Synthesis and structural confirmation of calibagenin and saxosterol

Article abstract of DOI:10.1016/j.tet.2021.132194

Calibagenin and saxosterol are cholesterol-based plant steroids isolated from Calibanus hookerii and Narthecium ossifragum, respectively. To date, the configurations of their 16- and 22-hydroxy groups have not yet been determined. In this study, all the four 16,22-stereoisomers were chemically synthesized. The ¹H and ¹³C NMR spectra were fully assigned using 2D NMR techniques, and the structures were determined unambiguously using X-ray crystallography. The H-18 and H-22 signals in the NMR spectra of the products are diagnostic for determining the configurations of the 16- and 22-hydroxy groups. A comparison of the NMR, [α]_D, and mp data of the four isomers with those of natural calibagenin and saxosterol confirmed that the configurations of the 16- and 22-hydroxy groups of the former and the later are identical to 16β,22S and 16β,22R, respectively.

Full text of DOI:10.1016/j.tet.2021.132194

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Synthesis and structural confirmation of calibagenin and saxosterol
Bunta Watanabe, Katsunori Makino, Masaharu Mizutani, Hikaru Takaya
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S0040-4020(21)00392-6
https://doi.org/10.1016/j.tet.2021.132194
TET 132194
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To appear in: Tetrahedron
Received Date: 9 February 2021
Revised Date: 14 April 2021
Accepted Date: 28 April 2021
Please cite this article as: Watanabe B, Makino K, Mizutani M, Takaya H, Synthesis and structural
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Synthesis and structural confirmation of calibagenin and saxosterol
Bunta Watanabe,a, * Katsunori Makino,a Masaharu Mizutani,b and Hikaru Takayaa
a Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
b Graduate School of Agricultural Science, Kobe University, Rokkodai, Nada, Kobe 657-8501,
Japan
*To whom correspondence should be addressed. Tel: +81-774-38-3233; Fax: +81-774-38-3229;
E-mail: bunta@scl.kyoto-u.ac.jp
Manuscript type: Article
1

Abstract
Calibagenin and saxosterol are cholesterol-based plant steroids isolated from Calibanus hookerii
and Narthecium ossifragum, respectively. To date, the configurations of their 16- and 22-hydroxy
groups have not yet been determined. In this study, all the four 16,22-stereoisomers were
chemically synthesized. The 1H and 13C NMR spectra were fully assigned using 2D NMR
techniques, and the structures were determined unambiguously using X-ray crystallography. The
H-18 and H-22 signals in the NMR spectra of the products are diagnostic for determining the
configuration of the 16- and 22-hydroxy groups. A comparison of the NMR, [α]D, and mp data of
the four isomers with those of natural calibagenin and saxosterol confirmed that the configurations
of the 16- and 22-hydroxy groups of the former and the later are identical to 16β,22S and 16β,22R,
respectively.
Keywords: calibagenin, saxosterol, 2D NMR, X-ray crystallography
2

1. Introduction
Calibagenin was isolated from Calibanus hookerii, a plant native to Tamaulipas in Mexico, in 1975,
and its structure was proposed to be one of the four isomers of cholest-5-en-3β,16,22-triol (1,
Figure 1), while the configuration of the hydroxy groups at C-16 and C-22 remained undetermined.1
In 1988, saxosterol was isolated from Narthecium ossifragum, a plant of Western Europe, and its
structure was also proposed to be one of the four isomers of 1.2 Stabursvik and Holen deduced the
absolute structure of saxosterol to be 16β,22R-1 based on the observation that the complete
acetylation of saxosterol required a more reactive reagent than that of calibagenin, presumably due
to the steric hindrance of the 16-hydroxy group (β vs. α), and the fact that 22R-hydroxycholesterol
has been isolated from N. ossifragum.2 They also speculated that the absolute structure of
calibagenin should be 16α,22R-1, albeit that the absolute structures of calibagenin and saxosterol
have not yet been determined unequivocally.
Figure 1. Structures of the four 16,22-stereoisomers of cholest-5-en-3β,16,22-triol (1). The
previously proposed structures of calibagenin (16α,22R-1) and saxosterol (16β,22R-1) are also
indicated.
In contrast to the study of Stabursvik and Holen, 16β,22S-1 is a frequently encountered structure in
plants. For example, 16β,22S-1 is a common aglycone found in schubertosides B and C from Allium
schubertii,3 one of the cholestane bisdesmosides from Galtonia candicans,4 spongioside A from
Dioscorea spongiosa,5 and karataviosides J and K from Allium karataviense.6 However, only the
mp, [α]D, and limited 1H NMR data have been reported in the cases of calibagenin and saxosterol,
and these data were not provided completely in the report of 16β,22S-1 mentioned above. As a
result, a structural assignment of calibagenin/saxosterol and 16β,22S-1 has not been yet
accomplished. Recently, 16β,22S-1 has been identified as an enzymatic reaction product of
cytochromes P450 involved in the biosynthesis of plant steroid diosgenin.7 Therefore, the synthesis
of all the four 16,22-stereoisomers of 1 may not only lead to the absolute structure determination of
calibagenin and saxosterol, but also provide a useful tool for the biosynthetic study of plant steroids
such as diosgenin.
3

In this study, we have developed a synthetic route to the four 16,22-stereoisomers of 1 with reliable
configurations, and their absolute structures were unambiguously determined using X-ray
crystallography. Moreover, we fully assigned their 1H and 13C NMR spectra and established
diagnostic chemical shifts that allow differentiating these isomers. Furthermore, a comparison of
the NMR, [α]D, and mp data of synthetic 1 with those of calibagenin and saxosterol confirmed the
absolute structures of these two natural products.
2. Results and Discussion
Our retrosynthetic strategy is outlined in Scheme 1. The introduction of the oxygen functionality at
C-16 of 1 was achieved by a hydroboration of the ꢀ16 double bond of 2. The presence of the C-18
methyl group on the β-face of the steroidal skeleton can be expected to force the borane to approach
from the opposite α-face. In fact, the hydroboration of ꢀ16 double bonds has been reported to
produce 16α-alcohols.8-12 Similarly, the reduction of C-16 carbonyl groups is known to afford
16β-alcohols as the major product.8,9,12 The 3α,5-cyclosteroidal structure of 2 is a protected form of
the ꢀ5 double bond of 3 during hydroboration. The side chain and the ꢀ16 double bond of 3 could be
constructed by the ene reaction between the (Z)-olefin 4, which is readily prepared in two steps
from commercially available dehydroepiandrosterone (6),14 and 4-methylpentanal (5). Houston and
co-workers previously obtained (22S)-3β,22-diacetoxycholest-5,16-diene, which exhibits
configurations identical to those of 22S-3, being the predominant product from a Me2AlCl-mediated
ene reaction between a (Z)-olefin similar to 4 (R5 = Ac) and 5, followed by acetylation.13
Scheme 1. Retrosynthetic strategy.
Our synthesis started with the ene reaction between 414 and aldehyde 515 to provide 22S-3 and
22R-3 in 72% and 16% yield, respectively (Scheme 2). To convert the major product 22S-3 into the
minor product 22R-3, 22S-3 was oxidized with Dess-Martin periodinane (DMP), and the resulting
ketone 7 was reduced using Li(sec-Bu)3BH to furnish 22R-3 in 89% yield. Pivaloylation of the
22-hydroxy group of 3 and hydrolysis of the TBDPS protecting group afforded 8 in 96–97% yield.
The obtained 8 was converted into 3α,5-cyclosteroid 2 in 69–75% yield by methanolysis of the
homoallylic mesylate in the presence of KOAc.16
4

Scheme 2. Ene reaction and subsequent introduction of the oxygen functionality at C-16.
Excess BH3•SMe2 (5 equiv.) was necessary to complete the hydroboration of 22S-2, and 16α,22S-9
was obtained in 87% yield. In the hydroboration of 22R-2 with five equivalents of BH3•SMe2,
16α,22R-9 was obtained in 72% yield, and 16α,22R-10, in which the pivaloyl group remained on
the 22-hydroxy group, was obtained in 5.6% yield. The NOE correlation between H-18 and H-16
confirmed the configuration of the 16-hydroxy groups of the hydroboration products to be α.
Prior to the inversion of the configuration of the 16-hydroxy group from α to β, the 22-hydroxy
group of 16α,22S-9 was re-pivaloylated to give 16α,22S-10 in 18% yield using 1.1 equiv. of PivCl
and 1.5 equiv. of DMAP. The remaining products were a mixture of 16α,22S-11, in which the
pivaloyl group was introduced on the 16α-hydroxy group, and bis-pivaloylated 16α,22S-12 (11/12 =
88/12, 79%). When 16α,22R-9 was treated with PivCl (2.7 equiv.) and DMAP (3.2 equiv.),
16α,22R-10 (45%), 16α,22R-11 (5.3%), and 16α,22R-12 (38%) were isolated. 16α,22S-10 and
16α,22R-10 were oxidized with DMP to give 22S-13 and 22R-13 in 89–94% yield (Scheme 2).
A reduction of the carbonyl group of 22S-13 with excess DIBAL at –78 °C provided 16αβ,22S-9
(α/β = 9/91) in 72% yield (Scheme 2). During the reaction, the pivaloyl group on the 22-hydroxy
group was also removed. While it is difficult to separate 16α,22S-9 and 16β,22S-9 by
chromatography on silica gel, their derivatization into the corresponding cyclic carbonates
16α,22S-14 and 16β,22S-14 made their chromatographic separation manageable. When 16αβ,22S-9
(α/β = 17/83) was treated with 1,1′-carbonyldiimidazole (CDI), 16α,22S-14 and 16β,22S-14 were
obtained in 13% and 70% yield, respectively, and bis-imidazolylcarbonylated 15 was also isolated
in 10% yield (Scheme 3). The NOE correlation between H-18 and H-16 in 16α,22S-14 as well as
that between H-17 and H-16 in 16β,22S-14 confirmed their configuration of the oxygen
5

functionality at C-16. The cyclic carbonates of 16α,22S-14 and 16β,22S-14 were reductively
cleaved with LiAlH4 to produce 16α,22S-9 and 16β,22S-9 in 91–92% yield (Scheme 3).
Scheme 3. Separation of 16α,22S-9 and 16β,22S-9 via cyclic carbonates 14.
In the reduction of the carbonyl group of 22R-13, treatment with LiAlH4 in refluxing THF gave
16β,22R-9 in 85% yield (Scheme 1). The configuration of the 16-hydroxy group of 16β,22R-9 was
confirmed by the NOE correlation between H-17 and H-16. When 22R-13 was treated with DIBAL
at –78 °C, β selectivity was somewhat lower (α/β = 6/94, 88%). Similar to 16α,22S-9 and 16β,22S-9,
16α,22R-9 and 16β,22R-9 are also difficult to separate by chromatography on silica gel. Thus,
derivatization into the cyclic carbonates was examined (Scheme 4). When 16αβ,22R-9 (α/β = 10/90)
was treated with CDI, cyclic carbonate 16β,22R-14 was obtained in 43% yield together with
tetrahydrofuran 16 (43%). In this operation, no attempt was made to isolate the cyclic carbonate
from the minor component 16α,22R-9. The NOE correlation between H-17 and H-16 confirmed the
configuration of the oxygen functionality at C-16 of 16β,22R-14 and 16.
Scheme 4. Formation of 16β,22R-14 and 16.
In order to improve access to 16β,22S-9 and 16β,22R-9, we investigated the reduction of diketone
17, which was derived from 16α,22S-9 in 94% yield (Scheme 2). The reduction of 17 with LiAlH4
at 0 ºC provided 16αβ,22S-9 (α/β = 12/88, 56%) and 16β,22R-9 (26%). 16αβ,22S-9 and 16β,22R-9
6

can be separated chromatographically, and the two isomers in 16αβ,22S-9 can be separated via the
cyclic carbonates as described above. Therefore, the oxidation–reduction of 16α,22S-9, which can
be easily obtained from the major ene-adduct 22S-3, can provide 16β,22S-9 and 16β,22R-9 without
the involvement of less accessible 16-monoketones such as 22S-13 and 22R-13.
The final step of the synthesis of 1 was the acid hydrolysis of 9. Each 16,22-stereoisomer of 9 was
treated with p-TsOH•H2O in refluxing aqueous 1,4-dioxane to provide 1 in more than 94% yield.
By choosing an appropriate solvent system for each stereoisomer of 1, single crystals suitable for
X-ray crystallography were obtained for all the four 16,22-stereoisomers of 1. As shown in Figure 2,
the absolute structures of synthetic 1 were confirmed unequivocally.
Figure 2. Molecular structures of the 16,22-stereoisomers of 1 with thermal ellipsoids at 30%
probability. Solvent molecules are omitted for clarity and carbon atoms 16 and 22 are labelled (cf.
Figure 1).
1H and 13C NMR spectra of 1 were analyzed in both CDCl3 and C5D5N. The results are summarized
in Tables S1 and S2. The H-18 signals of the 16α-isomers (0.70–0.74 ppm in CDCl3; 0.79 ppm in
C5D5N) were observed at considerably higher field than those of the 16β-isomers (0.92–0.94 ppm in
CDCl3; 1.20–1.21 ppm in C5D5N), which is probably due to deshielding of the 16β-hydroxy group.
7

This tendency has also been observed in 16-hydroxylated steroids bearing shorter side chains in
which stereogenic centers are not present.8,9,12 Noteworthily, the effect of the configuration of the
22-hydroxy group on the chemical shift of H-18 proved to be marginal. The H-16α signal (4.16–
4.23 ppm in CDCl3; 4.41–4.53 ppm in C5D5N) also appeared further upfield than that of H-16β
(4.35 ppm in CDCl3; 4.66–4.79 ppm in C5D5N), while the C-16 signals of the 16α-isomers (75.7–
76.6 ppm in CDCl3; 74.4–75.5 ppm in C5D5N) were observed at lower field than those of the
16β-isomers (72.0–72.6 ppm in CDCl3; 70.7–70.8 ppm in C5D5N). The chemical shift of H-22 did
not reflect the configuration of the 22-hydroxy group in CDCl3 (3.63–3.85 ppm for the
22S-isomers; 3.65–3.68 ppm for the 22R-isomers). However, in C5D5N, the H-22 signals of the
22S-isomers (4.19–4.26 ppm) appeared at higher field than those of the 22R-isomers (4.42–4.45
ppm). This observation is consistent with the report by Challinor and co-workers, in which the H-22
signals of helosides and bethosides with 22S-hydroxy groups were observed at 4.15–4.21 ppm,
whereas the H-22 signal of a closely related compound with a 22R-isomer was observed at 4.48
ppm.17 Since all these previously reported compounds bear 16β-hydroxy groups, we revealed in this
study that the configuration of the 16-hydroxy group only marginally affects the chemical shift of
H-22. The advanced Mosher method was required to confirm the absolute configurations of the
22-hydroxy groups of steroidal saponins from A. schubertii3 and A. karataviense.6 The chemical
shift of H-22 in C5D5N is thus a useful marker for the determination of the configuration of
22-hydroxy group of aglycones similar to 1 in steroidal saponins.
The 1H and 13C spectra of synthetic 16β,22S-1 in CDCl3 were, bar a few slight exceptions, in good
accordance with those of enzymatically prepared 16β,22S-17 (Table S3). In C5D5N, however, the 1H
and 13C spectra of synthetic 16β,22S-1 did not agree with those of 16β,22S-1 chemically derivatized
from naturally occurring karataviosides J and K, which was reported by Kuroda and co-workers.6
Their spectra also did not agree well with those of synthetic 16β,22R-1, 16α,22S-1, and 16α,22R-1
(Tables S4 and S5). Even though the reasons for these discrepancies are unclear at this point, the
spectra of 16β,22S-1 provided by Kuroda and co-workers6 should be treated carefully.
We also compared the 1H and 13C spectra of synthetic 16β,22S-1 in C5D5N to those of the natural
steroidal saponins in which the hydroxy groups at C-3 and/or C-16 of 16β,22S-1 are glycosylated
(Figure S1).3–6 As shown in Tables S6 and S7, glycosylation at the 3-hydroxy group shifts the H-6
and H-19 signals upfield (0.04–0.24 ppm), whereas the C-3 signals are shifted downfield (7.6–8.0
ppm). Similarly, glycosylation at the 16-hydroxy group shifts the H-16 and H-18 signals upfield
(0.22–0.30 ppm), whereas the C-16 signals are shifted downfield (11.7–12.2 ppm). With respect to
the chemical shift of H-22, which was proven to be a diagnostic marker for determining the
configuration of the 22-hydroxy group by this study, a consistent trend could not be established.
Therefore, a direct determination of the configuration of the 22-hydroxy group of steroidal saponins
8

bearing aglycones similar to 1 based on their NMR spectra is difficult at this moment.
The diagnostic data differentiating calibagenin, saxosterol, and synthetic 1 are summarized in Table
1. Based on the lower chemical shifts of the H-18 and H-16 signals of calibagenin and saxosterol,
the configurations of their 16-hydroxy groups were determined to be β. Although the H-22 chemical
shifts of calibagenin and saxosterol do not reflect their configurations, they are comparable to those
of 16β,22S-1 and 16β,22R-1. Fortunately, the [α]D value of calibagenin is about twice that of
saxosterol, and their values are in accordance with those of 16β,22S-1 and 16β,22R-1, respectively.
The mp of calibagenin and saxosterol are also comparable to those of 16β,22S-1 and 16β,22R-1.
Therefore, it can be concluded that calibagenin is 16β,22S-1, which is identical to the aglycone of
the above mentioned steroidal saponins.3-6 We also confirmed that the absolute structure of
saxosterol is 16β,22R-1, which is in agreement with the work of Stabursvik and Holen.2
Table 1. Comparison of the 1H NMR, [α]D, and mp data of calibagenin, saxosterol, and 1.
b
1H NMRa
[α]D
Mp
H-18
H-16
H-22
(°C)
Solventc
calibagenin 0.91 (s)
4.36 (m)d
4.35 (m)
3.56 (m)e
3.65 (m)
–56f
–27f
195–196 MeOH/H2O
182–183 MeOH
saxosterol
0.92 (s)
0.70 (s)
16α,22S-1
4.23 (ddd, 8.0, 6.7, 1.3) 3.85 (ddd, 8.2, 4.5, 2.4) –53.1g 230
4.16 (m)
3.68 (ddd, 9.0, 4.5, 2.4) –56.4h 118–119 MeOH
4.35 (ddd, 8.0, 6.9, 4.6) 3.63 (ddd, 9.7, 2.3, 2.3) –55.8i 189
MeOH/H2O
4.35 (ddd, 8.0, 7.2, 5.0) 3.65 (ddd, 8.6, 6.1, 6.1) –32.6i 185–186 MeOH
MeOH
16α,22R-1 0.74 (s)
16β,22S-1
0.94 (s)
16β,22R-1 0.92 (s)
a Recorded in CDCl3 at 60 MHz (calibagenin), 270 MHz (saxosterol), or 600 MHz (1); b Measured in CHCl3 (c 0.2 g/dL). c Solvent
used for recrystallization. d Assigned as H-22 in the original literature. e Assigned as H-16 in the original literature. f Measured at 20
ºC. g Measured at 19 ºC. h Measured at 17 ºC. i Measured at 18 ºC.
3. Conclusions
In this study, the four 16,22-stereoisomers of 1 were synthesized. During the synthesis, easy
separation of the 16α- and 16β-isomer of the 16,22-diols 9 was achieved via their derivatization into
cyclic carbonates 14, and a simple route to 16β,22S- and 16β,22R-isomers via the reduction of the
16,22-diketone 17 was developed. X-ray crystallographic data unambiguously determined the
structure of the isomers of synthetic 1. Consequently, the absolute structures of calibagenin and
saxosterol were determined to be 16β,22S-1 and 16β,22R-1, respectively. The spectroscopic data
presented in this study can thus be expected to serve as useful criteria for the configurational
determination of steroidal saponins bearing hydroxy groups at C-16 and C-22.
9

4. Materials and Methods
4.1. General.
Melting points (mp) were measured on an AS ONE ATM-01 melting point apparatus and are
uncorrected. Optical rotations were measured using a HORIBA SEPA-500 polarimeter. NMR
spectra were obtained using a Bruker AVANCEIII 600 or JEOL JNM-ECA600 spectrometer (600
MHz for 1H; 151 MHz for 13C). Chemical shifts are reported in parts per million relative to the
internal standards [tetramethylsilane (0.00 ppm) for 1H; the solvents CDCl3 (77.0 ppm) and C5D5N
(149.2 ppm) for 13C]. Assignment of the 1H and 13C NMR signals was based on 2D NMR
measurements (1H-1H COSY, NOESY, HSQC, and HMBC). HRMS were recorded on a JEOL
JMS-700 spectrometer. Reagents were used as received from commercial suppliers unless otherwise
stated; 414 and 515 were prepared according to previously reported procedures. Flash column
chromatography on silica gel was carried out using a Biotage Isolera One chromatograph with
SNAP Ultra cartridges (silica gel, 25 µm) or a Yamazen W-prep 2XY chromatograph with
UNIVERSAL Premium columns (silica gel, 30 µm). Single-crystal X-ray crystallographic analyses
were performed at the BL40XU and BL02B1 beam lines of SPring-8 using Si (111)
monochromated synchrotron radiation (0.78229 and 0.70060 Å) with a Rigaku Saturn 724+ CCD
detector.
4.2. (22S)- and (22R)-3β-(tert-butyldiphenylsiloxy)cholesta-5,16-dien-22-ol (22S- and 22R-3).
A Me2AlCl solution (1.0 M in hexane, 100 mL, 100 mmol) was added to anhydrous CH2Cl2 (200
mL) at –78 °C under an argon atmosphere. This mixture was treated dropwise with
4-methylpentanal 5 (10.2 g, 102 mmol) dissolved in anhydrous CH2Cl2 (50 mL), followed by
stirring for 30 min at the same temperature. The resulting mixture was treated dropwise with 4 (27.5
g, 51.0 mmol) dissolved in anhydrous CH2Cl2 (100 mL) at –78 °C; the mixture was gradually
warmed to 0 °C for 5 h under constant stirring. The reaction was quenched by dropwise addition of
MeOH/H2O (1/1, v/v, 50 mL), and subsequently diluted with CHCl3 (100 mL). The organic layer
was washed successively with 1 M HCl, water, a saturated aqueous solution of NaHCO3, and brine
(100 mL each), dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel (hexane/EtOAc = 95/5–85/15, v/v). 22S-3
(23.4 g, 72%) eluted after 22R-3 (5.37 g, 16%). 22S-3. Colorless oil. [α]D26 –46.9 (c 1.71 g/dL,
10

CHCl3). 1H NMR (600 MHz, CDCl3, δ): 0.84 (3H, s, H3-18), 0.86 (1H, m, H-1α), 0.88 (3H, d, J =
6.6 Hz, H3-26), 0.89 (1H, m, H-9α), 0.89 (3H, d, J = 6.6 Hz, H3-27), 1.00 (3H, d, J = 7.0 Hz, H3-21),
1.02 (3H, s, H3-19), 1.06 (9H, s, t-Bu), 1.17 (1H, m, Ha-24), 1.26 (1H, m, H-14α), 1.28 (1H, ddd, J
= 12.1, 12.1, 4.8 Hz, H-12α), 1.34 (1H, m, Hb-24), 1.43 (1H, m, Ha-23), 1.50 (1H, m, Hb-23), 1.51
(2H, m, H-7α and H-11β), 1.54 (2H, m, H-11α and H-25), 1.61 (1H, m, H-2β), 1.63 (1H, m, H-8β),
1.68 (1H, m, H-2α), 1.69 (1H, m, H-1β), 1.74 (1H, ddd, J = 12.1, 4.4, 2.6 Hz, H-12β), 1.86 (1H,
dddd, J = 14.9, 11.5, 1.7, 1.7 Hz, H-15β), 1.93 (1H, dddd, J = 17.4, 5.2, 5.2, 2.7 Hz, H-7β), 2.05
(1H, ddd, J = 14.9, 6.3, 3.2 Hz, H-15α), 2.15 (1H, ddd, J = 13.3, 4.7, 2.1 Hz, H-4α), 2.26 (1H, m,
H-20), 2.34 (1H, m, H-4β), 3.54 (1H, dddd, J = 11.0, 11.0, 4.7, 4.7 Hz, H-3α), 3.61 (1H, m, H-22),
5.14 (1H, m, H-6), 5.47 (1H, m, H-16), 7.36 (4H, m, Ph-3/5), 7.41 (2H, m, Ph-4), 7.68 (4H, m,
Ph-2/6). 13C NMR (151 MHz, CDCl3, δ): 14.1 (C-21), 16.7 (C-18), 19.1 (t-Bu), 19.3 (C-19), 20.6
(C-11), 22.58 (C-26), 22.64 (C-27), 27.0 (3C, t-Bu), 28.1 (C-25), 30.3 (C-8), 31.2 (C-15), 31.5
(C-7), 31.8 (C-2), 32.4 (C-23), 34.7 (C-12), 35.5 (C-24), 36.7 (C-10), 37.1 (C-1), 37.7 (C-20), 42.5
(C-4), 46.8 (C-13), 50.5 (C-9), 57.8 (C-14), 73.0 (C-22), 73.2 (C-3), 120.9 (C-6), 124.3 (C-16),
127.42 (2C, Ph-3/5), 127.44 (2C, Ph-3/5), 129.40 (Ph-4), 129.43 (Ph-4), 134.7 (Ph-1), 134.8 (Ph-1),
135.7 (2C, Ph-2/6), 135.8 (2C, Ph-2/6), 141.5 (C-5), 158.6 (C-17). HRMS-FAB-NBA (m/z): [M–
H]+ calcd for C43H61O2Si, 637.4441; found, 637.4431. 22R-3. Colorless oil. [α]D27 –46.6 (c 1.25
g/dL, CHCl3). 1H NMR (600 MHz, CDCl3, δ): 0.79 (3H, s, H3-18), 0.85 (1H, ddd, J = 14.2, 14.2,
4.3 Hz, H-1α), 0.89 (3H, d, J = 6.5 Hz, H3-26), 0.90 (3H, d, J = 6.5 Hz, H3-27), 0.90 (1H, m, H-9α),
0.97 (3H, d, J = 7.0 Hz, H3-21), 1.02 (3H, s, H3-19), 1.06 (9H, s, t-Bu), 1.26 (1H, m, Ha-24), 1.29
(1H, m, H-14α), 1.30 (1H, m, Ha-23), 1.36 (1H, ddd, J = 12.1, 12.1, 5.0 Hz, H-12α), 1.42 (1H, m,
Hb-24), 1.50 (1H, m, H-11β), 1.51 (1H, m, H-7α), 1.54 (1H, m, H-11α), 1.55 (1H, m, H-25), 1.59
(1H, m, H-2β), 1.61 (1H, m, H-8β), 1.64 (1H, m, Hb-23), 1.68 (1H, m, H-2α), 1.69 (1H, m, H-1β),
1.75 (1H, ddd, J = 12.1, 4.2, 2.6 Hz, H-12β), 1.87 (1H, m, H-15β), 1.93 (1H, dddd, J = 17.2, 5.2,
5.2, 2.8 Hz, H-7β), 2.07 (1H, ddd, J = 15.0, 6.6, 3.2 Hz, H-15α), 2.14 (1H, m, H-4α), 2.15 (1H, m,
H-20), 2.34 (1H, m, H-4β), 3.54 (1H, dddd, J = 11.1, 11.1, 4.5, 4.5 Hz, H-3α), 3.55 (1H, m, H-22),
5.14 (1H, m, H-6), 5.47 (1H, m, H-16), 7.36 (4H, m, Ph-3/5), 7.41 (2H, m, Ph-4), 7.68 (4H, m,
Ph-2/6). 13C NMR (151 MHz, CDCl3, δ): 16.2 (C-18), 18.0 (C-21), 19.1 (t-Bu), 19.3 (C-19), 20.7
(C-11), 22.4 (C-26), 22.9 (C-27), 27.0 (3C, t-Bu), 28.2 (C-25), 30.5 (C-8), 31.20 (C-15), 31.21
(C-23), 31.5 (C-7), 31.8 (C-2), 34.7 (C-12), 34.8 (C-24), 36.7 (C-10), 37.1 (C-1), 40.1 (C-20), 42.5
(C-4), 47.1 (C-13), 50.6 (C-9), 57.4 (C-14), 73.1 (C-3), 73.9 (C-22), 120.9 (C-6), 123.0 (C-16),
127.42 (2C, Ph-3/5), 127.45 (2C, Ph-3/5), 129.40 (Ph-4), 129.43 (Ph-4), 134.76 (Ph-1), 134.78
(Ph-1), 135.7 (2C, Ph-2/6), 135.8 (2C, Ph-2/6), 141.5 (C-5), 158.5 (C-17). HRMS-FAB-NBA (m/z):
[M–H]+ calcd for C43H61O2Si, 637.4441; found, 637.4442.
11

4.3. 3β-(tert-Butyldiphenylsiloxy)cholesta-5,16-dien-22-one (7).
A solution of 22S-3 (11.3 g, 17.7 mmol) in anhydrous CH2Cl2 (100 mL) was treated with DMP
(8.38 g, 19.8 mmol) at 0 °C, and the mixture was stirred for 2 h at room temperature. The reaction
mixture was diluted with CHCl3 (400 mL), washed successively with a saturated aqueous solution
of NaHCO3 and brine (200 mL each), dried over anhydrous MgSO4, filtered, and concentrated in
vacuo. The residue was purified by flash column chromatography on silica gel (hexane/EtOAc =
98/2, v/v) to give 7 (9.92 g, 88%) as a colorless oil. [α]D26 +6.5 (c 1.55 g/dL, CHCl3). 1H NMR (600
MHz, CDCl3, δ): 0.82 (3H, s), 0.85 (3H, d, J = 6.5 Hz), 0.86 (3H, d, J = 6.5 Hz), 1.02 (3H, s), 1.06
(9H, s), 1.13 (3H, d, J = 6.8 Hz), 3.17 (1H, q, J = 6.8 Hz), 3.54 (1H, dddd, J = 10.9, 10.9, 4.5, 4.5
Hz), 5.12–5.14 (1H, m), 5.34–5.35 (1H, m), 7.34–7.37 (4H, m), 7.39–7.42 (2H, m), 7.66–7.69 (4H,
m). 13C NMR (151 MHz, CDCl3, δ): 16.2, 16.8, 19.1, 19.3, 20.7, 22.2, 22.5, 27.0 (3C), 27.6, 30.5,
31.2, 31.5, 31.8, 33.0, 34.6, 36.7, 37.1, 38.2, 42.5, 45.7, 47.3, 50.5, 57.1, 73.2, 120.8, 125.1, 127.42
(2C), 127.44 (2C), 129.40, 129.43, 134.76, 134.77, 135.7 (4C), 141.5, 154.3, 211.6.
HRMS-FAB-NBA (m/z): [M–H]+ calcd for C43H59O2Si, 635.4284; found, 635.4287.
4.4. Reduction of 7.
A solution of 7 (9.00 g, 14.1 mmol) in anhydrous THF (150 mL) at –78 °C under an argon
atmosphere was treated with a solution of Li(sec-Bu)3BH (1.0 M in THF, 72 mL, 72 mmol). After 5
h of stirring at –78 °C, Li(sec-Bu)3BH (1.0 M in THF, 23 mL, 23 mmol) was added dropwise to the
mixture, and stirring was continued for a further 2 h at –78 °C. Then, the mixture was treated
dropwise at –78 °C with MeOH (20 mL), and at 0 °C with a 10% aqueous solution of NaOH (100
mL) and a 30% aqueous solution of H2O2 (100 mL). The mixture was stirred for 1 h at 0 °C and
then diluted with water (300 mL). The aqueous layer was extracted with EtOAc (1 × 300 mL, 2 ×
150 mL), and the combined organic layers were washed with brine (150 mL), dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (hexane/EtOAc = 95/5, v/v) to give 22R-3 (8.03 g, 89%) as a colorless
oil.
4.5. (22S)-22-(2,2-Dimethylpropanoyloxy)cholesta-5,16-dien-3β-ol (22S-8).
12

An ice-cold mixture of 22S-3 (8.71 g, 13.6 mmol) and DMAP (4.20 g, 34.4 mmol) in anhydrous
CH2Cl2 (100 mL) was treated with PivCl (3.4 mL, 28 mmol), and the mixture was stirred for 21 h at
room temperature. The solvent was evaporated, and 1 M HCl (150 mL) was added to the residue.
The aqueous layer was extracted with toluene (1 × 150 mL, 2 × 50 mL), and the combined organic
layers were washed successively with water, a saturated aqueous solution of NaHCO3, and brine (75
mL each), dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residual colorless
oil (10.3 g) was dissolved in anhydrous THF (100 mL) and treated with a TBAF solution (1 M in
THF, 18 mL, 18 mmol). After 22 h of stirring at room temperature under an argon atmosphere,
additional TBAF solution (1 M in THF, 18 mL, 18 mmol) was added to the mixture, before stirring
was continued for a further 2 days. The reaction was quenched with AcOH (2.0 mL, 35 mmol) and
the solvent was evaporated. The residue was purified by flash column chromatography on silica gel
(hexane/EtOAc = 80/20–60/40, v/v) to give 22S-8 (6.37 g, 96%) as a colorless solid. Mp 109 °C.
[α]D26 –44.4 (c 1.18 g/dL, CHCl3). 1H NMR (600 MHz, CDCl3, δ): 0.82 (3H, s), 0.83 (3H, d, J = 6.2
Hz), 0.84 (3H, d, J = 6.2 Hz), 0.99 (3H, d, J = 6.9 Hz), 1.05 (3H, s), 1.21 (9H, s), 3.53 (1H, dddd, J
= 11.2, 11.2, 4.5, 4.5 Hz), 4.99 (1H, ddd, J = 8.0, 8.0, 4.0 Hz), 5.36–5.38 (1H, m), 5.40–5.41 (1H,
m). 13C NMR (151 MHz, CDCl3, δ): 16.2, 17.6, 19.3, 20.7, 22.3, 22.7, 27.3 (3C), 27.8, 30.46, 30.48,
31.3, 31.5, 31.6, 34.3, 34.9, 36.4, 36.7, 37.2, 38.9, 42.3, 47.1, 50.7, 57.3, 71.7, 76.3, 121.5, 123.2,
141.0, 157.2, 178.1. HRMS-FAB-NBA (m/z): [M+Na]+ calcd for C32H52O3Na, 507.3814; found,
507.3812.
4.6. (22S)-22-(2,2-Dimethylpropanoyloxy)-6β-methoxy-3α,5-cyclocholest-16-ene (22S-2).
An ice-cold solution of 22S-8 (6.25 g, 12.9 mmol) and Et3N (2.60 g, 25.7 mmol) in toluene (100
mL) was treated with MsCl (1.50 mL, 19.4 mmol), before the mixture was stirred for 1 h at room
temperature. The reaction mixture was diluted with toluene (100 mL) and washed successively with
water (2 × 50 mL), 2 M HCl (50 mL), water (50 mL), a saturated aqueous solution of NaHCO3 (50
mL), and brine (50 mL). The organic layer was dried over anhydrous MgSO4, filtered, and
concentrated in vacuo. A mixture of the residual colorless solid (6.55 g) and KOAc (6.45 g, 65.7
13

mmol) in MeOH (200 mL) was stirred under reflux for 1 h and then cooled to room temperature.
The solvent was evaporated, and toluene (200 mL) was added to the residue. The organic layer was
washed successively with water (100 mL) and brine (50 mL), dried over anhydrous MgSO4, filtered,
and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel
(hexane/EtOAc = 98/2–90/10, v/v) to give 22S-2 (4.42 g, 69%) as a colorless oil. [α]D18 +34.7 (c
1.91 g/dL, CHCl3). 1H NMR (600 MHz, CDCl3, δ): 0.45 (1H, dd, J = 8.0, 5.0 Hz, H-4α), 0.66 (1H,
dd, J = 5.0, 3.9 Hz, H-4β), 0.83 (3H, d, J = 6.5 Hz, H3-26), 0.84 (3H, d, J = 6.5 Hz, H3-27), 0.85
(3H, s, H3-18), 0.86 (1H, m, H-1β), 0.90 (2H, m, H-3 and H-9α), 0.99 (3H, d, J = 6.9 Hz, H3-21),
1.06 (3H, s, H3-19), 1.13 (1H, m, Ha-24), 1.14 (1H, m, Hb-24), 1.15 (1H, m, H-7α), 1.21 (9H, s, Piv),
1.35 (1H, m, H-14α), 1.37 (1H, m, H-12α), 1.43 (1H, m, Ha-23), 1.48 (1H, m, H-25), 1.50 (1H, m,
H-11β), 1.52 (1H, m, H-1α), 1.53 (1H, m, H-2β), 1.54 (1H, m, H-11α), 1.66 (1H, m, Hb-23), 1.75
(1H, m, H-2α), 1.76 (1H, m, H-12β), 1.90 (1H, m, H-15β), 1.93 (1H, m, H-7β), 1.96 (1H, m, H-8β),
2.08 (1H, ddd, J = 14.8, 6.5, 3.2 Hz, H-15α), 2.32 (1H, m, H-20), 2.79 (1H, dd, J = 2.8, 2.3 Hz,
H-6α), 3.34 (3H, s, OMe), 5.00 (1H, ddd, J = 8.1, 8.1, 4.0 Hz, H-22), 5.39 (1H, m, H-16). 13C NMR
(151 MHz, CDCl3, δ): 13.0 (C-4), 16.7 (C-18), 17.8 (C-21), 19.2 (C-19), 21.4 (C-3), 22.31 (C-11),
22.32 (C-26), 22.7 (C-27), 24.9 (C-2), 27.3 (3C, Piv), 27.8 (C-25), 29.0 (C-8), 30.4 (C-23), 31.2
(C-15), 33.1 (C-1), 34.3 (C-24), 35.0 (C-7), 35.2 (C-12), 35.4 (C-10), 36.4 (C-20), 38.9 (Piv), 43.6
(C-5), 47.4 (C-13), 48.6 (C-9), 56.6 (OMe), 57.4 (C-14), 76.3 (C-22), 82.3 (C-6), 122.9 (C-16),
157.5 (C-17), 178.1 (Piv). HRMS-FAB-NBA (m/z): [M–H]+ calcd for C33H53O3, 497.3995; found,
497.3999.
4.7. (22R)-22-(2,2-Dimethylpropanoyloxy)-6β-methoxy-3α,5-cyclocholest-16-ene (22R-8).
Using the same procedure as for the synthesis of 22S-8, 22R-3 (3.39 g, 5.30 mmol) was converted
into 22R-8 (2.49 g, 97%). Colorless oil. [α]D26 –39.5 (c 1.06 g/dL, CHCl3). 1H NMR (600 MHz,
CDCl3, δ): 0.83 (3H, s), 0.851 (3H, d, J = 6.5 Hz), 0.853 (3H, d, J = 6.5 Hz), 1.00 (3H, d, J = 7.2
Hz), 1.04 (3H, s), 1.19 (9H, s), 3.53 (1H, dddd, J = 11.2, 11.2, 4.5, 4.5 Hz), 4.98 (1H, ddd, J = 8.2,
5.6, 3.7 Hz), 5.36–5.37 (1H, m), 5.46–5.47 (1H, m). 13C NMR (151 MHz, CDCl3, δ): 16.0, 16.4,
19.3, 20.7, 22.4, 22.7, 27.3 (3C), 27.6, 27.9, 30.5, 31.2, 31.58, 31.62, 34.5, 34.7, 35.8, 36.7, 37.1,
38.8, 42.3, 46.9, 50.6, 57.6, 71.7, 75.9, 121.5, 123.4, 141.1, 156.4, 178.3. HRMS-FAB-NBA (m/z):
[M+Na]+ calcd for C32H52O3Na, 507.3814; found, 507.3825.
14

4.8. (22R)-22-(2,2-Dimethylpropanoyloxy)-6β-methoxy-3α,5-cyclocholest-16-ene (22R-2).
Using the same procedure as for the synthesis of 22S-2, 22R-8 (2.31 g, 4.77 mmol) was converted
into 22R-2 (1.79 g, 75%). Colorless oil. [α]D18 +40.2 (c 3.19 g/dL, CHCl3). 1H NMR (600 MHz,
CDCl3, δ): 0.45 (1H, dd, J = 8.0, 5.0 Hz, H-4α), 0.66 (1H, dd, J = 5.0, 3.9 Hz, H-4β), 0.853 (3H, d,
J = 6.6 Hz, H3-27), 0.854 (3H, d, J = 6.6 Hz, H3-26), 0.86 (3H, s, H3-18), 0.86 (1H, m, H-1β), 0.89
(2H, m, H-3 and H-9α), 1.00 (3H, d, J = 7.0 Hz, H3-21), 1.05 (3H, s, H3-19), 1.14 (2H, m, H-7α and
Ha-24), 1.16 (1H, m, Hb-24), 1.19 (9H, s, Piv), 1.34 (1H, m, H-14α), 1.36 (1H, m, H-12α), 1.49 (1H,
m, H-25), 1.50 (1H, m, H-11β), 1.51 (1H, m, Ha-23), 1.52 (2H, m, H-1α and H-2β), 1.54 (1H, m,
H-11α), 1.56 (1H, m, Hb-23), 1.75 (1H, m, H-2α), 1.86 (1H, m, H-12β), 1.89 (1H, m, H-15β), 1.92
(1H, m, H-7β), 1.95 (1H, m, H-8β), 2.06 (1H, ddd, J = 14.9, 6.5, 3.2 Hz, H-15α), 2.43 (1H, m,
H-20), 2.79 (1H, dd, J = 2.7, 2.7 Hz, H-6α), 3.35 (3H, s, OMe), 4.99 (1H, ddd, J = 8.4, 6.0, 3.4 Hz,
H-22), 5.44 (1H, m, H-16). 13C NMR (151 MHz, CDCl3, δ): 13.1 (C-4), 16.4 (C-21), 16.7 (C-18),
19.2 (C-19), 21.4 (C-3), 22.3 (C-11), 22.4 (C-26), 22.7 (C-27), 24.9 (C-2), 27.3 (3C, Piv), 27.8
(C-23), 27.9 (C-25), 29.1 (C-8), 31.2 (C-15), 33.1 (C-1), 34.4 (C-24), 35.0 (C-12), 35.1 (C-7), 35.3
(C-10), 35.7 (C-20), 38.8 (Piv), 43.6 (C-5), 47.2 (C-13), 48.6 (C-9), 56.6 (OMe), 57.5 (C-14), 75.9
(C-22), 82.3 (C-6), 123.0 (C-16), 156.6 (C-17), 178.2 (Piv). HRMS-FAB-NBA (m/z): [M–H]+ calcd
for C33H53O3, 497.3995; found, 497.3997.
4.9. (22S)-6β-Methoxy-3α,5-cyclocholestane-16α,22-diol (16α,22S-9).
An ice-cold solution of 22S-2 (5.21 g, 10.4 mmol) in anhydrous THF (180 mL) under an argon
atmosphere was treated dropwise with a BH3•THF solution (1.0 M in THF, 54 mL, 54 mmol),
before the mixture was stirred for a further 18 h at room temperature. The reaction mixture was
treated dropwise at 0 °C with a 10% aqueous solution of NaOH (55 mL) and a 30% aqueous
solution of H2O2 (55 mL). After 30 min of stirring at the same temperature, the mixture was diluted
with water (100 mL) and extracted with EtOAc (1 × 100 mL, 2 × 50 mL). The combined organic
layers were washed with brine (100 mL), dried over anhydrous MgSO4, filtered, and concentrated
in vacuo. The residue was purified by flash column chromatography on silica gel (hexane/EtOAc =
15

28
85/15–70/30, v/v) to give 16α,22S-9 (3.92 g, 87%) as a colorless solid. Mp 160–161 °C. [α]D
+34.4 (c 1.06 g/dL, CHCl3). 1H NMR (600 MHz, CDCl3, δ): 0.45 (1H, dd, J = 8.0, 5.0 Hz, H-4α),
0.66 (1H, dd, J = 5.0, 4.0 Hz, H-4β), 0.74 (3H, s, H3-18), 0.86 (1H, ddd, J = 13.3, 11.6, 7.7 Hz,
H-1β), 0.89 (1H, m, H-9α), 0.90 (6H, d, J = 6.7 Hz, H3-26 and H3-27), 0.90 (1H, m, H-3), 0.92 (3H,
d, J = 6.7 Hz, H3-21), 1.02 (3H, s, H3-19), 1.15 (1H, m, Ha-24), 1.15 (1H, ddd, J = 13.3, 12.1, 2.9
Hz, H-7α), 1.32 (1H, m, H-12α), 1.36 (1H, m, Hb-24), 1.37 (1H, m, H-11β), 1.39 (1H, m, H-17α),
1.40 (1H, m, Ha-23), 1.44 (1H, m, H-11α), 1.50 (2H, m, H-1α and H-14α), 1.53 (1H, m, Hb-23),
1.54 (1H, m, H-2β), 1.56 (1H, m, H-25), 1.58 (1H, m, H-20), 1.60 (1H, m, H-15α), 1.69 (1H, m,
H-15β), 1.74 (1H, m, H-8β), 1.76 (1H, m, H-2α), 1.85 (1H, ddd, J = 13.3, 2.9, 2.9 Hz, H-7β), 1.93
(1H, ddd, J = 11.9, 3.2, 2.8 Hz, H-12β), 2.79 (1H, dd, J = 2.9, 2.9 Hz, H-6α), 2.86 (1H, br s, OH),
2.98 (1H, br s, OH), 3.33 (3H, s, OMe), 3.84 (1H, br s, H-22), 4.22 (1H, dd, J = 7.2, 7.2 Hz, H-16β).
13C NMR (151 MHz, CDCl3, δ): 12.5 (br, C-21), 13.1 (C-4), 14.3 (C-18), 19.2 (C-19), 21.3 (C-3),
22.46 (C-11), 22.54 (C-26), 22.7 (C-27), 24.9 (C-2), 28.2 (C-25), 30.1 (C-8), 32.0 (br, C-23), 33.2
(C-1), 35.0 (C-5), 35.1 (C-7), 35.8 (C-24), 36.1 (C-15), 37.3 (br, C-20), 40.4 (C-12), 43.3 (C-10),
44.3 (C-13), 47.8 (C-9), 53.5 (C-14), 56.6 (OMe), 63.1 (br, C-17), 73.9 (br, C-22), 75.5 (C-16), 82.2
(C-6). HRMS-FAB-NBA (m/z): [M+Na]+ calcd for C28H48O3Na, 455.3501; found, 455.3504.
4.10. (22R)-6β-Methoxy-3α,5-cyclocholestane-16α,22-diol (16α,22R-9) and
(22R)-22-(2,2-dimethylpropanoyloxy)-6β-methoxy-3α,5-cyclocholestan-16α-ol (16α,22R-10).
An ice-cold solution of 22R-2 (2.89 g, 5.79 mmol) in anhydrous THF (100 mL) was treated
dropwise under an argon atmosphere with a BH3•THF solution (1.0 M in THF, 30 mL, 30 mmol),
before the mixture was stirred for 17 h at room temperature. At 0 °C, the mixture was then
successively treated dropwise with a 10% aqueous solution of NaOH (30 mL) and a 30% aqueous
solution of H2O2 (30 mL), before the resulting mixture was stirred for 30 min at the same
temperature. The mixture was diluted with water (60 mL) and extracted with EtOAc (3 × 50 mL).
The combined organic layers were washed with brine (50 mL), dried over anhydrous MgSO4,
filtered, and concentrated in vacuo. The residue was purified by flash column chromatography on
silica gel (hexane/EtOAc = 85/15–70/30, v/v) to give 16α,22R-9 (1.81 g, 72%) and 16α,22R-10
(167 mg, 5.6%), respectively, as colorless solids. 16α,22R-9. Mp 119–120 °C. [α]D27 +28.3 (c 1.02
g/dL, CHCl3). 1H NMR (600 MHz, CDCl3, δ): 0.44 (1H, dd, J = 8.0, 5.1 Hz, H-4α), 0.66 (1H, dd, J
= 5.1, 3.7 Hz, H-4β), 0.78 (3H, s, H3-18), 0.86 (1H, m, H-1β), 0.88 (1H, m, H-9α), 0.89 (3H, d, J =
16

6.6 Hz, H3-26), 0.90 (3H, d, J = 6.6 Hz, H3-27), 0.90 (1H, m, H-3), 0.97 (3H, d, J = 6.8 Hz, H3-21),
1.01 (3H, s, H3-19), 1.14 (1H, ddd, J = 13.4, 12.1, 2.9 Hz, H-7α), 1.21 (1H, dd, J = 10.9, 6.0 Hz,
H-17α), 1.21 (1H, m, Ha-24), 1.28 (1H, ddd, J = 12.4, 12.4, 4.1 Hz, H-12α), 1.36 (1H, m, Ha-23),
1.37 (1H, m, H-11β), 1.38 (1H, m, Hb-24), 1.42 (1H, m, H-11α), 1.44 (1H, m, H-14α), 1.50 (1H, m,
H-1α), 1.53 (1H, m, H-2β), 1.55 (1H, m, H-25), 1.59 (1H, m, H-15α), 1.61 (1H, m, Hb-23), 1.72
(1H, m, H-15β), 1.75 (2H, m, H-8β and H-20), 1.76 (1H, m, H-2α), 1.86 (1H, ddd, J = 13.4, 2.9, 2.9
Hz, H-7β), 1.98 (1H, ddd, J = 12.4, 3.1, 3.1 Hz, H-12β), 2.78 (1H, dd, J = 2.9, 2.9 Hz, H-6α), 3.33
(3H, s, OMe), 3.66 (1H, ddd, J = 8.9, 4.4, 2.6 Hz, H-22), 4.15 (1H, dd, J = 6.9, 6.9 Hz, H-16β). 13C
NMR (151 MHz, CDCl3, δ): 13.1 (C-4), 13.7 (C-18), 15.5 (C-21), 19.2 (C-19), 21.3 (C-3), 22.46
(C-11), 22.53 (C-26), 22.8 (C-27), 24.9 (C-2), 28.2 (C-25), 30.0 (C-8), 31.1 (C-23), 33.2 (C-1), 35.0
(C-5), 35.1 (C-7), 35.5 (C-24), 36.7 (C-15), 40.4 (C-12), 40.8 (C-20), 43.3 (C-10), 45.1 (C-13), 47.8
(C-9), 53.2 (C-14), 56.6 (OMe), 63.8 (C-17), 76.6 (C-16), 76.7 (C-22), 82.2 (C-6).
HRMS-FAB-NBA (m/z): [M+Na]+ calcd for C28H48O3Na, 455.3501; found, 455.3503. 16α,22R-10.
Mp 128–129 °C. [α]D27 +36.5 (c 0.836 g/dL, CHCl3). 1H NMR (600 MHz, CDCl3, δ): 0.44 (1H, dd,
J = 7.9, 4.9 Hz, H-4α), 0.65 (1H, dd, J = 4.9, 4.1 Hz, H-4β), 0.74 (3H, s, H3-18), 0.857 (3H, d, J =
6.6 Hz, H3-27), 0.860 (3H, d, J = 6.6 Hz, H3-26), 0.86 (1H, m, H-1β), 0.88 (1H, m, H-9α), 0.89 (1H,
ddd, J = 7.9, 7.9, 4.1 Hz, H-3), 0.93 (3H, d, J = 6.8 Hz, H3-21), 1.01 (3H, s, H3-19), 1.13 (1H, ddd,
J = 13.5, 12.1, 2.8 Hz, H-7α), 1.16 (3H, m, H-17α and H2-24), 1.21 (9H, s, Piv), 1.27 (1H, ddd, J =
12.6, 12.6, 4.2 Hz, H-12α), 1.36 (1H, dddd, J = 12.6, 12.6, 12.6, 3.2 Hz, H-11β), 1.41 (1H, m,
H-11α), 1.45 (2H, m, H-14α and Ha-23), 1.49 (1H, m, H-1α), 1.51 (1H, m, H-25), 1.53 (2H, m,
H-2β and H-15α), 1.62 (1H, m, Hb-23), 1.72 (1H, m, H-15β), 1.74 (1H, m, H-8β), 1.75 (1H, m,
H-2α), 1.85 (1H, ddd, J = 13.5, 2.8, 2.8 Hz, H-7β), 1.86 (1H, qdd, J = 6.8, 6.8, 2.7 Hz, H-20), 1.93
(1H, ddd, J = 12.6, 3.2, 3.2 Hz, H-12β), 2.78 (1H, dd, J = 2.8, 2.8 Hz, H-6α), 3.33 (3H, s, OMe),
4.30 (1H, dd, J = 6.8, 6.8 Hz, H-16β), 5.10 (1H, ddd, J = 10.9, 2.7, 1.8 Hz, H-22). 13C NMR (151
MHz, CDCl3, δ): 13.0 (C-21), 13.1 (C-4), 13.5 (C-18), 19.2 (C-19), 21.3 (C-3), 22.4 (2C, C-11 and
C-26), 22.8 (C-27), 24.896 (C-23), 24.904 (C-2), 27.2 (3C, Piv), 27.8 (C-25), 29.9 (C-8), 33.2 (C-1),
35.0 (C-5), 35.1 (C-7), 35.6 (C-24), 37.5 (C-15), 38.2 (C-20), 38.9 (Piv), 40.3 (C-12), 43.3 (C-10),
44.4 (C-13), 47.9 (C-9), 53.4 (C-14), 56.6 (OMe), 62.1 (C-17), 76.6 (C-16), 77.3 (C-22), 82.2 (C-6),
178.6 (Piv). HRMS-FAB-NBA (m/z): [M+Na]+ calcd for C33H56O4Na, 539.4076; found, 539.4078.
4.11. (22S)-22-(2,2-Dimethylpropanoyloxy)-6β-methoxy-3α,5-cyclocholestan-16α-ol (16α,22S-10),
(22S)-16α-(2,2-dimethylpropanoyloxy)-6β-methoxy-3α,5-cyclocholestan-22-ol (16α,22S-11), and
(22S)-16α,22-bis(2,2-dimethylpropanoyloxy)-6β-methoxy-3α,5-cyclocholestane (16α,22S-12).
17

An ice-cold solution of 16α,22S-9 (715 mg, 1.65 mmol) and DMAP (304 mg, 2.49 mmol) in
anhydrous CH2Cl2 (15 mL) was slowly treated with PivCl (0.22 mL, 1.8 mmol), before the mixture
was stirred for 22 h at room temperature. The reaction was quenched by addition of a saturated
aqueous solution of NaHCO3 (40 mL). After 20 min of stirring at room temperature, the mixture
was extracted with CHCl3 (1 × 30 mL, 2 × 20 mL). The combined organic layers were washed with
brine (20 mL), dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel (hexane/EtOAc = 85/15–65/35, v/v) to give
16α,22S-10 (151 mg, 18%) as a colorless oil, which solidified during storage at room temperature.
Mp 52–53 °C. [α]D28 +23.4 (c 0.707 g/dL, CHCl3). 1H NMR (600 MHz, CDCl3, δ): 0.44 (1H, dd, J
= 8.0, 5.0 Hz, H-4α), 0.65 (1H, dd, J = 5.0, 4.0 Hz, H-4β), 0.77 (3H, s, H3-18), 0.85 (1H, ddd, J =
13.2, 11.9, 7.6 Hz, H-1β), 0.879 (3H, d, J = 6.6 Hz, H3-26), 0.880 (3H, d, J = 6.6 Hz, H3-27), 0.88
(1H, m, H-9α), 0.89 (1H, m, H-3), 1.00 (3H, d, J = 6.8 Hz, H3-21), 1.01 (3H, s, H3-19), 1.10 (1H,
dd, J = 11.4, 4.5 Hz, H-17α), 1.14 (1H, m, Ha-24), 1.15 (1H, m, Hb-24), 1.17 (1H, ddd, J = 13.5,
11.4, 2.9 Hz, H-7α), 1.22 (9H, s, Piv), 1.24 (1H, ddd, J = 12.4, 12.4, 4.7 Hz, H-12α), 1.36 (1H, dddd,
J = 13.7, 13.7, 12.4, 3.3 Hz, H-11β), 1.40 (1H, dddd, J = 13.7, 4.7, 4.7, 3.3 Hz, H-11α), 1.48 (2H, m,
H-1α and H-14α), 1.49 (1H, m, Ha-23), 1.52 (1H, m, H-2β), 1.53 (1H, m, H-25), 1.64 (1H, m,
H-15β), 1.65 (1H, m, H-15α), 1.70 (2H, m, H-20 and Hb-23), 1.75 (1H, m, H-2α), 1.77 (1H, dddd, J
= 11.4, 11.4, 11.4, 2.9 Hz, H-8β), 1.88 (1H, ddd, J = 13.5, 2.9, 2.9 Hz, H-7β), 1.93 (1H, ddd, J =
12.4, 3.3, 3.3 Hz, H-12β), 2.78 (1H, dd, J = 2.9, 2.9 Hz, H-6α), 3.33 (3H, s, OMe), 3.61 (1H, br s,
OH), 3.97 (1H, m, H-16β), 5.04 (1H, dd, J = 8.5, 5.3 Hz, H-22). 13C NMR (151 MHz, CDCl3, δ):
13.0 (br, C-21), 13.1 (C-4), 13.4 (C-18), 19.2 (C-19), 21.3 (C-3), 22.49 (C-26), 22.52 (C-11), 22.53
(C-27), 24.9 (C-2), 27.2 (3C, Piv), 27.9 (C-25), 29.8 (C-8), 30.9 (br, C-23), 33.3 (C-1), 34.9 (C-24),
34.99 (C-7), 35.04 (C-5), 36.2 (C-15), 38.7 (C-20), 39.2 (Piv), 40.6 (C-12), 43.3 (C-10), 44.7
(C-13), 47.7 (C-9), 53.3 (C-14), 56.6 (OMe), 63.9 (br, C-17), 76.6 (br, C-22), 76.8 (C-16), 82.3
(C-6), 179.8 (Piv). HRMS-FAB-NBA (m/z): [M+Na]+ calcd for C33H56O4Na, 539.4076; found,
539.4076. In this experiment, a mixture of 16α,22S-11 and 16α,22S-12 (691 mg, 11/12=88/12,
79%) was also obtained as a colorless oil. A part of them were further separated by flash column
18

chromatography on silica gel (hexane/EtOAc = 90/10–75/25, v/v) to use the following analysis.
16α,22S-11. Colorless oil, which solidified during storage at room temperature. Mp 132–135 °C.
[α]D28 –29.6 (c 0.678 g/dL, CHCl3). 1H NMR (600 MHz, CDCl3, δ): 0.43 (1H, dd, J = 8.0, 5.0 Hz,
H-4α), 0.65 (1H, dd, J = 5.0, 4.0 Hz, H-4β), 0.79 (3H, s, H3-18), 0.87 (1H, m, H-1β), 0.87 (3H, d, J
= 6.6 Hz, H3-26), 0.88 (3H, d, J = 6.6 Hz, H3-27), 0.89 (1H, m, H-9α), 0.91 (1H, m, H-3), 0.93 (3H,
d, J = 6.7 Hz, H3-21), 1.02 (3H, s, H3-19), 1.07 (1H, m, Ha-24), 1.10 (1H, m, H-7α), 1.21 (9H, s,
Piv), 1.29 (1H, m, Ha-23), 1.31 (1H, m, Hb-24), 1.34 (1H, m, H-12α), 1.39 (1H, m, H-14α), 1.40
(1H, m, H-11β), 1.44 (1H, m, H-15α), 1.46 (1H, m, H-11α), 1.51 (1H, m, H-1α), 1.52 (2H, m, Hb-23
and H-25), 1.54 (1H, m, H-2β), 1.55 (1H, m, H-20), 1.62 (1H, br s, OH), 1.66 (1H, dd, J = 11.1, 7.0
Hz, H-17α), 1.75 (1H, m, H-8β), 1.76 (1H, m, H-2α), 1.85 (1H, m, H-15β), 1.86 (1H, m, H-7β),
2.01 (1H, ddd, J = 12.1, 3.1, 3.1 Hz, H-12β), 2.76 (1H, dd, J = 2.8, 2.8 Hz, H-6α), 3.30 (3H, s,
OMe), 3.44 (1H, br d, J = 7.7 Hz, H-22), 4.89 (1H, dd, J = 7.0, 7.0 Hz, H-16β). 13C NMR (151
MHz, CDCl3, δ): 11.4 (C-21), 12.9 (C-4), 13.6 (C-18), 19.2 (C-19), 21.6 (C-3), 22.4 (C-11), 22.5
(C-26), 22.6 (C-27), 24.9 (C-2), 27.0 (3C, Piv), 28.1 (C-25), 29.8 (C-8), 32.2 (C-23), 33.2 (C-1),
34.5 (C-7), 34.6 (C-15), 35.4 (C-5), 35.6 (C-24), 38.5 (Piv), 38.9 (C-20), 40.3 (C-12), 43.2 (C-10),
43.6 (C-13), 47.7 (C-9), 53.7 (C-14), 56.5 (OMe), 58.1 (C-17), 72.9 (C-22), 80.1 (C-16), 82.1 (C-6),
178.1 (Piv). HRMS-FAB-NBA (m/z): [M+Na]+ calcd for C33H56O4Na, 539.4076; found, 539.4080.
16α,22S-12. Colorless oil. [α]D27 –31.7 (c 0.290 g/dL, CHCl3). 1H NMR (600 MHz, CDCl3, δ): 0.43
(1H, dd, J = 7.9, 5.2 Hz), 0.64 (1H, dd, J = 5.2, 3.8 Hz), 0.83 (3H, s), 0.86 (3H, d, J = 6.5 Hz), 0.87
(3H, d, J = 6.5 Hz), 1.02 (3H, s), 1.04 (3H, d, J = 6.5 Hz), 1.18 (9H, s), 1.19 (9H, s), 2.75 (1H, dd, J
= 2.7, 2.7 Hz), 3.30 (3H, s), 4.60 (1H, ddd, J = 8.0, 5.5, 1.6 Hz), 4.85 (1H, dd, J = 6.7, 6.7 Hz). 13C
NMR (151 MHz, CDCl3, δ): 12.9, 13.0, 13.5, 19.3, 21.7, 22.3, 22.4, 22.8, 24.9, 27.0 (3C), 27.2 (3C),
28.0, 29.1, 29.6, 33.3, 34.5, 34.6, 35.4, 35.5, 37.4, 38.5, 38.9, 40.4, 43.3, 43.9, 47.7, 53.7, 56.6, 57.7,
76.1, 80.4, 82.2, 177.3, 178.7. HRMS-FAB-NBA (m/z): [M+Na]+ calcd for C38H64O5Na, 623.4651;
found, 623.4653.
4.12. (22R)-16α-(2,2-Dimethylpropanoyloxy)-6β-methoxy-3α,5-cyclocholestan-22-ol (16α,22R-11),
and (22R)-16α,22-bis(2,2-dimethylpropanoyloxy)-6β-methoxy-3α,5-cyclocholestane (16α,22R-12).
An ice-cold solution of 16α,22R-9 (163 mg, 0.377 mmol) and DMAP (75.2 mg, 0.616 mmol) in
anhydrous CH2Cl2 (9.0 mL) was treated with a solution of PivCl (49.9 mg, 0.414 mmol) dissolved
in anhydrous CH2Cl2 (1.0 mL). After 16 h of stirring at room temperature, PivCl (0.07 mL, 0.6
19

mmol) and DMAP (73.2 mg, 0.599 mmol) were added to the mixture and stirring was continued for
a further 8 h. The reaction was quenched by addition of a saturated aqueous solution of NaHCO3
(10 mL). After 30 min of vigorous stirring at room temperature, the mixture was diluted with water
(30 mL) and extracted with CHCl3 (1 × 30 mL, 2 × 20 mL). The combined organic layers were
washed with brine (20 mL), dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The
residue was purified by flash column chromatography on silica gel (hexane/EtOAc = 85/15–60/40,
v/v). 16α,22R-12 (86.4 mg, 38%) eluted first, followed by 16α,22R-10 (87.2 mg, 45%) and then
16α,22R-11 (10.4 mg, 5.3%). 16α,22R-11. Colorless oil. [α]D27 –8.3 (c 0.165 g/dL, CHCl3). 1H
NMR (600 MHz, CDCl3, δ): 0.43 (1H, dd, J = 8.0, 5.2 Hz, H-4α), 0.65 (1H, dd, J = 5.2, 3.7 Hz,
H-4β), 0.82 (3H, s, H3-18), 0.87 (1H, m, H-1β), 0.88 (3H, d, J = 6.2 Hz, H3-27), 0.88 (1H, m, H-9α),
0.89 (3H, d, J = 6.2 Hz, H3-26), 0.90 (1H, m, H-3), 0.97 (3H, d, J = 6.8 Hz, H3-21), 1.02 (3H, s,
H3-19), 1.06 (1H, ddd, J = 13.3, 12.1, 2.8 Hz, H-7α), 1.09 (1H, m, Ha-24), 1.18 (9H, s, Piv), 1.29
(1H, m, Ha-23), 1.31 (2H, m, H-12α and H-14α), 1.37 (1H, m, H-15α), 1.38 (1H, m, Hb-24), 1.39
(1H, m, Hb-23), 1.40 (1H, m, H-11β), 1.44 (1H, m, H-11α), 1.47 (1H, dd, J = 11.4, 6.4 Hz, H-17α),
1.51 (2H, m, H-1α and H-25), 1.54 (1H, m, H-2β), 1.75 (1H, m, H-2α), 1.76 (1H, m, H-8β), 1.80
(1H, m, H-15β), 1.84 (1H, m, H-20), 1.85 (1H, ddd, J = 13.3, 5.8, 2.8 Hz, H-7β), 2.02 (1H, ddd, J =
12.3, 3.2, 3.2 Hz, H-12β), 2.75 (1H, dd, J = 2.8, 2.8 Hz, H-6α), 3.30 (3H, s, OMe), 3.50 (1H, br d, J
= 10.4 Hz, H-22), 4.96 (1H, dd, J = 6.4, 6.4 Hz, H-16β). 13C NMR (151 MHz, CDCl3, δ): 12.9
(C-4), 13.1 (C-21), 13.4 (C-18), 19.3 (C-19), 21.7 (C-3), 22.38 (C-26), 22.43 (C-11), 23.0 (C-27),
24.9 (C-2), 27.1 (3C, Piv), 28.2 (C-23), 28.8 (C-25), 29.7 (C-8), 33.3 (C-1), 34.6 (C-7), 35.2 (C-15),
35.5 (C-5), 35.7 (C-24), 38.5 (Piv), 40.2 (C-12), 41.7 (C-20), 43.3 (C-10), 44.2 (C-13), 47.8 (C-9),
53.5 (C-14), 56.6 (OMe), 57.9 (C-17), 73.8 (C-22), 80.0 (C-16), 82.2 (C-6), 178.3 (Piv).
HRMS-FAB-NBA (m/z): [M+Na]+ calcd for C33H56O4Na, 539.4076; found, 539.4079. 16α,22R-12.
Colorless oil. [α]D27 –13.4 (c 0.864 g/dL, CHCl3). 1H NMR (600 MHz, CDCl3, δ): 0.43 (1H, dd, J =
7.9, 5.2 Hz), 0.64 (1H, dd, J = 5.2, 3.8 Hz), 0.79 (3H, s), 0.86 (3H, d, J = 6.5 Hz), 0.87 (3H, d, J =
6.5 Hz), 0.97 (3H, d, J = 6.9 Hz), 1.01 (3H, s), 1.19 (18H, s), 2.74 (1H, dd, J = 2.6, 2.6 Hz), 3.29
(3H, s), 4.73 (1H, ddd, J = 10.7, 2.4, 2.4 Hz), 5.07 (1H, dd, J = 6.9, 6.9 Hz). 13C NMR (151 MHz,
CDCl3, δ): 12.9, 13.2, 13.3, 19.2, 21.7, 22.3, 22.4, 22.9, 24.9, 25.3, 27.09 (3C), 27.13 (3C), 28.6,
29.7, 33.2, 34.5, 35.1, 35.2, 35.5, 38.3, 38.4, 38.8, 40.2, 43.3, 44.0, 47.8, 53.6, 56.5, 57.8, 75.9, 80.0,
82.2, 177.7, 177.8. HRMS-FAB-NBA (m/z): [M+Na]+ calcd for C38H64O5Na, 623.4651; found,
623.4651.
4.13. (22S)-22-(2,2-Dimethylpropanoyloxy)-6β-methoxy-3α,5-cyclocholestan-16-one (22S-13).
20

A solution of 16α,22S-10 (101 mg, 0.195 mmol) in anhydrous CH2Cl2 (10 mL) was treated with
DMP (91.7 mg, 0.216 mmol). After 6.5 h of stirring at room temperature, the reaction mixture was
diluted with CHCl3 (40 mL) and washed with a saturated aqueous solution of NaHCO3 (20 mL).
The organic layer was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The
residue was purified by flash column chromatography on silica gel (hexane/EtOAc = 90/10–70/30,
v/v) to give 22S-13 (94.6 mg, 94%) as a colorless oil, which solidified during storage at room
temperature. Mp 118–120 °C. [α]D30 –91.0 (c 0.946 g/dL, CHCl3). 1H NMR (600 MHz, CDCl3, δ):
0.47 (1H, dd, J = 7.9, 5.2 Hz), 0.68 (1H, dd, J = 5.2, 3.8 Hz), 0.84 (3H, s), 0.88 (3H, d, J = 6.5 Hz),
0.89 (3H, d, J = 6.5 Hz), 0.99 (3H, d, J = 7.2 Hz), 1.04 (3H, s), 1.21 (9H, s), 1.98–2.03 (1H, m),
2.09 (1H, ddd, J = 12.5, 3.2, 3.2 Hz), 2.27 (1H, dd, J = 18.4, 8.1 Hz), 2.80 (1H, dd, J = 2.7, 2.7 Hz),
3.33 (3H, s), 5.43 (1H, ddd, J = 7.9, 5.8, 1.7 Hz). 13C NMR (151 MHz, CDCl3, δ): 11.3, 13.07,
13.10, 19.2, 21.3, 22.1, 22.49, 22.55, 24.9, 27.3 (3C), 27.9, 29.6, 30.2, 33.1, 34.6, 34.99, 35.03, 35.2,
38.3, 39.0, 39.5, 43.0, 43.4, 47.7, 50.7, 56.6, 64.2, 75.4, 81.9, 178.3, 218.2. HRMS-FAB-NBA
(m/z): [M+Na]+ calcd for C33H54O4Na, 537.3920; found, 537.3919.
4.14. (22R)-22-(2,2-Dimethylpropanoyloxy)-6β-methoxy-3α,5-cyclocholestan-16-one (22R-13).
A solution of 16α,22R-10 (167 mg, 0.323 mmol) in anhydrous CH2Cl2 (10 mL) was treated with
DMP (155 mg, 0.365 mmol), before the mixture was stirred for 2 h at room temperature. The
mixture was diluted with CHCl3 (40 mL) and washed with a saturated aqueous solution of NaHCO3
(20 mL). The organic layer was dried over anhydrous MgSO4, filtered, and concentrated in vacuo.
The residue was purified by flash column chromatography on silica gel (hexane/EtOAc = 90/10–
70/30, v/v) to give 22R-13 (148 mg, 89%) as a colorless oil. [α]D28 –71.7 (c 0.977 g/dL, CHCl3). 1H
NMR (600 MHz, CDCl3, δ): 0.47 (1H, dd, J = 7.9, 5.2 Hz), 0.68 (1H, dd, J = 5.2, 3.8 Hz), 0.85 (3H,
d, J = 6.5 Hz), 0.86 (3H, d, J = 6.5 Hz), 0.91 (3H, s), 0.95 (3H, d, J = 7.2 Hz), 1.05 (3H, s), 1.21
(9H, s), 2.10 (1H, ddd, J = 12.4, 3.1, 3.1 Hz), 2.24 (1H, dd, J = 18.2, 7.2 Hz), 2.30–2.36 (1H, m),
2.80 (1H, dd, J = 2.6, 2.6 Hz), 3.34 (3H, s), 5.46 (1H, ddd, J = 8.2, 6.2, 3.4 Hz). 13C NMR (151
MHz, CDCl3, δ): 13.1, 13.4, 14.5, 19.2, 21.2, 22.1, 22.3, 22.8, 24.9, 26.8, 27.3 (3C), 27.9, 29.5,
21

33.1, 34.0, 34.4, 34.9, 35.4, 38.85, 38.93, 39.7, 43.4, 43.5, 47.8, 51.1, 56.6, 64.2, 75.0, 81.9, 177.6,
217.6. HRMS-FAB-NBA (m/z): [M+Na]+ calcd for C33H54O4Na, 537.3920; found, 537.3922.
4.15. Reduction of 22S-13.
A solution of 22S-13 (121 mg, 0.235 mmol) in anhydrous CH2Cl2 (4.0 mL) at –78 °C under an
argon atmosphere was treated dropwise with a DIBAL solution (1.5 M in toluene, 0.80 mL, 1.2
mmol). After 3.5 h of stirring at the same temperature, additional DIBAL solution (1.5 M in toluene,
0.80 mL, 1.2 mmol) was added dropwise to the reaction mixture, before stirring was continued for 2
h at –78 °C. The mixture was then treated dropwise with water (2.0 mL), before the cooling bath
was removed. The mixture was diluted with EtOAc (30 mL) and dried over anhydrous MgSO4.
Insoluble materials were filtered off, and the filtrate was concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel (hexane/EtOAc = 75/25–60/40, v/v) to give
16αβ,22S-9 (73.3 mg, 72%, α/β = 9/91) as a colorless solid.
4.16. (22S)-16α,22-Carbonyldioxy-6β-methoxy-3α,5-cyclocholestane (16α,22S-14) and
(22S)-16β,22-carbonyldioxy-6β-methoxy-3α,5-cyclocholestane (16β,22S-14).
16αβ,22S-9 (1.38 g, 3.19 mmol, α/β = 17/83) and CDI (779 mg, 4.80 mmol) were dissolved in
toluene (220 mL), and the solution was stirred under reflux for 4 days before the mixture was
cooled to room temperature and concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (hexane/EtOAc = 90/10–70/30, v/v) to give 16α,22S-14 (188 mg,
13%) and 16β,22S-14 (1.03 g, 70%) as colorless solids. 16α,22S-14: Mp 212 °C. [α]D16 +57.0 (c
1.54 g/dL, CHCl3). 1H NMR (600 MHz, CDCl3, δ): 0.46 (1H, dd, J = 8.0, 5.2 Hz, H-4α), 0.66 (1H,
dd, J = 5.2, 3.7 Hz, H-4β), 0.79 (3H, s, H3-18), 0.87 (1H, ddd, J = 13.2, 11.8, 7.8 Hz, H-1β), 0.900
(3H, d, J = 6.6 Hz, H3-26), 0.901 (3H, d, J = 6.6 Hz, H3-27), 0.91 (1H, m, H-9α), 0.92 (1H, m, H-3),
22

1.00 (3H, d, J = 6.7 Hz, H3-21), 1.02 (3H, s, H3-19), 1.12 (1H, ddd, J = 13.3, 12.1, 2.8 Hz, H-7α),
1.16 (1H, m, Ha-24), 1.34 (1H, ddd, J = 13.1, 11.9, 3.7 Hz, H-12α), 1.40 (1H, m, H-11β), 1.46 (1H,
m, Hb-24), 1.48 (1H, m, Ha-23), 1.49 (2H, m, H-1α and H-11α), 1.53 (1H, m, H-14α), 1.55 (1H, m,
H-2β), 1.56 (1H, m, H-25), 1.62 (1H, dd, J = 10.3, 9.1 Hz, H-17α), 1.68 (1H, m, Hb-23), 1.75 (1H,
m, H-8β), 1.76 (1H, m, H-2α), 1.79 (1H, m, H-15β), 1.81 (1H, ddd, J = 13.3, 2.8, 2.8 Hz, H-7β),
1.86 (1H, ddd, J = 11.9, 3.2, 2.6 Hz, H-12β), 1.96 (1H, ddd, J = 14.2, 8.5, 2.4 Hz, H-15α), 1.96 (1H,
br s, H-20), 2.79 (1H, dd, J = 2.8, 2.8 Hz, H-6α), 3.33 (3H, s, OMe), 4.55 (1H, br s, H-22), 4.94 (1H,
br s, H-16β). 13C NMR (151 MHz, CDCl3, δ): 13.08 (C-4), 13.08 (br, C-21), 14.3 (br, C-18), 19.1
(C-19), 21.2 (C-3), 22.0 (C-11), 22.37 (C-26), 22.43 (C-27), 24.8 (C-2), 27.9 (C-25), 29.0 (br, C-23),
30.1 (C-8), 32.7 (br, C-15), 33.1 (C-1), 34.8 (br, C-20), 34.89 (C-7), 34.93 (C-5), 35.0 (C-24), 39.4
(C-12), 43.3 (C-10), 43.6 (C-13), 47.8 (C-9), 53.4 (C-14), 56.6 (OMe), 59.9 (br, C-17), 81.9 (C-6),
83.3 (br, C-22), 83.9 (br, C-16), 152.4 (br, C=O). HRMS-FAB-NBA (m/z): [M+Na]+ calcd for
C29H46O4Na, 481.3294; found, 481.3299. 16β,22S-14: Mp 183 °C. [α]D16 –46.0 (c 1.53 g/dL,
CHCl3). 1H NMR (600 MHz, CDCl3, δ): 0.46 (1H, dd, J = 8.0, 5.2 Hz, H-4α), 0.66 (1H, dd, J = 5.2,
3.7 Hz, H-4β), 0.85 (1H, m, H-9α), 0.88 (1H, m, H-1β), 0.90 (3H, d, J = 6.8 Hz, H3-26), 0.91 (1H,
m, H-3), 0.91 (3H, d, J = 6.8 Hz, H3-27), 0.92 (3H, s, H3-18), 1.01 (3H, d, J = 6.9 Hz, H3-21), 1.02
(1H, m, H-14α), 1.04 (3H, s, H3-19), 1.09 (1H, ddd, J = 13.7, 12.6, 2.7 Hz, H-7α), 1.17 (1H, ddd, J
= 17.6, 8.5, 3.9 Hz, H-12α), 1.27 (1H, m, Ha-24), 1.41 (1H, dd, J = 10.8, 7.4 Hz, H-17α), 1.46 (1H,
m, H-11β), 1.47 (1H, m, H-11α), 1.50 (1H, m, H-1α), 1.54 (1H, m, H-2β), 1.56 (3H, m, H2-23 and
Hb-24), 1.58 (1H, m, H-25), 1.59 (1H, ddd, J = 13.8, 13.8, 4.8 Hz, H-15β), 1.73 (1H, dddd, J = 12.1,
12.1, 7.9, 4.2 Hz, H-2α), 1.88 (1H, m, H-8β), 1.89 (1H, m, H-12β), 1.90 (1H, m, H-7β), 2.26 (1H,
ddd, J = 13.8, 8.3, 7.1 Hz, H-15α), 2.32 (1H, dqd, J = 10.8, 6.9, 3.9 Hz, H-20), 2.78 (1H, dd, J = 2.7,
2.7 Hz, H-6α), 3.33 (3H, s, OMe), 4.18 (1H, m, H-22), 4.85 (1H, ddd, J = 8.3, 7.4, 4.8 Hz, H-16α).
13C NMR (151 MHz, CDCl3, δ): 13.0 (C-4), 13.3 (C-18), 15.8 (C-21), 19.2 (C-19), 21.4 (C-3), 22.2
(C-11), 22.3 (C-26), 22.6 (C-27), 24.9 (C-2), 27.0 (br, C-23), 27.8 (C-25), 29.8 (C-8), 32.2 (C-20),
32.5 (C-15), 33.2 (C-1), 34.9 (C-7), 35.1 (C-5), 35.2 (C-24), 39.7 (C-12), 42.3 (C-13), 43.3 (C-10),
48.0 (C-9), 53.8 (C-14), 54.5 (br, C-17), 56.6 (OMe), 80.5 (C-16), 81.9 (C-6), 85.7 (br, C-22), 153.5
(C=O). HRMS-FAB-NBA (m/z): [M+Na]+ calcd for C29H46O4Na, 481.3294; found, 481.3296. In
this reaction, bis-imidazolylcarbonylated 15 (203 mg, 10%, 16α/16β = 33/67) was also obtained as
a colorless solid. The representative 1H NMR peaks of 15 are the following (600 MHz, CDCl3, δ).
For the 16β-isomer: 5.07 (1H, dd, J = 7.0, 7.0 Hz), 5.66 (1H, ddd, J = 7.6, 7.6, 4.1 Hz), 7.09 (1H, s),
7.37 (1H, s), 7.40 (1H, s), 8.09 (1H, s), 8.11 (1H, s). For the 16α-isomer: 4.95 (1H, dd, J = 7.0, 7.0
Hz), 5.26 (1H, dd, J = 6.7, 6.7 Hz), 6.99 (1H, s), 7.06 (1H, s), 7.16 (1H, s), 7.18 (1H, s), 7.92 (1H,
s), 7.95 (1H, s).
23

4.17. (22S)-6β-Methoxy-3α,5-cyclocholestane-16β,22-diol (16β,22S-9).
An ice-cold solution of 16β,22S-14 (151 mg, 0.329 mmol) in anhydrous THF (5.0 mL) was slowly
treated with LiAlH4 (64.9 mg, 1.71 mmol), before the mixture was stirred for 1 h at 0 °C. The
reaction mixture was successively treated dropwise with water (0.10 mL), a 10% aqueous solution
of NaOH (0.10 mL), and water (0.30 mL), before stirring was continued for 1 h at room
temperature. The mixture was diluted with EtOAc (30 mL) and dried over anhydrous MgSO4.
Insoluble materials were filtered off, and the filtrate was concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel (hexane/EtOAc = 75/25–60/40, v/v) to give
16β,22S-9 (129 mg, 91%) as a colorless solid. Mp 77 °C. [α]D23 +23.1 (c 1.29 g/dL, CHCl3). 1H
NMR (600 MHz, CDCl3, δ): 0.43 (1H, dd, J = 8.0, 5.1 Hz, H-4α), 0.64 (1H, dd, J = 5.1, 3.7 Hz,
H-4β), 0.82 (1H, ddd, J = 10.9, 10.9, 5.1 Hz, H-9α), 0.86 (1H, ddd, J = 13.4, 12.0, 7.8 Hz, H-1β),
0.90 (1H, m, H-3), 0.90 (3H, d, J = 6.6 Hz, H3-26), 0.91 (3H, d, J = 6.6 Hz, H3-27), 0.96 (3H, s,
H3-18), 0.97 (3H, d, J = 7.1 Hz, H3-21), 0.97 (1H, m, H-14α), 1.03 (3H, s, H3-19), 1.06 (1H, ddd, J
= 13.1, 11.9, 2.9 Hz, H-7α), 1.12 (1H, ddd, J = 12.3, 12.3, 4.9 Hz, H-12α), 1.17 (1H, m, Ha-24),
1.23 (1H, dd, J = 11.2, 7.0 Hz, H-17α), 1.26 (1H, ddd, J = 13.1, 13.1, 4.6 Hz, H-15β), 1.39 (1H, m,
Hb-24), 1.40 (1H, m, Ha-23), 1.41 (1H, m, H-11β), 1.43 (1H, m, H-11α), 1.48 (1H, m, Hb-23), 1.50
(1H, m, H-1α), 1.52 (1H, m, H-2β), 1.57 (1H, m, H-25), 1.73 (1H, dddd, J = 12.0, 12.0, 7.9, 4.2 Hz,
H-2α), 1.82 (1H, dddd, J = 11.9, 10.9, 10.9, 2.9 Hz, H-8β), 1.92 (1H, ddd, J = 13.1, 2.9, 2.9 Hz,
H-7β), 1.94 (1H, ddd, J = 12.3, 3.4, 3.4 Hz, H-12β), 2.24 (1H, ddd, J = 13.1, 7.9, 7.5 Hz, H-15α),
2.27 (1H, dqd, J = 11.2, 7.1, 2.2 Hz, H-20), 2.77 (1H, dd, J = 2.9, 2.9 Hz, H-6α), 2.90 (1H, br s,
OH), 3.32 (3H, s, OMe), 3.62 (1H, ddd, J = 9.4, 2.6, 2.2 Hz, H-22), 3.80 (1H, br s, OH), 4.35 (1H,
ddd, J = 7.9, 7.0, 4.6 Hz, H-16α). 13C NMR (151 MHz, CDCl3, δ): 13.0 (C-4), 13.4 (C-18), 16.2 (br,
C-21), 19.3 (C-19), 21.6 (C-3), 22.4 (C-11), 22.5 (C-26), 22.7 (C-27), 24.9 (C-2), 28.1 (C-25), 30.0
(C-23), 30.1 (C-8), 33.3 (C-1), 34.7 (C-7), 34.9 (C-20), 35.4 (C-5), 35.8 (C-15), 36.1 (C-24), 40.5
(C-12), 42.9 (C-13), 43.3 (C-10), 48.0 (C-9), 54.4 (C-14), 56.4 (OMe), 57.4 (C-17), 71.9 (C-16),
77.6 (C-22), 82.3 (C-6). HRMS-FAB-NBA (m/z): [M+Na]+ calcd for C28H48O3Na, 455.3501; found,
455.3503. Using the same procedure, 16α,22S-14 (154 mg, 0.336 mmol) was converted into
16α,22S-9 (134 mg, 92%).
4.18. (22R)-6β-Methoxy-3α,5-cyclocholestane-16β,22-diol (16β,22R-9).
24

A boiling suspension of LiAlH4 (56.0 mg, 1.48 mmol) in anhydrous THF (5.0 mL) was treated
dropwise with a solution of 22R-13 (148 mg, 0.287 mmol) in anhydrous THF (5.0 mL). The
resulting mixture was stirred under reflux for 1 h and then cooled to 0 °C. This mixture was
successively treated dropwise with water (0.10 mL), a 10% aqueous solution of NaOH (0.10 mL),
and water (0.30 mL), before stirring was continued for 1 h at room temperature. The mixture was
diluted with EtOAc (30 mL) and dried over anhydrous MgSO4. Insoluble materials were filtered off,
and the filtrate was concentrated in vacuo, before the residue was purified by flash column
chromatography on silica gel (hexane/EtOAc = 70/30–50/50, v/v) to give pure 16β,22R-9 (106 mg,
85%) as a colorless solid. Mp 149–150 °C. [α]D22 +55.0 (c 0.551 g/dL, CHCl3). 1H NMR (600 MHz,
CDCl3, δ): 0.43 (1H, dd, J = 8.0, 5.1 Hz, H-4α), 0.65 (1H, dd, J = 5.1, 3.7 Hz, H-4β), 0.81 (1H, ddd,
J = 10.7, 10.7, 5.5 Hz, H-9α), 0.86 (1H, ddd, J = 13.2, 11.3, 7.8 Hz, H-1β), 0.89 (1H, m, H-3), 0.90
(3H, d, J = 6.7 Hz, H3-26), 0.91 (3H, d, J = 6.7 Hz, H3-27), 0.93 (1H, m, H-14α), 0.95 (3H, s,
H3-18), 0.96 (3H, d, J = 6.7 Hz, H3-21), 1.02 (3H, s, H3-19), 1.06 (1H, dd, J = 11.0, 7.4 Hz, H-17α),
1.06 (1H, ddd, J = 13.4, 13.4, 2.9 Hz, H-7α), 1.11 (1H, ddd, J = 12.5, 11.7, 5.9 Hz, H-12α), 1.22
(1H, m, Ha-24), 1.25 (1H, ddd, J = 13.2, 13.2, 4.9 Hz, H-15β), 1.35 (1H, m, Ha-23), 1.40 (1H, m,
Hb-24), 1.41 (1H, m, H-11β), 1.43 (1H, m, H-11α), 1.46 (1H, m, Hb-23), 1.50 (1H, m, H-1α), 1.52
(1H, m, H-2β), 1.55 (1H, m, H-25), 1.74 (1H, dddd, J = 12.0, 12.0, 7.8, 4.2 Hz, H-2α), 1.82 (1H, m,
H-8β), 1.91 (1H, ddd, J = 13.4, 2.9, 2.9 Hz, H-7β), 2.00 (1H, ddd, J = 12.5, 3.3, 3.3 Hz, H-12β),
2.12 (1H, m, H-20), 2.25 (1H, ddd, J = 13.2, 7.8, 7.4 Hz, H-15α), 2.77 (1H, dd, J = 2.9, 2.9 Hz,
H-6α), 3.32 (3H, s, OMe), 3.66 (1H, ddd, J = 8.9, 5.4, 2.1 Hz, H-22), 4.32 (1H, ddd, J = 7.8, 7.4,
4.9 Hz, H-16α). 13C NMR (151 MHz, CDCl3, δ): 13.0 (C-4), 13.3 (C-18), 13.7 (C-21), 19.2 (C-19),
21.5 (C-3), 22.4 (2C, C-11 and C-26), 22.9 (C-27), 24.9 (C-2), 28.2 (C-25), 30.0 (C-8), 30.3 (C-23),
33.2 (C-1), 34.8 (C-7), 35.1 (C-24), 35.3 (C-5), 36.2 (C-15), 36.3 (C-20), 40.5 (C-12), 43.1 (C-13),
43.3 (C-10), 47.9 (C-9), 54.1 (C-14), 56.5 (OMe), 59.8 (C-17), 72.5 (C-16), 75.5 (C-22), 82.2 (C-6).
HRMS-FAB-NBA (m/z): [M+Na]+ calcd for C28H48O3Na, 455.3501; found, 455.3507.
4.19. (22R)-16β,22-Carbonyldioxy-6β-methoxy-3α,5-cyclocholestane (16β,22R-14) and
(22S)-6β-methoxy-3α,5-cyclofurostane (16).
25

16αβ,22R-9 (129 mg, 0.298 mmol, α/β = 10/90) and CDI (73.7 mg, 0.455 mmol) were dissolved in
toluene (20 mL), before the resulting solution was stirred under reflux for 4 days. The mixture was
then cooled to room temperature and concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel (hexane/EtOAc = 90/10–80/20, v/v) to give 16β,22R-14 (58.2
mg, 43%) as a colorless solid, and 16 (53.4 mg, 43%) as a colorless oil, respectively. 16β,22R-14:
Mp 132–133 °C. [α]D19 +33.1 (c 0.274 g/dL, CHCl3). 1H NMR (600 MHz, CDCl3, δ): 0.46 (1H, dd,
J = 8.0, 5.2 Hz, H-4α), 0.66 (1H, dd, J = 5.2, 3.7 Hz, H-4β), 0.84 (1H, m, H-9α), 0.88 (1H, m,
H-1β), 0.90 (3H, d, J = 6.5 Hz, H3-26), 0.90 (1H, m, H-3), 0.92 (3H, d, J = 6.5 Hz, H3-27), 0.93 (3H,
s, H3-18), 1.01 (1H, ddd, J = 14.1, 10.9, 6.8 Hz, H-14α), 1.03 (3H, d, J = 6.5 Hz, H3-21), 1.04 (3H,
s, H3-19), 1.10 (1H, ddd, J = 13.7, 12.7, 2.8 Hz, H-7α), 1.21 (1H, m, H-12α), 1.39 (1H, dd, J = 10.6,
7.9 Hz, H-17α), 1.39 (1H, m, Ha-24), 1.45 (1H, m, H-11β), 1.46 (1H, m, H-11α), 1.49 (1H, m,
H-1α), 1.50 (1H, m, Hb-24), 1.54 (1H, m, H-2β), 1.56 (1H, ddd, J = 13.5, 10.9, 5.5 Hz, H-15β), 1.57
(1H, m, H-25), 1.62 (1H, m, Ha-23), 1.74 (1H, dddd, J = 12.1, 12.1, 7.9, 4.2 Hz, H-2α), 1.80 (1H,
dddd, J = 14.1, 11.2, 5.1, 2.7 Hz, Hb-23), 1.87 (1H, m, H-8β), 1.89 (1H, ddd, J = 13.7, 2.8, 2.8 Hz,
H-7β), 1.99 (1H, ddd, J = 12.5, 3.3, 3.3 Hz, H-12β), 2.08 (1H, ddq, J = 10.6, 10.6, 6.5 Hz, H-20),
2.22 (1H, ddd, J = 13.5, 8.4, 6.8 Hz, H-15α), 2.79 (1H, dd, J = 2.8, 2.8 Hz, H-6α), 3.33 (3H, s,
OMe), 4.04 (1H, ddd, J = 10.6, 8.1, 2.7 Hz, H-22), 4.92 (1H, ddd, J = 8.4, 7.9, 5.5 Hz, H-16α). 13C
NMR (151 MHz, CDCl3, δ): 13.1 (C-4), 13.4 (C-18), 15.9 (C-21), 19.2 (C-19), 21.3 (C-3), 22.2
(C-26), 22.4 (C-11), 22.8 (C-27), 24.9 (C-2), 27.8 (C-25), 29.8 (C-8), 31.3 (C-23), 32.2 (C-15), 33.2
(C-1), 33.4 (C-24), 33.7 (C-20), 35.0 (C-7), 35.1 (C-5), 40.5 (C-12), 42.7 (C-13), 43.3 (C-10), 47.8
(C-9), 53.6 (C-14), 56.6 (OMe), 59.6 (C-17), 80.7 (C-16), 81.9 (C-6), 87.9 (C-22), 152.9 (C=O).
19
HRMS-FAB-NBA (m/z): [M+Na]+ calcd for C29H46O4Na, 481.3294; found, 481.3294. 16: [α]D
+20.0 (c 0.534 g/dL, CHCl3). 1H NMR (600 MHz, CDCl3, δ): 0.44 (1H, dd, J = 8.0, 5.1 Hz, H-4α),
0.65 (1H, dd, J = 5.1, 3.7 Hz, H-4β), 0.82 (1H, ddd, J = 10.5, 10.5, 5.8 Hz, H-9α), 0.86 (1H, ddd, J
= 13.4, 8.2, 3.7 Hz, H-1β), 0.88 (3H, s, H3-18), 0.886 (3H, d, J = 6.7 Hz, H3-26), 0.888 (3H, d, J =
6.7 Hz, H3-27), 0.89 (1H, m, H-3), 0.91 (3H, d, J = 7.2 Hz, H3-21), 1.03 (1H, ddd, J = 13.9, 11.0,
5.5 Hz, H-14α), 1.04 (3H, s, H3-19), 1.07 (1H, ddd, J = 13.3, 12.1, 2.9 Hz, H-7α), 1.11 (1H, m,
H-12α), 1.13 (1H, m, Ha-24), 1.28 (1H, ddd, J = 13.9, 12.2, 6.4 Hz, H-15β), 1.32 (1H, m, Ha-23),
1.33 (1H, m, Hb-24), 1.44 (1H, m, H-11β), 1.46 (1H, m, H-11α), 1.47 (1H, m, Hb-23), 1.51 (1H, dd,
J = 13.4, 7.7 Hz, H-1α), 1.53 (1H, dd, J = 12.2, 7.7 Hz, H-2β), 1.54 (1H, m, H-25), 1.73 (1H, dd, J
26

= 8.6, 2.7 Hz, H-17α), 1.74 (1H, m, H-2α), 1.76 (1H, ddd, J = 12.5, 3.5, 3.5 Hz, H-12β), 1.88 (1H,
m, H-8β), 1.90 (1H, ddd, J = 13.3, 2.9, 2.9 Hz, H-7β), 2.02 (1H, ddd, J = 12.2, 7.5, 5.5 Hz, H-15α),
2.19 (1H, m, H-20), 2.77 (1H, dd, J = 2.9, 2.9 Hz, H-6α), 3.32 (3H, s, OMe), 3.99 (1H, ddd, J = 8.6,
5.6, 4.6 Hz, H-22), 4.50 (1H, m, H-16α). 13C NMR (151 MHz, CDCl3, δ): 13.0 (C-4), 15.1 (C-18),
16.2 (C-21), 19.3 (C-19), 21.4 (C-3), 22.4 (C-11), 22.56 (C-26), 22.61 (C-27), 24.9 (C-2), 28.1
(C-23), 28.2 (C-25), 29.5 (C-8), 33.2 (C-1), 33.3 (C-15), 35.2 (2C, C-5 and C-7), 35.3 (C-20), 35.8
(C-24), 40.2 (C-12), 41.3 (C-13), 43.5 (C-10), 48.2 (C-9), 55.3 (C-14), 56.5 (OMe), 64.5 (C-17),
81.5 (C-16), 82.2 (C-6), 84.4 (C-22). HRMS-FAB-NBA (m/z): [M+Na]+ calcd for C28H46O2Na,
437.3396; found, 437.3396.
4.20. 6β-Methoxy-3α,5-cyclocholesta-16,22-dione (17).
A solution of 16α,22S-9 (502 mg, 1.16 mmol) in anhydrous CH2Cl2 (20 mL) was treated with DMP
(1.25 g, 2.95 mmol), before the mixture was stirred for 21 h at room temperature under an argon
atmosphere. The mixture was diluted with CHCl3 (80 mL) and washed with a saturated aqueous
solution of NaHCO3 (40 mL). The organic layer was dried over anhydrous MgSO4, filtered, and
concentrated in vacuo. The residue was purified by flash column chromatography on silica gel
(hexane/EtOAc = 90/10–70/30, v/v) to give 17 (465 mg, 94%) as a colorless oil, which solidified
during storage at room temperature. Mp 88–89 °C. [α]D30 –106 (c 1.04 g/dL, CHCl3). 1H NMR (600
MHz, CDCl3, δ): 0.48 (1H, dd, J = 7.9, 5.2 Hz), 0.69 (1H, dd, J = 5.2, 3.8 Hz), 0.84 (3H, s), 0.92
(3H, d, J = 6.4 Hz), 0.92 (3H, d, J = 6.4 Hz), 1.05 (3H, d, J = 6.8 Hz), 1.05 (3H, s), 2.20 (1H, dd, J
= 18.6, 7.9 Hz), 2.57 (1H, ddd, J = 17.5, 9.3, 5.5 Hz), 2.60–2.66 (2H, m), 2.71 (1H, ddd, J = 17.5,
9.8, 5.3 Hz), 2.81 (1H, dd, J = 2.7, 2.7 Hz), 3.34 (3H, s). 13C NMR (151 MHz, CDCl3, δ): 13.2, 13.3,
15.4, 19.2, 21.2, 22.1, 22.4 (2C), 24.8, 27.6, 29.7, 32.2, 33.1, 34.9, 35.4, 37.3, 39.1, 40.4, 42.1, 43.3,
43.5, 47.7, 51.1, 56.6, 66.4, 81.9, 214.1, 218.1. HRMS-FAB-NBA (m/z): [M+H]+ calcd for
C28H45O3, 429.3369; found, 429.3365.
4.21. Reduction of 17.
An ice-cold solution of 17 (89.0 mg, 0.208 mmol) in anhydrous THF (10 mL) was slowly treated
with LiAlH4 (43.8 mg, 1.15 mmol), before stirring was continued for 1 h at 0 °C. The mixture was
successively treated dropwise with water (0.10 mL), a 10% aqueous solution of NaOH (0.10 mL),
and water (0.30 mL), before the resulting mixture was stirred for 1 h at room temperature. The
27

mixture was diluted with EtOAc (30 mL) and dried over anhydrous MgSO4. Insoluble materials
were filtered off, and the filtrate was concentrated in vacuo, before the residue was purified by flash
column chromatography on silica gel (hexane/EtOAc = 70/30–55/45, v/v) to give 16αβ,22S-9 (50.5
mg, 56%, 16α/16β = 12/88) and pure 16β,22R-9 (23.8 mg, 26%) as colorless oils.
4.22. (22S)-Cholest-5-ene-3β,16α,22-triol (16α,22S-1).
A mixture of 16α,22S-9 (612 mg, 1.41 mmol), a catalytic amount of p-TsOH•H2O, 1,4-dioxane (70
mL), and water (35 mL) was stirred under reflux for 2 h. Then, the mixture was cooled to room
temperature, diluted with CHCl3 (280 mL), and washed successively with a saturated aqueous
solution of NaHCO3 (110 mL) and brine (110 mL). The organic layer was dried over anhydrous
MgSO4, filtered, and concentrated in vacuo, before the residue was purified by flash column
chromatography on silica gel (hexane/EtOAc = 60/40–50/50, v/v) to give 16α,22S-1 (588 mg, 99%)
as a colorless solid. Mp 230 °C (MeOH). [α]D19 –53.1 (c 0.202 g/dL, CHCl3). 1H NMR (600 MHz,
CDCl3, δ): 0.70 (3H, s, H3-18), 0.90 (6H, d, J = 6.6 Hz, H3-26 and H3-27), 0.93 (3H, d, J = 6.8 Hz,
H3-21), 1.00 (3H, s, H3-19), 1.01 (1H, m, H-9α), 1.09 (1H, ddd, J = 13.6, 13.6, 3.8 Hz, H-1α), 1.16
(1H, m, Ha-24), 1.34 (1H, m, H-12α), 1.36 (1H, m, Hb-24), 1.39 (1H, m, H-17α), 1.41 (1H, m,
Ha-23), 1.44 (1H, m, H-11β), 1.46 (2H, m, H-8β and H-14α), 1.50 (1H, m, H-2β), 1.54 (1H, m,
H-11α), 1.55 (1H, m, Hb-23), 1.56 (1H, m, H-25), 1.58 (1H, br s, OH-3β), 1.59 (2H, m, H-15α and
H-20), 1.60 (1H, m, H-7α), 1.64 (1H, ddd, J = 13.3, 13.3, 8.0 Hz, H-15β), 1.84 (2H, m, H-1β and
H-2α), 1.96 (2H, m, H-7β and H-12β), 2.23 (1H, m, H-4β), 2.30 (1H, ddd, J = 13.0, 4.8, 2.0 Hz,
H-4α), 2.59 (2H, br s, OH-16α and OH-22), 3.54 (1H, dddd, J = 11.3, 11.3, 4.8, 4.8 Hz, H-3α), 3.85
(1H, ddd, J = 8.2, 4.5, 2.4 Hz, H-22), 4.23 (1H, ddd, J = 8.0, 6.7, 1.3 Hz, H-16β), 5.35 (1H, m, H-6).
1H NMR (600 MHz, C5D5N, δ): 0.79 (3H, s, H3-18), 0.91 (3H, d, J = 6.5 Hz, H3-27), 0.93 (3H, d, J
= 6.5 Hz, H3-26), 1.02 (1H, ddd, J = 12.3, 11.2, 4.8 Hz, H-9α), 1.07 (3H, s, H3-19), 1.12 (1H, ddd, J
= 14.7, 13.3, 5.2 Hz, H-1α), 1.19 (3H, d, J = 6.2 Hz, H3-21), 1.33 (1H, ddd, J = 12.3, 11.8, 4.4 Hz,
H-12α), 1.34 (1H, m, Ha-24), 1.45 (1H, dddd, J = 13.2, 12.3, 12.3, 3.5 Hz, H-11β), 1.49 (1H, m,
H-8β), 1.52 (1H, m, H-11α), 1.56 (1H, m, Ha-23), 1.60 (1H, m, H-25), 1.60 (1H, ddd, J = 13.0, 13.0,
6.4 Hz, H-14α), 1.62 (1H, m, H-7α), 1.64 (1H, m, Hb-24), 1.74 (1H, ddd, J = 13.0, 13.0, 8.4 Hz,
H-15β), 1.78 (1H, m, H-20), 1.80 (1H, dd, J = 13.0, 6.8 Hz, H-17α), 1.81 (1H, m, H-2β), 1.83 (1H,
m, H-1β), 1.84 (1H, m, Hb-23), 1.92 (1H, ddd, J = 13.0, 6.4, 0.9 Hz, H-15α), 1.98 (1H, dddd, J =
17.3, 5.4, 4.9, 2.0 Hz, H-7β), 2.02 (1H, ddd, J = 11.8, 3.5, 2.6 Hz, H-12β), 2.10 (1H, m, H-2α), 2.62
28