C-24 stereochemistry of marine sterols: (22E)-24-ethyl-24-methylcholesta-5, 22-dien-3β-ol and 24-ethyl-24-methylcholest-5-en-3β-ol

Article abstract of DOI:10.1590/S0103-50532011000500026

The C-24 configurations of (22E)-24-ethyl-24-methylcholesta-5,22-dien- 3β-ol (1) and 24-ethyl-24-methylcholest-5-en-3β-ol (2), isolated from the Colombian Caribbean sponge Topsentia ophiraphidites, were determined to be R and S, respectively, by comparing their NMR data with those of stereodefined (24R)-and (24S)-samples that were synthesized in routes involving the orthoester Claisen rearrangement of Δ²³-22-allylic alcohols. This is the first synthetic study where the Claisen rearrangement is used to introduce a C-24 quaternary center in a stereospecific manner with acceptable yield. X-ray analysis of 1 conirmed these stereochemical assignments.

Full text of DOI:10.1590/S0103-50532011000500026

J. Braz. Chem. Soc., Vol. 22, No. 5, 997-1004, 2011.
Printed in Brazil - ©2011 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Short Report
S
C-24 Stereochemistry of Marine Sterols: (22E)-24-Ethyl-24-methylcholesta-5,22-dien-3b-ol
and 24-Ethyl-24-methylcholest-5-en-3b-ol
Shizue Echigo,a Leonardo Castellanos,b Carmenza Duque,*,b Hidehiro Uekusa,a
Noriyuki Haraa and Yoshinori Fujimotoa
aDepartment of Chemistry and Materials Science, Tokyo Institute of Technology,
Meguro, 152-8551, Tokyo, Japan
bDepartamento de Química, Universidad Nacional de Colombia,
AA 14490, Bogotá, Colombia
As configurações dos carbonos C-24 nos compostos (22E)-24-etil-24-metilcolesta-5,22-
dien-3b-ol (1) e 24-etil-24-metilcolest-5-en-3b-ol (2), isolados da esponja do caribe colombiano
Topsentia ophiraphidites foram determinadas como R e S, respectivamente, com base na comparação
dos seus dados de RMN com aqueles de amostras de configurações já definidas 24R e 24S, as
quais foram sintetizadas em rotas envolvendo ortoésteres provenientes do rearranjo de Claisen de
álcoois D23-22 alílicos. Este é primeiro estudo de síntese, em que o rearranjo de Claisen é utilizado
para introduzir um centro quaternário em C-24, de maneira estereoespecífica e com rendimento
aceitável. Análises por difração de raio X de 1 confirmaram essas atribuições estereoquímicas.
The C-24 configurations of (22E)-24-ethyl-24-methylcholesta-5,22-dien-3b-ol (1) and 24-ethyl-
24-methylcholest-5-en-3b-ol (2), isolated from the Colombian Caribbean sponge Topsentia
ophiraphidites, were determined to be R and S, respectively, by comparing their NMR data with
those of stereodefined (24R)- and (24S)-samples that were synthesized in routes involving the
orthoester Claisen rearrangement of D23-22-allylic alcohols. This is the first synthetic study where
the Claisen rearrangement is used to introduce a C-24 quaternary center in a stereospecific manner
with acceptable yield. X-ray analysis of 1 confirmed these stereochemical assignments.
Keywords: 24-ethyl-24-methylcholesterol, 24-ethyl-24-methylcholesta-5,22-dien-3b-ol,
Topsentia ophiraphidites, marine sterol, orthoester Claisen rearrangement
Introduction
24-ethyl-24-methylcholestanols.4 Recently, sterol 1 was
isolated from the marine sponge Psammocinia bulbosa
and its C-24 configuration was inferred as R without any
discussion on the stereochemistry.5 Furthermore, 24-ethyl-
24-methylcycloartane triterpenoids were isolated from
higher plants, Coelogyne uniflora (Orchidaceae)6 and
Skimmia wallichii (Rutaceae).7 In a previous paper we
reported the isolation of a number of multiply alkylated
sterols, including compounds 1 and 2, from the Colombian
Caribbean sponge, Topsentia ophiraphidites.8 In particular,
compound 1 was a major sterol constituent (ca. 40% of the
total sterol). With sizeable amounts of compounds 1 and
2 in hand, we undertook a study to elucidate their C-24
configurations. In this paper we report a stereochemical
determination at C-24 of 24-ethyl-24-methylsterols 1
and 2 (Figure 1), along with the 13C NMR data for the
stereodefined C-24 epimers of 1 and 2.
It is well known that marine organisms such as sponges
and corals are rich sources of uniquely modified sterols
in the side-chain.1 Among these sterols, C-24 dialkylated
sterols are relatively limited. Djerassi’s group reported the
isolation of (22E)-D22-24-ethyl-24-methylcholesterol (1),
(22E)-D22-24-ethyl-24-methylcholestanol, 24-ethyl-24-
methylcholesterol (2), and (22E)-D22,25-24-ethyl-24-
methylcholesterol from a sponge, Pseudoaxinyssa sp.2,3
Although they briefly mentioned the C-24 configurations
of these sterols, details of the stereochemical assignment
have not been reported.3 They also reported the preparation
of stereochemically unestablished C-24 diastereomers of 12
and stereochemically defined, C-24 diastereomers of
*e-mail: cduqueb@unal.edu.co, cduqueb@etb.net.co

998
C-24 Stereochemistry of Marine Sterols: (22E)-24-Ethyl-24-methylcholesta-5,22-dien-3b-ol
J. Braz. Chem. Soc.
29
30
28
27
24
26
HO
HO
1: 24ξ (natural)
1a: 24S
2: 24ξ (natural)
2a: 24R
C24
C24
1b: 24R
2b: 24S
C24
C24
Figure 1. Chemical structures of C-24 epimers of (22E)-24-ethyl-24- methylcholesta- 5,22-dien-3b-ol (1) and 24-ethyl-24-methylcholest-5-en-3b-ol (2).
C-24 configurations of 1 and 2 isolated from Topsentia ophiraphidites were not defined.
Results and Discussion
4a was assumed on the basis of the preferred transition state
conformation for the rearrangement reaction (Figure S1)
that is analogous to the conformation well established for
the 3,3-sigmatropic reactions of steroidal 23-ene-22-allylic
alcohols.16 The 24S configuration of 4a was ascertained by
the eventual conversion of 4a to the (24S)-diastereomer 1a
(vide infra). The product mixture containing 4a was reduced
with LiAlH4 and then the resulting alcohol mixture was
acetylated to facilitate the separation. Hydride reduction
of the purified acetate gave the primary alcohol 5a in 24%
yield from 3a. Deoxygenation of the C-29 hydroxy group
of 5a was achieved by super-hydride reduction of the
corresponding mesylate 6a to give the i-methyl ether 7a.
Removal of the i-methyl ether of compound 7a furnished
the (24S)-diastereomer 1a. Hydrogenation of 7a gave the
saturated (24R)-diastereomer 8a which was converted to the
(24R)-diastereomer 2a upon deprotection of the i-methyl
ether. Application of the same sequence of reactions to the
(22S)-allylic alcohol 3b, furnished the (24R)-diastereomer
1b via the intermediates 4b, 5b, 6b and 7b. Hydrogenation
of 7b gave 8b, which furnished the (24S)-diastereomer 2b
by regeneration of the D5-3b-hydroxy system.
In order to determine the C-24 configuration of 1
and 2, we decided to synthesize the stereochemically
defined (24S)- and (24R)- (22E)-D22-24-ethyl-24-
methylcholesterols (1a and 1b), and (24R)- and (24S)-
24-ethyl-24-methylcholesterols (2a and 2b). A method
based on Claisen rearrangement appeared to be attractive
for the stereoselective construction of the C-24 dialkyl
side chains, although the application of this method to the
construction of a C-24 quaternary center is unprecedented.
The Johnson orthoester procedure and the Ireland variant
of Claisen rearrangement are frequently employed for the
synthesis of stereo-defined C-24 mono-alkylated sterol
side-chains.9-11 We also utilized the orthoester Claisen
rearrangement for the stereoselective synthesis of (24R)-
and (24S)- (22E)-24-isopropenyl-22-dehydrocholesterol
and 24-isopropenylcholesterol, another multiply alkylated
sterol of T. ophiraphidites.12
The synthetic route to 1a and 1b, and 2a and 2b is shown
in Figure 2. The required starting materials, (22R)-allylic
alcohol 3a and (22S)-allylic alcohol 3b, for the orthoester
Claisen rearrangement were obtained stereoselectively
from the corresponding enone13-15 by reduction using
DIBAL-H and L-selectride, respectively.14 Treatment of 3a
with triethyl orthoacetate and propionic acid in refluxing
xylene gave a mixture of the desired rearranged product
4a (not isolated as such) and by-products containing the
22-propionate ester of 3a. The C-24 epimer of 4a was not
formed in the rearrangement reaction, since careful TLC
and NMR analysis revealed that compounds 4a and 5a were
free from 4b and 5b, respectively. The 24S configuration of
The 1H and 13C NMR spectroscopic data of the synthetic
diastereomers 1a and 1b are listed in Tables 1 and 2,
respectively, together with those of the sterol 1 isolated
from T. ophiraphidites. The 1H and 13C signals of 1a and
1b were unambiguously assigned on the basis of 2-D (H-H
COSY, HMQC and HMBC) NMR spectra. For example,
in the HMQC spectrum of 1a, H-22 (d 5.058) and H-23
(d 5.144) were correlated with C-22 (d 134.9) and C-23
(d 134.6), respectively, while in the HMBC spectrum,
correlation peaks were observed from 21-H3 (d 1.017) to

Vol. 22, No. 5, 2011
Echigo et al.
999
HO
HO
R
S
OMe
OMe
3a
3b
i
i
CO2Et
CO2Et
S
R
4a
4b
ii-v
ii
CH2OR
CH2OR
5a: R = H
6a: R = Ms
5b: R = H
6b: R = Ms
vi
vi
vii
vii
ix
ix
R
S
S
R
8a
7a
7b
8b
viii
viii
viii
viii
2a
1a
1b
2b
Reagents: i) MeC(OEt)3, propionic acid, ii) LiAlH4, iii) Ac2O, Py, iv) separation, v) LiAlH4, vi) MsCl, Py,
vii) Super hydride, viii) p-TsOH/aq. dioxane, ix) H2, Pd/C.
Figure 2. Synthesis of sterols 1a, 1b, 2a and 2b.
C-17, C-20 and C-22, H3-26/H3-27 (d 0.785 and 0.803) to
C-24 and C-25, H3-29 (d 0.739) to C-24 and C-28, and H3-
30 (d 0.795) to C-23, C-24, C-25 and C-28. The 1H NMR
data of the epimers were closely similar to each other.
However, careful comparison of the spectra revealed that
1a or 1b showed an appreciable difference in the chemical
shifts of 29-H3 (Dd 0.011 ppm), and natural 1 seemed to be
identical with the (24R)-diastereomer 1b in this regard. As
shown in Table 2, the 13C data were much more diagnostic
for the identification of the diastereomers 1a and 1b. The
C-24 signal showed the largest difference (0.19 ppm)
between the diastereomers and the C-29 and C-28 signals

1000
C-24 Stereochemistry of Marine Sterols: (22E)-24-Ethyl-24-methylcholesta-5,22-dien-3b-ol
J. Braz. Chem. Soc.
Table 1. 1H NMR spectroscopic data (400 MHz, in CDCl3) for compounds 1, 1a, 1b, 2, 2a and 2b
No.
3
natural-1
3.53 (m)
(24S)-1a
3.53 (m)
(24R)-1b
3.53 (m)
natural-2
3.52 (m)
5.35 (m)
0.678 (s)
1.009 (s)
(24R)-2a
3.52 (m)
5.35 (m)
0.678 (s)
1.008 (s)
(24S)-2b
3.52 (m)
5.35 (m)
0.678 (s)
1.008 (s)
6
5.35 (m)
5.35 (m)
5.35 (m)
18
19
20
21
22
23
26
27
29
30
0.698 (s)
0.698 (s)
0.699 (s)
1.010 (s)
1.011 (s)
1.011 (s)
2.05 (tq, 8.4, 6.1)
1.018 (d, 6.1)
2.05 (tq, 8.4, 6.1)
1.017 (d, 6.1)
2.05 (tq, 8.3, 6.4)
1.019 (d, 6.4)
0.925 (d, 6.6)
0.923 (d, 6.6)
0.925 (d, 6.6)
5.054 (dd, 15.7, 8.4) 5.058 (dd, 16.0, 8.4) 5.060 (dd, 15.9, 8.3)
5.142 (d, 15.8)
0.779 (d, 7.0)a
0.801 (d, 7.1)a
0.748 (t, 7.9)
0.796 (s)
5.144 (d, 16.0)
0.785 (d, 7.0)a
0.803 (d, 7.0)a
0.739 (t, 7.6)
0.795 (s)
5.146 (d, 15.9)
0.779 (d, 7.1)a
0.802 (d, 7.1)a
0.750 (t, 7.9)
0.799 (s)
0.793 (d, 7.1)a
0.796 (d, 7.1)a
0.755 (t, 7.6)
0.674 (s)
0.786 (d, 7.1)a
0.797 (d, 6.8)a
0.749 (t, 7.6)
0.674 (s)
0.793 (d, 6.8)a
0.796 (d, 7.1)a
0.755 (t, 7.6)
0.675 (s)
aAn upfield resonance was tentatively assigned to H3-26.
Table 2. 13C NMR spectroscopic data (100 MHz, in CDCl3) for compounds 1, 1a, 1b, 2, 2a and 2b
No.
1
natural-1
37.3
31.7
71.8
42.3
140.8
121.7
31.9
31.9
50.2
36.5
21.1
39.7
42.2
56.9
24.3
28.9
56.1
12.1
19.4
40.8
21.3
134.8
134.7
35.7
41.1
17.3a
17.9a
31.8
8.7
(24S)-1a
37.3
31.7
71.8
42.3
140.8
121.7
31.9
31.9
50.2
36.5
21.1
39.7
42.2
56.9
24.4
28.9
56.1
12.1
19.4
40.8
21.4
134.9
134.6
35.9
41.1
17.3a
18.0a
31.6
8.5
(24R)-1b
37.3
31.7
71.8
42.3
140.8
121.7
31.9
31.9
50.2
36.5
21.1
39.7
42.2
56.9
24.3
28.9
56.1
12.1
19.4
40.8
21.3
134.8
134.7
35.7
41.1
17.3a
17.9a
31.7
8.6
natural-2
37.3
31.7
71.8
42.3
140.8
121.7
31.9
31.9
50.1
36.5
21.1
39.7
42.3
56.8
24.3
28.3
56.0
11.8
19.4
36.6
18.9
29.0
32.2
36.8
33.3
17.1a
17.2a
28.6
7.9
(24R)-2a
37.3
31.7
71.8
42.3
140.8
121.7
31.9
31.9
50.1
36.5
21.1
39.8
42.3
56.8
24.3
28.3
56.0
11.8
19.4
36.7
18.9
29.1
32.2
36.8
33.3
17.1a
17.2a
28.7
8.0
(24S)-2b
37.3
31.7
71.8
42.3
140.8
121.7
31.9
31.9
50.2
36.5
21.1
39.8
42.3
56.8
24.3
28.3
56.0
11.8
19.4
36.6
18.9
29.1
32.3
36.8
33.3
17.2a
17.2a
28.6
7.9
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
18.3
18.3
18.3
20.4
20.3
20.4
aAn upfield resonance was tentatively assigned to C-26 (The C-26 and C-27 signals were not necessarily correlated to the H3-26 and H3-27 signals because
of very close shifts of these signals).

Vol. 22, No. 5, 2011
Echigo et al.
1001
were different by more than 0.10 ppm, and this much
difference would be sufficient for the identification of the
diastereomers. The 13C data of natural 1 met with those of
the (24R)-diastereomer 1b. It was, therefore, concluded
that the sterol 1 isolated from T. ophiraphidites has 24R
configuration.Thesterol1isolatedfromPseudoaxinyssasp.3
was also identified as the (24R)-diastereomer 1b, since
conformation (Figure S1) proposed for the orthoester
Claisen rearrangement employed for the stereoselective
creation of the C-24 quaternary center.
Conclusions
The present study established the 24R configuration
of the marine sterol 1, isolated from T. ophiraphidites,
Pseudoaxinyssa sp., and Psammocinia bulbosa, and the
24S configuration of the marine sterol 2, isolated from
T. ophiraphidites and Pseudoaxinyssa sp. The C-24
configurations for the sterols 1 and 2, originally proposed
by Djerassi’s group,3 was confirmed with full experimental
details. The findings that sterols 1 and 2 possess the same
C-24 orientation (24aF for the methyl group) supported
the view that the sterol 2 is a biosynthetic precursor of
the corresponding D22-sterol 1 in sponges. Djerassi and
co-workers3 suggested that stigmasta-5,24-dien-3b-ol
could be a progenitor of the unique sterols, 1 and 2.
Stereochemical studies on the other stereochemically
undefined multiply-alkylated sterols isolated from
T. ophiraphidites are in progress in our laboratory.
1
the published H NMR data (adjusted by the addition of
0.005 ppm) was much closer to those of 1b, rather than
those of 1a. The sterol 1 isolated from P. bulbosa could be
assigned as the (24R)-diastereomer 1b, since the reported
13C shifts of C-24, C-28 and C-29 resembled the values of
1b rather than those of 1a.
The 1H and 13C NMR spectroscopic data of the sterol 2
isolated from T. ophiraphidites and the synthetic
24-diasteromers 2a and 2b are listed in Tables 1 and 2,
1
respectively. The H and 13C signals of 2a and 2b were
unambiguously assigned as described for 1a and 1b. For
example, in the HMBC spectrum of 2a, H3-21 (d 0.923)
was correlated with C-17, C-20 and C-22, H3-26/H3-27
(d 0.786 and 0.797) with C-24 and C-25, H3-29 (d 0.749)
with C-24 and C-28, and H3-30 (d 0.674) with C-23, C-24,
C-25 and C-28. The 1H NMR signals, in particular H3-26
and H3-29, of 2 were in excellent agreement with those of
the (24S)-diastereomer 2b, but not with those of the (24R)-
diastereomer 2a. This was confirmed by comparison of the
13C NMR data. The C-30 signal showed the largest chemical
shift difference (Dd 0.11) between the diastereomers, and
the C-20 and C-28 signals exhibited 0.09 and 0.07 ppm
difference, respectively. Figure S2 illustrates a graphic
representation of the systematic 13C shift comparison. It is
obvious from Figure S2 that the sterol 2 is identical with
2b, rather than 2a. The minor deviation observed in some
signals of 2 and 2b could be within an error level in the
NMR measurements. Hence, the 24S configuration of the
sterol 2 isolated from T. ophiraphidites was unequivocally
established. Comparison of melting points and [a]D values
(see Experimental) also supported this stereochemical
assignment. The C-24 configuration of 24-ethyl-24-
methylcholesterol isolated from Pseudoaxinyssa sp.2
Experimental
Melting points were determined on aYazawa BY-1 hot-
stage micro melting point apparatus and are uncorrected.
NMR spectra were obtained on a JEOL JNM LA-400
1
(400 MHz for H, 100 MHz for 13C) spectrometer in
CDCl3 solution (4-10 mg sample per test tube) with
tetramethylsilane as an internal reference at ca. 25 oC. 13C
chemical shifts are referred to the solvent signal (d 77.00).
EI and FAB MS spectra were recorded with a JEOL JMS-
AX700 spectrometer. Optical rotations were measured on
a JASCO DIP-360 polarimeter.
(22R,23E)-6b-Methoxy-3a,5-cyclo-5a-ergost-23-en-22-ol(3a)
and (22S,23E)-6b- methoxy-3a,5-cyclo-5a-ergost-23-en-
22-ol (3b)
DIBAL-H (1.01 mol L-1 in toluene, 0.70 mL, 0.71 mmol)
was added to a solution of 6b-methoxy-3a,5-cyclo-5a-
ergost-23-en-22-one14 (490 mg, 1.15 mmol) in dry THF
(10 mL) at -78 oC under N2, and the mixture was stirred
at the same temperature for 30 min. Addition of ether
and extractive work-up gave a crude product which was
chromatographed on silica gel with hexane-EtOAc (15:1)
to give the (22R)-alcohol 3a (308 mg, 63%) as a white
solid, mp 128-129 oC. 1H NMR: d 0.44 (dd, J 8.0, 5.1 Hz,
4a-H), 0.65 (dd, J 5.1, 4.4 Hz, 4b-H), 0.73 (s, H3-18), 0.96
(d, J 6.4 Hz, H3-21), 1.01 (d, J 6.8 Hz, H3-26, H3-27), 1.03
(s, H3-19), 1.62 (d, J 1.2 Hz, H3-28), 2.23 (sep, J 6.8 Hz,
1
was also assigned as S, since the reported H NMR data
(recorded in benzene-d6) met with those (recorded in
benzene-d6) of (24S)-2b.
Finally, we carried out X-ray analysis for a crystalline
sample of the sterol 1 isolated from T. ophiraphidites. The
molecular structure and the C-24 stereochemistry of 1 were
established as shown in Figure S3. The 24R configuration
of 1 was inferred by assuming the absolute configuration
of natural sterols. The results proved the 24R configuration
of the rearranged product 4b (therefore, 4a should have
24S configuration) and correctness of the transition state

1002
C-24 Stereochemistry of Marine Sterols: (22E)-24-Ethyl-24-methylcholesta-5,22-dien-3b-ol
J. Braz. Chem. Soc.
H-25), 2.78 (brt, J 2.8 Hz, H-6), 3.33 (s, OMe), 4.47 (dd,
J 7.8, 1.2 Hz, H-22), 5.33 (brd, J 7.8 Hz, H-23). 13C NMR:
d 12.16, 12.29, 13.07, 14.16, 19.28, 21.27, 21.37, 21.46,
22.77, 24.18, 24.94, 27.92, 30.56, 33.34, 35.09, 35.23,
36.68, 40.20, 41.88, 42.73, 43.37, 47.97, 52.67, 56.38,
56.56, 70.54, 82.41, 124.85, 142.72. Analysis calc. for
C29H48O2: C, 81.25; H, 11.29. Found: C, 81.31; H, 11.56.
L-Selectride (1.0 mol L-1 in THF, 8.64 mL, 8.64 mmol)
was added to a solution of 6b-methoxy-3a,5-cyclo-5a-
ergost-23-en-22-one (1.23 g, 2.88 mmol) in dry THF
(25 mL) at –78 oC under N2, and the mixture was stirred
at the same temperature for 5 h. Addition of ether and
extractive work-up gave a crude product which was
chromatographed on silica gel with hexane-ether (4:1) to
give the (22S)-alcohol 3b (784 mg, 64%) as a colorless
oil. 1H NMR: d 0.43 (dd, J 8.1, 5.1 Hz, H-4a), 0.65 (dd,
J 5.1, 4.4 Hz, H-4b), 0.74 (s, H3-18), 1.00-1.02 (H3-21,
H3-26, H3-27, H3-19), 1.70 (brs, H3-28), 2.26 (sep, J 6.8 Hz,
H-25), 2.77 (brt, J 2.3 Hz, H-6), 3.32 (s, OMe), 4.42 (dd,
J 9.5, 3.7 Hz, H-22), 5.28 (brd, J 9.3 Hz, H-23). 13C NMR:
d 12.20, 12.53, 13.04, 14.07, 19.25, 21.35, 21.43, 21.50,
22.72, 24.35, 24.92, 27.87, 30.45, 33.32, 34.97, 35.22,
37.04, 40.15, 42.04, 43.03, 43.33, 47.97, 53.24, 56.08,
56.52, 69.55, 82.34, 120.42, 146.45. Analysis calc. for
C29H48O2: C, 81.25; H, 11.29. Found: C, 81.44; H, 11.51.
2.77 (m, H-6), 3.32 (s, OMe), 3.63 (m, H2-29), 5.14 (dd,
J 15.9, 8.6 Hz, H-22), 5.27 (d, J 15.9 Hz, H-23). 13C NMR:
d 12.40, 13.06, 17.20, 17.73, 18.41, 19.26, 21.01, 21.43,
22.72, 24.21, 24.92, 29.02, 30.45, 33.32, 35.05, 35.20, 37.13,
40.14, 40.30, 40.73, 41.85, 42.68, 43.35, 48.01, 56.08, 56.52,
56.57, 60.28, 82.37, 134.77, 135.27. HREIMS m/z: 456.3967
calc. for C31H52O2 [M+]. Found: 456.4011.
(22E,24S)-24-Ethyl-6b-methoxy-24-methyl-3a,5-cyclo-
5a-cholest-22-ene (7a)
MsCl (15 mL, 194 mmol) was added to a solution of 5a
(30.6 mg, 67 mmol) in pyridine (0.30 mL) and the mixture
was stirred for 10 min at room temperature. Extractive (ether)
work-up gave a crude mesylate 6a as an oil. A solution of
the mesylate in dry THF was treated with Super-hydride
(1.0 mol L-1 inTHF, 370 mL, 370 mmol) at room temperature
under N2 for 30 min. Extractive (ether) work-up gave a crude
product which was chromatographed on silica gel with
hexane-EtOAc (20:1) to yield the 22-ene 7a (14.9 mg, 50%)
as an oil. 1H NMR: d 0.43 (dd, J 8.1, 5.1 Hz, H-4a), 0.65
(dd, J 5.1, 4.4 Hz, H-4b), 0.73 (s, H3-18), 0.74 (d, J 7.4 Hz,
H3-29), 0.79 (d, J 7.6 Hz, H3-26), 0.80 (s, H3-30), 0.81 (d,
J 5.8 Hz, H3-27), 1.01 (d, J 6.6 Hz, H3-21), 1.03 (s, H3-19),
2.07 (m, H-20), 2.77 (brt, 2.8 Hz, H-6), 3.32 (s, OMe),
5.06 (dd, J 15.7, 8.4 Hz, H-22), 5.14 (d, J 15.9 Hz, H-23).
13C NMR: d 8.52, 12.46, 13.08, 17.33, 17.99, 18.25, 19.30,
21.36, 21.50, 22.77, 24.26, 24.97, 29.03, 30.50, 31.62, 33.36,
35.09, 35.29, 35.87, 40.22, 40.87, 41.09, 42.68, 43.41, 48.09,
56.28, 56.55, 56.67, 82.45, 134.59, 134.93. HREIMS m/z:
440.4018 calc. for C31H52O [M+]. Found: 440.4067.
(22E,24S)-24-(2-Hydroxyethyl)-6b-methoxy-24-methyl-
3a,5-cyclo-5a-cholest-22-ene (5a)
Asolutionofthe(22R)-alcohol3a(155mg, 0.362mmol),
triethyl orthoacetate (0.23 mL, 1.25 mmol) and propionic
acid (12 mL, 0.161 mmol) in xylene (5.0 mL) was heated at
reflux under N2 for 3 h. The mixture was directly subjected
to silica gel column chromatography. Elution with hexane-
EtOAc (15:1) gave the crude rearranged product (140 mg)
which was mainly composed of the desired 4a and
22-O-propionate side-product. The mixture was dissolved
in dry THF (2.0 mL) and LiAlH4 (24.0 mg, 0.632 mmol)
was added and after 30 min stirring, it was worked up in the
usual manner. The resulting mixture (62.4 mg) was mixed
with Ac2O (0.25 mL) and pyridine (0.50 mL) and allowed
to stand overnight at room temperature. Extractive (ether)
work-up gave a crude product which was chromatographed
over silica gel with hexane-EtOAc (20:1) to give 29-acetate
(40.5 mg) as an oil. LiAlH4 (6.0 mg, 158 mmol) was added
to a solution of the acetate in THF (1.0 mL) and the mixture
was stirred for 10 min at room temperature. Extractive (ether)
work-up gave the primary alcohol 5a (36.0 mg, 24% from
3a) as a colorless oil. 1H NMR: d 0.43 (dd, J 8.1, 5.1 Hz,
H-4a), 0.65 (dd, J 5.1, 4.4 Hz, H-4b), 0.73 (s, H3-18), 0.81
(d, J 7.1 Hz, H3-26), 0.83 (d, J 7.1 Hz, H3-27), 0.89 (s, H3-30),
1.01 (d, J 6.8 Hz, H3-21), 1.02 (s, H3-19), 2.06 (m, H-20),
(22E,24S)-24-Ethyl-24-methylcholesta-5,22-dien-3b-ol
(1a)
A solution of the 22-ene 7a (7.3 mg, 16.6 mmol)
in dioxane (0.5 mL) and H2O (1.15 mL) containing
o
•
p-TsOH H2O (12 mg, 63.1 mmol) was heated at 105 C for
3 h. Extractive (ether) work-up gave a crude product which
was crystallized from MeOH to yield the (24S)-22-ene 1a
o
25
(5.4 mg, 76%) as white needles, mp 159-161 C. [a]D
–41.6° (c, 0.50, CHCl3). H and 13C NMR spectroscopic
data are listed in Tables 1 and 2, respectively. EIMS m/z:
426 [M+, trace], 383 [M-isopropyl], 367, 273, 257, 231, 213.
HRFABMS m/z: 409.3834 calc. for C30H49 [MH+–H2O].
Found: 409.3784.
1
(24R)-24-Ethyl-6b-methoxy-24-methyl-3a,5-cyclo-5a-
cholestane (8a)
A solution of the 22-ene 7a (10.5 mg, 23.8 mmol)
in AcOEt (1.0 mL) was hydrogenated in the presence
of 10% Pd/C (5.8 mg) overnight. Dilution with hexane
and filtration through a pad of silica gel afforded an oily

Vol. 22, No. 5, 2011
Echigo et al.
1003
saturated product 8a (7.4 mg, 70%). 1H NMR : d 0.43 (dd,
J 8.1, 5.1 Hz, H-4a), 0.65 (dd, J 5.1, 4.4 Hz, H-4b), 0.67
(s, H3-30), 0.72 (s, H3-18), 0.75 (t, J 7.6 Hz, H3-29), 0.79
(d, J 7.1 Hz, H3-26), 0.80 (d, J 7.1 Hz, H3-27), 0.92 (d, J
6.6, H3-21), 1.02 (s, H3-19), 2.77 (brt, J 2.8 Hz, H-6), 3.33
(s, OMe). 13C NMR: d 7.95, 12.22, 13.07, 17.05, 17.15,
18.87, 19.29, 20.25, 21.50, 22.77, 24.21, 24.97, 28.37,
28.69, 29.05, 30.48, 32.26, 33.31, 33.36, 35.06, 35.30,
36.75, 36.82, 40.28, 42,77, 43.39, 48.03, 56.19, 56.54,
56.55, 82.45. HREIMS m/z: 442.4172 calc. for C31H54O
[M+]. Found: 442.4156.
H3-19), 2.77 (brt, J 2.6 Hz, 6-H), 3.32 (s, OMe), 5.05 (dd, J
15.6, 8.0 Hz, H-22), 5.25 (brd, J 15.6 Hz, H-23). 13C NMR:
d 8.67, 12.46, 13.08, 17.26, 17.88, 18.29, 19.29, 21.29,
21.49, 22.77, 24.24, 24.97, 28.96, 30.49, 31.76, 33.36,
35.09, 35.29, 35.70, 40.22, 40.84, 41.07, 42.68, 43.41,
48.09, 56.23, 56.54, 56.67, 82.44, 134.61, 134.88. HREIMS
m/z: 440.4018 calc. for C31H52O [M+]. Found: 440.4014.
(22E,24R)-24-Ethyl-24-methylcholesta-5,22-dien-3b-ol
(1b)
The 22-ene 7b (25.8 mg) was treated as described
for the conversion of 7a to 1a. The crude product was
chromatographed on silica gel with hexane-EtOAc
(7:1) and crystallized from MeOH to afford 1b (15 mg,
60%) as white needles, mp 164-165 °C [mp of natural 1,
(24R)-24-Ethyl-24-methylcholest-5-en-3b-ol (2a)
Compound 8a (5.9 mg, 13.3 mmol) was treated as
described for the conversion of 7a to 1a. Crystallization
of a crude product from MeOH gave the (24R)-epimer 2a
25
165-166.5 °C (from methanol)8]. [a]D –49.8° (c, 2.0,
(4.7 mg, 82%) as white needles, mp 159-161 °C. [a]D
CHCl3) (lit.8 –45.4°for 1). The EI-MS data were essentially
identical with those of 1a. 1H and 13C NMR data are listed
in Tables 1 and 2, respectively. Analysis calc. for C30H50O:
C, 84.44; H, 11.81. Found: C, 84.68; H, 11.49.
25
–30.8° (c, 0.42, CHCl3). H and 13C NMR spectroscopic
1
data are listed in Tables 1 and 2, respectively. EIMS m/z:
428 (M+), 413, 410, 395, 385, 367, 343, 317, 273, 255,
231, 213. HREIMS m/z: 428.4018 calc. for C30H52O [M+].
Found: 428.4020.
(24S)-24-Ethyl-6b-methoxy-24-methyl-3a,5-cyclo-5a-
cholestane (8b)
(22E,24R)-24-(2-Hydroxyethyl)-6b-methoxy-24-methyl-
3a,5-cyclo-5a-cholest-22-ene (5b)
The 22-ene 7b (24.8 mg) was hydrogenated as described
for 8a to afford the saturated compound 8b (24.4 mg, 98%)
as an oil. 1H NMR: d 0.43 (dd, J 7.8, 5.1 Hz, H-4a), 0.65
(dd, J 5.1, 4.4 Hz, H-4b), 0.67 (s, H3-30), 0.71 (s, H3-18),
0.75 (t, J 7.7 Hz, H3-29), 0.77 (d, J 7.8 Hz, H3-26), 0.79
(d, J 7.1 Hz, H3-27), 0.92 (d, J 6.3 Hz, H3-21), 1.02 (s, H3-
19), 2.77 (brt, J 2.7 Hz, H-6), 3.32 (s, OMe). 13C-NMR:
d 7.94, 12.22, 13.07, 17.12, 17.15, 18.91, 19.29, 20.37,
21.50, 22.77, 24.21, 24.97, 28.38, 28.62, 29.03, 30.49,
32.27, 33.29, 33.36, 35.08, 35.31, 36.67, 36.81, 40.28,
42.78, 43.39, 48.05, 56.18, 56.55, 82.45. HRFABMS m/z:
443.4253 calc. for C31H55O [MH+]. Found: 443.4277.
Treatment of the (22S)-alcohol 3b (414 mg) as described
for 3a gave a product (377 mg) which was composed of
the desired 4b and 22-O-propionate side-product. The
mixture was reduced with LiAlH4 (28.7 mg) in ether (5 mL)
and the product was chromatographed on silica gel with
hexane-ether (10:1) to give a primary alcohol 5b (105 mg,
1
24% from 3b) as an oil. H NMR: d 0.43 (dd, J 8.0, 5.1
Hz, H-4a), 0.65 (dd, J 5.1, 4.4 Hz, H3-4b), 0.73 (s, H3-18),
0.80 (d, J 7.6 Hz, H3-26), 0.83 (d, J 7.6 Hz, H3-27), 0.88 (s,
H3-30), 1.01 (d, J 6.6 Hz, H3-21), 1.03 (s, H3-19), 2.07 (m,
H-20), 2.77 (brt, J 2.6 Hz, H-6), 3.32 (s, OMe), 5.13 (dd, J
15.6, 8.3 Hz, H-22), 5.25 (brd, J 15.6 Hz, H-23). 13C NMR:
d 12.42, 13.06, 17.15, 17.61, 18.58, 19.28, 21.16, 21.47,
22.74, 24.25, 24.95, 28.99, 30.47, 33.35, 35.07, 35.26,
36.97, 40.14, 40.19, 40.78, 42.04, 42.71, 43.39, 48.05,
56.11, 56.55, 56.59, 60.41, 82.40, 134.62, 135.37. HREIMS
m/z: 456.3967 calc. for C31H52O2 [M+]. Found: 456.3948.
(24S)-24-Ethyl-24-methylcholest-5-en-3b-ol (2b)
Compound 8b (22.9 mg) was converted to 2b as
described for the conversion of 8a to 2a. Recrystallization
of the product from MeOH gave 2b (15 mg, 68%) as white
needles, mp 147-148°C (mp of natural 2, 145-146 °C (from
methanol)8), [a]D25 −35.9° (c, 1.7, CHCl3) (lit.7 –34.6° for
2). The EIMS data were essentially identical with those
of 2a. 1H and 13C NMR data are listed in Tables 1 and 2,
respectively.Analysis calc. for C30H52O: C, 84.04; H, 12.23.
Found: C, 84.04; H, 12.53.
(22E,24R)-24-Ethyl-6b-methoxy-24-methyl-3a,5-cyclo-
5a-cholest-22-ene (7b)
The primary alcohol 5b (100 mg) was converted to the
22-ene 7b (47.5 mg, 49%) via the mesylate 6b as described
for the conversion of 5a to 7a. 7b: an oil. 1H NMR: d 0.43
(dd, J 8.0, 5.1 Hz, H-4a), 0.65 (dd, J 5.1, 4.4 Hz, H-4b ),
0.73 (s, H3-18), 0.78 (d, J 7.1 Hz, H3-26), 0.80 (s, H3-30),
0.80 (d, J 6.8 Hz, H3-27), 1.01 (d, J 7.1 Hz, H3-21), 1.02 (s,
X-ray study of natural sterol (1)
Crystals for X-ray analysis were obtained by allowing
to stand a warmed MeOH solution of 1 with slow

1004
C-24 Stereochemistry of Marine Sterols: (22E)-24-Ethyl-24-methylcholesta-5,22-dien-3b-ol
J. Braz. Chem. Soc.
evaporation of the solvent through a small hole in the
References
•
•
cap. C30H52O 1/2MeOH 1/2H2O, orthorhombic, P212121,
Z = 8, a = 70.8949(17), b = 7.7590(2), c = 10.0415(2) Å,
V = 5523.6(2) Å3, T = 123 K, Dx = 1.086 gcm-3,
l(CuKa) = 1.54184 Å, m = 0.490 mm-1. A number of 9298
reflections were collected by the oscillation photograph
method using Rigaku R-AXIS RAPID Imaging Plate
camera and Lp and semi-empirical absorption corrections
were applied. The structure was solved by direct methods
using SIR-97. The positional and anisotropic thermal
parameters were refined by full-matrix least squares
SHELXL-97. The positions of hydrogen atoms were
calculated geometrically and refined using the riding model.
Final R = 0.090 for 6737 unique reflections with |Io| > 2s(Io).
One methyl group of the terminal isopropyl group has a
disordered structure in both independent molecules. The
occupancy factors are 0.60:0.40 and 0.52:0.48, respectively.
Sterol molecules are linked by hydrogen bonding directly
or via methanol and water molecules.
1. Kerr, R. G.; Baker, B. J.; Nat. Prod. Rep. 1991, 8, 465.
2. Li, X.; Djerassi, C.; Tetrahedron Lett. 1983, 24, 665.
3. Tam, H. T. B.; Kokke, W. C. M. C.; Proudfoot, J. R.; Djerassi,
C.; Steroids 1985, 45, 263.
4. Giner, J. L.; Zimmerman, M. P.; Djerassi, C.; J. Org. Chem.
1988, 53, 5895.
5. Mandeau, A.; Masson, V.; Menou, J. L.; Debitus, C.; Biochem.
System. Ecol. 2006, 34, 92.
6. Majumder, P. L.; Pal, S.; Phytochemistry 1990, 29, 2717.
7. Kostova, I.; Simeonov, M.; Iossifova, T.; Tappe, R.; Pardeshi,
N.; Budzikiewicz, H.; Phytochemistry 1996, 43, 643.
8. Calderon, G. J.; Castellanos, L.; Duque, C.; Echigo, S.; Hara,
N.; Fujimoto, Y.; Steroids 2004, 69, 93.
9. Giner, J. -L.; Li, X.; Tetrahedron 2000, 56, 9575.
10. Khripach, V. A.; Zhabinskii, V. N.; Konstantinova, O. V.;
Khripach, N. B.; Tetrahedron Lett. 2000, 41, 5765.
11. Khripach, V. A.; Zhabinskii, V. N.; Konstantinova, O. V.;
Khripach, N. B.; Antonchick, A. P.; Schneider, B.; Steroids
2002, 67, 597.
Supplementary Information
12. Echigo, A.; Hara, N.; Carderon, G. J.; Duque, C.; Fujimoto,Y.;
Chem. Pharm. Bull. 2006, 54, 1473.
Supplementary data associated with this paper are
available free of charge at http://jbcs.sbq.org.br as a PDF
file.
13. Hazra, B. G.; Kumar, T. P.; Pore, V. S.; J. Chem. Res. (S) 1996,
536.
14. Takahashi, T.; Ootake, A.; Yamada, H.; Tsuji, H.; Tetrahedron
Lett. 1985, 26, 69.
Acknowledgments
15. Shen, Z. W.; Zhou, W. S.; J. Chem. Soc., Perkin Trans. 1 1990,
1765.
This work was supported in part by a Grant from
Colciencias to L. C. and C. D.
16. Wiersig, J. R.;Waespe-Sarcevic, N.; Djerassi, C.; J. Org. Chem.
1979, 44, 3374.
Submitted: March 19, 2010
Published online: February 8, 2011

J. Braz. Chem. Soc., Vol. 22, No. 5, S1-S11, 2011.
Printed in Brazil - ©2011 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Supplementary Information
SI
C-24 Stereochemistry of Marine Sterols: (22E)-24-Ethyl-24-methylcholesta-5,22-dien-3b-ol
and 24-Ethyl-24-methylcholest-5-en-3b-ol
Shizue Echigo,a Leonardo Castellanos,b Carmenza Duque,*,b Hidehiro Uekusa,a
Noriyuki Haraa and Yoshinori Fujimotoa
aDepartment of Chemistry and Materials Science, Tokyo Institute of Technology,
Meguro, 152-8551, Tokyo, Japan
bDepartamento de Química, Universidad Nacional de Colombia, AA 14490, Bogotá, Colombia
Figure S1. Conformation of a preferred transition state for the orthoester Claisen rearrangement of compound 3a.
Figure S2. Comparison of the 13C NMR data of compounds 2, 2a and 2b. The values for Dd (2a)-d (2) and Dd (2b)-d (2) are shown in the left and right
graphs, respectively.
Figure S3. ORTEP drawing based on X-ray crystallographic analysis of compound 1.
*e-mail: cduqueb@unal.edu.co, cduqueb@etb.net.co

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C-24 Stereochemistry of Marine Sterols: (22E)-24-Ethyl-24-methylcholesta-5,22-dien-3b-ol
J. Braz. Chem. Soc.
Figure S4. 1H NMR spectrum of compound 1.
Figure S5. 13C NMR spectrum of compound 1.

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Figure S6. 1H NMR spectrum of compound 1a.
Figure S7. 13C NMR spectrum of compound 1a.

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C-24 Stereochemistry of Marine Sterols: (22E)-24-Ethyl-24-methylcholesta-5,22-dien-3b-ol
J. Braz. Chem. Soc.
Figure S8. 1H NMR spectrum of compound 1b.
Figure S9. 13C NMR spectrum of compound 1b.

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Figure S10. 1H NMR spectrum of compound 2.
Figure S11. 13C NMR spectrum of compound 2.

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C-24 Stereochemistry of Marine Sterols: (22E)-24-Ethyl-24-methylcholesta-5,22-dien-3b-ol
J. Braz. Chem. Soc.
Figure S12. 1H NMR spectrum of compound 2a.
Figure S13. 13C NMR spectrum of compound 2a.

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Figure S14. 1H NMR spectrum of compound 2b.
Figure S15. 13C NMR spectrum of compound 2b.

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C-24 Stereochemistry of Marine Sterols: (22E)-24-Ethyl-24-methylcholesta-5,22-dien-3b-ol
J. Braz. Chem. Soc.
Figure S16. 1H NMR spectrum of compound 5a.
Figure S17. 13C NMR spectrum of compound 5a.

Vol. 22, No. 5, 2011
Echigo et al.
S9
Figure S18. 1H NMR spectrum of compound 5b.
Figure S19. 13C NMR spectrum of compound 5b.

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C-24 Stereochemistry of Marine Sterols: (22E)-24-Ethyl-24-methylcholesta-5,22-dien-3b-ol
J. Braz. Chem. Soc.
Figure S20. HMBC spectrum of compound 1a.
Figure S21. HMBC spectrum of compound 1b.

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Echigo et al.
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Figure S22. HMBC spectrum of compound 2a.
Figure S23. HMBC spectrum of compound 2b.