Short synthesis of 16β-hydroxy-5α-cholestane-3,6-dione a novel cytotoxic marine oxysterol

Article abstract of DOI:10.1016/j.steroids.2006.03.002

The first and short synthesis of 16β-hydroxy-5α-cholestane-3,6-dione 1 a metabolite from marine algae, has been achieved in six steps from readily available diosgenin 5. Selective deoxygenation of primary alcohol of triol 6 has been accomplished in one step using Et₃SiH and catalytic amount of B(C₆F₅)₃ to produce compound 9 in high yield. Oxidation of 11 with PCC, allowed the introduction of 3,6-ene-dione functionality, and further catalytic hydrogenation and deprotection furnished the 3,6-diketo steroid 1.

Full text of DOI:10.1016/j.steroids.2006.03.002

steroids 7 1 ( 2 0 0 6 ) 599–602
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Short synthesis of 16␤-hydroxy-5␣-cholestane-3,6-dione
a novel cytotoxic marine oxysterol
a
b
a,∗
Micka e¨ l Denanc e´ , Mich e` le Guyot , Mohammad Samadi
a
D e´ partement de Chimie, LIMBP, IPEM, Universit e´ Paul Verlaine de Metz, 1 Bd Arago,
Metz Technop oˆ le, 57078 Metz, France
b
Laboratoire de Chimie des Substances Naturelles, associ e´ au CNRS, USM 502,
Mus e´ um National d’Histoire Naturelles, 63 rue Buffon, 75005 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The first and short synthesis of 16␤-hydroxy-5␣-cholestane-3,6-dione 1 a metabolite from
marine algae, has been achieved in six steps from readily available diosgenin 5. Selective
deoxygenation of primary alcohol of triol 6 has been accomplished in one step using Et3SiH
and catalytic amount of B(C F ) to produce compound 9 in high yield. Oxidation of 11 with
Received 23 January 2006
Received in revised form
2
March 2006
6
5 3
Accepted 7 March 2006
PCC, allowed the introduction of 3,6-ene-dione functionality, and further catalytic hydro-
Published on line 18 April 2006
genation and deprotection furnished the 3,6-diketo steroid 1.
©
2006 Elsevier Inc. All rights reserved.
Keywords:
Oxysterols
Diosgenin
Selective reduction
Boron catalyst
1
.
Introduction
mon as natural products. To date in addition to 1, only three
examples were found in the literature: 5␣-cholestane-3,6-
dione 2 and 11-hydroxy-5␣-cholestane-3,6-dione 3 were iso-
lated from the red alga Acantophora spicifera [9,10] and the
20-hydroxy-5␣-cholest-22-ene-3,6-dione 4 from the red alga
Hypnea musciformis which exhibits PPE elastase inhibition [11]
(Fig. 1).
Due to very low natural abundance of 1 and as a part of
our program directed toward the synthesis of bioactive marine
natural products, the design of a total synthesis was required
for further biological evaluation and medicinal exploitation.
We undertook the synthesis of 1, starting from diosgenin 5 as
a useful starting material both for its commercial availability
and for the presence of functional groups in positions suitable
for conversion to 1.
Oxysterols represent a class of potent regulatory molecules
with remarkably diverse, important biological actions [1,2].
They exhibit a number of biological activities, including
inhibition of cellular proliferation and cytotoxicity associ-
ated with induction of apoptosis [3–5]. A large of num-
ber of oxysterols also occur as marine natural products
with unusual functionalization [6,7]. Among them the 16␤-
hydroxy-5␣-cholestane-3,6-dione 1, was isolated from the red
alga Jania rubens. It exhibits a cytotoxic activity towards KB
cells with an ID50 of 0.5 ␮g/ml. Its structure and the rel-
ative configuration of hydroxyl group at C-16 were estab-
lished by extensive spectroscopic analyses [8]. It is impor-
tant to mention that the 3,6-diketo steroids are not com-
∗
Corresponding author. Fax: +33 387315801.
E-mail address: samadi@sciences.univ-metz.fr (M. Samadi).
0039-128X/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2006.03.002

6
00
steroids 7 1 ( 2 0 0 6 ) 599–602
2.2.2. 3ˇ, 16ˇ-bis[(triethylsilyl)oxy]-cholest-5-ene (9)
To a solution of triol 6 (251 mg, 0.6 mmol) and B(C6F5)3 (15 mg,
0.029 mmol) in dry CH2Cl2 was added Et3SiH (6 mmol, 1 mL)
dropwise. The reaction was stirred for 6 days at room temper-
ature. Et3N (0.03 mL) was added and the solvent was removed
under reduced pressure. The residue was purified by column
chromatography on silica gel using cyclohexane–EtOAc (99:1)
as eluent to give compound 9 (344 mg, 91%) as a colorless oil:
2
0
R = 0.36 (cyclohexane–EtOAc, 98:2). [˛] = −8.6 (c = 0.7, CHCl3).
f
D
1
H NMR (CDCl3): ı = 5.35 (d, J = 5 Hz, 1H, H-6), 4.35 (m, 1H, H-
1
3
2
6), 3.47 (m, 1H, H-3), 1.01 (s, 3H, 19-CH3), 0.98 (d, J = 6.6 Hz,
H, 21-CH3), 0.93 (t, 18H, Si-CH2-CH3), 0.90 (d, J = 6.6 Hz, 6H,
6-CH3 and 27-CH3), 0.88 (s, 3H, 18-CH3), 0.58 (q, 12H, Si-CH2-
CH3) ppm. 13C NMR (CDCl3) ı = 141.7, 120.9, 72.6, 72.3, 62.1, 54.8,
Fig. 1 – Structures of 3,6-diketo steroids.
50.3, 42.9, 42.3, 39.9, 39.8, 37.8, 37.4, 36.6, 36.4, 32.2, 31.9, 31.6,
30.0, 28.2, 24.5, 22.9, 22.4, 20.7, 19.4, 18.1, 12.9, 7.1, 6.8, 5.2,
4.9 ppm. HRMS: calcd. for C39H75O2Si2 [MH] 631.5306, found
631.5322.
2
.
Experimental
2
.1.
General
2.2.3. 16ˇ-[(triethylsilyl)oxy]-cholest-5-ene-3˛-ol (11)
To a solution of 9 (380 mg, 0.603 mmol) in dry THF (5 mL) at
◦
All of the reactions were carried out under an argon atmo-
sphere. All reagents were obtained from commercial suppli-
ers and used without further purification. THF was freshly
distilled from sodium/benzophenone. Methylene chloride
was distilled from CaH2. Flash chromatography was car-
ried out using silica gel 60 F254 (Merck) with mixtures
of ethyl acetate and cyclohexane as eluent unless spec-
ified otherwise. TLC analyses were performed on thin-
layer analytical Plates 60 F254 (Merck). 1H and 13C NMR
spectra were recorded with a Bruker Advance 250 and
0 C, was added TBAF·3H O (210 mg, 0.665 mmol). The mixture
2
was stirred at this temperature for 1 h and THF was removed
under reduced pressure. The residue was purified by column
chromatography on silica gel using cyclohexane–EtOAc (80:20)
as eluent to give compound 11 (305 mg, 98%) as a colorless
2
0
oil: R = 0.43 (cyclohexane–EtOAc, 70:30); [˛] = −10.8 (c = 0.5,
f
D
−
1 1
CHCl ); IR (neat) 3465, 2954, 1645 cm ; H NMR (CDCl ) ı = 5.34
3
3
(d, J = 5 Hz, 1H, 6-H), 4.35 (m, 1H, 16-H), 3.50 (m, 1H, 3-H), 1.01
(s, 3H, 19-CH ), 0.98 (d, J = 6.6 Hz, 3H, 21-CH3), 0.93 (t, 9H, Si-
CH -CH ), 0.90 (d, J = 6.6 Hz, 6H, 26-CH and 27-CH ), 0.88 (s,
3
2
3
3
3
1
3
4
00 MHz spectrometer, respectively. IR spectra were recorded
3 2 3 3
3H, 18-CH ), 0.58 (q, 6H, Si-CH -CH ); C NMR (CDCl ) ı = 140.9,
with a Mattson 3000 spectrometer. HRMS (positive mode)
was measured on a JEOL 700 spectrometer (Ecole Normale
Sup e´ rieure Paris) and EIMS, CIMS on a Nermag R 10-10.
Optical rotations were measured with a Perkin-Elmer 341
polarimeter with a sodium lamp (˛ = 589 nm) in a 10 cm
microcell.
121.4, 72.6, 71.7, 62.1, 54.7, 50.2, 42.3, 42.2, 39.9, 39.8, 37.7, 37.2,
36.5, 36.4, 32.2, 31.9, 31.6, 30.1, 28.2, 24.5, 22.9, 22.4, 20.7, 19.4,
18.0, 12.9, 7.1, 5.2 ppm. HRMS: m/z calcd for C33H59OSi [MH-
2
H O] 499.4335, found 499.4373.
2.2.4. 16ˇ-[(triethylsilyl)oxy]-cholest-ene-3,6-dione (12)
To a solution of 11 (300 mg, 0.581 mmol) in dry CH2Cl2 (5 mL),
was added PCC (873 mg, 4.01 mmol) under argon atmosphere.
The solution was stirred at room temperature for 15 h and the
solvent was removed under reduce pressure. The residue was
triturated with ether, filtered through Celite and concentrated
under reduced pressure. The residue was purified by column
chromatography on silica gel using cyclohexane–EtOAc (90:10)
2
.2.
Synthesis
2
.2.1. (25R)-Cholest-5-en-3ˇ,16ˇ,27-triol (6)
This compound was prepared according to the literature [13]
as follows: to a boiling mixture of diosgenin 5 (1.2 g, 2.89 mmol)
and freshly prepared zinc amalgam (40 g) in ethanol (120 mL),
was added dropwise concentrated hydrochloric acid (40 mL).
Following the addition of the hydrochloric acid, the solu-
tion was refluxed for 30 min and filtered. The cooled filtrate
was poured into ice water (1 L). The obtained solid was fil-
tered and purified by column chromatography on silica gel
using cyclohexane–EtOAc as eluent to give triol 6 (619 mg,
as eluent to give 12 (178 mg, 58%) as a colorless oil: R = 0.32
f
2
0
(cyclohexane–EtOAc 85:15). [˛]D = +5.7 (c 0.5, CHCl3). MS (EI)
−
1
1
m/z = 526; IR (neat) 2954, 1724, 1701 cm
.
H NMR (CDCl3)
ı = 6.16 (s, 1H, 4-H), 4.36 (m, 1H, 16-H), 1.13 (s, 3H, 18-CH3), 0.98
(s, 3H, 19-CH3), 0.97 (d, J = 6.6 Hz, 3H, 21-CH3), 0.95 (t, 9H, Si-
CH2CH3), 0.90 (d, J = 6.6 Hz, 6H, 26-CH3 and 27-CH3), 0.57 (q,
6H, Si-CH2-CH3) ppm. 13C NMR (CDCl3): ı = 202.1, 199.4, 160.9,
125.5, 72.2, 62.0, 54.6, 51.0, 46.8, 42.5, 39.8, 39.3, 37.4, 36.3, 36.1,
35.5, 34.0, 33.9, 29.9, 28.2, 24.4, 22.9, 22.4, 20.5, 17.9, 17.5, 12.9,
7.1, 5.2 ppm.
◦
◦
5
1%) as a white solid: m.p. 176–178 C (lit. [13] 176–178 C).
Rf = 0.27 (cyclohexane–EtOAc, 50:50). 1H NMR (CDCl3): ı = 5.34
d, J = 5 Hz, 1H, 6-H), 4.34 (m, 1H, 16-H), 3.49 (m, 1H, 3-
(
H), 3.46 (d, J = 6 Hz, 2H, 26-H), 1.01 (s, 3H, 19-CH3), 0.98 (d,
J = 6.6 Hz, 3H, 21-CH3), 0.90 (d, J = 6.6 Hz, 3H, 27-CH3), 0.88 (s,
3
6
3
1
H, 18-CH3) ppm. 13C NMR (CDCl3) ı = 140.9, 121.4, 72.5, 71.8,
8.4, 61.5, 54.5, 50.1, 42.3, 42.2, 39.9, 37.2, 36.7, 36.5, 36.0,
5.6, 33.4, 31.8, 31.6, 31.5, 29.7, 23.7, 20.7, 19.4, 18.2, 16.6,
3.1 ppm.
2.2.5. 16ˇ-hydoxy-5˛-cholestane-3, 6-dione (1)
A mixture of ene dione 12 (150 mg, 0.284 mmol) and 10%
Pd/C (15 mg) in EtOH (2 mL) was stirred at room tempera-
ture under 1 atm of hydrogen for 2 h. The reaction was fil-

steroids 7 1 ( 2 0 0 6 ) 599–602
601
tered through Celite, and the filtrate was concentrated under
reduced pressure to give the crude dione 13, which was used
for the next step without purification. To a solution of 13
and are in agreement with those reported by Gevorgyan et
al. [17], in that the reversible coordination of the ate-complex
+
−
EtSi3 / HB(C6F5)3, [20] which induces the reduction of the pri-
mary silyl ether of 10 to form 9, seems to be the slowest step
over the reduction process and silylation of alcohols, and it is
completely sterically controlled [21]. It is important to mention
that, this is the first example of application of this methodol-
ogy in poly oxygenated natural products.
(
0
150 mg) in dry THF (2 mL) was added TBAF·3H2O (134 mg,
◦
.424 mmol). The reaction was stirred for 2 h at 40 C. The mix-
ture was cooled and the solvent was removed under reduce
pressure. The residue was purified by column chromatog-
raphy on silica gel using cyclohexane–EtOAc (80:20) to give
◦
compound 1 (111 mg, 94%) as a white solid. m.p. 153–155 C;
Having successfully prepared compound 9, we next turned
to introduce the oxygen at C-6 in order to install the 3,6-
dione functionality. In this respect the procedure describing
the direct conversion of the steroidal 4-en-3␤ alcohols into
steroidal 4-en-3,6-diones using PCC oxidation seemed to us
the method of choice [22]. To obtain the free alcohol at C-
3 for this purpose, we found that the silyl ether at C-3 of
compound 9 could be selectively removed with TBAF at room
temperature for 1h to give 11 in 98% yield, whereas the depro-
tection of the silyl ether at C-16 requires higher temperature
reaction, which is due to steric hindrance. Oxidation of 11
with PCC in CH2Cl2 afforded in one step the unsaturated 3,6-
dione 12 in 58% yield. Finally to accomplish the synthesis,
catalytic hydrogenation of 12 in the presence of Pd/C, pro-
duced the 5␣-reduced compound 13 as a single isomer, fol-
lowed by deprotection of the remaining silyl ether with TBAF,
furnished the desired 3,6-diketo steroid 1 in 94% yield (over
two steps) (Scheme 2). The synthetic compound 1 obtained
here was identical by NMR, TLC, and MS with natural 16␤-
hydroxy-5␣-cholestane-3,6-dione. The only difference lies in
the optical rotation, which is likely due to some impurities in
the natural compound [23].
In summary, the first and short synthesis of 16␤-hydroxy-
5␣-cholestane-3,6-dione 1 has been accomplished, in six steps
and in 25% overall yield from readily available diosgenin. This
synthesis fully confirms the structure and relative stereo-
chemistry previously proposed for the natural product. The
first application of Et3SiH/B(C6F5)3 system in selective reduc-
tion of primary alcohol of triol 1 is worthy mentioning, and will
allow further exploration of this methodology to other poly
oxygenated natural products. Further biological evaluation of
1 is currently underway.
◦
20
lit. [8] 125–126 C; R = 0.29 (cyclohexane–EtOAc, 85:15). [˛]
=
f
D
2
0
+
3.8 (c = 0.5, CHCl3); lit. [˛] = −2 (c = 0.1, CH2Cl2); IR (KBr)
D
3
521, 2954, 1712 cm 1. 1H NMR (CDCl3) ı = 4.38 (m, 1H, 16-
−
H), 0.98 (d, J = 6.6 HZ, 3H, 21-CH3), 0.97 (s, 3H, 19-CH3), 0.90
s, 3H, 18-CH3), 0.89 (d, J = 6.6 Hz, 6H, 26-CH3 and 27-CH3)
ppm. 13C NMR (CDCl3): ı = 211.1, 208.7, 72.0, 61.4, 57.5, 54.4,
(
5
3
3.5, 46.4, 42.8, 41.1, 39.4, 38.0, 37.5, 37.3, 37.0, 36.2, 36.1,
6.0, 29.8, 28.0, 24.1, 22.7, 22.6, 21.3, 18.1, 13.1, 12.5 ppm.
HRMS m/z calculated for C27H45O3 [MH] 417.3369, Found
17.3363.
4
3
.
Results and discussion
Diosgenin 5 was converted by Clemmensen reduction to triol
[12–14]. Selective tosylation of the primary alcohol with p-
toluenesulfonyl chloride in pyridine produced compound 7
15] in 30% yield. Reduction of 7 with LiAlH4 in ether gave
6
[
the diol 8 [15], followed by protection of hydroxyl groups with
Et3SiCl in DMF to produce the protected compound 9 in 90%
(
from 7). Given the poor yield obtained during tosylation of
triol 6, we envisaged another approach to remove the OH at C-
7. It has been recently reported that B(C6F5)3 in the presence
2
of Et3SiH was an effective catalyst for selective reduction of
the primary alcohol through its silyl ether by the ate complex
+
−
Et3Si / HB(C6F5)3 [16,17]. Application of this methodology to
triol 6 with 10 equiv of Et3SiH in the presence of catalytic
amount of B(C6F5)3 furnished the desired compound 9 in one
step (91%). On the other hand, treatment of 6 with 4 equiv of
Et3SiH in presence of B(C6F5)3 as catalyst gave 10 [18] within
5
h as a major product (Scheme 1) [19]. These results indicate
Scheme 1 – (i): Zn–Hg, HCl, EtOH, reflux (51%); (ii): 1.2 equiv of TsCl, Pyridine, rt, 24 h (30%); (iii): 1.1 equiv LiAlH4, THF, reflux;
(
(
iv): 2.2 equiv of Et3SiCl, 4.4 equiv of imidazol, DMF, rt (90% from 7); v: 10 equiv Et3SiH, B(C6F5)3 (5 mol%), CH2Cl2, rt, 6 days
91%); vi: 4 equiv Et3SiH, B(C6F5)3 (5 mol%), CH2Cl2, rt, 5 h.

6
02
steroids 7 1 ( 2 0 0 6 ) 599–602
[
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2
[
[
[
[
[
[
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Scheme 2 – (i): 1.1 equiv of TBAF, THF, rt, 1 h (98%); (ii): 7
equiv of PCC, CH2Cl2, rt, 15 h (58%); (iii): H2, Pd/C 10%, EtOH,
◦
rt, 2 h; (iv): 1.5 equiv of TBAF, THF, 40 C, 2 h (94% from 12).
[
[
16] Gevorgyan V, Liu JX, Rubin M, Benson S, Yamamoto Y. A
novel reduction of alcohols and ethers with a
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Acknowledgments
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novel B(C6F5)3 catalyzed reduction of alcohols and
cleavage of aryl and alkyl ethers with hydrosilanes. J Org
Chem 2000;65:6179–86.
The authors thank Lionel Dubost (Service de spectrom e´ trie
de masse, MNHN, Paris) and Nicole Morin (ENS, Paris) for low
and high-resolution mass spectra. This work was supported
by grant from the Conseil R e´ gional de Lorraine.
[
18] Compound 10 was characterized after deprotection of silyl
1
13
ethers with TBAF, whose H and C NMR spectrum was
identical to 6.
r e f e r e n c e s
[
19] For silylation of alcohols using hydrosilane/B(C6F5)3 see:
Blackwell JM, Foster KL, Beck VH, Piers WE. B(C6F5)3-catalyzed
silation of alcohols: a mild, general method for synthesis of
silyl ethers. J Org Chem 1999;64:4887–92.
[
[
1] Schroepfer Jr GJ. Oxysterols: modulators of cholesterol
metabolism and other processes. Physiol Rev
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−
[
20] For the formation of the ate complex Et3Si / HB(C6F5)3 see:
2
000;80:361–554.
Parks DJ, Blackwell JM, Piers WE. Studies on the mechanism of
B(C6F5)3-catalyzed hydrosilation of carbonyl functions. J Org
Chem 2000;65:3090–8.
2] Guardiola F, Dutta PC, Codony R, Savage GP, editors.
Cholesterol and phytosterol oxidation products: analysis,
occurrence and biological effects. Champaign, IL: AOCS
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5] Panini SR, Sinensky MS. Mechanisms of oxysterol-induced
apoptosis. Curr Opin Lipidol 2001;12:529–53.
[
21] It was reported that the simple primary silyl ethers were
reduced within 20 h. The reduction of the primary silyl
ether of 10 requires at least 6 days reaction time. This is
probably due to the steroid skeleton which induces an
additional steric hindrance, so affecting the coordination
[
[
+ −
of the ate Et3Si / HB(C6F5)3 complex to the primary silyl
ether of 10. For more details of reaction mechanism, see
Ref. [17].
[
[
22] Li SH, Li TS. Steroidal 5-en-3-ones, intermediates of the
transformation of steroidal 5-en-3b-ols to steroidal 4-en-3
[
[
6-diones oxidized by pyridinium dichromate and
6] Faulkner DJ. Marine natural products. Nat Prod Rep
pyridinium chlorochromate. Steroids 1998;63:76.
2
002;19:1–48, and previous reviews in this series cited
23] The 1H NMR spectra of the isolated natural 1 shows some
therein.
impurities, for this reason the reported [8] optical rotation
of the natural material is slightly different from that of
[
[
7] D’Auria MV, Minale L, Riccio R. Polyoxygenated steroids of
marine origin. Chem Rev 1993;93:1839–95.
8] Ktari L, Blond A, Guyot M. 16␤-Hydroxy-5␣-cholestane-3,6-
dione, a novel cytotoxic oxysterol from the red alga Jania
rubens. Bioorg Med Chem Lett 2000;10:2563–5.
◦
◦
our synthetic substance (−2 versus +3.8 ).