporated. Water (20 ml) was added to the
oily residue and the resulting emulsion was extracted with DCM (3 × 20 ml). The combined
organic layers were dried (MgSO4) and the solvent was evaporated. The crude product was
purified by flash chromatography on a silica gel (chloroform–hexane–methanol 20:20:1) to
afford a pure 3,6,20-trione 8 (240 mg, 58%) as a white solid, m.p. 175–176 °C (chloroform–
20
hexane), [α]D + 73 (c 0.31, CHCl3), RF 0.28 (silica gel; chloroform–hexane–methanol
20:20:1). MS (ESI): 369.2 (100, M + Na), 715.5 (20, 2 × M + Na). HRMS (ESI): for C21H30O4Na
calculated 369.2036, found 369.2035. 1H NMR: 0.63 (3 H, s, CH3-18); 1.03 (3 H, s, CH3-19);
1.22 and 1.70 (2 × 1 H, 2 × m, CH2-15); 1.33 and 1.81 (2 × 1 H, 2 × m, CH2-16); 1.42 and
1.76 (2 × 1 H, 2 × m, CH2-11); 1.40 (1 H, m, CH-14); 1.41 (1 H, m, CH-9); 1.45 and 2.03 (2 ×
1 H, 2 × m, CH2-1); 1.49 and 2.10 (2 × 1 H, 2 × m, CH2-12); 1.73 (1 H, m, CH-8); 2.01 and
2.39 (2 × 1 H, 2 × m, CH2-7); 2.10 (3 H, s, CH3-21); 2.53 (1 H, CH-17α); 2.60 (1 H, m,
CH-5α); 2.48 and 2.66 (2 × 1 H, 2 × m, CH2-4); 3.51 (1 H, J = 3.0, OH); 4.22 (1 H, m,
CH-2β). 13C NMR: 13.55 (CH3-18); 14.01 (CH3-19); 21.93 (CH2-11); 22.94 (CH2-16); 24.31
(CH2-15); 31.57 (CH3-21); 35.22 (CH2-4); 37.47 (CH-8); 38.49 (CH2-12); 42.73 (CH2-10);
44.48 (C-13); 46.34 (C-7); 47.92 (CH2-1); 53.47 (CH-9); 56.67 (CH-14); 58.73 (CH-5); 63.40
(CH-17); 72.11 (CH-2); 207.57 (CO-6); 208.88 (CO-20); 210.97 (CO-3).
20
Regioisomer 9 (24 mg, 6%) was isolated as an oil, [α]D + 19 (c 0.07, CHCl3), RF 0.15
(silica gel; chloroform–hexane–methanol 20:20:1). MS (ESI): 369.2 (100, M + Na), 715.5 (20,
2 × M + Na). HRMS (ESI): for C21H30O4Na calculated 369.2036, found 369.2035. 1H NMR:
0.64 (3 H, s, CH3-18); 0.73 (3 H, s, CH3-19); 1.21 and 1.69 (2 × 1 H, 2 × m, CH2-15); 1.35
and 1.82 (2 × 1 H, 2 × m, CH2-16); 1.42 and 1.76 (2 × 1 H, 2 × m, CH2-11); 1.26 (1 H, m,
CH-14); 1.39 (1 H, m, CH-9); 2.36 and 2.10 (2 × 1 H, 2 × m, CH2-1); 1.48 and 2.13 (2 × 1 H,
2 × m, CH2-12); 1.73 (1 H, m, CH-8); 2.05 and 2.43 (2 × 1 H, 2 × m, CH2-7); 2.14 (3 H, s,
CH3-21); 2.56 (1 H, CH-17α); 1.76 and 2.48 (2 × 1 H, 2 × m, CH2-4); 2.79 (1 H, dd, J = 12.6,
3.0, CH-5α); 3.55 (1 H, J = 3.6, OH); 4.15 (1 H, m, CH-3β). 13C NMR: 13.52 (CH3-18); 14.49
(CH3-19); 21.62 (CH2-11); 22.97 (CH2-16); 24.31 (CH2-15); 31.23 (CH2-4); 31.64 (CH3-21);
37.62 (CH-8); 38.38 (CH2-12); 44.33 (C-13); 46.46 (CH2-10); 46.55 (C-7); 50.75 (CH2-1);
53.38 (CH-9); 55.47 (CH-5); 56.76 (CH-14); 63.40 (CH-17); 74.69 (CH-3); 208.00 (CO-6);
209.03 (CO-20); 209.77 (CO-2).
Trialkylsilyloxycyclohexene 11 or 12
The silylating reagent was added to a stirred solution of cyclohexanone (10; 100 mg, 1 mmol)
in 5 ml of solvent and base under argon atmosphere by a syringe via a rubber septum in
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452
Marek, Klepetářová, Elbert:
one portion. The ratios of the reagents, temperatures and reaction times are indicated in
Table I. The reaction was quenched by adding 10 ml of a 10% water solution of sodium
bicarbonate. The mixture was extracted thrice with 15 ml of chloroform, combined chloro-
form extracts were dried by MgSO4, the solvent was evaporated and the oily residue was ap-
plied on a flash silica gel column pretreated with ammonia (the silica gel was mixed with
the mobile phase containing 1% of conc. water solution of ammonia and left to stand for
10 min prior to pouring onto the column). After elution with a 10:1 hexane–ethyl acetate
mixture, the fractions containing enolate 11 or 12 were evaporated and the oily residue was
analyzed by NMR.
tert-Butyldimethylsilyloxy cyclohexene (12). 1H NMR: 0.13 (6 H, s, 2 × Me); 0.92 (9 H, s,
t-Bu); 1.49–1.53 (2 H, m, CH2); 1.64–1.68 (2 H, m, CH2); 2.00–2.01 (4 H, m, 2 × CH2);
4.86–4.89 (1 H, m, CH). It is in agreement with the published data10,11 (the shift δ 0.01
given for nine hydrogens of tert-butyl group in ref.10 is most likely a typographic error).
Reaction of 2α-Hydroxy-5α-pregnane-3,6,20-trione (8) with TBDMSiOTf.
General Conditions
The silylating reagent was added to a solution of 3,6,20-trione 8 (20 mg, 58 µmol) and
triethyl amine in 2 ml of DCM stirred under inert argon atmosphere by a syringe through
a rubber septum. The progress of the reaction was monitored by TLC on aluminium oxide
plates. After the reaction completion as monitored by TLC, 10 ml of DCM were added and
the reaction mixture was poured into 5 ml of a 10% water solution of sodium bicarbonate.
The organic layer was separated and the water phase was washed with two 5 ml portions
of DCM. The combined DCM extracts were dried over MgSO4, the solvent was evaporated
and the oily mixture was separated on a chromatography column filled with silica gel or
aluminium oxide. The following compounds were separated as chromatographically pure.
2α-tert-Butyldimethylsilyloxy-5α-pregnane-3,6,20-trione (15): white solid, m.p. 174–176 °C,
20
[α]D + 49 (c 0.21, CHCl3), RF 0.43 (silica gel; hexane–ethyl acetate–methanol 30:10:1). MS
(ESI): 483.3 (100, M + Na), 484.3 (30, M + H + Na), 943.6 (10, 2 × M + Na). HRMS (ESI): for
C
27H44O4SiNa calculated 483.2901, found 483.2901. 1H NMR: 0.05 and 0.16 (2 × 3 H, 2 × s,
(CH3)2Si); 0.63 (3 H, s, CH3-18); 0.93 (9 H, s, (CH3)3CSi); 1.05 (3 H, s, CH3-19); 1.25 and
1.65 (2 × 1 H, 2 × m, CH2-15); 1.32 and 1.68 (2 × 1 H, 2 × m, CH2-16); 1.45 and 1.77 (2 ×
1 H, 2 × m, CH2-11); 1.42 (1 H, m, CH-14); 1.43 (1 H, m, CH-9); 1.67 and 2.26 (2 × 1 H, 2 ×
m, CH2-1); 1.47 and 2.10 (2 × 1 H, 2 × m, CH2-12); 1.86 (1 H, m, CH-8); 2.03 and 2.36 (2 ×
1 H, 2 × m, CH2-7); 2.16 (3 H, s, CH3-21); 2.40 and 2.63 (2 × 1 H, 2 × m, CH2-4); 2.54 (1 H,
CH-17α); 2.65 (1 H, m, CH-5α); 4.27 (1 H, dd, J = 12.0, 6.6, CH-2β). 13C NMR: –5.22 and
–4.33 ((CH3)2Si); 13.60 (CH3-18); 14.18 (CH3-19); 18.72 (SiC); 22.03 (CH2-11); 23.05
(CH2-16); 24.37 (CH2-15); 26.02 ((CH3)3C); 31.62 (CH3-21); 36.26 (CH2-4); 37.68 (CH-8);
38.56 (CH2-12); 42.78 (CH2-10); 44.53 (C-13); 46.44 (C-7); 49.01 (CH2-1); 53.69 (CH-9);
56.72 (CH-14); 58.37 (CH-5); 63.47 (CH-17); 73.83 (CH-2); 208.07 (CO-6); 208.55 (CO-20);
209.05 (CO-3).
2α,20-Bis(tert-butyldimethylsilyloxy)-5α-pregn-20-ene-3,6-dione (16): white solid, m.p.
20
127–130 °C, [α]D + 13 (c 0.25, CHCl3), RF 0.52 (silica gel; chloroform–hexane–methanol
20:20:1). MS (ESI): 597.4 (100, M + Na), 1171.9 (50, 2 × M + Na). HRMS (ESI): for
C
33H58O4Si2Na calculated 597.3766, found 597.3766. 1H NMR: 0.04 and 0.14 (2 × 3 H, 2 × s,
(CH3)2SiOC-2); 0.18 (6 H, s, (CH3)2SiOC-20); 0.66 (3 H, s, CH3-18); 0.91 and 0.94 (2 × 9 H,
2 × s, (CH3)3CSi); 1.03 (3 H, s, CH3-19); 1.23 and 1.73 (2 × 1 H, 2 × m, CH2-15); 1.45 and
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Double Bond on the Steroid Skeleton
453
1.75 (2 × 1 H, 2 × m, CH2-11); 1.25 (1 H, m, CH-14); 1.35 (1 H, m, CH-9); 1.80 and 2.02 (2 ×
1 H, 2 × m, CH2-16); 1.63 and 2.39 (2 × 1 H, 2 × m, CH2-1); 1.47 and 2.05 (2 × 1 H, 2 × m,
CH2-12); 1.88 (1 H, m, CH-8); 2.03 and 2.38 (2 × 1 H, 2 × m, CH2-7); 2.45 and 2.60 (2 ×
1 H, 2 × m, CH2-4); 2.58 (1 H, m, CH-5α); 2.62 (1 H, CH-17α); 4.04 and 4.11 (2 × 1 H, br s
and d, J = 1.2, CH2-21); 4.28 (1 H, dd, J = 12.0, 6.6, CH-2β). 13C NMR: –5.18 and –4.31 (2 ×
(CH3)2Si); 13.37 (CH3-18); 14.21 (CH3-19); 18.40 and 18.74 (2 × SiC); 22.09 (CH2-11); 24.32
(CH2-16); 24.61 (CH2-15); 26.04 ((CH3)3C); 36.35 (CH2-4); 38.15 (CH-8); 38.41 (CH2-12);
42.95 (CH2-10); 43.93 (C-13); 46.67 (C-7); 49.08 (CH2-1); 53.03 (CH-9); 56.03 (CH-14); 56.75
(CH-5); 58.46 (CH-17); 73.92 (CH-2); 90.22 (CH2-21); 159.43 (C-20); 208.65 (CO-6); 208.72
(CO-3).
2,3,6-Tris(tert-butyldimethylsilyloxy)-5α-pregna-2,6-dien-20-one (18): oil, RF 0.23 (aluminium
oxide; hexane–ethyl acetate 50:1). HRMS (ESI): for C39H72O4Si3Na calculated 711.4631,
found 711.4630. 1H NMR: 0.12 (3 H, s, (CH3)2SiOC-6); 0.19 and 0.20 (2 × 3 H, 2 × s,
(CH3)2SiOC-2(3)); 0.66 (3 H, s, CH3-18); 0.81 (3 H, s, CH3-19); 0.94 and 0.94 (9 H and 18 H,
2 × s, (CH3)3CSi); 1.90 and 2.30 (2 × 1 H, 2 × m, CH2-4); 2.12 (3 H, s, CH3-21); 2.35 (1 H,
dd, J = 15.5, 3.9, CH-5α); 2.55 (1 H, t, J = 8.7, CH-17α); 5.76 (1 H, m, CH2-7). 13C NMR:
–4.14, –3.53, –3.40 and –2.98 (3 × (CH3)2Si); 12.52 (CH3-18); 13.78 (CH3-19); 18.53, 18.59
and 18.65 (3 × SiC); 21.37 (CH2-11); 23.08 (CH2-16); 24.38 (CH2-15); 26.09, 26.25 and 26.27
((CH3)3C); 31.82 (CH2-21); 35.60 (CH2-4); 35.76 (CH-8); 39.31 (CH2-12); 42.94 (CH2-10);
44.27 (C-13); 45.01 (CH2-1); 48.05 (CH-9); 51.73 (CH-5); 55.81 (CH-14); 63.87 (CH-17);
104.54 (C-7); 131.14 and 131.76 (C-2 and C-3); 151.37 (C-6); 209.57 (C-20).
2,3,6-Tris(tert-butyldimethylsilyloxy)-5α-pregna-3,5-dien-20-one (19): oil, RF 0.17 (aluminium
oxide; hexane–ethyl acetate 50:1). HRMS (ESI): for C39H72O4Si3Na calculated 711.4631,
found 711.4630. 1H NMR: 0.10, 0.13 and 0.14 (3 × 9 H, 3 × s, (CH3)2SiOC); 0.64 (3 H, s,
CH3-18); 0.90, 0.91 and 0.96 (3 × 9 H, 3 × s, (CH3)3CSi); 0.96 (3 H, s, CH3-19); 1.20 and 2.08
(2 × 1 H, 2 × m, CH2-1); 2.13 (3 H, s, CH3-21); 2.55 (1 H, t, J = 8.7, CH-17α); 4.24 (1 H, dd,
J = 9.6, 6.3, CH-2); 5.76 (1 H, m, C-4). 13C NMR: –4.07, –3.68, –3.58 and –3.13 (3 ×
(CH3)2Si); 13.53 (CH3-18); 18.53, 18.60 and 18.70 (3 × SiC); 20.19 (CH3-19); 21.50 (CH2-11);
23.01 (CH2-16); 24.63 (CH2-15); 26.11, 26.22 and 26.27 (3 × (CH3)3C); 31.73 (CH3-21); 35.76
(CH-8); 36.62 (CH2-10); 37.13 (C-7); 38.95 (CH2-12); 44.27 (C-13); 46.16 (CH2-1); 48.05
(CH-9); 57.12 (CH-14); 63.87 (CH-17); 68.13 (C-2); 103.95 (CH2-4); 119.87 (CH-5); 140.50
(C-3); 149.62 (C-6); 209.53 (CO-20).
2α-Hydroxy-20-tert-butyldimethylsilyloxy-5α-pregn-20-ene-3,6-dione (20): white solid, m.p.
90–93 °C, RF 0.35 (silica gel; chloroform–hexane–methanol 20:20:1). 1H NMR: 0.18 (6 H, s,
(CH3)2SiO); 0.67 (3 H, s, CH3-18); 0.94 (9 H, s, (CH3)3CSi); 1.05 (3 H, s, CH3-19); 1.21 and
1.75 (2 × 1 H, 2 × m, CH2-15); 1.46 and 1.77 (2 × 1 H, 2 × m, CH2-11); 1.26 (1 H, m,
CH-14); 1.37 (1 H, m, CH-9); 1.76 and 2.02 (2 × 1 H, 2 × m, CH2-16); 1.45 and 2.55 (2 ×
1 H, 2 × m, CH2-1); 1.48 and 2.08 (2 × 1 H, 2 × m, CH2-12); 1.88 (1 H, m, CH-8); 2.03 and
2.40 (2 × 1 H, 2 × m, CH2-7); 2.45 and 2.60 (2 × 1 H, 2 × m, CH2-4); 2.62 (1 H, CH-17α);
2.70 (1 H, m, CH-5α); 3.48 (1 H, d, J = 3.3, OH); 4.03 and 4.10 (2 × 1 H, br s and d, J = 0.9,
CH2-21); 4.25 (1 H, m, CH-2β). 13C NMR: –4.29 ((CH3)2Si); 13.44 (CH3-18); 14.12 (CH3-19);
18.40 (SiC); 22.04 (CH2-11); 24.37 (CH2-16); 24.57 (CH2-15); 26.07 ((CH3)3C); 35.36 (CH2-4);
38.01 (CH-8); 38.35 (CH2-12); 42.96 (CH2-10); 42.96 (C-13); 46.63 (C-7); 49.97 (CH2-1);
54.04 (CH-9); 56.18 (CH-14); 56.96 (CH-5); 59.10 (CH-17); 72.51 (CH-2); 90.40 (CH2-21);
159.41 (C-20); 208.11 (CO-6); 211.32 (CO-3).
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Marek, Klepetářová, Elbert:
2,3-Bis(tert-butyldimethylsilyloxy)-5α-pregn-2-ene-6,2-dione (14)
At r.t., 265 µl (1.2 mmol) of TBDMSiOTf were added to a solution of 3,6,20-trione 8 (20 mg,
58 µmol) in 1 ml of DCM and 175 µl (1.3 mmol) of triethyl amine stirred under argon
atmosphere by a syringe through a rubber septum. After 1 h, additional 160 µl (1.2 mmol)
of triethyl amine were added and the reaction mixture was treated with 1.2 mmol of
TBDMSiOTf. The acidity of the reaction mixture was checked as follows: the reaction mix-
ture was sampled by capillary on pH indicator paper and, after the evaporation of the sol-
vent, the paper was moistened with water. Another 5-µl portion of TBDMSiOTf was added if
necessary until the pH 3 to 5 was indicated on the paper. The reaction mixture was then
stirred until TLC (aluminium oxide; hexane–ethyl acetate 10:1) revealed only traces of the
starting compound 8, RF 0.1 and one major product with RF 0.4, which strongly quenched
UV254 fluorescence. For the workup, 10 ml of DCM were added and the reaction mixture
was poured into 5 ml of a 10% water solution of sodium bicarbonate. The organic layer was
separated and the water layer was washed with two 5 ml portions of DCM. The combined
organic layers were dried by MgSO4 and the solvent was evaporated. The crude product
was purified by column chromatography on aluminium oxide (hexane–ethyl acetate 10:1).
Enol 14 (22 mg, 66%) was obtained as white crystals after the evaporation of the solvents.
After crystallization from diethyl ether, the m.p. was 180–182 °C.
Single Crystal X-ray Structure Analysis
The diffraction data of single crystals of 7 (colorless, 0.13 × 0.54 × 0.86 mm), 8 (colorless,
0.08 × 0.19 × 0.43 mm) and 15 (Fig. 2) (colorless, 0.12 × 0.13 × 0.85 mm) were collected on
Xcalibur X-ray diffractometer with CuKα (λ = 1.54180 Å) at 150 K. The structures were
solved by direct methods with SIR92 12 and refined by full-matrix least-squares on F with
CRYSTALS 13. The hydrogen atoms were all located in a difference map, but those attached
to carbon atoms were repositioned geometrically. All of the hydrogen atoms were initially
refined with soft restraints on the bond lengths and angles to regularize their geometry,
after which the positions were refined with riding constraints. The other, non-hydrogen
atoms were refined with anisotropic displacement parameters.
Crystal data for 7: C24H36O4, monoclinic, space group P21, a = 11.3272(6) Å, b =
7.5692(3) Å, c = 13.2246(7) Å, β = 111.279(6)°, V = 1056.54(10) Å3, Z = 2, M = 388.55, 10467
reflections measured, 4284 independent reflections. Final R = 0.042, wR = 0.049, GOF =
1.052 for 4058 reflections with I > 2σ(I) and 255 parameters.
Crystal data for 8: C21H30O4, monoclinic, space group P21, a = 7.3381(7) Å, b =
10.1698(8) Å, c = 12.2865(10) Å, β = 102.941(9)°, V = 893.62(13) Å3, Z = 2, M = 346.47, 8563
reflections measured, 3328 independent reflections. Final R = 0.060, wR = 0.071, GOF =
0.997 for 2794 reflections with I > 2σ(I) and 227 parameters.
Crystal data for 15: C27H44O4Si, monoclinic, space group C2, a = 33.535(4) Å, b =
6.6145(12) Å, c = 12.0723(17) Å, β = 91.202(10)°, V = 2677.3(7) Å3, Z = 4, M = 460.73, 17625
reflections measured, 5250 independent reflections. Final R = 0.053, wR = 0.062, GOF =
1.042 for 4696 reflections with I > 2σ(I) and 291 parameters.
CCDC 803973 (for 7), 803974 (for 8) and 803975 (for 15) contain the supplementary
crystallographic data for this paper. These data can be obtained free of charge via
www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic
Data Centre, 12, Union Road, Cambridge, CB2 1EZ, UK; fax: +44 1223 336033; or
deposit@ccdc.cam.ac.uk).
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 5, pp. 443–456

Double Bond on the Steroid Skeleton
455
FIG. 2
ORTEP drawing 7, 8 and 15. Thermal ellipsoids are drawn at the 50% probability level
CONCLUSIONS
The attempts of the formation of a dimethyl dioxolene ring on the steroid
skeletone were not successful. Notwithstanding that, we succeeded in find-
ing the conditions for the transformation of the 2,3-diol group of common
brassinosteroids to the 2,3-bis(tert-butyldimethylsilyloxy) enol group, a
suitable precursor for regio- and stereoselective labeling of brassinosteroids
by deuterium or tritium.
This work was performed as part of a research project of the Institute of Organic Chemistry
and Biochemistry (Z40550506) and was supported by Grant IAA400550801 of the Grant Agency of
the Academy of the Sciences of the Czech Republic.
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