Stereo and chemical course of acid-catalyzed double bond migration of cholesta-5,7-dien-3β-ol to 5α-cholesta-8,14-dien-3β-ol

Article abstract of DOI:10.1039/b001726o

Acid-catalyzed double bond migration of steroid 5,7-dienes to the 5α-8,14-dienes, a well-known reaction in steroid chemistry, was reinvestigated by using cholesta-5,7-dien-3β-ol 1, and the stereo and chemical course of this reaction was detailed. Treatment of 1 with 36% hydrochloric acid in refluxing ethanol for 3 h afforded a 6:11:65:16:2 mixture of dienols (93%): 5α- and 5β-cholesta-6,8(14)-dien-3β-ols 7a,b, 5α- and 5β-cholesta-8,14-dien-3β-ols 2a,b, and 5α-cholesta-14,16-dien-3β-ol 10a, along with a mixture of enones (1.7%): 5β-cholest-8(14)-,5β-cholest-14- and 14-epi-5β-cholest-8-en-3-ones 11-13. The experiments using 7a,b and 2a,b suggested the reaction sequence: 1→7a,b?7,14-dienols 8a,b?2a,b?8(14),15-dienols 9a,b?10a,b, in which 8a,b?9a,b should be also implicated. The initial step, 1 to 7a,b proceeded irreversibly with the stereoselectivity, ca. 7:3 of 5α-H to 5β-H. Dienols 7a, 8a, 2a and 10a with 5α-H were identified, which were equilibrated at 6:0:92:2. Among dienols with 5β-H, only 7b and 2b were identified, which were equilibrated at 2:98, and this interconversion proceeded in competition with an intramolecular hydride shift from the C-3α to C-6α of 7b, leading to the formation of a mixture of enones 11-13. The considerable difference in activation energies between 1→7a,b and 7a,b→8a,b/11-13 realized the predominant formation of 7a,b: by treatment at 30°C for 44 h, 1 gave a 53:27:5:15 mixture (94%) of 7a, 7b, 8a and 2a. The Royal Society of Chemistry 2000.

Full text of DOI:10.1039/b001726o

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Stereo and chemical course of acid-catalyzed double bond
migration of cholesta-5,7-dien-3ꢀ-ol to 5ꢁ-cholesta-8,14-dien-3ꢀ-ol
Hideharu Seto,*a Shozo Fujioka,a Hiroyuki Koshino,a Suguru Takatsutob and Shigeo Yoshidaa
1
a The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351-0198,
Japan
b Department of Chemistry, Joetsu University of Education, Joetsu-shi, Niigata 943-8512,
Japan
Received (in Cambridge, UK) 2nd March 2000, Accepted 13th April 2000
Published on the Web 15th May 2000
Acid-catalyzed double bond migration of steroid 5,7-dienes to the 5α-8,14-dienes, a well-known reaction in
steroid chemistry, was reinvestigated by using cholesta-5,7-dien-3β-ol 1, and the stereo and chemical course of
this reaction was detailed. Treatment of 1 with 36% hydrochloric acid in refluxing ethanol for 3 h afforded a
6:11:65:16:2 mixture of dienols (93%): 5α- and 5β-cholesta-6,8(14)-dien-3β-ols 7a,b, 5α- and 5β-cholesta-8,14-
dien-3β-ols 2a,b, and 5α-cholesta-14,16-dien-3β-ol 10a, along with a mixture of enones (1.7%): 5β-cholest-8(14)-,
5β-cholest-14- and 14-epi-5β-cholest-8-en-3-ones 11–13. The experiments using 7a,b and 2a,b suggested the reaction
sequence: 1→7a,b↔7,14-dienols 8a,b↔2a,b↔8(14),15-dienols 9a,b↔10a,b, in which 8a,b↔9a,b should be also
implicated. The initial step, 1 to 7a,b proceeded irreversibly with the stereoselectivity, ca. 7:3 of 5α-H to 5β-H.
Dienols 7a, 8a, 2a and 10a with 5α-H were identified, which were equilibrated at 6:0:92:2. Among dienols with
5β-H, only 7b and 2b were identified, which were equilibrated at 2:98, and this interconversion proceeded in
competition with an intramolecular hydride shift from the C-3α to C-6α of 7b, leading to the formation of a mixture
of enones 11–13. The considerable difference in activation energies between 1→7a,b and 7a,b→8a,b/11–13 realized
the predominant formation of 7a,b: by treatment at 30 ЊC for 44 h, 1 gave a 53:27:5:15 mixture (94%) of 7a, 7b, 8a
and 2a.
Introduction
Acid-catalyzed double bond migration of a 5,7-diene to the
8,14-diene with 5α-H in steroids, e.g., cholesta-5,7-dien-3β-ol
(7-dehydrocholesterol: 1) to 5α-cholesta-8,14-dien-3β-ol 2a, is a
well-known reaction,1,2 and the resulting 5α-8,14-dienes have
been employed to prepare the biosynthetic intermediates and
their labelled compounds useful for the investigation of steroid
biosynthesis of mammals, plants, etc.3–6 In recent years, we elu-
cidated that fackel-J79, a dwarf mutant of Arabidopsis thaliana
with unique phenotypes, had a defect of sterol C-14 reductase
which reduces the 14-double bond in brassinosteroid bio-
synthesis and thus accumulated three abnormal sterols, 2a and
its (24R)-24-methyl and -ethyl congeners, i.e., campesta-8,14-
dienol 5 and stigmasta-8,14-dien-3β-ol 6 (sitosta-8,14-dienol)
(Scheme 1).7 This was clearly verified by GC-MS analysis using
the synthetic 5α-8,14-dienols as the reference markers. How-
ever, in the synthesis, we could not attain the reported high
yields under conventional reaction conditions, and found that
these reactions were accompanied by the formation of several
minor products, some of which were suspected to be the reac-
tion intermediates and their C-5 epimers. Since there is no
paper describing the actual features of this reaction including
the chemical and stereo-selectivities, and the 8,14-diene func-
tionality on the steroid should be a promising starting point for
introducing a variety of substituents and functional groups
to the C and D rings to assemble complex steroidal natural
products, we undertook a study on the acid-catalyzed double
bond migration of steroid 5,7-diene to the 5α-8,14-diene
by using commercially available cholesta-5,7-dien-3β-ol 1 as a
reaction substrate.
Scheme 1
C-3α to C-6α observed on 5β-cholesta-6,8(14)-dien-3β-ol 7b
leading to the formation of 5β-cholest-8(14)-, 5β-cholest-14-
and 14-epi-5β-cholest-8-en-3-ones 11–13, is also presented in
this context.
Results and discussion
When cholesta-5,7-dien-3β-ol 1, campesta-5,7-dienol 38 and
stigmasta-5,7-dienol 49 were subjected to acid-catalyzed diene
migration from the 5,7-diene to the corresponding 8,14-diene
by following the conventional procedure,2 the yields of 5α-8,14-
dienols, 2a, 5 and 6, were 55, 53 and 50%, respectively. There-
fore, to clarify whether the unsatisfactory yields compared to
those reported are inevitable or not, we first analyzed the
In this paper we detail the stereo and chemical course of this
reaction on the basis of product analysis and equilibrium
experiments, which enabled us to establish other acidic
conditions to give steroid 6,8(14)-dienes predominantly. An
intriguing reaction, an intramolecular hydride shift from the
DOI: 10.1039/b001726o
J. Chem. Soc., Perkin Trans. 1, 2000, 1697–1703
This journal is © The Royal Society of Chemistry 2000
1697

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Table 1 Reaction of dienols 1, 7a,b and 2a,b, with 36% hydrochloric acid in ethanol
Reaction
temperature
Reaction
time/h
Combined
yield (%)b
Entry
1
Substrate
Product ratio (%)a
1
reflux
3
7a (5), 7b (11), 2a (64), 2b (16), 10a (2)
a mixture of 11, 12 and 13 (2)
7a (53), 7b (27), 8a (5), 2a (15)
7a (7), 2a (90), 10a (3)
7a (6), 2a (92), 10a (2)
7b (4), 2b (56), 11 (24), 12 (14), 13 (2)
7b (1), 2b (63), 11 (20), 12 (14), 13 (2)
95
2
3
4
5
6
1
30 ЊC
reflux
reflux
reflux
reflux
44
3
3
8
8
94
93
93
96
85
7a
2a
7b
2b
a Ratios were calculated from 1H NMR spectra in Entries 2–4, and by combination of the combined yields of dienol and enone mixtures, and their
1H NMR spectra in Entries 1, 5 and 6. b After flash chromatography.
Fig. 1 Retention times/min of dienols identified in this work on
HPLC. Simplified spectrum of a dienol mixture obtained by Entry 1 in
Table 1 is shown above, where the length of each bar reflects the
amount; others are shown below. Abbreviation Cho means cholest-5-
en-3β-ol which is a contaminant in commercial 1.
plete consumption of the starting material 1 indicated that the
initial reaction of 1 leading to 6,8(14)-dienes 7a,b was irrevers-
ible. Therefore, the stereoselectivity of this reaction was calcu-
lated as 69:31 of 5α-H to 5β-H, which is obviously determined
kinetically at the initial protonation at C-5 of 1.
In order to increase the stereoselectivity biased to 5α-H by
kinetic control, 1 was treated with 36% hydrochloric acid in a
mixed solution of ethanol and benzene, 5:1, at 30 ЊC (a co-
solvent, benzene, was necessary due to the low solubility of 1
in ethanol). After 44 h, the starting material was completely
consumed, as monitored by 1H NMR spectroscopy, and a
53:27:5:15 mixture of 7a, 7b, 5α-cholesta-7,14-dien-3β-ol 8a
and 2a was obtained in 94% combined yield (Entry 2). Separ-
ation by HPLC gave 7a and 7b in 46 and 25% yields along with
8a (4.8%) and 2a (11%). The calculated stereoselectivity, 73:27
of 5α-H to 5β-H, was nearly the same as that attained under
refluxing conditions, but the product distribution was com-
pletely different: i.e., 6,8(14)-dienols 7a,b predominantly
formed. This indicates that the initial step, migration of the 5,7-
diene in 1 to the 6,8(14)-diene in 7a,b, proceeds much faster
than further migrations or the intramolecular hydride shift of
7b, and that 7b is more stable than 7a. Other attempts to
prevent the formation of 8a and 2a, and to further enhance
the 5α-H selectivity, resulted in near failure. Among them, a
cationic resin, Dowex 50W, gave a similar result with some
enhancement of the stereoselectivity. Treatment of 1 with
Dowex 50W resin in ethanol at 70 ЊC for 51 h gave a
55:22:11:12 mixture of 7a, 7b, 8a and 2a in 96% combined
yield, thus the stereoselectivity of 5α-H to 5β-H being 78:22.
After separation, the isolated yields of 7a, 7b, 8a and 2a were
48, 21, 6.8 and 11%, respectively.
products formed from 1. A solution of 1 (1.30 mmol) and 36%
hydrochloric acid (1.0 cm3) in ethanol (20 cm3) was stirred at
refluxing temperature for 3 h. After the usual aqueous work-up
and flash chromatography on silica gel, a 6:11:65:16:2 mix-
ture of dienols, 5α-cholesta-6,8(14)-dien-3β-ol 7a, 5β-cholesta-
6,8(14)-dien-3β-ol 7b, 5α-cholesta-8,14-dien-3β-ol 2a, 5β-
cholesta-8,14-dien-3β-ol 2b and 5α-cholesta-14,16-dien-3β-ol
10a, was obtained in 93% combined yield along with 1.7% of a
mixture of enones, 5β-cholest-8(14)-en-3-one 11, 5β-cholest-14-
en-3-one 12 and 14-epi-5β-cholest-8-en-3-one 13 (Entry 1 in
Table 1). The ratio of 7a, 7b, 2a, 2b, 10a was estimated from the
1H NMR spectrum. The dienol mixture could be separated by
high-performance liquid chromatography (HPLC), giving 7a,
7b, 2a, 2b and 10a in 6.4, 9.0, 55, 14 and 2.2% isolated yields,
respectively. Cholest-5-en-3β-ol was also isolated in 3.2% yield;
this is a contaminant of commercial 1. Fig. 1 shows a simplified
HPLC spectrum of the dienol mixture, shown above, along
with retention times of 1 and 8a, the latter of which was
obtained under other conditions (vide infra). Since the form-
ation of enones 11–13 was assumed to be the result of an
intramolecular hydride shift of 7b as discussed later, the com-
Further insight into the kinetic or thermodynamic corre-
lation among dienols was obtained from reactions of the
isolated 7a, 2a, 7b and 2b under the same acidic conditions at
refluxing temperature. With 3 h treatment, both 7a and 2a gave
a mixture of 7a, 2a and 10a in nearly the same ratio (Entries
1698
J. Chem. Soc., Perkin Trans. 1, 2000, 1697–1703

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Scheme 2 Events taking place in treatment of cholesta-5,7-dien-3β-ol 1 with 36% hydrochloric acid in refluxing ethanol. Compounds in brackets
are not identified. Protonation and deprotonation positions (/): a: 5α-/14α-; b: 5β-/14α-; c: 6-/15-; d: 15-/6-; e: 7-/9α-; f: 9α-/7-; g: 9α-/16-; h: 16-/9α-;
i: 8α-/17α-; j: 17α-/8α-; k: 7-/16-; l: 16-/7-; m: 14-/3β-OH, an intramolecular hydride shift of 3α-H to the C-6α position.
3 and 4), which indicated that dienols 7a, 8a, 2a, 9a, 10a with
5α-H were equilibrated at a ratio of 6:0:92:0:2. On the other
hand, the reaction of 7b proceeded much more slowly than that
of 7a. After 3 h, ca. 45% of 7b still remained, and after 8 h 7b
afforded a 4:56:24:14:2 mixture of 7b, 2b and 11–13 (Entry 5),
while 2b gave a 1:63:20:14:2 mixture of 7b, 2b and 11–13 after
8 h (Entry 6). Assuming that dienols 8b, 9b and 10b should be
implicated like 8a, 9a and 10a with 5α-H, these results suggest
that dienols 7b, 8b, 2b, 9b, 10b with 5β-H are equilibrated at
2:0:98:0:0, although this process is impeded by an irreversible
conversion of 7b to enones 11–13. On the basis of these data, all
events taking place in treatment of 1 with 36% hydrochloric
acid in refluxing ethanol can be eventually summarized as
illustrated in Scheme 2.
intramolecular shift of α-H of a hydroxy function under acidic
conditions.10
The structures of compounds, 7a,b, 2b, 10a, 11–13, 5 and 6
were verified by MS, and 1H and 13C NMR spectroscopy. NMR
experiments on these compounds except for 5 and 6, as well as
the known 8a11 and 2a2 for reference, were carried out with 600
MHz by PFG-DQFCOSY, PFG-HMQC, PFG-HMBC and
NOE difference experiments (400 MHz), by which all reson-
ances were completely assigned (Experimental section). The
stereochemistries of C-5 and C-9 of these compounds were
respectively deduced from NOE difference experiments cen-
tered on 19-H3, 5-H and 9-H. The NOE effects were observed
between 19-H3 and 5-H in 7b, 2b and 11–13 with 5β-H, but not
observed in 7a, 8a, 2a and 10a with 5α-H. No effect was
observed at 9-H of 7a,b, 8a, 2a,b, 10a, 11 and 12 with 9α-H
when the 19-H3 was irradiated. Similarly, NOE correlations
between 8-H and both of 18-H3 and 19-H3 in 10a and 12 demon-
strated their 8β-configuration, and correlation between 14-H
and 18-H3 in 13 indicated the 14β-configuration. Some selected
With respect to the organic reaction mechanism, conversion
of 7b to enones 11–13, is of special interest, which doubtless
involves an intramolecular hydride shift, as depicted in Scheme
3. A 1,4-hydride-shift from the C-3α to C-6α occurs via a con-
1
1H NMR data are shown in Table 2. The H and 13C NMR
spectra of (24R)-24-methyl and -ethyl congeners of 2a (5 and 6),
were reasonably similar to those of 2a.
In conclusion, a well-known reaction in steroid chemistry,
acid-catalyzed double bond migration of steroid 5,7-dienes to
the 5α-8,14-dienes was reinvestigated by using cholesta-5,7-
dien-3β-ol 1. The product analysis and equilibrium experiments
suggested the reaction sequence: 1→7a,b↔8a,b↔2a,b↔9a,
b↔10a,b, in which 8a,b↔9a,b should also be implicated. The
initial step, 1 to 6,8(14)-dienol 7a,b, proceeded irreversibly with
stereoselectivity of ca. 7:3 of 5α-H to 5β-H. Dienols 7a, 8a, 2a
and 10a with 5α-H were identified, which were equilibrated at
6:0:92:2. On the other hand, among dienols with 5β-H, only
7b and 2b were identified, which were equilibrated at 2:98. The
conversion of 7b to 8b competed with an intramolecular
hydride shift from the C-3α to C-6α, resulting in the form-
ation of enones 11–13. These results proved that moderate but
not high yields of 5α-8,14-dienols, 2a, 5 and 6, from 5,7-dienols,
1, 3 and 4, were reasonable for this acidic migration reaction.
The considerable difference in activation energies between
1→7a,b and 7a,b→8a,b or 11–13 realized the reaction condi-
tions for the predominant formation of 6,8(14)-dienols 7a,b
from 1.
Scheme 3 Acid-catalysed intramolecular hydride shift of 5β-cholesta-
6,8(14)-dien-3β-ol 7b via an unstable conformer 7b-B.
former 7b-B, which is one of the unstable conformers of 7b
satisfying the stereoelectric requirement for the reaction, to give
intermediary 14, 5β-cholest-7-en-3-one or its 14-epimer, which
then isomerizes to the 8(14)-ene 11 and 14-ene 12, and 14-epi-
5β-cholest-8-en-3-one 13 by acid. The suitability of 7b-A as a
Experimental
General
Melting points (mp) were determined on a Yanagimoto micro-
melting point apparatus and are uncorrected. NMR measure-
ments were performed on a Bruker AC-300, JEOL JNM-A400
or JEOL JNM-A600 spectrometer. All spectra were recorded
using standard pulse sequences. Chemical shifts were recorded
as δ values in parts per million (ppm) relative to tetramethyl-
silane (δ 0 ppm) for 1H or to the solvent (CDCl3) (δ 77.0 ppm)
for 13C as an internal reference. All J-values are given in Hz.
1
stable conformer of 7b in a solution was deduced from the H
NMR spectrum: 3α-H resonates at δ 4.07 (br s, ω1/2 ca. 7 Hz)
and has no large coupling constants due to an anti-vicinal coup-
ling, indicating its equatorial orientation. It is well documented
that secondary alcohols serve as hydride donors by their
α-positioned hydrides as observed in the Meerwein–Ponndorf–
Verley process, but no example has been reported so far of an
J. Chem. Soc., Perkin Trans. 1, 2000, 1697–1703
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Table 2 Selected 1H-NMR Data (δ: multiplicity, J in Hz) of dienols, 7a,b, 8a, 2a,b and 10a, and enones 11–13
Atom
Compound
3-H or 4α-H
18-H3
19-H3
Olefinic protons
7a
7b
8a
2a
2b
10a
11
12
13
3.68: tt
0.89: s
0.89: s
0.83: s
0.82: s
0.83: s
0.97: s
0.87: s
0.93: s
0.90: s
0.64: s
0.76: s
0.79: s
0.99: s
1.12: s
0.92: s
0.90: s
1.03: s
1.14: s
5.26: dd
J 9.8, 1.5
5.52: dd
J 9.8, 5.4
5.50: dd
J 3.4, 2.0
5.36: dd
J 2.5, 2.0
5.39: br s
6.13: dd
J 9.8, 2.9
6.09: d
J 9.8
J 10.7, 4.9
4.09: br s
ω1/2 ca. 7
3.60: tt
J 11.2, 4.4
3.63: tt
J 10.7, 4.9
3.89: m
ω1/2 ca. 16
3.59: tt
J 11.2, 4.9
2.82: dd
J 14.7, 14.2
2.68: dd
J 14.7, 14.2
2.33: ddd
J 14.2, 9.8, 1.0
5.75: m
3-H
5.81: dd
J 2.0, 2.0
—
5.96: d
J 2.0
4α-H
5.20: m
—
Mass spectra, EI-MS and HR-EI-MS, were obtained with
JEOL-SX102 mass spectrometer. Analytical thin-layer
5α-Cholesta-6,8(14)-dien-3β-ol 7a. Colorless needles, mp
90–98 ЊC (MeOH); δH (600 MHz) 0.64 (3H, s, 19-H3), 0.864
(3H, d, J 6.8 Hz, 26-H3), 0.867 (3H, d, J 6.8 Hz, 27-H3), 0.89
(3H, s, 18-H3), 0.94 (3H, d, J 6.8 Hz, 21-H3), 1.07 and 1.38
(each 1H, each m, 22-H2), 1.10 and 1.17 (each 1H, each m, 24-
H2), 1.14 (1H, m, 1α-H), 1.14 and 1.36 (each 1H, each m, 23-
H2), 1.19 (1H, m, 17-H), 1.26 (1H, ddd, J 12.7, 12.7 and 3.4
Hz, 12α-H), 1.38 (1H, m, 4β-H), 1.44 and 1.90 (each 1H, each
m, 16-H2), 1.47 (1H, m, 11β-H), 1.47 (1H, m, 20-H), 1.48 (1H,
m, 2β-H), 1.52 (1H, m, 25-H), 1.62 (1H, m, 11α-H), 1.69 (1H,
ddd, J 13.2, 3.4 and 3.4 Hz, 1β-H), 1.78 (1H, m, 4α-H), 1.88
(1H, m, 2α-H), 1.92 (1H, m, 9-H), 2.01 (1H, ddd, J 12.7, 3.4
and 3.4 Hz, 12β-H), 2.06 (1H, br dm, J 13.2 Hz, 5-H), 2.30
(1H, m, 15β-H), 2.39 (1H, m, 15α-H), 3.68 (1H, tt, J 10.7 and
4.9 Hz, 3-H), 5.26 (1H, dd, J 9.8 and 1.5 Hz, 6-H), 6.13 (1H,
dd, J 9.8 and 2.9 Hz, 7-H); δC (150 MHz) 11.31 (C-19), 18.88
(C-21), 19.24 (C-18), 19.73 (C-11), 22.56 (C-26), 22.79 (C-27),
23.68 (C-23), 24.93 (C-15), 27.35 (C-16), 28.02 (C-25), 31.48
(C-2), 34.77 (C-20), 35.15 (C-1), 35.68 (C-10), 35.88 (C-22),
36.59 (C-4), 36.76 (C-12), 39.52 (C-24), 43.65 (C-13), 44.75
(C-5), 48.15 (C-9), 55.92 (C-17), 71.44 (C-3), 125.23 (C-8),
125.75 (C-7), 129.38 (C-6), 147.54 (C-14); EI-MS m/z 384
(Mϩ, 11%), 366 (Mϩ Ϫ H2O, 48), 351 (Mϩ Ϫ H2O Ϫ CH3,
38), 271 (Mϩ Ϫ C8H17, 28), 253 (Mϩ Ϫ C8H17 Ϫ H2O, 100), 199
(36); HR-EI-MS m/z Mϩ: Found, 384.3389. Calc. for C27H44O,
384.3392.
5β-Cholesta-6,8(14)-dien-3β-ol 7b. Colorless needles, mp
100 ЊC (MeOH); δH (600 MHz) 0.76 (3H, s, 19-H3), 0.866 (3H,
d, J 6.8 Hz, 26-H3), 0.869 (3H, d, J 6.8 Hz, 27-H3), 0.89 (3H, s,
18-H3), 0.95 (3H, d, J 6.4 Hz, 21-H3), 1.08 and 1.38 (each 1H,
each m, 22-H2), 1.13 (2H, 24-H2), 1.17 and 1.37 (each 1H, each
m, 23-H2), 1.21 (1H, m, 17-H), 1.34 (1H, m, 12α-H), 1.44 and
1.89 (each 1H, each m, 16-H2), 1.45–1.55 (2H, 11-H2), 1.48 (1H,
m, 4α-H), 1.48 (1H, m, 20-H), 1.53 (1H, m, 25-H), 1.58 (2H,
1-H2), 1.58 (2H, 2-H2), 1.76 (1H, m, 4β-H), 2.04 (1H, ddd,
J 12.2, 3.4 and 3.4 Hz, 12β-H), 2.14 (1H, ddd, J 12.7, 5.4 and
4.9 Hz, 5-H), 2.28 and 2.38 (each 1H, each m, 15-H2), 2.43 (1H,
m, 9-H), 4.09 (1H, m, ω1/2 ca. 7 Hz, 3-H), 5.52 (1H, dd, J 9.8
and 5.4 Hz, 6-H), 6.09 (1H, d, J 9.8 Hz, 7-H); δC (150 MHz)
18.92 (C-21), 19.05 (C-18), 19.46 (C-11), 22.55 (C-26), 22.78
(C-27), 23.06 (C-19), 23.67 (C-23), 24.92 (C-15), 27.38 (C-16),
28.01 (C-25), 28.07 (C-2), 28.17 (C-1), 34.21 (C-9), 34.73
(C-20), 34.53 (C-10), 35.90 (C-22), 36.59 (C-4), 37.43 (C-12),
39.09 (C-5), 39.52 (C-24), 43.59 (C-13), 55.99 (C-17), 66.31 (C-
3), 124.46 (C-7), 124.86 (C-8), 129.82 (C-6), 147.04 (C-14); EI-
MS m/z 384 (Mϩ, 19%), 366 (Mϩ Ϫ H2O, 76), 351 (Mϩ Ϫ H2O Ϫ
CH3, 40), 271 (Mϩ Ϫ C8H17, 41), 253 (Mϩ Ϫ C8H17 Ϫ H2O,
a
chromatography (TLC) was conducted on micro-slides coated
with Merck Kieselgel KG60F-254; the developed plates were
stained with 10% (w/v) vanillin in concentrated sulfuric acid
at 180 ЊC. All reactions were carried out under a nitrogen
atmosphere. Flash chromatography was conducted using
silica gel FL-60D [Fuji Silysia Chemical Ltd.] as the
adsorbent. High-performance liquid chromatography (HPLC)
was conducted with Senshu Pak PG-S60-5251 (20 mm
i.d. × 25 cm; Senshu Scientific Co.) at a flow rate of 9.0
cm3 minϪ1; the peaks were detected by a refractive index
detector. The ratios of mixed solvents were v/v. Commercial
cholesta-5,7-dien-3β-ol 1 (7-dehydrocholesterol: Sigma) was
purified by flash chromatography on silica gel before use,
and still contained ca. 3% of cholest-5-en-3β-ol. The ratios
of compound mixtures were estimated from the 1H NMR
spectra.
Acid treatment of cholesta-5,7-dien-3ꢀ-ol 1
With 36% hydrochloric acid (HCl) at reflux temperature. A
solution of cholesta-5,7-dien-3β-ol 1 (500 mg, 1.30 mmol) and
36% HCl (1.0 cm3) in EtOH (20 cm3) was refluxed for 3 h. After
removal of EtOH under reduced pressure, the residue was
diluted with water and extracted with Et2O. The extracts were
successively washed with saturated aqueous NaHCO3 and
brine, dried over Na2SO4 and evaporated. The residue was sub-
jected to flash chromatography using hexane–AcOEt (4:1) as
eluent. The fraction eluted at Rf 0.61 gave compounds (8.5 mg,
1.7%) which had no ultra-violet absoption by 254 nm irradi-
ation on TLC, and which were found to be a mixture of
5β-cholest-8(14)-en-3-one 11, 5β-cholest-14-en-3-one 12 and
1
14-epi-5β-cholest-8-en-3-one 13 from the H NMR spectrum.
The isolation and structural assignment of 11–13 were per-
formed from the experiment on treatment of 5β-cholesta-
6,8(14)-dien-3β-ol 7b with 36% HCl, as mentioned below.
The fraction eluted at Rf 0.18–0.27 gave a dienol mixture (465
mg, 93%) consisting of 5α-cholesta-6,8(14)-dien-3β-ol 7a, 5β-
cholesta-6,8(14)-dien-3β-ol 7b, 5α-cholesta-8,14-dien-3β-ol 2a,
5β-cholesta-8,14-dien-3β-ol 2b and 5α-cholesta-14,16-dien-3β-
ol 10a in a ratio of 6:11:65:16:2. This mixture was subjected
to HPLC using hexane–AcOEt (4:1) as a mobile phase.
Elutions at different Rts as shown in Fig. 1 gave 7b (45 mg,
9.0%), 5α-cholest-5-en-3β-ol (16 mg, 3.2%), 2b (71 mg, 14%),
7a (32 mg, 6.4%), 10a (11 mg, 2.2%) and 2a (273 mg, 55%),
successively.
1700
J. Chem. Soc., Perkin Trans. 1, 2000, 1697–1703

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100), 199 (50); HR-EI-MS m/z Mϩ: Found, 384.3389. Calc. for
C27H44O, 384.3392.
281 (Mϩ Ϫ C6H13 Ϫ H2O, 100); HR-EI-MS m/z Mϩ: Found,
384.3400. Calc. for C27H44O, 384.3392.
5α-Cholesta-8,14-dien-3β-ol 2a. Colorless scales, mp 111–
113 ЊC (MeOH) [lit.,2 mp 116–117 ЊC (MeOH)]; δH (600 MHz)
0.82 (3H, s, 18-H3), 0.868 (3H, d, J 6.8 Hz, 26-H3), 0.871 (3H, d,
J 6.8 Hz, 27-H3), 0.94 (3H, d, J 6.4 Hz, 21-H3), 0.99 (3H, s,
19-H3), 1.05 and 1.38 (each 1H, each m, 22-H2), 1.08 and 1.20
(each 1H, each m, 24-H2), 1.18 and 1.37 (each 1H, each m,
23-H2), 1.26 (1H, ddd, J 13.2, 13.2 and 3.9 Hz, 1α-H), 1.38 (1H,
m, 4β-H), 1.40 (1H, m, 12α-H), 1.44 and 1.58 (each 1H, each m,
6-H2), 1.48 (1H, m, 2β-H), 1.50 (1H, m, 5-H), 1.52 (1H, m,
17-H), 1.53 (1H, m, 25-H), 1.61 (1H, m, 20-H), 1.68 (1H, m, 4α-
H), 1.85 (1H, ddd, J 13.2, 3.9 and 3.0 Hz, 1β-H), 1.88 (1H, m,
2α-H), 2.03 (1H, m, 12β-H), 2.07 and 2.35 (each 1H, each
m, 16-H2), 2.10 and 2.35 (each 1H, each m, 7-H2), 2.18 (1H, m,
11β-H), 2.22 (1H, m, 11α-H), 3.63 (1H, tt, J 10.7 and 4.9 Hz,
3-H), 5.36 (1H, dd, J 2.5 and 2.0 Hz, 15-H); δC (150 MHz) 15.66
(C-18), 18.34 (C-19), 18.87 (C-21), 21.84 (C-11), 22.53 (C-26),
22.78 (C-27), 23.73 (C-23), 25.28 (C-6), 27.99 (C-7), 27.99
(C-25), 31.69 (C-2), 34.04 (C-20), 35.31 (C-1), 35.90 (C-16),
36.10 (C-22), 36.52 (C-10), 36.93 (C-12), 38.30 (C-4), 39.50
(C-24), 45.02 (C-13), 40.93 (C-5), 57.22 (C-17), 71.00 (C-3),
117.41 (C-15), 123.07 (C-8), 140.84 (C-9), 151.08 (C-14); EI-MS
m/z 384 (Mϩ, 100%), 369 (Mϩ Ϫ CH3, 15), 351 (Mϩ Ϫ CH3 Ϫ
H2O, 30), 271 (Mϩ Ϫ C8H17, 20); HR-EI-MS m/z Mϩ: Found,
384.3395. Calc. for C27H44O, 384.3392.
5β-Cholesta-8,14-dien-3β-ol 2b. Colorless needles, mp 50–
53 ЊC (MeOH); δH (600 MHz) 0.83 (3H, s, 18-H3), 0.869 (3H, d,
J 6.8 Hz, 26-H3), 0.871 (3H, d, J 6.8 Hz, 27-H3), 0.94 (3H, d,
J 6.3 Hz, 21-H3), 1.06 and 1.38 (each 1H, each m, 22-H2), 1.10
and 1.18 (each 1H, each m, 24-H2), 1.12 (3H, s, 19-H3), 1.17 and
1.38 (each 1H, each m, 23-H2), 1.37 (1H, m, 12α-H), 1.46 and
1.58 (each 1H, each m, 2-H2), 1.53 (1H, m, 17-H), 1.53 (1H, m,
25-H), 1.56 and 1.70 (each 1H, each m, 6-H2), 1.58 and 1.67
(each 1H, each m, 1-H2), 1.63 (1H, m, 20-H), 1.68 (1H, m,
5-H), 1.70 (2H, 4-H2), 1.98 and 2.33 (each 1H, each m, 7-H2),
2.03 (1H, m, 12β-H), 2.07 (1H, m, 11α-H), 2.08 (1H, m, 16β-
H), 2.24 (1H, m, 11β-H), 2.37 (1H, m, 16α-H), 3.89 (1H, m,
ω1/2 ca. 16 Hz, 3-H), 5.39 (1H, br s, 15-H); δC (150 MHz)
15.33 (C-18), 18.84 (C-21), 22.54 (C-26), 22.77 (C-11), 22.77
(C-27), 23.69 (C-23), 24.87 (C-6), 24.87 (C-7), 25.09 (C-19,
br), 27.99 (C-25), 30.96 (C-1), 31.16 (C-2), 33.97 (C-20),
35.85 (C-16), 36.09 (C-22), 36.92 (C-4), 36.92 (C-12), 36.98
(C-10, br), 39.29 (C-5, br), 39.50 (C-24), 45.07 (C-13), 57.20
(C-17), 67.33 (C-3), 117.57 (C-15), 123.54 (C-8), 138.92
(C-9, br), 150.77 (C-14); EI-MS m/z 384 (Mϩ, 100%), 369
(Mϩ Ϫ CH3, 44), 351 (Mϩ Ϫ CH3 Ϫ H2O, 35), 271 (Mϩ Ϫ
C8H17, 9); HR-EI-MS m/z Mϩ: Found, 384.3398. Calc. for
C27H44O, 384.3392.
With 36% HCl at 30 ؇C. A solution of 1 (250 mg, 0.65 mmol)
and 36% HCl (0.5 cm3) in EtOH (10 cm3) and benzene (2 cm3)
Њ
was stirred at 30 C for 44 h. The same work-up and flash
chromatography as described above gave a 53:27:5:15 dienol
mixture (235 mg, 94%) of 7a, 7b, 5α-cholesta-7,14-dien-3β-ol
8a and 2a. Separation by HPLC using hexane–AcOEt (4:1) as a
mobile phase gave 7b (63 mg, 25%), 7a (115 mg, 46%), 2a (28
mg, 11%) and 8a (Rt 32.2 min: 12 mg, 4.8%), successively.
5α-Cholesta-7,14-dien-3β-ol 8a. Colorless plates, mp 78–
80 ЊC (MeOH) [lit.,11 mp 103–105 ЊC (MeOH)]; δH (600 MHz)
0.79 (3H, s, 19-H3), 0.83 (3H, s, 18-H3), 0.868 (3H, d, J 6.8 Hz,
26-H3), 0.871 (3H, d, J 6.8 Hz, 27-H3), 0.92 (3H, d, J 6.0 Hz,
21-H3), 1.05 and 1.36 (each 1H, each m, 22-H2), 1.07 (1H, m,
1α-H), 1.14 and 1.36 (each 1H, each m, 23-H2), 1.15 (2H,
24-H2), 1.25 (1H, m, 4β-H), 1.31 (1H, m, 12α-H), 1.41 (1H, m,
2β-H), 1.42 (1H, m, 5-H), 1.49 (1H, m, 11β-H), 1.53 (1H, m,
25-H), 1.57 (1H, m, 17-H), 1.58 (1H, m, 20-H), 1.60 (1H, m,
11α-H), 1.72 (1H, m, 4α-H), 1.72 (1H, m, 9-H), 1.78–1.90 (2H,
6-H2), 1.81 (1H, m, 2α-H), 1.85 (1H, m, 1β-H), 1.90 (1H, m,
16-H), 2.03 (1H, ddd, J 12.2, 3.4 and 3.4 Hz, 12β-H), 2.31 (1H,
ddd, J 15.6, 6.8 and 3.4 Hz, 16-H), 3.60 (1H, tt, J 11.2 and 4.4
Hz, 3-H), 5.50 (1H, dd, J 3.4 and 2.0 Hz, 15-H), 5.75 (1H, m,
7-H); δC (150 MHz) 12.37 (C-19), 16.50 (C-18), 18.92 (C-21),
20.97 (C-11), 22.55 (C-26), 22.81 (C-27), 23.75 (C-23), 28.01
(C-25), 30.28 (C-6), 31.53 (C-2), 33.88 (C-10), 34.12 (C-20),
35.16 (C-16), 36.06 (C-22), 36.80 (C-1), 37.92 (C-4), 39.50
(C-24), 39.68 (C-5), 40.16 (C-12), 46.47 (C-13), 49.76 (C-9),
58.70 (C-17), 70.95 (C-3), 119.45 (C-15), 120.17 (C-7), 134.51
(C-8), 152.07 (C-14); EI-MS m/z 384 (Mϩ, 39%), 366 (Mϩ Ϫ
H2O, 74), 351 (Mϩ Ϫ H2O Ϫ CH3, 53), 271 (Mϩ Ϫ C8H17, 46),
253 (Mϩ Ϫ C8H17 Ϫ H2O, 100); HR-EI-MS m/z Mϩ: Found,
384.3406. Calc. for C27H44O, 384.3392.
With Dowex resin. Purchased resin Dowex-50W-X2 (Hϩ
form), was used after being successively washed with dist. H2O,
2 M NaOH, dist. H2O and 2 M HCl, then dist. H2O until the
filtrate was neutral, and finally washed with ethanol. A hetero-
geneous mixture of 1 (250 mg, 0.65 mmol) and Dowex resin
(500 mg) in EtOH (10 cm3) was stirred at 70 ЊC for 51 h. The
resin was filtered off through a glass filter and washed with
Et2O. The filtrate was concentrated in vacuo, and the residue
was subjected to the purification and separation procedure as
described above. Flash chromatography gave a 55:22:11:12
dienol mixture (239 mg, 96%) of 7a, 7b, 8a and 2a. HPLC gave
7b (52 mg, 21%), 7a (119 mg, 48%), 2a (27 mg, 11%) and 8a
(17 mg, 6.8%), successively.
5α-Cholesta-14,16-dien-3β-ol 10a. Colorless wax; δH (600
MHz) 0.60 (1H, ddd, J 12.2, 11.2 and 3.9 Hz, 9-H), 0.84 (3H, d,
J 6.8 Hz, 26-H3), 0.85 (3H, d, J 6.8 Hz, 27-H3), 0.86 (1H, m,
12α-H), 0.92 (3H, s, 19-H3), 0.97 (3H, s, 18-H3), 0.97 (1H, ddd,
J 13.2, 13.2 and 3.4 Hz, 1α-H), 1.05 (3H, d, J 6.3 Hz, 21-H3),
1.14 (1H, m, 5-H), 1.15 (2H, 24-H2), 1.26 (2H, 23-H2), 1.33 and
1.59 (each 1H, each m, 4-H2), 1.38 and 1.50 (each 1H, each m,
22-H2), 1.39 (1H, m, 11β-H), 1.40 and 1.48 (each 1H, each m,
6-H2), 1.45 and 1.80 (each 1H, each m, 2-H2), 1.48 and 1.94
(each 1H, each m, 7-H2), 1.50 (1H, m, 25-H), 1.60 (1H, m,
11α-H), 1.75 (1H, ddd, J 13.2, 3.4 and 3.4 Hz, 1β-H), 1.96 (1H,
m, 12β-H), 2.21 (1H, m, 8-H), 2.29 (1H, m, 20-H), 3.59 (1H, tt,
J 11.2 and 4.9 Hz, 3-H), 5.81 (1H, dd, J 2.0 and 2.0 Hz, 15-H),
5.96 (1H, d, J 2.0 Hz, 16-H); δC (150 MHz) 12.42 (C-19), 18.72
(C-18), 21.32 (C-11), 22.66 (C-26), 22.66 (C-27), 22.99 (C-21),
25.56 (C-23), 27.92 (C-25), 28.47 (C-6), 29.72 (C-7), 31.46
(C-2), 31.46 (C-20), 35.55 (C-8), 35.96 (C-10), 36.16 (C-12),
37.38 (C-1), 38.13 (C-4), 38.25 (C-22), 39.20 (C-24), 44.71
(C-5), 53.57 (C-13), 57.47 (C-9), 71.25 (C-3), 117.62 (C-15),
120.98 (C-16), 158.79 (C-14), 164.10 (C-17); EI-MS m/z 384
(Mϩ, 13%), 366 (Mϩ Ϫ H2O, 30), 299 (Mϩ Ϫ C6H13, 45),
Campesta-8,14-dienol 5
Campesta-5,7-dienol 3 was prepared from campesterol supplied
by Tama Biochemical Co., Ltd. according to a method reported
by Kircher and Rosenstein.8 A solution of 3 (7.2 mg, 0.018
mmol) and 36% HCl (0.025 cm3) in EtOH (0.5 cm3) was
refluxed for 3 h. After the same work-up as that for treatment
of 1 with 36% HCl and flash chromatography using hexane–
AcOEt (4:1), the product mixture was subjected to HPLC
using hexane–AcOEt (4:1) as a mobile phase. The elution at Rt
32.0 min gave campesta-8,14-dienol 5 (3.8 mg, 53%): colorless
scales, mp 111–113 ЊC (MeOH); δH (300 MHz) 0.78 (3H, d,
J 6.9 Hz, 26-H3), 0.81 (3H, d, J 7.0 Hz, 27-H3), 0.82 (3H, s,
18-H3), 0.86 (3H, d, J 6.8 Hz, 28-H3), 0.93 (3H, d, J 6.2 Hz,
21-H3), 0.99 (3H, s, 19-H3), 3.63 (1H, tt, J 10.9 and 4.7 Hz,
3-H), 5.36 (1H, br s, 15-H); δC (75 MHz) 15.38, 15.70, 18.26,
18.37, 18.89 and 20.19 (C-18, -19, -21, -26, -27 and -28), 21.88,
25.32, 26.61, 30.21, 31.74, 33.67, 35.35, 35.92, 36.98 and 38.35
(C-1, -2, -4, -6, -7, -11, -12, -16, -22 and -23), 32.46, 34.17, 38.90,
40.98 and 57.22 (C-5, -17, -20, -24 and -25), 36.57 and 45.08
J. Chem. Soc., Perkin Trans. 1, 2000, 1697–1703
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(C-10 and -13), 71.03 (C-3), 117.42 (C-15), 123.12 (C-8),
140.86 (C-9), 151.14 (C-14); EI-MS m/z 398 (Mϩ, 100%), 380
(Mϩ Ϫ H2O, 73), 365 (Mϩ Ϫ H2O Ϫ CH3, 68), 271 (Mϩ Ϫ
C9H19, 17), 253 (Mϩ Ϫ C9H19 Ϫ H2O, 20); HR-EI-MS m/z
(Mϩ): Found, 398.3551. Calc. for C28H46O, 398.3549.
23.75 (C-23), 23.91 (C-7), 25.95 (C-15), 26.77 (C-6), 27.10
(C-16), 28.02 (C-25), 34.44 (C-20), 35.87 (C-9), 35.96 (C-22),
36.21 (C-1), 36.42 (C-10), 37.15 (C-12), 37.72 (C-2), 39.55
(C-24), 42.13 (C-4), 42.80 (C-13), 44.51 (C-5), 56.99 (C-17),
125.40 (C-8), 143.35 (C-14), 213.18 (C-3); EI-MS m/z 384 (Mϩ,
100%), 369 (Mϩ Ϫ CH3, 26), 351 (65), 271 (Mϩ Ϫ C8H17, 30);
HR-EI-MS m/z (Mϩ): Found, 384.3391. Calc. for C27H44O,
384.3392.
Stigmasta-8,14-dien-3ꢀ-ol 6
Stigmasta-5,7-dien-3β-ol 4 was prepared from stigmast-5-en-
3β-ol supplied by Tama Biochemical Co., Ltd. according to a
method reported by Kircher.9 A solution of 4 (13.4 mg, 0.032
mmol) and 36% HCl (0.05 cm3) in EtOH (1 cm3) was refluxed
for 3 h. The same work-up and purification procedure as
described above gave stigmasta-8,14-dien-3β-ol 6 (6.7 mg,
50%): Rt 30.0 min; colorless scales, mp 105–106 ЊC (MeOH);
δH (300 MHz) 0.82 (3H, s, 18-H3), 0.82 and 0.84 (each 3H, each
d, each J 7.2 Hz, 26- and 27-H3), 0.95 (3H, d, J 6.1 Hz, 21-H3),
0.99 (3H, s, 19-H3), 0.85 (3H, t, J 6.8 Hz, 29-H3), 3.63 (1H, tt,
J 10.8 and 4.8 Hz, 3-H), 5.36 (1H, br s, 15-H); δC (75 MHz)
11.98, 15.70, 18.37, 18.96, 19.03 and 19.78 (C-18, -19, -21, -26,
-27 and -29), 21.87, 23.04, 25.31, 26.00, 26.61, 31.73, 33.90,
35.35, 35.94, 36.97 and 38.35 (C-1, -2, -4, -6, -7, -11, -12, -16,
-22, -23 and -28), 29.15, 34.40, 40.98, 45.89 and 57.15 (C-5, -17,
-20, -24 and -25), 36.56 and 45.07 (C-10 and -13), 71.03 (C-3),
117.42 (C-15), 123.11 (C-8), 140.87 (C-9), 151.14 (C-14); EI-MS
m/z 412 (Mϩ, 100%), 394 (Mϩ Ϫ H2O, 72), 379 (Mϩ Ϫ H2O Ϫ
CH3, 64), 271 (Mϩ Ϫ C10H21, 18), 253 (Mϩ Ϫ C10H21 Ϫ H2O,
19); HR-EI-MS m/z (Mϩ): Found, 412.3714. Calc. for C29H48O,
412.3705.
5β-Cholest-14-en-3-one 12. Colorless amorphous powder;
δH (600 MHz) 0.870 (3H, d, J 6.8 Hz, 26-H3), 0.872 (3H, d, J 6.8
Hz, 27-H3), 0.92 (3H, d, J 6.0 Hz, 21-H3), 0.93 (3H, s, 18-H3),
1.03 (3H, s, 19-H3), 1.05 and 1.38 (each 1H, each m, 22-H2),
1.08–1.22 (2H, 24-H2), 1.17 and 1.37 (each 1H, each m, 23-H2),
1.32 (1H, ddd, J 13.2, 13.2 and 3.4 Hz, 12α-H), 1.33 and 1.94
(each 1H, each m, 6-H2), 1.42 (1H, m, 1β-H), 1.42 and 1.50
(each 1H, each m, 11-H2), 1.47 (1H, m, 7α-H), 1.53 (1H, m,
9-H), 1.53 (1H, m, 25-H), 1.55 (1H, m, 17-H), 1.60 (1H, m,
20-H), 1.73 (1H, m, 7β-H), 1.82 (1H, m, 5-H), 1.93 and 2.31
(each 1H, each m, 16-H2), 2.02 (1H, ddd, J 14.7, 4.9 and 2.4 Hz,
4β-H), 2.06 (1H, ddd, J 13.2, 2.4 and 2.4 Hz, 12β-H), 2.08 (1H,
m, 1α-H), 2.12 (1H, m, 8-H), 2.16 (1H, m, 2β-H), 2.34 (1H,
ddd, J 14.7, 14.7 and 5.4 Hz, 2α-H), 2.68 (1H, dd, J 14.7
and 14.2 Hz, 4α-H), 5.20 (1H, m, 15-H); δC (150 MHz) 16.88
(C-18), 18.95 (C-21), 22.04 (C-11), 22.40 (C-19), 22.57 (C-26),
22.80 (C-27), 23.57 (C-7), 23.68 (C-23), 26.30 (C-6), 28.04
(C-25), 33.89 (C-20), 34.83 (C-8), 35.08 (C-10), 35.65 (C-16),
36.06 (C-22), 36.85 (C-1), 37.28 (C-2), 39.53 (C-24), 40.35
(C-9), 42.26 (C-4), 42.43 (C-12), 44.27 (C-5), 47.21 (C-13),
58.82 (C-17), 117.61 (C-15), 154.90 (C-14), 213.19 (C-3);
EI-MS m/z 384 (Mϩ, 9%), 271 (Mϩ Ϫ C8H17, 100), 253 (13);
HR-EI-MS m/z (Mϩ): Found, 384.3386. Calc. for C27H44O,
384.3392.
Treatment of dienols, 7a,b and 2a,b, with 36% HCl at reflux
temperature
Treatment of 7a. A solution of 7a (15 mg, 0.039 mmol)
and 36% HCl (0.05 cm3) in EtOH (1.0 cm3) was refluxed for
3 h. After the same work-up as that for treatment of 1 with
36% HCl, flash chromatography using hexane–AcOEt (4:1) as
an eluent gave a 7:90:3 mixture of 7a, 2a and 10a (14 mg,
93%).
14-epi-5β-Cholest-8-en-3-one 13. Colorless amorphous
powder; δH (600 MHz) 0.864 (3H, d, J 6.4 Hz, 26-H3), 0.867
(3H, d, J 6.8 Hz, 27-H3), 0.90 (3H, s, 18-H3), 0.93 (3H, d, J 6.4
Hz, 21-H3), 1.01 and 1.37 (each 1H, each m, 22-H2), 1.07 and
1.98 (each 1H, each m, 15-H2), 1.14 (3H, s, 19-H3), 1.14 (2H,
24-H2), 1.15 and 1.37 (each 1H, each m, 23-H2), 1.27 and 1.75
(each 1H, each m, 16-H2), 1.37 (1H, m, 12β-H), 1.37 (1H, m,
17-H), 1.40 and 1.97 (each 1H, each m, 6-H2), 1.49 (1H,
m, 20-H), 1.52 (1H, m, 25-H), 1.58 (1H, m, 1β-H), 1.62 (1H, m,
12α-H), 1.73 and 2.15 (each 1H, each m, 7-H2), 1.79 (1H, dd,
J 8.3 and 8.3 Hz, 14-H), 1.85 and 2.00 (each 1H, each m,
11-H2), 1.92 (1H, m, 5-H), 2.09 (1H, m, 1α-H), 2.11 (1H, m,
2α-H), 2.22 (1H, m, 2β-H), 2.25 (1H, ddd, J 14.2, 5.4 and 2.0
Hz, 4β-H), 2.33 (1H, ddd, J 14.2, 9.8 and 1.0 Hz, 4α-H); δC (150
MHz) 19.84 (C-21), 21.17 (C-11), 22.57 (C-26), 22.80 (C-27),
23.11 (C-18), 24.16 (C-6), 24.42 (C-23), 26.10 (C-7), 26.68
(C-19), 28.01 (C-25), 28.50 (C-16), 30.67 (C-15), 33.55 (C-20),
35.64 (C-22), 35.68 (C-1), 35.68 (C-12), 36.69 (C-10), 38.48
(C-2), 39.50 (C-24), 41.21 (C-13), 42.72 (C-5), 42.89 (C-4),
52.22 (C-17), 54.11 (C-14), 130.15 (C-9), 132.63 (C-8), 213.44
(C-3); EI-MS m/z 384 (Mϩ, 100%), 369 (Mϩ Ϫ CH3, 44), 351
(48), 314 (95), 271 (Mϩ Ϫ C8H17, 46); HR-EI-MS m/z (Mϩ):
Found, 384.3396. Calc. for C27H44O, 384.3392.
Treatment of 7b. A solution of 7b (55 mg, 0.14 mmol) and
36% HCl (0.11 cm3) in EtOH (2.2 cm3) was refluxed. After 3 h,
1
the H NMR spectrum showed that ca. 45% of the starting
material 7b still remained; thus, the reaction was further con-
tinued for 8 h. After the same work-up as that for treatment of 1
with 36% HCl at refluxing temperature, flash chromatography
using hexane–AcOEt (4:1) as an eluent gave an enone mixture
consisting of 11–13 (21 mg, 38%) and a 6:94 mixture of 7b and
2b (32 mg, 58%). The enone mixture was subjected to HPLC
using hexane–AcOEt (100:1) as a mobile phase, affording a
88:12 mixture (Rt 13.4 min, 7.8 mg, 14%) of 5β-cholest-14-en-
3-one 12 and 14-epi-5β-cholest-8-en-3-one 13, and 5β-cholest-
8(14)-en-3-one 11 (Rt 14.0 min, 12 mg, 22%). This mixture was
separated by HPLC using hexane–iPrOH (100:1) to give 12
(Rt 8.6 min, 5.0 mg), 13 (Rt 9.2 min, 1.1 mg) and their mixture
(4.2 mg).
5β-Cholest-8(14)-en-3-one 11. Colorless foam; δH (600 MHz)
0.869 (3H, d, J 6.8 Hz, 26-H3), 0.872 (3H, d, J 6.8 Hz, 27-H3),
0.87 (3H, s, 18-H3), 0.90 (3H, s, 19-H3), 0.95 (3H, d, J 6.4 Hz,
21-H3), 1.08 and 1.39 (each 1H, each m, 22-H2), 1.14 (2H,
24-H2), 1.15 and 1.36 (each 1H, each m, 23-H2), 1.16 (1H, m,
17-H), 1.21 (1H, m, 12α-H), 1.28 and 1.84 (each 1H, each m,
6-H2), 1.40 and 1.85 (each 1H, each m, 16-H2), 1.47 (1H, m,
20-H), 1.49 (1H, ddd, J 14.2, 14.2 and 4.9 Hz, 1β-H), 1.53 (1H,
m, 25-H), 1.58 (2H, 11-H2), 1.83 (1H, m, 5-H), 1.93 (1H, m,
7α-H), 2.01 (1H, m, 12β-H), 2.02 (1H, m, 1α-H), 2.08 (1H, ddd,
J 14.7, 3.9 and 2.4 Hz, 4β-H), 2.21 and 2.27 (each 1H, each m,
15-H2), 2.24 (1H, m, 2β-H), 2.26 (1H, m, 7β-H), 2.42 (1H, ddd,
J 14.7, 14.2 and 5.4 Hz, 2α-H), 2.52 (1H, m, 9-H), 2.82 (1H, dd,
J 14.7 and 14.2 Hz, 4α-H); δC (150 MHz) 18.14 (C-18), 19.11
(C-21), 19.84 (C-11), 22.57 (C-26), 22.80 (C-27), 22.99 (C-19),
Treatment of 2a. A solution of 2a (15 mg, 0.039 mmol) and
36% HCl (0.05 cm3) in EtOH (1.0 cm3) was refluxed for 3 h.
After the same work-up as that for treatment of 1 with 36%
HCl, flash chromatography using hexane–AcOEt (4:1) as
eluent gave a 6:92:2 mixture of 7a, 2a and 10a (14 mg, 93%).
Treatment of 2b. A solution of 2b (35 mg, 0.091 mmol) and
36% HCl (0.07 cm3) in EtOH (1.4 cm3) was refluxed for 8 h. The
same work-up and column chromatography as those for treat-
ment 7b gave an enone mixture consisting of 11–13 (11 mg,
31%) and a 2:98 mixture of 7b and 2b (19 mg, 54%). The HPLC
of the enone mixture using hexane–AcOEt (100:1) as eluent
gave a 86:14 mixture of 12 and 13 (4.7 mg, 13%) and 11 (5.6
mg, 16%).
1702
J. Chem. Soc., Perkin Trans. 1, 2000, 1697–1703

View Article Online
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Acknowledgements
We gratefully acknowledge generous gifts of campesterol and
stigmast-5-en-3β-ol from Tama Biochemical Co., Ltd. This
work was supported in part by Grant-in-Aid for Scientific
Research (C) to H. Seto (No. 09672183) and Scientific Research
(B) to S. Fujioka (No. 10460050) from the Ministry of
Education, Science, Sports and Culture of Japan.
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