Synthesis of 3β,6α-dihydroxy-5α-cholan-23-one

Article abstract of DOI:10.1016/j.tet.2003.08.020

Evidence for the presence of 3β,6α-dihydroxy-5α -chol-9(11)-en-23-one in the aglycone mixture from the starfish Marthasterias glacialis is provided by the synthesis of 3β,6α-dihydroxy-5α -cholan-23-one (19) and its identification in the hydrogenated aglycone mixture. The side-chain is constructed from the 23,24-dinorcholanol (13) by reaction of the 22-tosylate (16) with the acetylide anion, followed by hydration of the resulting 23-yne (17).

Full text of DOI:10.1016/j.tet.2003.08.020

Tetrahedron 59 (2003) 8623–8628
Synthesis of 3b,6a-dihydroxy-5a-cholan-23-one
*
Lutfun Nahar and Alan B. Turner
Japp Laboratory, Department of Chemistry, University of Aberdeen, Meston Walk, Aberdeen AB24 3UE, Scotland, UK
Received 25 May 2003; revised 21 July 2003; accepted 13 August 2003
Abstract—Evidence for the presence of 3b,6a-dihydroxy-5a-chol-9(11)-en-23-one in the aglycone mixture from the starfish Marthasterias
glacialis is provided by the synthesis of 3b,6a-dihydroxy-5a-cholan-23-one (19) and its identification in the hydrogenated aglycone mixture.
The side-chain is constructed from the 23,24-dinorcholanol (13) by reaction of the 22-tosylate (16) with the acetylide anion, followed by
hydration of the resulting 23-yne (17).
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
in the aglycone mixture from the Atlantic starfish,
Marthasterias glacialis, was originally adduced from
GCMS studies,4 and in this paper we discuss additional
evidence for the cholanes from analytical studies and
synthetic work.
23-Oxocholestane conjugates were first isolated from an
Atlantic starfish more than 30 years ago.1,2 Since then there
have been numerous reports of the isolation and character-
isation of related saponins from marine organisms which
continue up to the present day.3 The commonly observed
hydroxylation of 23-oxocholestanes such as marthasterone
1 at tertiary positions in the side-chain (C-20 and C-25)
leads to enones by dehydration and also provides an
opportunity for degradation of the cholestane side-chain
by retro-aldol cleavage (Scheme 1). Both of these reactions
could occur in vivo or during the acid hydrolysis procedures
normally used to obtain the aglycones. In particular, the
20-oxopregnane 4 and 23-oxocholane 5 can be expected to
arise from the side-chain hydroxylated 23-oxocholestanes 2
and 3. Evidence for the presence of pregnanes and cholanes
2. Results and discussion
The synthesis of 23-oxocholanes from cholic acids did not
look appealing, so a route involving the addition of a two-
carbon fragment to the readily available bisnorcholanals
was devised (Scheme 2) and carried out initially in the 4-en-
3-one series. 22-Hydroxy-23,24-dinorchol-4-en-3-one (7)5,6
was obtained in 68% yield by reduction of the commercially
available 3-oxo-23,24-dinorchol-4-en-22-al (6) using one
hydride equivalent of NaBH4 at rt.
Scheme 1. Possible origin of 20-oxopregnane (4) and 23-oxocholane (5) from 23-oxocholestanes via acid-catalysed retro-aldol cleavage.
Keywords: starfish; steroid; dinorcholane; oxocholestane; NMR.
*
Corresponding author. Tel.: þ44-1224-272919; fax: þ44-1224-272921; e-mail: l.nahar@abdn.ac.uk
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.08.020

8624
L. Nahar, A. B. Turner / Tetrahedron 59 (2003) 8623–8628
Scheme 2. (i) NaBH4 (1 equiv.), EtOH, rt, 40 min. (ii) NaBH4 (2 equiv.), EtOH, rt, 2 h. (iii) Ac2O, Py, rt, 15 h. (iv) DDQ (1 equiv.), dioxane, rt, 24 h. (v) p-TsCl
(4 equiv.) Py, rt, 36 h. (vi) LiCuCH·EDA (13.6 equiv.), DMSO, 508C, 3 h. (vii) AcOH, Hg-resin, H2O, reflux, 5 h.
When two hydride equivalents of NaBH4 were used for the
reduction of the aldehyde 6, the ketonic carbonyl group at
C-3 was also reduced and 3b,22-dihydroxy-23,24-
dinorchol-4-ene (8)5 was obtained in 65% yield. Its 1H
NMR showed signals at dH 0.66 (s, 3H, CH3), 1.01 (s, 3H,
CH3) and 1.00 (d, J¼6.5 Hz, 3H, CH3) for three methyl
groups: 18-CH3, 19-CH3 and 21-CH3, respectively. The
resonances for the oxymethylene protons at C-22 were
observed at dH 3.33 (dd, J¼6.8, 10.3 Hz, 1H, OCH2) and
3.59 (dd, J¼3.1, 10.3 Hz, 1H, OCH2) and that for the
olefinic proton (at C-4) was at dH 5.24 (d, J¼1.7 Hz, olefinic
CH). The 13C NMR spectrum showed signals for an olefinic
methine (dC 124.3, C-4), an olefinic quaternary carbon (dC
148.7, C-5), an oxymethine (dC 69.1, C-3), and an
oxymethylene group (dC 69.0, C-22). Acetylation of 8
with acetic anhydride in pyridine afforded 3b,22-diacetoxy-
23,24-dinorchol-4-ene (9) in 88% yield. To confirm the
identity of diol 8, a selective allylic oxidation was
performed using DDQ in dioxane at room temperature to
produce 22-hydroxy-23,24-dinorchol-4-en-3-one (7) in
50% yield.
carbonyl group (1666 and 1620 cm21). Hydration of the
triple bond using Hg-resin prepared from Zeo-Karb by
Newman’s method8 as catalyst afforded chol-4-ene-3,23-
dione (12) in 50% yield. The overall yield of the dione 12
from the bisnorcholenal 6 was 11%.
This route was then applied in the 3b,6a-dihydroxy-5a-
cholane series for the synthesis of the starfish steroid
(Scheme 3). Ethynylation of the bisnorcholane tosylate 164
with an equimolar amount of lithium acetylide/ethane-
diamine complex in anhydrous dimethylsulfoxide gave the
alkyne as before, but the crude product was found to consist
of a mixture of partially and fully deacetylated material. It
was found most convenient to reacetylate the crude product
with acetic anhydride in pyridine before purification, when
the alkyne 17 was obtained in 65% yield. Hydration of the
alkyne using the resin catalyst as before gave the 23-
oxocholane diacetate 18 in 73% yield, and subsequent
hydrolysis using methanolic potassium hydroxide gave the
crystalline 3b,6a-dihydroxy-5a-cholan-23-one (19), in
95% yield. In their mass spectra, both of the 23-oxocholanes
18 and 19 exhibited characteristic loss of acetone from the
side-chain by McLafferty rearrangement9 to afford the base
peak. This was evident in the mass spectrum of diacetate 18
from both the parent ion and from the daughter ions formed
by loss of acetic acid. In all cases the abundance of the
molecular ion was low under electron impact.
Tosylation of the alcohol 7 with toluene-p-sulfonyl chloride
in pyridine gave the 22-tosyloxy-23,24-dinorchol-4-en-3-
one (10)5,7 in 72% yield. Selective displacement of the
22-tosyl group by the ethynyl group was achieved using a
suspension of lithium acetylide/ethylenediamine complex in
dry dimethylsulfoxide. No products of addition of the
ethynyl group to the unsaturated ketone system were
detected. The crude product was purified by preparative
thin-layer chromatography (TLC) to give the 23-yne 11 as a
colourless glass (46%, Rf 0.56, silica gel, benzene/
EtOAc¼3:1). Its IR spectrum showed stretching frequencies
characteristic of the acetylenic C–H bond (3300 cm21) and
the CuC bond (2130 cm21), as well as the conjugated
The gas chromatographic retention time (tR 3.56) and the Rf
value on TLC of the synthetic compound 19 were identical
to that of a minor component in a sample of the aglycone
mixture from M. glacialis which had been subjected to
prolonged hydrogenation over Pd/C. As shown previously,
the 9(11)-double bond is reduced under these conditions4
so this provides further evidence for the presence of

L. Nahar, A. B. Turner / Tetrahedron 59 (2003) 8623–8628
8625
Scheme 3. (i) NaBH4 (1 equiv.) EtOH, rt, 40 min. (ii) Ac2O, Py, rt, 24 h. (iii) p-TsCl (3.4 equiv.), Py, rt, 44 h. (iv) LiCuCH·EDA (13.9 equiv.), DMSO, 408C,
2 h. (v) AcOH, Hg-resin, H2O, reflux, 7 h. (vi) KOH (2 M), MeOH, rt, 27 h.
3b,6a-dihydroxy-5a-chol-9(11)-en-23-one in the original
aglycone mixture. No evidence has been found for enzymic
catalysis of the retro-aldol reaction leading to the C-24
sterol, but the work of Kitagawa10 suggests that this type of
side-chain cleavage takes place only during the acid
hydrolysis used for the isolation of the aglycones.
were recorded on a Varian Unity INOVA 400 MHz NMR
spectrometer. Chemical shifts (d) are reported in ppm
downfield from TMS, using the middle resonance of CDCl3
1
(7.25 ppm for H and 77.23 ppm for 13C) as an internal
standard and coupling constants (J) in Hz. Data are reported
as follows: chemical shift, multiplicity (s¼singlet, d¼
doublet, m¼multiplet) coupling constant(s), integration and
peak assignment. Elemental analyses were performed in
Butterworth Laboratories Limited, Middlesex. The MS
were recorded at the EPSRC Central Mass Spectrometry
Service at Swansea.
3. Experimental
3.1. General
3.2. Synthesis of chol-4-en-3,23-dione (12)
The steroid starting materials [stigmasterol, stigmasta-
dieneone, and 3-oxo-22,23-dinorchol-4-en-22-al] were
purchased from Sigma. Most chemicals and solvents were
analytical grade and used without further purification. The
purity of the products and the reaction time were supported
by TLC performed on silica gel (Merck type 60) and
visualised under UV illumination and/or by I2 vapour. The
gas chromatographic (GC) was conducted on 2 m£1.5 mm
columns packed with 2.5% silicone on OV-1 (3%) at 2508C
with a nitrogen flow rate of 16 ml min21 on a Perkin–Elmer
F-11 instrument. Quoted retention times are relative to
pregnenolone (tR 1.0). Mps were determined on a Kofler
hot-stage apparatus and are uncorrected. Infrared spectra
(wave numbers in cm21) were recorded Nicolet Avatar 320
or ATI Mattson Genesis Series FT-IR spectrometer as films
or KBr discs. Nuclear magnetic resonance (NMR) spectra
3.2.1. 22-Hydroxy-23,24-dinorchol-4-en-3-one (7). To a
stirred ethanolic solution of 3-oxo-23,24-dinorchol-4-en-
22-al 6 (200 mg, 0.60 mmol in 50 ml), NaBH4 (23 mg,
0.60 mmol, 1 equiv.) was added (Scheme 1). Stirring was
continued for 1 h at rt. When no starting material remained
(checked by TLC), H2O (40 ml) was added to the solution,
and extracted with EtOAc (3£30 ml). The combined
extracts were dried over MgSO4 and concentrated in
vacuo to give a slowly solidifying yellow oil (190 mg).
The product was purified by column chromatography
(neutral Al2O3, activity 1) using step gradient elution
(petroleum-ether/EtOAc¼75:25, 65:35) to yield the title
compound 7 (134 mg, 68%) as a colourless solid, mp 134–
1368C (lit.5 mp 138–1418C). IR (KBr): n 3427s (O–H),

8626
L. Nahar, A. B. Turner / Tetrahedron 59 (2003) 8623–8628
2933s (C–H), 1673vs (CvO), 1617m (CvC), 1443m,
1230m and 1046m (alcoholic C–O) cm21 (lit.6 IR). 1H
NMR (CDCl3): d 0.69 (s, 3H, 18-CH3), 1.08 (d, J¼6.8 Hz,
3H, 21-CH3), 1.14 (s, 3H, 19-CH3), 3.33 (dd, J¼6.8,
10.3 Hz, 1H, 22-OCH2), 3.59 (dd, J¼3.4, 10.6 Hz, 1H,
22-OCH2), 5.68 (br s, 1H, 4-CH) (lit.6 1H NMR). 13C NMR
(CDCl3): d 199.6 (C-3), 171.5 (C-5), 123.8 (C-4), 68.0
(C-22) 55.7 (C-17), 53.8 (C-14), 52.4 (C-9), 42.5 (C-13),
39.5 (C-12), 38.7 (C-10), 38.6 (C-20), 35.7 (C-8), 35.6
(C-1), 34.0 (C-2), 32.9 (C-6), 32.0 (C-7), 27.7 (C-16), 24.3
(C-15), 21.0 (C-11), 17.4 (C-19), 16.7 (C-21), 12.1 (C-18)
(lit.8 13C NMR: selected peaks).
room temperature was added dropwise a solution of DDQ
(55 mg, 0.24 mmol, 1 equiv.) in dioxane (1 ml). After 24 h,
a white precipitation was found (Scheme 1). The solid was
filtered off, the orange solution was concentrated and
washed several times with petroleum-ether (40–608C) to
yield a colourless solid (40 mg, 50%). The white solid was
recrystallised from EtOH. The compound was identified as
compound 7 by mixed melting point determination (134–
1368C) and comparison of its IR and NMR data with that of
the authentic material described above.
3.2.5. 22-Tosyloxy-23,24-dinorchol-4-en-3-one (10).
p-Toluenesulfonyl chloride (175 mg, 0.92 mmol, 4 equiv.)
was added to a stirred solution (1 ml) of 7 (75 mg,
0.23 mmol in dry pyridine) at 08C The mixture was allowed
to stand for 36 h at room temperature (Scheme 1). The
reaction mixture was poured into cold H2O. The resulting
white precipitate was separated by filtration and washed
with H2O. It was then washed with ether and dried under
reduced pressure at room temperature to give the title
compound 10 (80 mg, 72%) as a white amorphous solid, mp
158–1608C (lit. mp5 169–1728C). IR (KBr): n 2940s
(C–H), 1672vs (CvO), 1627m (CvC), 1447m, 1360m,
3.2.2. 3b,22-Dihydroxy-23,24-dinorchol-4-ene (8). To a
stirred solution of 3-oxo-23,24-dinorchol-4-en-22-al 6
(200 mg, 0.60 mmol) in EtOH (50 ml) at room temperature
was added NaBH4 (46 mg, 1.20 mmol, 2 equiv.) and stirring
was continued for 2 h (Scheme 1). A pale yellow solution
was formed. Then H2O (20 ml) was added to the solution,
and extracted with EtOAc (3£20 ml). The organic extracts
were separated and dried over MgSO4. Evaporation of the
solvent gave the title compound 9 as a white amorphous
solid (190 mg). Recrystallisation from EtOH gave a white
solid (132 mg, 66%), mp 197–1998C (EtOH) (lit.5 mp 196–
2058C). IR (KBr): n 3275br (O–H), 2940s (C–H), 2864
(C–H), 1659m (CvC), 1445m, 1361w, 1270m and 1059m
(alcoholic C–O). 1H NMR (CDCl3): d 0.66 (s, 3H, 18-Me),
1.00 (d, J¼6.8 Hz, 3H, 21-Me), 1.01 (s, 3H, 19-Me), 3.33
(dd, J¼6.8, 10.3 Hz, 1H, 22-OCH2), 3.59 (dd, J¼3.1,
10.3 Hz, 1H, 22-OCH2), 4.11 (br t, J¼1.7 Hz, 1H, 3-OCH),
5.24 (d, J¼1.7 Hz, 1H, 4-CH). 13C NMR (CDCl3): d 147.7
(C-5), 123.3 (C-4), 68.1 (C-3), 68.0 (C-22), 55.9 (C-17),
54.5 (C-14), 52.5 (C-9), 42.6 (C-13), 39.7 (C-12), 38.8
(C-10), 37.4 (C-20), 36.0 (C-8), 35.4 (C-1), 33.1 (C-6), 32.2
(C-7), 29.6 (C-2), 27.7 (C-16), 24.3 (C-15), 21.0 (C-11),
19.0 (C-19), 16.7 (C-21), 12.1 (C-18). MS (FAB) m/z: 333
[MþH]þ, 355 [MþNa]þ.
1
and 1176m (O–SO2) cm21 (lit.7 IR). H NMR (CDCl3): d
0.64 (s, 3H, 18-Me), 0.94 (d, J¼6.8 Hz, 3H, 21-Me), 1.13 (s,
3H, 19-Me), 3.75 (dd, J¼6.8, 9.5 Hz, 1H, 22-OCH2), 3.93
(dd, J¼3.1, 9.5 Hz, 1H, 22-OCH2), 5.68 (s, 1H, 4-CH), 22-
Tosyl: 7.74 (d, J¼8.2 Hz, 2H, 2£Ph-H), 7.30 (d, J¼8.2 Hz,
2H, 2£Ph-H), 2.41 (s, 3H, Ph-Me). 13C NMR (CDCl3): d
199.5 (C-3), 171.3 (C-5), 123.9 (C-4), 75.5 (C-22), 55.5
(C-17), 53.7 (C-14), 51.8 (C-9), 42.5 (C-13), 39.3 (C-12),
38.6 (C-10), 36.2 (C-20), 35.7 (C-8), 35.6 (C-1), 34.0 (C-2),
32.9 (C-6), 32.0 (C-7), 27.4 (C-16), 24.1 (C-15), 21.0
(C-11), 17.4 (C-19), 16.7 (C-21), 12.1 (C-18), 22-OTs:
144.6 (C-10), 133.2 (C-40), 129.8 (C-20 and C-60), 127.9 (C-30
and C-50), 21.7 (C-70). MS (FAB) m/z 485 [MþH]þ, 507
[MþNa]þ.
3.2.3. 3b,22-Diacetoxy-23,24-dinorchol-4-ene (9). Acetyl-
ation of 8 (50 mg, 0.15 mmol) with Ac2O (0.5 ml) in
pyridine (0.5 ml) at room temperature for 15 h, followed by
addition of H2O (1.0 ml) gave a colourless precipitate
(Scheme 1). This was collected and dried in vacuo to give
the title compound 9 (55 mg, 88%), mp 178–1818C. IR
(KBr): n 3470br (O–H), 2940s (C–H), 2865 (C–H), 1742vs
(CvO), 1648m (CvC), 1446m, 1372m, 1239s (acetate
C–O) and 1032m. 1H NMR (CDCl3): d 0.66 (s, 3H, 18-Me),
0.96 (d, J¼6.8 Hz, 3H, 21-Me), 1.02 (s, 3H, 19-Me), 2.01 (s,
6H, 3-OCO–Me and 22-OCO–Me), 3.72 (dd, J¼7.5,
10.6 Hz, 1H, 22-OCH2), 4.03 (dd, J¼3.4, 10.6 Hz, 1H,
22-OCH2), 5.18 (m, 2H, 3-OCH and 4-CH). 13C NMR
(CDCl3): d 171.4 (22-OCO–Me), 171.1 (3-OCO–Me),
149.5 (C-5), 119.1 (C-4), 70.9 (C-3), 69.5 (C-22), 55.8
(C-17), 54.2 (C-14), 52.8 (C-9), 42.7 (C-13), 39.6 (C-12),
37.8 (C-8), 37.3 (C-10), 35.9 (C-20), 35.1 (C-1), 32.9 (C-6),
32.2 (C-7), 27.7 (C-2), 25.1 (C-16), 24.3 (C-15), 21.5
(3-OCO–Me), 21.1 (22-OCO–Me), 21.0 (C-11), 18.8
(C-21), 17.1 (C-19), 12.1 (C-18). MS (FAB) m/z 417
[MþH]þ, 439 [MþNa]þ. HRMS (CI): found: [MþNH4]þ,
434.3273. C26H44NO4 requires 434.3270.
3.2.6. Chol-4-en-23-yn-3-one (11). A solution of tosylate
10 (79 mg, 0.16 mmol) in dry DMSO (2 ml) was added
dropwise to a stirred suspension of lithium acetylide/
ethylenediamine
complex
(200 mg,
2.17 mmol,
13.6 equiv.), in dry DMSO (0.5 ml) (Scheme 1). The
brown solution was heated to 408C for 5 min and then at
508C for 3 h. Dilute HCl (2 ml) was cautiously added to the
cooled mixture (vigorous exothermic reaction occurred),
followed by H2O (5 ml). The aqueous layer was decanted
from the gummy product, which was washed with H2O to
remove DMSO, before being dissolved in CHCl3 (10 ml,
washed with H2O, and dried (MgSO4). The pale brown gum
(61 mg), obtained by evaporation of the CHCl3, was purified
by preparative TLC (Rf 0.56) eluted with a mixture (3:1) of
benzene and EtOAc to give the 23-yne 11 (25 mg, 46%) as a
colourless glass. IR (KBr): n 3270, 1660s (CvO) cm21. 1H
NMR (CDCl3): d 0.73 (s, 3H, 18-CH3), 0.85 (d, J¼6.5 Hz,
3H, 21-CH3), 1.21 (s, 3H, 19-CH3), 2.15 (s, 1H, 23-CuCH),
5.74 (s, 1H, 4-CH). 13C NMR (CDCl3): d 200.7 (C-3), 172.2
(C-5), 124.8 (C-4), 84.3 (C-23), 70.3 (C-24), 56.8 (C-17),
55.9 (C-14), 54.8 (C-9), 43.5 (C-13), 39.6 (C-12), 38.5
(C-10), 36.7 (C-20), 36.3 (C-8), 35.8 (C-1), 35.0 (C-2), 33.9
(C-6), 33.2 (C-7), 29.0 (C-16), 26.6 (C-22), 25.2 (C-15),
22.0 (C-11), 19.6 (C-21), 18.4 (C-19), 13.1 (C-18). MS (EI):
m/z 338 (Mþ 18%), 271 (M2C5H7, 11), 229 (M2C8H13,
3.2.4. 22-Hydroxy-23,24-dinorchol-4-en-3-one (7). To a
stirred solution of 8 (80 mg, 0.24 mmol) in dioxane (3 ml) at

L. Nahar, A. B. Turner / Tetrahedron 59 (2003) 8623–8628
8627
10), 215 (11), 159 (11), 148 (10), 147 (18), 131 (13), 124
(100). HRMS (EI): found: Mþ, 338.2613. C24H34O requires
338.2609.
10.6 Hz, 1H, 22-OCH2), 4.62 (m, 2H, 3-OCH and 6-OCH).
13C NMR (CDCl3):
172.4 (22-OCOCH3), 171.9
d
(6-OCOCH3), 171.6 (3-OCOCH3), 74.1 (C-3), 73.3 (C-6),
70.5 (C-22), 56.8 (C-17), 54.5 (C-14), 53.8 (C-9), 49.5
(C-5), 43.8 (C-13), 40.5 (C-12), 38.6 (C-10), 37.9 (C-20),
37.6 (C-8), 36.8 (C-1), 35.1 (C-2), 29.3 (C-4), 28.6 (C-16),
28.2 (C-7), 25.2 (C-15), 22.5 (3-OCOCH3 and 6-OCOCH3),
22.3 (22-OCOCH3), 22.1 (C-11), 18.1 (C-19), 14.3 (C-21),
13.1 (C-18). MS (ESI): m/z 499 [MþNa]þ and 975
[2MþNa]þ. HRMS (CI): found: [MþNH4]þ, 494.3477.
C28H48NO6 requires 494.3482.
3.2.7. Chol-4-ene-3,23-dione (12). A solution of 23-yn-3-
one 11 (22 mg, 0.065 mmol) in acetic acid (1.0 ml) and
water (0.1 ml) containing Hg-resin (190 mg) prepared from
Zeo-Karb 2256 was heated at reflux for 5 h (Scheme 1). The
Hg-resin was removed by filtration and washed with EtOH.
The filtrates were evaporated in vacuo to give the crude
product (22 mg), which was purified by preparative TLC (Rf
0.47) eluting with a mixture (3:1) of benzene/EtOAc to give
3,23-dione 12 (11.5 mg, 50%) as colourless needles, mp
156–1638C, raised upon recrystallisation from EtOH to mp
165–1678C. UV: lmax 240 nm (1 16800). IR (KBr): n 1705s
(CvO), 1665s (CvO) cm21. 1H NMR (CDCl3): d 0.77 (s,
3H, 18-CH3), 0.98 (d, J¼6.5 Hz, 3H, 21-CH3), 1.19 (s, 3H,
19-CH3), 2.12 (s, 3H, 23-CO–CH3), 5.73 (m, 1H, 4-CH).
13C NMR (CDCl3): d 209.8 (C-23), 200.7 (C-3), 172.2
(C-5), 124.8 (C-4), 56.9 (C-17), 56.0 (C-14), 54.8 (C-9),
43.7 (C-13), 39.6 (C-12), 38.5 (C-10), 36.9 (C-20), 36.4
(C-8), 35.8 (C-1), 35.0 (C-2), 33.9 (C-6), 33.6 (C-22), 33.2
(C-7), 29.3 (C-24), 29.1 (C-16), 25.3 (C-15), 22.0 (C-11),
17.6 (C-19), 14.6 (C-21), 13.1 (C-18). MS (EI): 356 (Mþ,
16%), 299 (32), 298 (100), 283 (18), 229 (14), 175 (25), 174
(10), 124 (70), 85 (50), 83 (90). HRMS (EI): found: Mþ,
356.2717. C24H36O2 requires 356.2715.
3.3.3. 3b,6a-Diacetoxy-22-tosyloxy-23,24-dinor-5a-
cholane (16). A solution of the alcohol 14 (100 mg,
0.23 mmol) and p-TsCl (150 mg, 0.79 mmol, 3.4 equiv.)
in dry pyridine (0.8 ml) was left at room temperature for
44 h (Scheme 2). Water (2 ml) was added and the resulting
gummy precipitate was separated and washed with H2O and
then dried in vacuo to give the title compound 16 (85 mg,
63%). The crude product was used directly for ethynylation.
3.3.4. 3b,6a-Diacetoxy-5a-cholan-23-yne (17). A solution
of the tosylate 16 (80 mg, 0.14 mmol) in dry DMSO
(1.5 ml) was added dropwise to a stirred suspension of
lithium acetylide/ethylenediamine complex (180 mg,
1.95 mmol, 13.9 equiv.) in dry DMSO (0.5 ml). The
resulting brown solution was warmed to 408C for 2 h,
before being cooled in iced water and treated dropwise with
dilute HCl (2 ml) followed by water (5 ml). The crude
product, isolated by extraction with chloroform, was a
brown gum (54 mg), the NMR spectrum of which showed
no tosyl group but indicated partial deacetylation. It was
therefore reacetylated by dissolution in pyridine (1 ml)
containing acetic anhydride (0.3 ml) for 18 h at 208C.
Addition of water (0.5 ml) followed by evaporation gave a
brown gum (62 mg), which was purified by preparative TLC
(Rf 0.63) and eluted with a mixture (3:1) of benzene and
EtOAc to give the 23-yne 17 (39 mg, 65%) as a colourless
gum. 1H NMR (CDCl3): d 0.62 (s, 3H, 18-CH3), 0.84 (s, 3H,
19-CH3), 1.02 (d, J¼6.5 Hz, 3H, 21-CH3), 1.98 (s, 6H,
3-OCOCH3 and 6-OCOCH3), 2.01 (s, 1H, 23-CuCH), 4.63
(m, 2H, 3-OCH and 6-OCH). 13C NMR (CDCl3): d 171.9
(6-OCOCH3), 171.6 (3-OCOCH3), 84.4 (C-23), 74.1 (C-3),
73.3 (C-6), 70.2 (C-24), 57.0 (C-17), 55.9 (C-14), 54.5
(C-9), 49.5 (C-5), 43.6 (C-13), 40.5 (C-12), 38.6 (C-10),
37.9 (C-20), 37.6 (C-8), 36.2 (C-1), 35.1 (C-2), 29.4 (C-4),
29.0 (C-7), 28.2 (C-16), 26.6 (C-22), 25.1 (C-15), 22.5
(3-COCH3), 22.3 (6-COCH3), 22.1 (C-11), 20.0 (C-19),
14.3 (C-21), 13.1 (C-18). MS (ESI): m/z 465 [MþNa]þ and
907 [2MþNa]þ. MS (EI): m/z 382 (M260, 10%), 323
(35%), 322 (100%), 228 (30%), 215 (22%), 214 (25%), 213
(45%). HRMS (CI): Found: [MþNH4]þ, 460.3429.
C28H46NO4 requires 460.3427.
3.3. Synthesis of 3b,6a-dihydroxy-5a-cholan-23-one (19)
3.3.1. 3b,6a-Diacetoxy-22-hydroxy-23,24-dinor-5a-
cholane (14). Borohydride reduction (Scheme 2) of
3b,6a-diacetoxy-23,24-dinor-5a-cholan-22-al 13 (200 mg,
0.46 mmol) was performed as for compound 6. After the
usual work-up the title compound 14 (140 mg, 70%) was
obtained as a white amorphous solid, mp 152–1548C, raised
upon recrystallisation from EtOH/EtOAc to mp 176–1778C
(lit.4 mp 173–1748C). IR (film): n 3533m (O–H), 2936s
(C–H), 1731vs (CvO), 1471m, 1444m, 1377m, 1247vs
(ester C–O), 1160m and 1029m (alcoholic C–O) cm21. 1H
NMR (CDCl3): d 0.63 (s, 3H, 18-CH3), 0.84 (s, 3H, 19-
CH3), 0.99 (d, J¼6.5 Hz, 3H, 21-CH3), 1.98 (s, 6H,
3-OCO–Me and 6-OCO–Me), 3.32 (dd, J¼3.4, 10.9 Hz,
1H, 22-OCH2), 3.58 (dd, J¼6.8, 10.3 Hz, 1H, 22-OCH2),
4.62 (m, 2H, 3-OCH and 6-OCH). 13C NMR (CDCl3): d
171.9 (6-OCOCH3), 171.6 (3-OCOCH3), 74.2 (C-3), 73.3
(C-6), 69.0 (C-22), 56.9 (C-17), 54.5 (C-14), 53.5 (C-9),
49.5 (C-5), 43.7 (C-13), 40.6 (C-12), 38.7 (C-10), 37.9
(C-20), 37.6 (C-8), 35.2 (C-1), 35.1 (C-2), 29.4 (C-4), 28.7
(C-7), 28.2 (C-16), 25.2 (C-15), 22.5 (3-COCH3), 22.3
(6-COCH3), 22.1 (C-11), 17.7 (C-19), 14.3 (C-21), 13.1
(C-18). HRMS (CI): found: [MþNH4]þ, 452.3379.
C26H46NO5 requires 452.3376.
3.3.2. 3b,6a,22-Triacetoxy-23,24-dinor-5a-cholane (15).
Acetylation of the alcohol 14 (30 mg, 0.069 mmol) using
Ac2O (0.5 ml) in pyridine as before gave the triacetate as a
colourless gum (Rf 0.75, from a mixture (3:1) of benzene
3.3.5. 3b,6a-Diacetoxy-5a-cholan-23-one (18). A solution
of the above alkyne 17 (38 mg, 0.08 mmol) in acetic acid
(2 ml) and water (0.2 ml) containing Hg-resin6 (274 mg)
was heated under reflux with stirring for 7 h (Scheme 3).
The resin was filtered off and washed with ethanol.
Evaporation of the filtrate and washings gave an oil
(42 mg), which was purified by preparative TLC (Rf 0.50)
eluted with a mixture (3:1) of benzene and EtOAc to give
the 23-one 18 (28 mg, 73%) as a colourless oil. IR (KBr): n
1
and ethyl acetate as eluent, 32 mg, 97%) (Scheme 2). H
NMR (CDCl3): d 0.63 (s, 3H, 18-CH3), 0.84 (s, 3H,
19-CH3), 0.96 (d, J¼6.8 Hz, 3H, 21-CH3), 1.98 (s, 6H,
3-OCO–Me and 6-OCO–Me), 2.00 (s, 3H, 22-OCOCH3),
3.72 (dd, J¼7.5, 10.6 Hz, 1H, 22-OCH2), 4.02 (dd, J¼3.4,

8628
L. Nahar, A. B. Turner / Tetrahedron 59 (2003) 8623–8628
1725vs (acetate CvO), 1705vs (ketonic CvO), 1365m,
1
318.2556. C24H40O3 and C21H34O2 require 376.2977 and
318.2558.
1235s (acetate C–O), 1020m, 955m and 895m cm21. H
NMR (CDCl3): d 0.69 (s, 3H, 18-CH3), 0.99 (d, J¼6.8 Hz,
3H, 21-CH3), 2.03 (s, 6H, 3-OCOCH3 and 6-OCOCH3),
2.11 (s, 3H, 23-COCH3), 4.63 (m, 2H, 3-OCH and 6-OCH).
13C NMR (CDCl3): d 210.6 (C-23), 171.9 (6-OCOCH3),
171.6 (3-OCOCH3), 74.2 (C-3), 73.4 (C-6), 56.9 (C-17),
54.6 (C-14), 53.5 (C-9), 49.6 (C-5), 43.8 (C-13), 40.6
(C-12), 38.6 (C-10), 38.2 (C-20), 37.8 (C-8), 35.3 (C-1),
35.1 (C-2), 30.9 (C-7), 29.5 (C-24), 29.4 (C-4), 28.7 (C-7),
28.2 (C-16), 25.3 (C-15), 22.4 (3-COCH3), 22.4
(6-COCH3), 22.1 (C-11), 20.8 (C-19), 14.7 (C-21), 13.1
(C-18). MS (EI): m/z 460 (Mþ, 1%), 402 (M2Me2CO, 4%),
400 (M2AcOH, 3%), 342 (13), 340 (7), 282 (7), 213 (15),
161 (35), 43 (100). HRMS (EI): found: Mþ, 460.3191.
C28H44O5 requires 460.3188.
Acknowledgements
EPSRC National Mass Spectrometry Service Centre
(Department of Chemistry, University of Wales Swansea,
Swansea, UK) for HRMS and MS analysis. Elemental
analyses for Butterworth Laboratories Limited, Middlesex.
Daresbury for structural analysis.
References
1. Mackie, A. M.; Lasker, R.; Grant, P. T. Comp. Biochem.
Physiol. 1968, 26, 415–418. Mackie, A. M.; Turner, A. B.
Biochem. J. 1970, 117, 543–550.
3.3.6. 3b,6a-Dihydroxy-5a-cholan-23-one (19). A sol-
ution of the diacetate 18 (20 mg) in methanolic KOH
(1.5 M; 2.0 ml) was kept at room temperature under
nitrogen for 27 h (Scheme 3). After evaporation to dryness
and addition of H2O, the dihydroxyketone 19 was extracted
with CHCl3, dried (MgSO4) and rotary evaporated to obtain
a colourless solid (15 mg, 92%), mp 181–1868C, raised
upon recrystallisation from EtOH/EtOAc to mp 212–
2. Turner, A. B.; Smith, D. H. S.; Mackie, A. M. Nature 1971,
233, 209–210.
3. Blunt, J. W.; Copp, B. R.; Munro, M. H. G.; Northcote, P. T.;
Prinsep, M. R. Nat. Prod. Rep. 2003, 20, 1–48. Levina, E. V.;
Andriyashchenko, P. V.; Kalinovskii, A. I.; Stonik, V. A. Russ.
Chem. Bull. 2001, 50, 313–315.
1
2158C. H NMR (CDCl3): d 0.66 (s, 3H, 18-CH3), 0.78 (s,
4. Smith, D. S. H.; Turner, A. B.; Mackie, A. M. J. Chem. Soc.,
Perkin Trans. 1 1973, 1745–1754.
3H, 19-CH3), 0.88 (d, J¼6.2 Hz, 3H, 21-CH3), 2.08 (s, 3H,
24-CH3), 3.54 (m, 1H, 3-OCH), 3.38 (m, 1H, 6-OCH). 13C
NMR (CDCl3): d 210.4 (C-23), 72.3 (C-3), 70.5 (C-6), 57.2
(C-17), 54.8 (C-14), 52.7 (C-9), 51.9 (C-5), 43.7 (C-13),
42.6 (C-12), 40.7 (C-10), 38.3 (C-20), 37.3 (C-8), 35.3
(C-1), 33.7 (C-22), 33.3 (C-2), 32.1 (C-4), 31.7 (C-16), 30.7
(C-7), 29.4 (C-24), 25.2 (C-15), 22.2 (C-11), 20.7 (C-19),
14.5 (C-21), 13.1 (C-18). MS (EI): m/z 376 (Mþ, 1.5%), 358
(7), 319 (16), 318 (100), 303 (19), 285 (11), 213 (15) 161
(18). HRMS (EI): found: Mþ and M258, 376.2976 and
5. Morita, K. Bull. Chem. Soc. Jpn 1959, 32, 227–232.
6. Katoch, R.; Shilpa, S. K.; Deodhar, K. D.; Girish, K. T.
Tetrahedron 1999, 55, 1741–1754.
7. Schering, A. G. Chem. Abstr. 1968, 69, 87386b.
8. Newman, M. S. J. Am. Chem. Soc. 1953, 75, 4740–4742.
9. Field, L. D.; Sternhell, S.; Kalman, J. R. Organic Structures
from Spectra; 2nd ed.; Wiley: New York, 2000 p 27.
10. Kitagawa, I.; Kobayashi, M.; Sugawara, T.; Yosioka, I.
Tetrahedron Lett. 1975, 11, 967–970.