Synthesis of (20S)-[7,7,21,21-2H4]-3β-(tert- butyldimethylsilanyloxy)-20-methyl-pregn-5-en-21-ol, a useful intermediate for the preparation of deuterated isotopomers of sterols

Article abstract of DOI:10.1016/S0039-128X(02)00172-1

(20S)-[7,7,21,21-²H₄]-3β-(tert- Butyldimethylsilanyloxy)-20-methyl-pregn-5-en-21-ol, an intermediate for the preparation of deuterated isotopomers of sterols to be used as standards for biomedical studies, was prepared by reduction with dichloroaluminum deuteride of ethyl (20S)-3β-(tert-butyldimethylsilanyloxy)-7-oxo-pregn-5-en-20-carboxylate. Using controlled experimental conditions, it has also been shown that the dichloroaluminum hydride reduction of a 7-keto steroid affords the corresponding 7β-hydroxy derivative in a highly stereoselective manner.

Full text of DOI:10.1016/S0039-128X(02)00172-1

Steroids 68 (2003) 193–198
Synthesis of (20S)-[7,7,21,21-2H4]-3␤-(tert-butyldimethylsilanyloxy)-20-
methyl-pregn-5-en-21-ol, a useful intermediate for the preparation of
deuterated isotopomers of sterolsଝ
Pierangela Ciuffreda, Silvana Casati, Deborah Bollini, Enzo Santaniello∗
Dipartimento di Scienze Precliniche LITA Vialba, Via G.B. Grassi, 74-20157 Milano, Italy
Received 5 September 2002; received in revised form 5 November 2002; accepted 11 November 2002
Abstract
(20S)-[7,7,21,21-2H4]-3␤-(tert-Butyldimethylsilanyloxy)-20-methyl-pregn-5-en-21-ol, an intermediate for the preparation of deuterated
isotopomers of sterols to be used as standards for biomedical studies, was prepared by reduction with dichloroaluminum deuteride
of ethyl (20S)-3␤-(tert-butyldimethylsilanyloxy)-7-oxo-pregn-5-en-20-carboxylate. Using controlled experimental conditions, it has also
been shown that the dichloroaluminum hydride reduction of a 7-keto steroid affords the corresponding 7␤-hydroxy derivative in a highly
stereoselective manner.
© 2002 Elsevier Science Inc. All rights reserved.
Keywords: Deuterated isotopomers; Deuterated sterols; (20S)-[7,7,21,21-2H4]-3␤-(tert-Butyldimethylsilanyloxy)-20-methyl-pregn-5-en-21-ol;
Dichloroaluminum deuteride; 7␤-Hydroxysterols
1. Introduction
easy to control, since the labeled 27-hydroxycholesterol 1
prepared by Clemmensen reduction of 2 contained five to
nine deuterium atoms [4].
The synthesis of multideuterated sterols is highly needed
to achieve the most accurate gas chromatography–mass
spectrometry (GC–MS) analysis of biologically rele-
vant steroids. This can be useful, for example in bile
acids biosynthesis for metabolites such as 24-, 25- or
27-hydroxycholesterols [1]. In connection with studies re-
lated to the evaluation of serum 27-hydroxycholesterol levels
in patients with primary biliary cirrhosis [2], it became nec-
essary to prepare deuterated 27-hydroxycholesterol for in-
travenous infusions aimed to evaluate the 27-hydroxylation
pathway in bile acid production [3]. Syntheses of deuterated
27-hydroxycholesterol (1) from two isoprenoids, krypto-
genin (2) and diosgenin (3), for biomedical use have been
already described [4,5] (Fig. 1). However, these prepa-
rations suffer of several disadvantages, such as the very
limited availability of kryptogenin (2) and yields from both
2 and 3 that are not reproducible or low in general. Further-
more, starting from 2, the deuterium incorporation is not
We, therefore, designed a more versatile approach to the
synthesis of deuterated isotopomers of 27-hydroxycholes-
terol, that could be extended to any other labeled sterol with
a functionalised side chain. The synthesis should proceed via
a deuterated C-22 steroid suitably protected at 3␤ position
that becomes a versatile intermediate for the construc-
tion of any required side chain. (20S)-[7,7,21,21-2H4]-20-
Methyl-pregn-5-en-3␤,21-diol (4a) fulfills all the above
requirements as one of the possible deuterated C-22 steroid
intermediates for the synthesis of labeled C-27 sterols [6]
(Fig. 2).
2. Experimental
2.1. General
Melting points were recorded on Stuart Scientific SMP3
instrument and are uncorrected. All reagents were pur-
chased from Sigma Chemical Co. (USA). Tetrahydrofuran
(THF) and diethyl ether were distilled from sodium/benzo-
phenone. Cholenic acid (5) was purchased from Steraloids
(USA).
ଝ
Dedicated to the memory of Dr. Eliahu Caspi, Principal Scientist at
The Worcester Foundation for Experimental Biology (Mass., USA).
∗
Corresponding author. Tel.: +39-02-50319691;
fax: +39-02-50319631.
E-mail address: enzo.santaniello@unimi.it (E. Santaniello).
0039-128X/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.
doi:10.1016/S0039-128X(02)00172-1

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P. Ciuffreda et al. / Steroids 68 (2003) 193–198
Fig. 1. Structure of 27-hydroxycholesterol 1, kryptogenin 2 and diosgenin 3.
All reactions were monitored by TLC on Silica Gel 60
The multiplicity of signals as doublets is indicated in the
text as d. The complete 13C signal assignments were made
using DEPT experiments for the unequivocal identification
of primary, secondary, tertiary and quaternary carbon atoms
and using two-dimensional techniques, such as HSQC.
F254 precoated plates with a fluorescent indicator (Merck).
Flash chromatography [7] was performed using Silica Gel
60 (230–400 mesh, Merck).
Mass spectra were recorded on a particle beam quadrupo-
lar mass spectrometer Hewlett-Packard 5988A spectrometer
equipped with an interface PB 5998A and a low pressure
HPLC HP1050. The mass spectrometric analysis was per-
formed in positive ion chemical ionization (PICI) with CH4
as chemical reactant gas at an electron energy of 240 eV
and with a source temperature of 250 ◦C. The particle beam
desolvation chamber temperature was 50 ◦C, the helium
pressure was 50 psi and the source pressure 0.9 Torr. Mass
spectra were acquired over 50–540 mass unit ranges at
0.78 scan s−1. Mass spectral data are given as m/z (relative
abundance).
2.3. Ethyl (20S)-3β-(tert-butyldimethylsilanyloxy)-
pregn-5-en-20-carboxylate (6)
A mixture of 5 (2 g, 5.7 mmol), absolute ethanol (100 ml)
and 96% sulphuric acid (5 ml) was refluxed for 10 h. After
cooling, the mixture was partially evaporated, neutralized
with a saturated solution of NaHCO3 and extracted with
CH2Cl2 (3 × 50 ml). The organic layer was dried (Na2SO4)
and evaporated. The residue (2.08 g, 98%) was sufficiently
pure by TLC (hexane/EtOAc 6:4) to be used in the next step
without purification.
To a solution of the above ester in THF (60 ml), imidazole
(990 mg; 14.5 mmol) and tert-butyldimethylchlorosilane
(1.0 g; 6.6 mmol) were sequentially added. After 12 h under
continuous stirring at room temperature, the mixture was
poured into water and extracted with CH2Cl2 (3 × 50 ml).
The organic phase was dried over Na2SO4 and evaporated
at reduced pressure. The product was purified by flash chro-
matography (hexane/EtOAc 95:5) to give compound 6 as
a white solid (2.5 g, 95%). Mp 129–130 ◦C (from hexane);
2.2. NMR experiments
All NMR spectra were recorded on a Bruker AM-500
spectrometer operating at 500.13 and 125.76 MHz for H
1
and 13C, respectively, in CDCl3 solutions. The sample tem-
perature was 303 K. Chemical shifts are reported as δ (ppm)
relative to CHCl3 fixed at 7.24 ppm for 1H-NMR spectra and
relative to CDCl3 fixed at 77.00 ppm for 13C-NMR. Cou-
pling constants (J) are given in Hz and the 1H signals were
1
selected H-NMR signals: δ 0.03 (6H, s, Si(CH3)2), 0.66
1
assigned by H-homodecoupling and COSY experiments.
(3H, s, 18-CH3), 0.86 (9H, s, SiC(CH3)3), 0.97 (3H, s,
19-CH3), 1.15 (3H, d, J = 7.0 Hz, 21-CH3), 1.22 (3H, t,
J = 7.0 Hz, COOCH2CH3), 2.13 (1H, ddd, J = 2.8, 4.9
and 13.3 Hz, 4␣-H), 2.23 (1H, ddddd, J = 2.1, 2.1, 2.1,
11.2 and 13.3 Hz, 4␤-H), 2.37 (1H, dq, J = 7.0 and 9.8 Hz,
20-H), 3.45 (1H, dddd, J = 4.9, 4.9, 11.2 and 11.2 Hz,
3␣-H), 4.08 (2H, q, J = 7.0 Hz, COOCH2CH3), 5.28 (1H,
ddd, J = 2.1, 2.1 and 4.9 Hz, 6-H). 13C-NMR: δ −3.92
(Si(CH3)2), 12.71 (CH2CH3), 14.95 (C-18), 17.80 (C-21),
18.93 (SiC(CH3)3), 20.09 (C-19), 21.68 (C-11), 25.04
(C-15), 26.61 (SiC(CH3)3), 27.82 (C-16), 30.38 (C-2),
32.54 (C-7), 32.75 (C-8), 37.24 (C-10), 38.02 (C-1), 40.26
Fig. 2. Structure of (20S)-[7,7,21,21-2H4]-20-methyl-pregn-5-en-3␤,21-
diol (4a) and (20S)-[7,7,21,21-2H4]-3␤-(tert-butyldimethylsilanyloxy)-20-
methyl-pregn-5-en-21-ol (4b).

P. Ciuffreda et al. / Steroids 68 (2003) 193–198
195
(C-12), 43.08 (C-13), 43.33 (C-20), 43.48 (C-4), 50.83
(C-9), 53.52 (C-17), 57.02 (C-14), 60.63 (CH2CH3), 73.27
(C-3), 121.71 (C-6), 142.21 (C-5), 177.56 (COOEt). MS:
489 (M+ + 1, 78), 473 (39), 431 (35), 357 (100). Analysis
calculated for C30H52O3Si (488.37): C, 73.71; H, 10.72.
Found: C, 73.59; H, 10.86.
21.88 (C-11), 23.24 (C-26), 23.49 (C-27), 24.49 (C-23),
26.51 (SiC(CH3)3), 26.99 (C-15), 28.67 (C-25), 29.24
(C-16), 32.42 (C-2), 36.38 (C-20), 36.87 (C-22), 37.09
(C-1), 39.03 (C-10), 39.41 (C-12), 40.16 (C-4), 43.22
(C-24), 43.75 (C-13), 46.08 (C-8), 50.63 (C-9), 50.68
(C-17), 55.17 (C-14), 72.01 (C-3), 126.49 (C-6), 166.53
(C-5), 201.14 (C-7). MS: 515 (M+ + 1, 100), 499 (33),
457 (22), 383 (45). Analysis calculated for C33H58O2Si
(514.42): C, 76.98; H, 11.35. Found: C, 76.73; H, 11.22.
Lithium aluminum hydride (LiAlH4, 105 mg, 2.75 mmol)
was cautiously added to a stirred mixture of anhydrous alu-
minum chloride (1.05 g; 7.75 mmol) in dry ether (12 ml) at
0 ◦C under nitrogen. The mixture was refluxed for 30 min,
cooled to room temperature, then a solution of compound
9 (500 mg, 0.95 mmol) in dry THF (5 ml) was added drop-
wise. The reaction mixture was stirred at room temperature
for 5 min and than poured into ice. The aqueous layer was
extracted with CH2Cl2 (3×15 ml) and the organic layer was
washed with brine (6 ml), dried over Na2SO4, evaporated
and chromatographed (hexane/EtOAc 9:1) to give com-
pound 10 (480 mg, 98%). Mp 125–126 ◦C (from hexane);
2.4. Ethyl (20S)-3β-(tert-butyldimethylsilanyloxy)-
7-oxo-pregn-5-en-20-carboxylate (7)
To a solution of compound 6 (2.5 g, 5.2 mmol) in ben-
zene (250 ml, dried over sodium) pyridinium chlorochro-
mate (PCC, 25 g, 116 mmol) and molecular sieves (1.5 g,
type 4 Å) were added. The reaction mixture was refluxed
under nitrogen for 24 h, cooled and filtered through a pad
of celite that was washed with hexane/EtOAc 1:1. The fil-
trate was dried over Na2SO4 and evaporated to dryness.
The resulting crude reaction mixture was purified by flash
chromatography (hexane/EtOAc 9:1) to give compound 7
as a white solid (1.7 g; 65%). Mp 173–174 ◦C; selected
1H-NMR signals: δ 0.04 (6H, s, Si(CH3)2), 0.58 (3H, s,
18-CH3), 0.77 (9H, s, SiC(CH3)3), 1.06 (3H, s, 19-CH3),
1.08 (3H, d, J = 7.0 Hz, 21-CH3), 1.13 (3H, t, J = 7.0 Hz,
COOCH2CH3), 3.50 (1H, dddd, J = 4.9, 4.9, 11.2 and
11.2 Hz, 3␣-H), 4.00 (2H, q, J = 7.0 Hz, COOCH2CH3),
5.56 (1H, d, J = 2.1 Hz, 6-H). 13C-NMR: δ −3.94
(Si(CH3)2), 12.77 (CH2CH3), 14.88 (C-18), 17.85 (C-21),
17.91 (C-19), 18.72 (SiC(CH3)3), 21.75 (C-11), 26.45
(SiC(CH3)3), 26.96 (C-15), 27.97 (C-16), 32.33 (C-2),
36.99 (C-1), 38.92 (C-10), 39.13 (C-12), 43.06 (C-20),
43.15 (C-4), 43.74 (C-13), 45.89 (C-8), 50.23 (C-9), 50.50
(C-17), 52.31 (C-14), 60.59 (CH2CH3), 71.86 (C-3), 126.32
(C-6), 166.59 (C-5), 177.23 (COOEt), 202.39 (C-7). MS:
503 (M+ + 1, 100), 487 (12), 445 (14), 371 (23). Analysis
calculated for C30H50O4Si (502.80): C, 71.66; H, 10.02.
Found: C, 71.45; H, 10.12.
1
selected H-NMR signals: δ 0.03 (6H, s, Si(CH3)2), 0.66
(3H, s, 18-CH3), 0.83 (3H, d, J = 6.3 Hz, 26-CH3), 0.835
(3H, d, J = 6.3 Hz, 27-CH3), 0.86 (9H, s, SiC(CH3)3),
0.90 (3H, d, J = 6.3 Hz, 21-CH3), 1.01 (3H, s, 19-CH3),
2.17 (1H, ddd, J = 2.8, 4.9 and 13.3 Hz, 4␣-H), 2.26 (1H,
dddd, J = 2.1, 2.1, 11.2 and 13.3 Hz, 4␤-H), 3.47 (1H,
dddd, J = 4.9, 4.9, 11.2 and 11.2 Hz, 3␣-H), 3.80 (1H, ddd,
J = 2.1, 2.1 and 7.7 Hz, 7␣-H), 5.23 (1H, dd, J = 2.1 and
2.1 Hz, 6-H). 13C-NMR: δ −3.92 (Si(CH3)2), 12.50 (C-18),
18.92 (SiC(CH3)3), 19.47 (C-19), 19.87 (C-21), 21.74
(C-11), 23.25 (C-26), 23.50 (C-27), 24.51 (C-23), 26.60
(SiC(CH3)3), 27.07 (C-15), 28.70 (C-25), 29.24 (C-16),
32.73 (C-2), 36.41 (C-20), 36.89 (C-22), 37.19 (C-10),
37.75 (C-1), 40.19 (C-12), 40.28 (C-8), 41.63 (C-4), 42.97
(C-24), 43.62 (C-13), 49.02 (C-9), 56.15 (C-17), 56.69
(C-14), 72.96 (C-3), 74.13 (C-7), 125.71 (C-6), 144.92
(C-5). MS: 515 (M+ −1, 18), 499 (38), 385 (40), 367 (100),
325 (52). Analysis calculated for C33H60O2Si (516.44): C,
76.68; H, 11.70. Found: C, 76.52; H, 11.66.
2.5. Reduction of 3β-(tert-butyldimethylsilanyloxy)-
cholest-5-en-7-one (9) to 3β-(tert-butyldimethylsilanyloxy)-
cholest-5-en-7β-ol (10)
2.6. (20S)-[7,7,21,21-2H4]-3β-(tert-
Butyldimethylsilanyloxy)-20-methyl-pregn-5-en-21-ol (4b)
3␤-(tert-Butyldimethylsilanyloxy)-cholest-5-en-7-one (9)
was obtained by allylic oxidation of 3␤-(tert-butyldimethyl-
silanyloxy)-cholesterol (830 mg, 1.66 mmol) with PCC.
The reaction was carried out and worked-up as described
for compound 7. Chromatographic purification of the crude
mixture (hexane/EtOAc 9:1) afforded compound 9 as a white
solid (530 mg, 62%). Mp 215–216 ◦C (from methanol); se-
lected 1H-NMR signals: δ 0.03 (6H, s, Si(CH3)2), 0.65 (3H,
s, 18-CH3), 0.83 (3H, d, J = 6.3 Hz, 26-CH3), 0.84 (3H, d,
J = 6.3 Hz, 27-CH3), 0.86 (9H, s, SiC(CH3)3), 0.89 (3H,
d, J = 6.3 Hz, 21-CH3), 1.16 (3H, s, 19-CH3), 3.50 (1H,
dddd, J = 4.9, 4.9, 11.2 and 11.2 Hz, 3␣-H), 5.63 (1H, d,
J = 2.1 Hz, 6-H). 13C-NMR: δ −3.97 (Si(CH3)2), 12.64
(C-18), 17.91 (C-19), 17.97 (C-21), 18.85 (SiC(CH3)3),
Anhydrous aluminum chloride (3.5 g, 25.8 mmol) was
poured into dry ether (25 ml) at 0 ◦C and lithium alu-
minum deuteride (LiAlD4, 350 mg; 8.3 mmol) was added
cautiously keeping the temperature at 0 ◦C under nitrogen.
The mixture was refluxed for 30 min, cooled to room tem-
perature and 7 (1.7 g, 3.4 mmol) in dry THF (10 ml) was
added dropwise to the mixture. After refluxing for 90 min,
the mixture was poured into ice, the aqueous solution was
extracted with CH2Cl2 (3 × 30 ml). The organic layer was
washed with brine (10 ml), dried over anhydrous Na2SO4,
evaporated and chromatographed (hexane/EtOAc 8:2) to
give compound 4b (987 mg, 72%). Mp 155–156 ◦C (from

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P. Ciuffreda et al. / Steroids 68 (2003) 193–198
hexane) ([8], 153.5–155.5 ◦C); selected 1H-NMR signals: δ
0.03 (6H, s, Si(CH3)2), 0.67 (3H, s, 18-CH3), 0.86 (9H, s,
SiC(CH3)3), 0.97 (3H, s, 19-CH3), 1.03 (3H, d, J = 7.0 Hz,
21-CH3), 2.14 (1H, ddd, J = 2.8, 4.9 and 13.3 Hz, 4␣-H),
2.24 (1H, dd, J = 2.1, 11.2 and 13.3 Hz, 4␤-H), 3.45
(1H, dddd, J = 4.9, 4.9, 11.2 and 11.2 Hz, 3␣-H), 5.28
(1H, d, J = 2.1 Hz, 6-H). 13C-NMR: δ −3.99 (Si(CH3)2),
12.57 (C-18), 17.36 (C-21), 18.92 (SiC(CH3)3), 20.09
(C-19), 21.71 (C-11), 25.05 (C-15), 26.61 (SiC(CH3)3),
28.36 (C-16), 32.41 (C-2), 32.74 (C-8), 37.24 (C-10), 38.05
(C-1), 39.22 (C-20), 40.32 (C-12), 43.09 (C-13), 43.49
(C-4), 50.82 (C-9), 53.06 (C-17), 57.17 (C-14), 73.31 (C-3),
121.65 (C-6), 142.39 (C-5). The compound 4b was >99.0%
isotopically pure (1H-NMR, 13C-NMR and MS). MS: 449
(M+ − 1, 13), 435 (29), 393 (22), 319 (70), 301 (100).
Analysis calculated for C28H46 D4O2Si (450.81): C, 74.60;
H, 12.07. Found: C, 74.43; H, 11.96.
32.66 (C-8), 37.21 (C-10), 38.02 (C-1), 39.39 (C-20),
40.29 (C-12), 43.07 (C-13), 43.39 (C-4), 50.83 (C-9), 53.11
(C-17), 57.17 (C-14), 68.32 (C-22), 73.37 (C-3), 121.77
(C-6), 142.16 (C-5). MS: 445 (M+ − 1, 12), 431 (20), 389
(16), 315 (79), 297 (100).
3. Results and discussion
We have decided to synthesize (20S)-[7,7,21,21-2H4]-3␤-
(tert-butyldimethylsilanyloxy)-20-methyl-pregn-5-en-21-ol
(4b), since a silyl ether is a protecting group stable under
basic, slightly acidic, reducing or oxidizing conditions and
is, therefore, compatible with most of chemical elaborations
necessary for the construction of the side chain [9].
Commercially available cholenic acid 5 was transformed
into the corresponding ethyl ester and protected at the
3␤-hydroxy group as tert-butyldimethylsilyl ether (TB-
DMS) to afford compound 6. Lithium aluminum deuteride
reduction of ester 6 would introduce in the product only
two deuterium atoms, but these are not sufficient for a
deuterated sterol to be used as standard for GC–MS anal-
ysis. A tetradeuterated intermediate such as, for instance,
compound 4b would be more suitable for the above pur-
pose. We planned the synthesis of the 7-keto derivative
7 that could be reduced with dichloroaluminum deuteride
(generated from lithium aluminum deuteride and aluminum
chloride in ether) to the corresponding [7-2H2]-steroid, ac-
cording to a reported method [10] that has been used for
the synthesis of 19-hydroxy-[7-2H2]-androstenedione for
human metabolism studies [11]. Applied to compound 7,
the procedure could be particularly convenient since the
simultaneous reduction of the ester and keto groups could
allow the introduction of four deuterium atoms in one
step.
2.7. (20S)-3β-(tert-Butyldimethylsilanyloxy)-
20-methyl-pregn-5-en-21-ol (11)
The reaction was carried out as described for the prepara-
tion of 4b and, starting from ethyl (20S)-3␤-(tert-butyldime-
thylsilanyloxy)-pregn-5-en-20-carboxylate (6), compound
11 was obtained. Mp 153–154 ◦C (from hexane) ([8],
1
153.5–155.5 ◦C); selected H-NMR signals: δ 0.03 (6H, s,
Si(CH3)2), 0.67 (3H, s, 18-CH3), 0.86 (9H, s, SiC(CH3)3),
0.97 (3H, s, 19-CH3), 1.03 (3H, d, J = 7.0 Hz, 21-CH3),
1.48 (1H, m, 7␣-H), 1.94 (1H, m, 7␤-H), 2.14 (1H, ddd,
J = 2.8, 4.9 and 13.3 Hz, 4␣-H), 2.24 (1H, ddddd, J = 2.1,
2.1, 2.1, 11.2 and 13.3 Hz, 4␤-H), 3.34 (1H, dd, J = 7.0
and 10.5 Hz, 22b-H), 3.45 (1H, dddd, J = 4.9, 4.9, 11.2 and
11.2 Hz, 3␣-H), 3.61 (1H, dd, J = 3.5 and 10.5 Hz, 22a-H),
5.28 (1H, ddd, J = 2.1, 2.1 and 4.9 Hz, 6-H). 13C-NMR:
δ −3.99 (Si(CH3)2), 12.52 (C-18), 17.35 (C-21), 18.89
(SiC(CH3)3), 20.03 (C-19), 21.69 (C-11), 25.09 (C-15),
26.55 (SiC(CH3)3), 28.35 (C-16), 32.54 (C-7), 32.57 (C-2),
The 7-keto compound 7 was obtained in 65% yield from
compound 6 by an allylic oxidation with excess PCC [11]
(Scheme 1).
Scheme 1.

P. Ciuffreda et al. / Steroids 68 (2003) 193–198
197
Scheme 2.
According to Blair et al. [10], the dichloroaluminum deu-
teride reduction of compound 7 should require conditions
that could lead to undesired side reaction, mainly cleavage
of the silyl-protecting group. Therefore, we decided to study
the LiAlH4/AlCl3 reaction on a model silyl 7-keto ster-
oid, 3␤-(tert-butyldimethylsilanyloxy)-cholest-5-en-7-one
9, that was prepared by allylic oxidation of 3␤-(tert-butyl-
dimethylsilanyloxy)-cholesterol 8 with PCC [12]. We found
that the initial step of the reduction was the previously unre-
ported conversion to the 7␤-hydroxy compound 10, that was
obtained in 98% yield at room temperature (5 min), with no
detectable amount of the 7␣-isomer. The ␤-configuration of
compound 10 was assigned by the analysis of the 1H-NMR
spectrum. Specifically, the value of 2.1 Hz, corresponding
to the coupling constant between 6-H and 7-H, is consistent
with a 6-H/7␣-H system. A value of about 5 Hz would be
expected for the 6-H/7␤-H counterpart, that is characterized
by a large dihedral angle between the two hydrogen atoms.
It should be reminded that previous methods for the stereo-
selective reduction of a 7-keto to a 7␤-hydroxy steroid can be
achieved at room temperature with NaBH4–CeCl3–MeOH
[13] or at −78 ◦C with in situ generated LiAlH(t-BuO)3
[14]. We propose, therefore, to add dichloroaluminum hy-
dride to the reagents that can allow a highly stereoselective
preparation of 7␤-hydroxy steroids. The 7␤-hydroxy com-
pound 10 could then be reduced with dichloroaluminum
hydride to the derivative 8 by additional reflux for 1.5 h
(Scheme 2).
therefore, these experimental conditions could be safely ap-
plied to the ketoester 7 bearing a silyl protecting group as
well. The one-step preparation of the tetradeuterated com-
pound 4b from the ketoester 7 was indeed achieved in 72%
yield (Scheme 3).
1
Comparison of the H-NMR spectrum of 4b and unla-
beled 11, confirmed the presence of four deuterium atoms
at 22 position and 7, for the lack of two dd at 3.61 and
3.34 ppm indicative of the protons at C-22 and for the ab-
sence of signals corresponding to 7␣-H (1.48 ppm) and 7␤-H
(1.94 ppm). Furthermore, a careful inspection of the spec-
tra gave information about the position more affected by
the deuterium substitution at C-7. In fact, the multiplicity
of 6-H, a ddd at 5.28 ppm (J = 2.1, 2.1 and 4.9 Hz) for
11, becomes d (J = 2.1 Hz) in 4b, as a consequence of the
disappearance of J with 7␣-H (2.1 Hz) and 7␤-H (4.9 Hz).
The residual J = 2.1 Hz is due to the allylic coupling of
6-H with 4␤-H. Moreover, the complex signal of 4␤-H in
11, a ddddd at 2.24 ppm becomes a ddd for the absence of
homoallylic coupling with 7␣-H (J = 2.1 Hz) and 7␤-H
(J = 2.1 Hz). In addition, the 4␣-H signal at 2.14 ppm is a
ddd for the presence of a long range J with 2␣-H (2.8 Hz),
part of a sterically fixed “W” arrangement of atoms. Finally,
in the 13C-NMR spectrum of 4b no signals were observed
for C-7 (32.54 ppm) and C-22 (68.38 ppm) (Fig. 3).
The comparative study of mass spectra of 4b and of
non deuterated analogue 11 confirms the clean incorpora-
tion of four deuterium atoms in compound 4b. The most
abundant ions present in the spectrum of unlabeled 11
[445 (M − H), 431 (M − CH3), 389 (M − C(CH3)3), 315
The direct reduction of the 7-keto derivative 9 to 3␤-silyl
derivative 8 was achieved in refluxing ether for 1.5 h and,
Scheme 3.

198
P. Ciuffreda et al. / Steroids 68 (2003) 193–198
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(M−tert-butyldimethylsilyloxy), 297 (315−H2O)] became
[449 (M − H), 435 (M − CH3), 393 (M − C(CH3)3), 319
(M − tert-butyldimethylsilyloxy), 301 (319 − H2O) in 4b
with nearly identical intensity of peaks (see Section 2). We,
therefore, conclude that the deuterated compound 4b can
now be proposed as a valuable intermediate for the synthesis
of deuterated isotopomers of biologically significant sterols.
Acknowledgments
We thank Mr. Andrea Lorenzi (Dipartimento di Chimica,
Biochimica e Biotecnologie per la Medicina) for record-
ing the mass spectra. This work was supported by the Ital-
ian National Council for Research (CNR, Target Project in
Biotechnology).