A novel strategy for the synthesis of bromo-substituted cholestenone and its new application to a synthesis of 1α-hydroxycholesterol

Article abstract of DOI:10.3184/174751916X14652251037643

A novel and efficient method was developed for the preparation of bromo-substituted cholestenone, including 6β-bromocholestenone and 2α,6β-dibromocholestenone. The key step in this synthesis is a very mild method for the transformation of 5α,6β-dibromocholesterol into the 6β-bromocholestenone by treatment with NaClO/NaBr/TEMPO, followed by dehydro-bromination with Et₃N. Bromination at C-2 with NBS/BPO (benzoyl peroxide) gave the 2α,6β-dibromo cholestenone. This was used in a synthesis of 1α-hydroxycholesterol.

Full text of DOI:10.3184/174751916X14652251037643

JOURNAL OF CHEMICAL RESEARCH 2016
VOL. 40 JULY, 407–409
RESEARCH PAPER 407
A novel strategy for the synthesis of bromo-substituted cholestenone and its
new application to a synthesis of 1α-hydroxycholesterol
Bin Suna, Can Jina,b and Weike Sua*
aNational Engineering Research Center for Process Development of Active Pharmaceutical Ingredients, Collaborative Innovation Center of Yangtze River Delta
Region Green Pharmaceuticals, Zhejiang University of Technology, Hangzhou 310014, P.R. China
bKey Laboratory for Green Pharmaceutical Technologies and Related Equipment of Ministry of Education, College of Pharmaceutical Sciences, Zhejiang
University of Technology, Hangzhou 310 014, P.R. China
A novel and efficient method was developed for the preparation of bromo-substituted cholestenone, including 6β-bromocholestenone and
2α,6β-dibromocholestenone. The key step in this synthesis is a very mild method for the transformation of 5α,6β-dibromocholesterol
into the 6β-bromocholestenone by treatment with NaClO/NaBr/TEMPO, followed by dehydro-bromination with Et3N. Bromination at C-2
with NBS/BPO (benzoyl peroxide) gave the 2α,6β-dibromocholestenone. This was used in a synthesis of 1α-hydroxycholesterol.
Keywords: steroids, bromo-substituted cholestenone, oxidation, 1α-hydroxycholesterol
Bromo-substituted cholestenones are suitable precursors for
synthesising specifically labelled cholesterols containing tritium in
defined positions.1 In addition, according to recent research, they
markedly inhibit body weight gain and body fat accumulation, as
well as reducing the levels of serum triglyceride and cholesterol in
animals, without any clinical abnormalities.2 However, the earlier
synthesis of bromo-substituted cholestenone via bromination of the
steroidal α,β-unsaturated ketone with Br2 (liq.) always leads to a
complicated mixture of 2-, 4- and 6-substituted products,3 which
limits the practical application of this method. Efficient methods
for the preparation of regio- and stereoselectively bromo-substituted
cholestenone are rarely reported. It is well known that 1α-OH-D3
is the active form of parent VD3 and it has been shown to exert
a comparable biological activity to 1α,25-(OH)2-D3,4 which is
recognised as possessing the highest activity of all the metabolites
of vitamin D3.5 Cholesta-1,4,6-trien-3-one is a key intermediate
in the synthesis of 1α-OH-D3. The previous synthesis of cholesta-
1,4,6-trien-3-one was via a DDQ-mediated dehydrogenation of
cholesterol.6 However, the large amount of DDQ which is used can
lead to a dark coloured solution and also to increased environmental
concern.
We now report a novel synthesis of bromo-substituted
cholestenones (Scheme 1), including 6β-bromocholestenone 4 and
2α,6β-dibromocholestenone 5 in an excellent yield and regio- and
stereoselectivity. This method also avoids the defects existing in
traditional method of bromination of steroidal α,β-unsaturated
ketones with Br2 (liq.), such as tedious work-up process, low yield
and non-selectivity. The key step was performed under very mild
conditions involving the oxidation of 5α,6β-dibromocholesterol to
the 5α,6β-dibromoketone by treatment with NaClO/NaBr/TEMPO,
Et3N
Br2, AcONa
AcOH
NaClO, TEMPO
NaBr
O
H
O
H
O
Br
Br
Br
Br
1
2
3
Br
NBS/BPO
O
O
Br
Br
5
4
Scheme 1 Synthesis of bromo-substituted cholestenone from cholesterol.
O
NaOH H O
2
,
Br
LiBr/Li2CO3
2
O
O
O
6
7
Br
5
OH
NH3(liq.)/Li
HO
8
Scheme 2 Synthesis of 1α-hydroxycholesterol from 2α,6β-dibromocholestenone.
* Correspondent. E-mail: pharmlab@zjut.edu.cn

408 JOURNAL OF CHEMICAL RESEARCH 2016
Table 1 Optimisation of the bromination reaction (4 → 5)a
followed by dehydrobromination with Et3N to obtain the 6β-bromo-
cholestenone. Bromination at C-2 with NBS/BPO gave the 2α,6β-
dibromocholestenone. In addition, we have applied this bromo-
substituted cholestenone to the synthesis of 1α-hydroxycholesterol.
We have found that the trienone 6 was obtained easily by
dehydrobromination of 5 with LiBr/Li2CO3. It was then converted
into 1α-hydroxycholesterol 8 by successive treatment with alkaline
hydrogen peroxide and Li/NH3 (liq.) (Scheme 2).
Entry Solvent
NBS (equiv.)
1.2
Temp/°C
40
40
40
40
40
75
55
75
40
40
40
40
25
30
35
45
Yield/%b
80.2
N.R.
N.R.
N.R.
N.R.
N.R.
N.R.
N.R.
72.2
81.3
84.5
83.2
N.R.
N.R.
N.R.
88.3
86.2
1
Dioxane
CH2Cl2
CH3CN
Acetone
EtOAc
CH3CN
2b
3b
4b
5b
6b
7b
8b
9
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1
1.5
2.0
2.5
2.0
2.0
2.0
2.0
2.0
Acetone
EtOAc
Results and discussion
The 5α,6β-dibromocholesterol 2 was prepared via bromination of
cholesterol in ether by addition of bromine in acetic acid.7 It was
observed that the dry dibromide, even highly purified with several
crystallisations, easily decomposed at room temperature. Because
of this instability, the dibromide was isolated by vacuum filtration,
washed with cold methanol and then the moist dibromide was used
in further experiments directly without drying.
Dioxane
Dioxane
Dioxane
Dioxane
Dioxane
Dioxane
Dioxane
Dioxane
Dioxane
10
11
12
13
14
15
16
17
In the next reaction sequence, the dibromide 2 was transformed
into 6β-bromocholestenone 4 (Scheme 1). NaClO/NaBr/TEMPO
has been introduced as an effective system for oxidation of primary
alcohols to aldehydes or secondary alcohols to ketones,8–10 as an
alternative to the traditional metal oxidants. This led us to test
the transformation of dibromide 2 to the intermediate 5α,6β-
dibromoketone 3. It was found that oxidation of the 3-hydroxy
group of the dibromide to the corresponding ketone 3 was achieved
by reacting 2 in dichloromethane with sodium hypochlorite in
the presence of catalytic amounts of TEMPO and NaBr. Using
this system the dibromide 2 was oxidised completely after 1 h
stirring. During conventional work up it was noticed that the 5α,6β-
dibromoketone 3 easily decomposed (turning purplish) when the
organic layer was concentrated under reduced pressure at room
temperature, suggesting that the dibromocholestanone was unstable
and difficult to isolate. The solution was therefore used directly in
the following step. When TLC monitoring indicated that none of
the dibromide 2 remained in the solution, the organic layer was
washed, separated and dried, and elimination of the C-5 bromine
atom was accomplished easily by treatment with triethylamine at
room temperature. The usual work up and recrystallisation from
methanol furnished 6β-bromocholestenone 4 in 67% yield (from
cholesterol).
50
aReagents and conditions: 6β-bromocholestenone 4 (2 mmol), NBS (2–4 mmol), 4 h.
bIsolated yield.
cExtending reaction time to 12 h.
a slightly lower chemical yield was obtained when the temperature
was increased to 50 °C (Table 1, entries 16 and 17).
The 2α,6β-dibromocholestenone 5 was further processed to
produce 1α-hydroxycholesterol. Initially, 5 was transformed into
the trienone 6 by treatment with LiBr/Li2CO3 (Scheme 2), which
is a novel system for this dehydrohalogenation.11,12 It was found to
be necessary to use eight equivalents of LiBr and 10 equivalents of
Li2CO3 for the complete conversion of 5. The action of an organic
base such as triethylamine or pyridine on dibromocholestenone 5
did not give the desired trienone 6.
In the next sequence of reactions, the trienone 6 was converted
into 1α,2α-epoxycholesta-4,6-dien-3-one
7
stereo-selectively
by oxidation with hydrogen peroxide under basic conditions
(Scheme 2). After crystallisation from methanol, the monoepoxide
7 was obtained in 72% yield (from 2α,6β-dibromocholestenone 5).
Finally, the target product 1α-hydroxycholesterol was obtained in
56.5% yield via reduction of 7 with Li/NH3 (liq.) (Scheme 2).
Next, the 6β-bromocholestenone 4 was transformed into 2α,6β-
dibromocholestenone (5) using the cheap and easily available
N-bromosuccinimide (NBS) (Scheme 1). We first studied the
reaction in different solvents at 40 °C in the presence of a small
amount of benzoyl peroxide (BPO) as a radical starter. The
results are presented in Table 1. Although various solvents such
as toluene, EtOAc, CH2Cl2, THF, dioxane and CH3CN were tried
for this transformation, unexpectedly, we found that the reaction
gave the desired product only when it was carried out in dioxane
(Table 1, entry 1). It did not proceed satisfactorily or at all when
carried out in the other solvents which were screened even at an
elevated temperature (Table 1, entries 2–8). We then studied
the effect of different amounts of NBS on this transformation. A
generally increasing trend was observed when the amount of NBS
was increased from 1 equiv. to 2.5 equiv. and the highest yield
was obtained when 2 equiv. of NBS were used (Table 1, entry 11).
Improvement was not observed beyond this (Table 2, entry 12).
Temperature is an important variable in bromination reactions
with free-radical initiation. In the search for a suitable temperature
for the bromination reaction, the reaction of 4 and NBS in dioxane
was examined at different temperatures. It was observed that
experiments carried out at a lower temperature failed to give the
target product (Table 1, entries 13–15), which may indicate that a
low temperature did not initiate this radical reaction. The highest
yield was obtained when the reaction was conducted at 45 °C, and
Conclusion
In conclusion, we here report an efficient and environmentally-
friendly method for the synthesis of bromo-substituted cholestenone.
There are no reports of the synthesis of 6β-bromocholestenone
(4) and 2α,6β-dibromocholestenone (5) employing our methods.
Compounds 4 and 5 were obtained in high yield and selectivity.
We also provide a method for synthesis of 1α-hydroxycholesterol
from 2α,6β-dibromocholestenone and thus offer a new application
of bromo-substituted cholestenones.
Experimental
Melting points were determined using a digital melting point apparatus.
1H and 13C NMR spectra were recorded on a Varian spectrometer at
working frequencies 400 and 100 MHz respectively in CDCl3 using TMS
as internal standards. All chemical shifts were reported as δ values (ppm)
relative to TMS and observed coupling constants (J) are given in Hertz
(Hz). Mass spectra were measured with a HRMS-ESI instrument or a low-
resolution MS instrument using ESI ionisation. Cholesterol was provided
by Hangzhou Xiasha Biochemical Science and Technology Co. Ltd., and
all other reagents were purchased from commercial sources and without
purification before use. Column chromatography was performed on silica
gel (200–300 mesh) and the elution was carried out using n-hexane/ethyl
acetate.

JOURNAL OF CHEMICAL RESEARCH 2016 409
Synthesis of 6β-bromocholestenone (4)
brine, dried over anhydrous sodium sulphate and then concentrated under
reduced pressure. Recrystallisation from methanol provided the epoxide 7
(0.86 g, 72%).
The 5α,6β-dibromocholesterol 2 was synthesised according to a literature
method.7 A well-stirred solution of cholesterol 1 (5.0 g, 12.9 mmol) in ether
25 (mL) was treated with another solution prepared by dissolving sodium
acetate (2.0 mmol, 0.16 g) and then Br2 (liq.) (2.27 g, 14.2 mmol) in acetic
acid (20 mL). The mixture was stirred at 25 °C for 1.5 h (TLC control,
n-hexane/EtOAc 4:1, v/v). The dibromide was separated from the solution
and the solid was washed with ice-cold methanol (25 mL) three times.
The moist 5α,6β-dibromocholesterol 2 was used directly without drying
because of its chemical instability. A solution of 5α,6β-dibromocholesterol
2 in CH2Cl2 (50 mL), was treated with TEMPO (0.040 g, 0.26 mmol),
sodium bromide (0.13 g, 1.29 mmol), and a saturated sodium bicarbonate
aqueous solution (10 mL). The two-phase mixture was stirred for 15 min,
and 2 was dissolved completely. Then 5% sodium hypochlorite aqueous
solution (30 mL) was slowly added dropwise at 25 °C. The mixture was
allowed to stir for 1 h (TLC control, n-hexane/EtOAc 4:1, v/v). When the
reaction finished, the organic layer was separated and washed with 10%
aqueous HCl and brine. The CH2Cl2 layer was dried over anhydrous sodium
sulphate and then Et3N (6 mL) was added. After being stirred for 5 h at
room temperature, the mixture was successively washed with HCl solution
(10% wt) and brine. The CH2Cl2 layer was dried over anhydrous sodium
sulphate and then concentrated under reduced pressure. Recrystallisation
from methanol provided the 6β-bromocholestenone 4 (4.0 g，67%, from
cholesterol).
1α,2α-Epoxycholesta-4,6-dien-3-one (7): White crystals; m.p.
1
108.2–110.5 °C (from methanol) (lit.14 109.3–110.9 °C). H NMR (400
MHz, CDCl3) δ 6.06 (s, 2H), 5.63 (m, 1H), 3.59 (d, J = 4.0 Hz, 1H), 3.43
(dd, J = 4.0, J = 2.0 Hz, 1H), 3.22 (t, J = 9.6 Hz, 1H), 2.10 (m, 1H), 1.95–1.24
(m, 14H), 1.18 (s, 3H), 1.16–0.98 (m, 4H), 0.93 (d, J = 6.4 Hz, 3H), 0.87 (d,
J = 1.6 Hz, 3H), 0.86 (d, J = 2.0 Hz, 3H), 0.77 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 194.4, 158.7, 140.4, 127.5, 119.2, 59.4, 55.9, 54.7, 53.3, 46.0, 43.0,
39.5, 39.3, 38.9, 37.6, 36.1, 35.8, 28.2, 28.1, 23.9, 23.8, 22.9, 22.6, 21.3, 18.7,
18.6, 12.0; MS (ESI) m/z 397 [M + H]+.
Synthesis of 1α-hydroxycholesterol (8)
A three-necked flask was equipped with a dropping funnel, a cold-finger
with liquid N2 and an inlet tube connected to an ammonia source. Nitrogen
was swept through the system for 15 min and then the flask was immersed
in a dry ice/acetone bath. Ammonia (50 mL) was collected in the flask and
lithium wire (1.2 g) was cut into short pieces and added. After 1 h stirring,
a solution of 1α,2α-epoxycholest-4,6-dien-3-one 7 (0.8 g, 2 mmol) in dry
THF (30 mL) was added dropwise to the dry ice/acetone cooled solution
over 1 h. Then the cooling bath was removed and the mixture was allowed
to warm to –40 °C for 0.5 h. The flask was dipped into a cooling bath and
anhydrous ammonium chloride (8 g) was added over 2 h. The mixture
turned white and pasty. Most of the ammonia was removed in a stream
of nitrogen. The residue was diluted with ether (40 mL) and then washed
with brine, dried over anhydrous sodium sulphate, and finally concentrated
under reduced pressure. The residue was purified by chromatography to
give a white solid 0.46 g (56%).
1α-Hydroxycholesterol (8): White solid; m.p. 159.2–161.8 °C (lit.6
162–163 °C). Column chromatography: n-hexane/ethyl acetate 3/7, v/v.
1H NMR (400 MHz, CDCl3) δ 5.59 (m, 1H), 3.98 (m, 1H), 3.84 (m, 1H),
2.43–1.05 (m, 26H), 1.03 (s, 3H), 0.91 (d, J = 6.4 Hz, 3H), 0.87 (d, J = 1.6
Hz, 3H), 0.85 (d, J = 1.6 Hz, 3H), 0.68 (s, 3H) 13C NMR (100 MHz, CDCl3)
δ 136.7, 125.3, 73.0, 66.6, 56.8, 56.3, 42.6, 42.1, 41.7, 39.9, 38.6, 36.6, 36.2,
32.3, 32.2, 30.1, 28.6, 28.5, 24.8, 24.3, 23.3, 23.0, 20.8, 20.0, 19.3, 14.7, 12.4;
HRMS for C27H46NaO2 [M + Na]+ calcd 425.3391; found: 425.3390.
6β-Bromocholestenone (4): White crystals; m.p. 131.5–132.6 °C (from
methanol) (lit.13 132 °C); 1H NMR (400 MHz, CDCl3) δ 5.88 (s, 1H), 4.97
(m, 1H), 2.55 (m, 1H), 2.39 (m, 1H), 2.23 (d, J = 16.0 Hz, 1H), 2.05 (m, 1H),
1.86 (m, 1H), 1.74–1.55 (m, 4H), 1.53 (s, 3H), 1.52–0.94 (m, 15H), 0.92 (d,
J = 6.4 Hz, 3H), 0.87 (d, J = 1.2 Hz, 3H), 0.85 (d, J = 1.6 Hz, 3H), 0.77 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 199.2, 165.5, 126.8, 56.2, 55.3, 53.1,
52.5, 52.4, 42.6, 41.0, 39.6, 38.5, 38.0, 36.2, 35.8, 34.3, 30.8, 28.2, 28.1, 24.2,
24.0, 22.9, 22.7, 22.3 21,1, 18.8, 12.2; MS (ESI) m/z 463 [M + H]+, 485 [M
+ Na]+.
Synthesis of 2α,6β-dibromocholestenone (5)
A solution of 6β-bromocholestenone 4 (3.0 g, 6.5 mmol) in dioxane (30 mL)
was treated with NBS (2.31 g, 13 mmol) and BPO (0.016 g, 0.065 mmol)
at room temperature. The reaction mixture was heated to 45 °C, and
the reaction was finished when the dark yellow solution turned buff.
After cooling, water (30 mL) was added and the mixture was extracted
with CH2Cl2 (3 × 50 mL). The combined organic layers were washed
successively with NaHSO3 solution (5% wt) and brine. The extract was
dried over anhydrous sodium sulphate and then concentrated under reduced
pressure. 2α,6β-Dibromocholestenone 5 (3.1 g, 88%) was obtained and
recrystallised from EtOH.
We are grateful to the National Natural Science Foundation of
China (21406200) and Collaborative Innovation Center of Yangtze
River Delta Region Green Pharmaceuticals for financial help.
2α,6β-Dibromocholestenone (5): White crystals; m.p. 160.2–162.5 °C
1
(from EtOH) (lit.13 162–163 °C); H NMR (400 MHz, CDCl3) δ 5.98
Received 28 February 2016; accepted 7 April 2016
Paper 1603950 doi: 10.3184/174751916X14652251037643
Published online: 15 June 2016
(s, 1H), 4.94 (m, 1H), 4.89 (dd, J = 4.8 Hz, J = 14.4 Hz, 1H), 2.60 (dd,
J = 4.8 Hz, J = 12.8 Hz, 1H), 2.27–1.96 (m, 4H), 1.85 (m, 1H), 1.62 (s, 3H),
1.57–0.96 (m, 19H), 0.92 (d, J = 6.4 Hz, 3H), 0.87 (d, J = 0.8 Hz, 3H), 0.85
(d, J = 1.2 Hz, 3H), 0.77 (s, 3H) 13C NMR (100 MHz, CDCl3) δ 191.2, 165.9,
124.5, 56.2, 55.1, 52.8, 51.0, 50.0, 49.9, 42.6, 41.8, 40.8, 39.6, 39.4, 36.2, 35.8,
30.4, 28.2, 28.1, 24.2, 23.9, 23.0 22.9, 22.7, 21.1, 18.8, 12.2; MS (ESI) m/z
543 [M + H]+, 565 [M + Na]+.
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