Synthesis of deuterated isotopomers of 7α- and (25R,S)-26- hydroxycholesterol, internal standards for in vivo determination of the two biosynthetic pathways of bile acids

Article abstract of DOI:10.1016/S0039-128X(03)00116-8

Deuterated isotopomers of 7α- and (25R,S)-26-hydroxycholesterol, internal standards for in vivo determination of the two biosynthetic pathways of bile acids formation from cholesterol, were prepared from [2,2,3,4,4,6- ²H₆]-cholestero

Full text of DOI:10.1016/S0039-128X(03)00116-8

Steroids 68 (2003) 733–738
Synthesis of deuterated isotopomers of 7␣- and
(25R,S)-26-hydroxycholesterol, internal standards for in vivo
determination of the two biosynthetic pathways of bile acids
Pierangela Ciuffreda, Silvana Casati, Laura Alessandrini, Giancarlo Terraneo, Enzo Santaniello∗
Dipartimento di Scienze Precliniche LITA Vialba, Universitá degli Studi di Milano, Via G.B. Grassi, 74-20157 Milan, Italy
Received 29 April 2003; received in revised form 30 June 2003; accepted 14 July 2003
Abstract
Deuterated isotopomers of 7␣- and (25R,S)-26-hydroxycholesterol, internal standards for in vivo determination of the two biosyn-
thetic pathways of bile acids formation from cholesterol, were prepared from [2,2,3,4,4,6-2H6]-cholesterol and (20S)-[7,7,21,21-2H4]-3␤-
(tert-butyldimethylsilyl)oxy-20-methylpregna-5-en-21-ol, respectively.
© 2003 Elsevier Inc. All rights reserved.
Keywords: Deuterated isotopomers; 7␣-hydroxycholesterol; (25R)-26-hydroxycholesterol; Bile acids; Cholesterol metabolism
1. Introduction
and versatile approach that relies on the construction of the
side chain and can therefore be extended to the preparation
of deuterated 2b and any other labeled sterols as well.
It is now commonly accepted that two main metabolic
pathways are involved in the formation of bile acids, the clas-
sical microsomal pathway that starts from 7␣-hydroxylation
of cholesterol [1] and an alternative 27-hydroxylation that
may occur also in extrahepatic tissues [2,3]. The rate of the
“in vivo” formation of bile acids via the “27-hydroxylation”
has been determined measuring the natural/deuterated
(25R)-26-hydroxycholesterol ratio in plasma by mass
spectrometry analysis [4]. In connection with a program
aimed to a simultaneous evaluation in human healthy
subjects of 7␣- and 27-hydroxylation pathways by gas
chromatographic/mass spectrometric analysis of deuter-
ated isotopomer, it became necessary to prepare deuterated
7␣- and (25R)-26-hydroxycholesterol for intravenous in-
fusions. For the synthesis of a deuterated isotopomer of
7␣-hydroxycholesterol (1a), we could rely on commer-
cially available hexadeuterated cholesterol, that could also
be prepared via a multiple exchange methodology [5].
(25R)-26-Hydroxycholesterol (2a) labeled with five to nine
deuterium atoms has been prepared from kryptogenin [4]
but this triterpene is no longer commercially available. Al-
ternatively, the deuterated compound 2b (Fig. 1) can be
prepared from diosgenin [6] but we preferred a more general
2. Experimental
2.1. General
Melting points were recorded on Stuart Scientific SMP3
instrument and are uncorrected. All reagents were purchased
from Sigma (USA). [2,2,3,4,4,6-2H6]-Cholesterol was ob-
tained from CDN Isotopes (Canada) and (20S)-[7,7,21,21-2
H4]-3␤-(tert-butyldimethylsilyl)oxy-20-methylpregna-5-en-
21-ol (5) was prepared according to [7]. Solvents were
purified and dried in the usual way; chromatography eluents
were petroleum ether (40–60 ◦C b.p.)/AcOEt.
All reactions were monitored by TLC on Silica Gel 60
F254 precoated plates with a fluorescent indicator (Merck).
Flash chromatography [8] was performed using silica gel
60 (230–400 mesh, Merck). In the experimental protocol,
unless differently specified, work-up consists in three ex-
tractions with CH2Cl2, washing of the organic solution with
water, drying with anhydrous Na2SO4 and evaporation of
the solvent at reduced pressure.
Mass spectra were carried out using a Hewlett-Packard
5988A instrument that was set at 70 eV ion energy, 0.1 mA
emission current, and 280 ◦C transfer line temperature. Syn-
thetic [2,2,3,4,4,6-2H6]-cholest-5-en-3␤,7␣-diol (1b) and
∗
Corresponding author. Tel.: +39-02-50319691;
fax: +39-02-50319631.
E-mail address: Enzo.Santaniello@unimi.it (E. Santaniello).
0039-128X/$ – see front matter © 2003 Elsevier Inc. All rights reserved.
doi:10.1016/S0039-128X(03)00116-8

734
P. Ciuffreda et al. / Steroids 68 (2003) 733–738
(0.068 g, 56%) as white crystals. M.p. 159 ◦C (from ethanol)
R
R
([10] 157–158 ◦C); H NMR: δ 0.68 (3H, s, 18-CH3), 0.84
1
(3H, d, J = 7.0 Hz, 26-CH3), 0.85 (3H, d, J = 7.0 Hz,
27-CH3), 0.91 (3H, d, J = 6.3 Hz, 21-CH3), 1.09 (3H, s,
19-CH3), 7.42 (2H, dd, J = 7.0 and 7.0 Hz, m-Ph H), 7.65
(1H, dd, J = 7.0 and 7.0 Hz, p-Ph H), 8.02 (2H, d, J =
7.0 Hz, o-Ph H).
R
R
OH
R
HO
HO
OH
R
R
R
R
R
1
2
a. R = H
b. R = D
2.2.3. [2,2,3,4,4,6-2H6]-Cholest-5-en-3β,7α-diol (1b)
To a stirred solution of the 7-oxo-derivative 4 (0.068 g,
0.13 mmol) in dry THF (2 ml), cooled to −78 ◦C under a
nitrogen atmosphere, a 1 M solution in THF of l-selectride
(0.40 ml, 0.40 mmol) was slowly added. The reaction was
kept under stirring for 6 h, then it was warmed to room tem-
perature and quenched with water (1 ml). To this solution
6 M NaOH (1 ml) and 30% H2O2 (1 ml) were added. The
solution was evaporated at reduced pressure and after usual
work-up the crude product was purified by flash chromatog-
raphy on silica gel. Elution with petroleum ether/AcOEt
(80:20) gave compound 1b as white powder (0.045 mg,
86%). M.p. 185 ◦C (from methanol) ([10] 184–186 ◦C); 1H
NMR: δ 0.67 (3H, s, 18-CH3), 0.84 (3H, d, J = 7.0 Hz,
26-CH3), 0.845 (3H, d, J = 7.0 Hz, 27-CH3), 0.91 (3H, d,
J = 6.3 Hz, 21-CH3), 0.98 (3H, s, 19-CH3), 3.83 (1H, d,
J = 1.4 Hz, 7␤-H). MS: 462 (100).
Fig. 1. Structure of unlabeled and labeled 7␣-hydroxycholesterol (1) and
(25R)-26-hydroxycholesterol (2).
[7,7,22,22-2H4]-cholest-5-en-3␤,26-diol (2b) were analysed
after derivatisation with trimethylsilylimidazole:piperidine
to obtain the bis-trimethylsilyl (TMS) ether. GC-MS anal-
ysis was performed with an SPB5 (Supelco Inc.) capillary
column 0.32 mm i.d., 0.25 ␮m film thickness, 30 m long,
operating at 20 ml/min helium flow rate. Column temper-
ature was programmed from 180 to 300 ◦C at 10 ◦C/min.
Mass spectra are given as m/z (relative abundance).
All NMR spectra were recorded on a Bruker AM-500
spectrometer operating at 500.13 MHz in CDCl3 solutions
at 303 K. Chemical shifts are reported as δ (ppm) relative
to CHCl3 fixed at 7.24 ppm and coupling constants (J) are
given in Hertz.
2.2.3.1. Cholest-5-en-3β,7α-diol (1a). 1H NMR: δ 0.67
(3H, s, 18-CH3), 0.84 (3H, d, J = 7.0 Hz, 26-CH3), 0.845
(3H, d, J = 7.0 Hz, 27-CH3), 0.91 (3H, d, J = 6.3 Hz,
21-CH3), 0.98 (3H, s, 19-CH3), 2.25 (1H, ddd, J = 1.4,
11.2 and 13.3 Hz, 4␤-H), 2.33 (1H, ddd, J = 1.4, 4.9 and
13.3 Hz, 4␣-H), 3.57 (1H, dddd, J = 4.9, 4.9, 11.2 and
11.2 Hz, 3␣-H), 3.83 (1H, dd, J = 1.4 and 4.9 Hz, 7␤-H),
5.58 (1H, dd, J = 1.4 and 4.9 Hz, 6-H). MS: 456 (100).
2.2. Synthesis of [2,2,3,4,4,6-2H6]-cholest-5-en-3β,
7α-diol (1b)
2.2.1. [2,2,3,4,4,6-2H6]-Cholest-5-en-3β-yl benzoate (3)
To a solution of [2,2,3,4,4,6-2H6]-cholesterol (0.10 g,
0.25 mmol) in CH2Cl2 (3 ml) pyridine (0.1 ml, 1.25 mmol)
was added and the external temperature was lowered to 0 ◦C.
Benzoyl chloride (0.07 ml, 0.6 mmol) was slowly added and
the solution stirred overnight. The reaction was neutralized
by addition of 1 M HCl and then worked-up to afford the
benzoate 3 as white crystals (0.12 g, 94%). M.p. 165–166 ◦C
(from acetone) ([9] 168 ◦C); 1H NMR: δ 0.68 (3H, s,
18-CH3), 0.84 (3H, d, J = 7.0 Hz, 26-CH3), 0.845 (3H, d,
J = 7.0 Hz, 27-CH3), 0.91 (3H, d, J = 6.3 Hz, 21-CH3),
1.05 (3H, s, 19-CH3), 7.42 (2H, dd, J = 7.0 and 7.0 Hz,
m-Ph H), 7.66 (1H, dd, J = 7.0 and 7.0 Hz, p-Ph H), 8.02
(2H, d, J = 7.0 Hz, o-Ph H).
2.3. Synthesis of (20S)-[7,7,21,21-2H4]-3β-
(tert-butyldimethylsilyl)oxy-21-iodo-20-methylpregna-
5-ene (7)
Toluene p-sulphonyl chloride (0.34 , 1.79 mmol) was
added at 0 ◦C to a stirred solution of (20S)-[7,7,21,21-2H4]
-3␤-(tert-butyldimethylsilyl)oxy-20-methylpregna-5-en-21-
ol (5) (1.0 g, 2.2 mmol) in pyridine (20 ml). The reac-
tion was kept at room temperature overnight. After usual
work-up, the tosylate 6 was directly used for the next
2.2.2. [2,2,3,4,4,6-2H6]-Cholest-5-en-7-oxo-3β-yl
benzoate (4)
step without further purification. H NMR: δ 0.03 (6H, s,
1
Si(CH3)2), 0.61 (3H, s, 18-CH3), 0.86 (9H, s, SiC(CH3)3),
0.95 (3H, s, 19-CH3), 0.96 (3H, d, J = 7.0 Hz, 21-CH3),
2.43 (3H, s, Ph CH3), 3.44 (1H, dddd, J = 4.9, 4.9, 11.2
and 11.2 Hz, 3␣-H), 5.27 (1H, d, J = 2.1 Hz, 6-H), 7.33
(2H, d, J = 7.7 Hz, Ph H), 7.76 (2H, d, J = 7.7 Hz, Ph H).
To a solution of the above tosylate 6 in dry acetone (20 ml)
lithium iodide (1.1 g, 8.14 mmol) was added and the mixture
refluxed under nitrogen atmosphere in the dark. After 24 h,
acetone was evaporated at reduced pressure, water was added
and the reaction worked up. The resulting crude product was
Pyridinium chlorochromate (PCC, 1.26 g, 5.9 mmol) and
molecular sieves (3 Å, 11.7 mg) were added to a solution of
benzoate 3 (0.12 g, 0.24 mmol) in dry benzene (6 ml) and the
mixture refluxed under a nitrogen atmosphere for 24 h. The
reaction was filtered through a pad of celite and washed with
hot CH2Cl2. The filtrate was dried on Na2SO4 and evapo-
rated at reduced pressure to give a crude product as a brown
solid that was purified by flash chromatography on silica gel.
Elution with petroleum ether/AcOEt 9:1 afforded ketone 4

P. Ciuffreda et al. / Steroids 68 (2003) 733–738
735
purified by flash chromatography to give the title compound
7 as white powder (1.4 g, 90%). M.p. 182 ◦C (from diethyl
ether–acetone) ([11] 184.5–186.5 ◦C); 1H NMR: δ 0.03 (6H,
s, Si(CH3)2), 0.69 (3H, s, 18-CH3), 0.87 (9H, s, SiC(CH3)3),
0.97 (3H, s, 19-CH3), 1.00 (3H, d, J = 7.0 Hz, 21-CH3),
3.45 (1H, dddd, J = 4.9, 4.9, 11.2 and 11.2 Hz, 3␣-H), 5.29
(1H, d, J = 2.1 Hz, 6-H). MS: 503 (M+-t-butyl, 100), 433
(M+-I, 15), 429 (41), 411 (55), 375 (60).
Non-deuterated iodo derivative 7 has been prepared
from unlabeled 5 as described above. NMR: δ 0.03 (6H, s,
Si(CH3)2), 0.69 (3H, s, 18-CH3), 0.87 (9H, s, SiC(CH3)3),
0.97 (3H, s, 19-CH3), 1.00 (3H, d, J = 7.0 Hz, 21-CH3),
3.14 (1H, dd, J = 4.9 and 9.1 Hz, 22a-H), 3.31 (1H, dd,
J = 2.1 and 9.1 Hz, 22b-H), 3.45 (1H, dddd, J = 4.9, 4.9,
11.2 and 11.2 Hz, 3␣-H), 5.29 (1H, ddd, J = 2.1, 2.1 and
4.9 Hz, 6-H). MS: 449 (M+-t-butyl, 82), 429 (M+-I, 10),
425 (39), 371 (100).
m, part XXꢀ of AAꢀXXꢀ system, 2-CH2), 3.08–3.11 (2H, m,
part AAꢀ of AAꢀXXꢀ system, 3-CH2), 3.49 (3H, s, OCH3),
5.76 (1H, s, 4-H), 7.52 (2H, dd, J = 7.0 and 7.0 Hz, m-Ph
H), 7.62 (1H, dd, J = 7.0 and 7.0 Hz, p-Ph H), 7.88 (2H,
d, J = 7.0 Hz, o-Ph H);
2.4.3. 4-Benzenesulfonyl-2-methyl-butan-1-ol (10)
To a solution of compound 9 (3.5 g, 14.7 mmol) in
H2O/THF 1:1 (150 ml) perchloric acid (20 ml) was added
dropwise and the solution refluxed for 2 h. The mixture is
quenched with a saturated solution of FeSO4 and an usual
work-up afforded the crude aldehyde which was used with-
out further purification. To a solution of the aldehyde in
methanol (80 ml) kept at 0 ◦C, NaBH4 (2.4 g, 64 mmol) was
slowly added and the reaction stirred to slowly reach room
temperature. After neutralization with 1 M HCl, methanol
was removed at reduced pressure and the aqueous solu-
tion worked-up. Alcohol 10 was obtained as a colorless oil
1
(3.2 g, 94%) [13]. H NMR: δ 0.85 (3H, d, J = 7.0 Hz,
CH3), 1.57 (1H, dddd, J = 7.0, 9.0, 9.0 and 11.9 Hz, 3a-H),
1.69 (1H, ddddq, J = 5.6, 6.3, 6.3, 7.0 and 7.0 Hz, 2-H),
1.83 (1H, dddd, J = 6.3, 6.3, 11.9 and 11.9 Hz, 3b-H),
3.09–3.19 (2H, m, part AAꢀ of AAꢀXY system, 4-CH2),
3.38 (1H, dd, J = 6.3 and 10.5 Hz, 1a-H), 3.45 (1H, dd,
J = 5.6 and 10.5 Hz, 1b-H), 7.54 (2H, dd, J = 7.0 and
7.0 Hz, m-Ph H), 7.63 (1H, dd, J = 7.0 and 7.0 Hz, p-Ph
H), 7.88 (2H, d, J = 7.0 Hz, o-Ph H).
2.4. Synthesis of (4-benzenesulfonyl-2-methyl-butoxy)-
tert-butyldimethylsilane (11)
2.4.1. 4-Benzenesulfonyl-butan-2-one (8)
Methyl vinyl ketone (1.7 ml, 21.2 mmol) was slowly
added to a solution of distilled water (20 ml), sodium
benzenesulfinate (2.3 g, 14.1 mmol) and KH2PO4 (1.9 g,
14.1 mmol). The resulting mixture was stirred at room tem-
perature in the dark for two days, then filtered and the solid
washed with methanol (3 × 10 ml). The solid was crystal-
lized from methanol to give butanone 8 as white crystals
(4.45 g, 99%). M.p. 93 ◦C (from diethyl ether) ([12] 91 ◦C).
1H NMR: δ 2.16 (3H, s, CH3), 2.91 (2H, t, J = 7.0 Hz,
3-CH2), 3.35 (2H, t, J = 7.0 Hz, 4-CH2), 7.56 (2H, dd,
J = 7.7 and 7.7 Hz, m-Ph H), 7.65 (1H, dd, J = 7.7 and
7.7 Hz, p-Ph H), 7.88 (2H, d, J = 7.7 Hz, o-Ph H).
2.4.4. (4-Benzenesulfonyl-2-methyl-butoxy)-tert-
butyldimethylsilane (11)
Imidazole (2.34 g, 34.5 mmol) and tert-butyldimethyl-
chlorosilane (2.5 g, 16.6 mmol) were sequentially added to a
solution of compound 10 (3.2 g, 13.8 mmol) in THF (60 ml)
at room temperature. The solution was stirred at room tem-
perature overnight and then water was added. An usual
work-up afforded a crude product which was purified by
flash chromatography on silica gel eluting with petroleum
ether/AcOEt (8:2). Silyl ether 11 was obtained as colorless
oil (4.1 g, 90%) and used as such for the next step. 1H
NMR: δ 0.03 (6H, s, Si(CH3)2), 0.79 (9H, s, SiC(CH3)3),
0.82 (3H, d, J = 7.0 Hz, CH3), 1.54 (1H, ddt, J = 7.0,
8.4 and 14.0 Hz, 3a-H), 1.63 (1H, ddddq, J = 5.6, 6.3,
6.3, 7.0 and 7.0 Hz, 2-H), 1.78 (1H, ddt, J = 6.3, 8.4 and
14.0 Hz, 3b-H), 3.12 (2H, t, J = 8.4 Hz, 4-CH2), 3.29 (1H,
dd, J = 6.3 and 10.5 Hz, 1a-H), 3.41 (1H, dd, J = 5.6 and
10.5 Hz, 1b-H), 7.54 (2H, dd, J = 7.0 and 7.0 Hz, m-Ph
H), 7.63 (1H, dd, J = 7.0 and 7.0 Hz, p-Ph H), 7.88 (2H,
d, J = 7.0 Hz, o-Ph H).
2.4.2. (4-Methoxy-3-methyl-but-3-ene-1-sulfonyl)-
benzene (9)
To a stirred suspension of (methoxymethyl)triphenylphos-
phonium chloride (28.8 g, 84 mmol) in dry THF (120 ml) un-
der a nitrogen atmosphere, a solution of 1.6 M n-butyllithium
in hexane (52 ml, 84 mmol) was slowly added at 0 ◦C. After
10 min, a solution of the butanone 8 (4.45 g, 21 mmol) in
THF (25 ml) was added dropwise and kept at 0 ◦C under
stirring for 20 min. The reaction was stopped and neutral-
ized by addition of a 10% aqueous solution of NH4Cl.
After work-up, the resulting product was purified by flash
chromatography on silica gel, eluting with petroleum
ether/AcOEt (9:1) to give enol ether 9 (mixture of E and Z
1
isomers) as a colorless oil (3.5 g, 70%). H NMR: minor
2.5. Synthesis of [7,7,22,22-2H4]-cholest-5-en-3β,
26-diol (2b)
isomer δ 1.45 (3H, s, CH3), 2.37–2.40 (2H, m, part XXꢀ
of AAꢀXXꢀ system, 2-CH2), 3.12-3.15 (2H, m, part AAꢀ of
AAꢀXXꢀ system, 3-CH2), 3.43 (3H, s, OCH3), 5.71 (1H, s,
4-H), 7.54 (2H, dd, J = 7.0 and 7.0 Hz, m-Ph H), 7.63 (1H,
dd, J = 7.0 and 7.0 Hz, p-Ph H), 7.89 (2H, d, J = 7.0 Hz,
o-Ph H); major isomer δ 1.46 (3H, s, CH3), 2.25–2.28 (2H,
2.5.1. [7,7,22,22-2H4]-3β,26-Bis(tert-butyldimethylsilyl)
oxy-cholest-5-en-3␤,26-diol (13)
A solution of n-butyllithium 1.6 M in hexane (7.8 ml,
12.5 mmol) was added dropwise under nitrogen to a solution

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P. Ciuffreda et al. / Steroids 68 (2003) 733–738
of diisopropylamine (1.7 ml, 12.5 mmol) in dry THF (20 ml)
at 0 ◦C. The stirred solution was cooled to −78 ◦C and a
solution of the sulfone 11 (4.1 g, 12.5 mmol) in dry THF
(15 ml) was added dropwise. The reaction was stirred at
−78 ◦C for 20 min, then a solution of the 22-iodocompound
5 (1.4 g, 2.5 mmol) and hexamethylphosphoric triamide
(HMPTA, 0.2 ml, 1.5 mmol) in dry THF (10 ml) was added
dropwise, very slowly. Then the temperature was brought
to 0 ◦C and the solution was stirred for an additional 3 h.
The reaction mixture was neutralized with saturated aque-
ous ammonium chloride and worked-up to give a crude
product. The residue was purified by flash chromatography
on silica gel elution with petroleum ether/AcOEt (95:5) to
give a diastereoisomeric mixture of sulfones 12 as an oil
(1.5 g, 80%).
0.98 (3H, s, 19-CH3), 3.40 (1H, dd, J = 2.1 and 9.8 Hz,
26a-H), 3.48 (1H, dd, J = 4.7 and 9.8 Hz, 26b-H), 3.52
(1H, dddd, J = 4.9, 4.9, 11.2 and 11.2 Hz, 3␣-H), 5.32 (1H,
d, J = 2.1 Hz, 6-H). MS: 460 (100), 445 (26), 421 (58).
2.5.2.1. Cholest-5-en-3β,26-diol (2a). δ 0.66 (3H, s,
18-CH3), 0.88 (3H, d, J = 6.3 Hz, 27-CH3), 0.90 (3H, d,
J = 6.3 Hz, 21-CH3), 0.98 (3H, s, 19-CH3), 3.40 (1H, dd,
J = 2.1 and 9.8 Hz, 26a-H), 3.48 (1H, dd, J = 4.7 and
9.8 Hz, 26b-H), 3.52 (1H, dddd, J = 4.9, 4.9, 11.2 and
11.2 Hz, 3␣-H), 5.32 (1H, ddd, J = 2.1, 2.1 and 4.9 Hz,
6-H). MS: 456 (100), 441 (28), 417 (38).
3. Results and discussion
A freshly prepared 6% Na–Hg amalgam (6 g) was added
to a solution of the compound 12 (1.5 g, 2 mmol) in ab-
solute ethanol (30 ml) under nitrogen at room temperature.
After 6 h other amalgam (6 g) was added to complete the
reaction that was stirred for 24 h. After this time, the amal-
gam was filtered off and the solution neutralized with sat-
urated aqueous ammonium chloride. The work-up provided
a crude product that was purified by flash chromatography
on silica gel by elution with petroleum ether/AcOEt (98:2)
to afford the title compound 13 as a white powder (0.86 g,
3.1. [2,2,3,4,4,6-2H6]-7α-Hydroxycholesterol (1b)
A sample of hexadeuterated 7␣-hydroxycholesterol was
prepared following the procedure described by Ourisson
and coworkers [10], i.e. stereoselective reduction of the ꢀ5
7-keto steroid to the corresponding 7␣-hydroxy derivative
by means of an appropriate reducing agent. Commercially
available [2,2,3,4,4,6-2H6]-cholesterol was transformed
into the corresponding benzoate 3 that was treated with an
excess of pyridinium chlorochromate. Allylic oxidation af-
forded the corresponding 7-keto derivative 4 that was stere-
ospecifically reduced with l-selectride to the corresponding
7␣-hydroxy derivative in 45% yield (Scheme 1).
Mass spectra of deuterated 7␣-hydroxycholesterol 1b as
bis-trimethylsilyl ether (Mr 552) shows as the most abun-
dant peak the fragment with m/z 462 [M − 90]+. This
confirms that all the deuterium atoms present in starting
[2,2,3,4,4,6-2H6]-cholesterol are maintained in the synthetic
sample of 7␣-hydroxycholesterol 1b. Furthermore, compar-
ison of the 1H NMR spectrum of 1b and unlabeled derivative
1a confirmed the presence of deuterium atoms at positions
3, 4, 4, 6. This was evident for the absence of two ddd at 2.25
and 2.33 ppm due to protons at 4␣-H and 4␤-H and of sig-
nals corresponding to 3␣-H (3.57 ppm) and 6-H (5.58 ppm).
Moreover, the multiplicity of 7␤-H, a dd at 3.83 ppm (J =
1.4 and 4.9 Hz) for non-deuterated compound 1a becomes
d (J = 1.4 Hz) in 1b, as a consequence of the disappear-
ance of coupling constant with 6-H. The two hydrogens at
the position 2 could not be clearly seen in the spectrum.
74%). M.p. 97 ◦C (from MeOH) ([14] 95 ◦C); H NMR: δ
1
0.02 (6H, s, Si(CH3)2), 0.04 (6H, s, Si(CH3)2), 0.65 (3H,
s, 18-CH3), 0.84 (3H, d, J = 6.3 Hz, 27-CH3), 0.86 (9H,
s, SiC(CH3)3), 0.87 (9H, s, SiC(CH3)3), 0.90 (3H, d, J =
6.3 Hz, 21-CH3), 0.97 (3H, s, 19-CH3), 3.34 (1H, dd, J =
2.1 and 9.8 Hz, 26a-H), 3.41 (1H, dd, J = 4.7 and 9.8 Hz,
26b-H), 3.46 (1H, dddd, J = 4.9, 4.9, 11.2 and 11.2 Hz,
3␣-H), 5.29 (1H, d, J = 2.1 Hz, 6-H).
2.5.2. [7,7,22,22-2H4]-Cholest-5-en-3β,26-diol (2b)
Into a solution of compound 13 (0.8 g, 1.2 mmol) in
THF (50 ml), a solution of tetrabutylammonium fluoride
1 M in THF (1.8 ml, 1.8 mmol) was slowly dropped. The
solution was stirred at room temperature for 24 h and then
evaporated at reduced pressure to give a crude product
that was purified by flash chromatography on silica gel.
Elution with petroleum ether/AcOEt (8:2) afforded the
deuterated (25R,S)-26-hydroxycolesterol (2b) as a white
powder (0.44 g, 90%). M.p. 176 ◦C (from AcOEt) ([15]
1
177–178 ◦C); H NMR: δ 0.66 (3H, s, 18-CH3), 0.88 (3H,
d, J = 6.3 Hz, 27-CH3), 0.90 (3H, d, J = 6.3 Hz, 21-CH3),
D
D
D
D
PCC
L-selectride
1b
BzO
D
BzO
O
D
D D
D
D D
D
4
3
Scheme 1. Synthesis of [2,2,3,4,4,6-2H6]-cholest-5-en-3␤,7␣-diol (1b) (PCC: pyridinium chlorochromate).

P. Ciuffreda et al. / Steroids 68 (2003) 733–738
737
D
D
D
D
OTs
OH
TsCl/Py
D
D
SiO
SiO
D
D
6
5
D
D
I
LiI
D
SiO
D
7
Scheme 2. Synthesis of (20S)-[7,7,21,21-2H4]-3␤-(tert-butyldimethylsilyl)oxy-21-iodo-20-methylpregna-5-ene (7) (TsCl: toluene p-sulphonyl chloride).
3.2. [7,7,22,22-2H4]-(25R,S)-26-Hydroxycholesterol (2b)
[7,7,22,22-2H4]-(25R,S)-26-hydroxycholesterol (2b). The
synthetic approach for the preparation of the C-5 synthon
is shown in the Scheme 3.
[7,7,22,22-2H4]-(25R,S)-26-Hydroxycholesterol was pre-
pared via a deuterated C-22 steroid suitably protected at 3␤
position, namely (20S)-[7,7,21,21-2H4]-3␤-(tert-butyldi-
methylsilyl)oxy-20-methyl-pregna-5-en-21-ol (5) that we
have recently prepared [7]. For the chemical elaboration
that is necessary for the construction of the side chain, the
tetradeuterated intermediate C-22 alcohol was converted
into the corresponding iodide 7 via an intermediate tosy-
late 6 (Scheme 2). The comparison of the mass spectra of
labeled and unlabeled iodo derivative 7 confirmed the pres-
ence of four deuterium atoms and, therefore, the labeling at
C-21 had been retained in the iodination step.
The C-5 synthon required for the construction of the
side chain has to contain two important functional groups
such as a phenylsulfone moiety that is needed for the
coupling of the C-5 synthon to the steroidal iodide and
the primary hydroxyl group corresponding to that present
in the side chain of (25R)-26-hydroxycholesterol. This
hydroxy group is protected as tert-butyldimethylsilyloxy
moiety, that is identical to the protection of the 3␤-hydroxy
group of the tetradeuterated intermediate 5. This pro-
tection could present the advantage that the simultane-
ous cleavage of two silyl groups may directly afford
The starting material for the preparation of the C-5 syn-
thon was the sulfone 8 readily obtained from methyl vinyl
ketone and sodium phenylsulfonate [12]. A Wittig reac-
tion with methoxymethyltriphenylphosphonium chloride
afforded the vinyl ether 9 as E–Z mixture that, after acid
hydrolysis and sodium borohydride reduction of the inter-
mediate aldehyde, afforded the alcohol 10. Silylation of this
alcohol gave the required silylated sulfone 11 (68% yield
from the phenyl sulfonyl ketone 8), that was used for the fol-
lowing coupling step with the steroidal iodide 7. The steroid
sulfone 12 so obtained contains the right number of carbon
atoms corresponding to (25R,S)-26-hydroxycholesterol and
the four deuterium that were supposed to be maintained in
the positions 7 and 22 not involved in the synthetic steps.
Removal of the phenylsulfonyl moiety was accomplished
by treatment with Na–Hg amalgam (74% from 7) to afford
compound 13. The simultaneous cleavage of the two silyl
groups (90%) afforded the required sample of tetradeuter-
ated (25R,S)-26-hydroxycholesterol (2b) (Scheme 4).
Comparison of the mass spectra of 2b and non-deuterated
analogue 2a confirms the presence of four deuterium atoms
in compound 2b. The most abundant ions present in the
OCH3
O
1. HClO4
(CH3OCH2)PPh3Cl
2. NaBH4
PhO2S
PhO2S
9
8
OH
OSi
TBDMSiCl
PhO2S
PhO2S
11
10
Scheme 3. Synthesis of (4-benzenesulfonyl-2-methyl-butoxy)-tert-butyldimethylsilane (11) (TBDMSiCl: tert-butyldimethylchlorosilane).

738
P. Ciuffreda et al. / Steroids 68 (2003) 733–738
D
D
D
D
SO2Ph
OSi
I
11, LDA
D
SiO
D
7
12
D
D
TBAF
Na/Hg, EtOH
2b
OSi
D
SiO
D
13
Scheme 4. Synthesis of ([7,7,22,22-2H4]-cholest-5-en-3␤,26-diol (2b) (LDA: lithium diisopropylamide, TBAF: tetrabutylammonium fluoride).
spectrum of unlabeled 2a [456 (100), 441 (28), 417 (38)]
becomes [460 (100), 445 (26), 421 (58)] in 2b with nearly
identical intensity of peaks. Therefore, it can be concluded
that no loss of deuterium, mainly from the position 22, oc-
curred in the iodination step.
[3] Del Puppo M, Galli Kienle M, Crosignani A, Petroni ML, Amati B,
Zuin M, et al. Cholesterol metabolism in primary biliary cirrhosis
during simvastatin and UDCA administration. J Lipid Res 2001;
42:437–41.
[4] Javitt NB, Kok E, Lloyd J, Benscath A, Field FH. Cholest-
5-ene-3␤,26-diol: synthesis and biomedical use of a deuterated
compound. Biom Mass Spectrom 1982;9:61–3.
[5] Gruenke LD, Craig JC. The synthesis of cholesterol-2-2-4-4-6-d5. J
Labelled Compd Radiopharm 1979;16:495–500.
[6] Ni Y, Kim H-S, Wilson WK, Kisic A, Schroepfer Jr GJ. A revisitation
of the Clemmensen reduction of diosgenin. Characterization of
byproducts and their use in the preparation of (25R)-26-hydroxy-
sterols. Tetrahedron Lett 1993;34:3687–90.
[7] Ciuffreda P, Casati S, Bollini D, Santaniello E. Synthesis of (20S)-
[7,7,21,21-2H4]-3␤-(tert-butyldimethylsilanyloxy)-20-methyl-pregn-
5-en-21-ol, an useful intermediate for the preparation of deuterated
isotopomers of sterols. Steroids 2003;68:193–8.
[8] Still WC, Kahn M, Mitra A. Rapid chromatographic technique for
preparative separations with moderate resolution. J Org Chem 1978;
43:2923–5.
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7␤- and 7␣-hydroxycholesterols. Synth Commun 1987;17:1279–86.
[11] Dygos JH, Desai BN. Steroidal ethers. Analogues of 7-ketochole-
sterol. J Org Chem 1979;44:1590–4.
4. Conclusion
We have described the preparation of deuterated iso-
topomers of 7␣- and (25R,S)-26-hydroxycholesterol bear-
ing the isotope labeling in fixed positions of the steroid
molecules. The deuterated samples are now available for
intravenous infusions to healthy human subjects in or-
der to evaluate the relative amounts of plasma 7␣- and
(25R)-26-hydroxycholesterol by mass spectrometry analy-
sis. This methodological approach will also make possible
the evaluation of the 7␣- and the 27-hydroxylation in the
bile acids metabolic pathways in different physiologic and
pathologic conditions.
Acknowledgements
[12] Julia M, Badet B. Synthèse d’aldéhydes ou de cétones par alkylation
de carbanions ␤-carbonylés saturés ou éthyléniques masqués. Bulletin
de la Société Chimique de France 1975;5/6:1363–6.
[13] Ferraboschi P, Grisenti P, Manzocchi A, Santaniello E. New chemo-
enzymatic synthesis of (R)- and (S)-4-(phenylsulfonyl)-2-methyl-1-
butanol: a chiral C5 isoprenoid synthon. J Org Chem 1990;55:
6214–6.
This work has been supported by the University of Milan
(Fondi FIRST 2002) and MURST (COFIN 2002064429).
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