Stereoselective synthesis of (22R)- and (22S)-castasterone/ponasterone a hybrid compounds and evaluation of their molting hormone activity

Article abstract of DOI:10.1016/j.steroids.2004.04.005

Two stereoisomers of a castasterone/ponasterone A hybrid compound, the (20R,22R) and (20R,22S)-isomers of 2α,3α,20,22-tetrahydroxy- 5α-cholestan-6-one, were synthesized stereoselectively and their binding activity to the ecdysteroid receptor was determined. From the concentration-response curve for the inhibition of the incorporation of tritiated ponasterone A into ecdysteroid receptor containing insect cells, the concentration (IC₅₀) required to inhibit 50% of the incorporation of radioactivity into cells was evaluated. The IC₅₀ values of the (22R)- and (22S)-isomers were determined to be 0.30 and 38.9 μM against Kc cells, respectively, indicating that the (22R)-isomer is about 100 times more potent than the corresponding (22S)-isomer. IC₅₀ values of these compounds against lepidopteran Sf-9 cells were determined to be 0.36 and 12.9 μM, respectively. The molting hormonal effect was examined in a Chilo suppressalis integument system and the 50% effective concentration for the stimulation of N-acetylglucosamine incorporation into the cultured integument was determined to be 2.7 μM for the (22R)-isomer, while the (22S)-isomer was inactive. On the other hand, both isomers did not show brassinolide-like activity in the rice lamina inclination assay.

Full text of DOI:10.1016/j.steroids.2004.04.005

Steroids 69 (2004) 483–493
Stereoselective synthesis of (22R)- and (22S)-castasterone/ponasterone A
hybrid compounds and evaluation of their molting hormone activity
Bunta Watanabe, Yoshiaki Nakagawa∗, Takehiko Ogura, Hisashi Miyagawa
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 6068502, Japan
Received 9 January 2004; received in revised form 5 April 2004; accepted 8 April 2004
Abstract
Two stereoisomers of a castasterone/ponasterone A hybrid compound, the (20R,22R) and (20R,22S)-isomers of 2␣,3␣,20,22-
tetrahydroxy-5␣-cholestan-6-one, were synthesized stereoselectively and their binding activity to the ecdysteroid receptor was deter-
mined. From the concentration–response curve for the inhibition of the incorporation of tritiated ponasterone A into ecdysteroid receptor
containing insect cells, the concentration (IC50) required to inhibit 50% of the incorporation of radioactivity into cells was evaluated. The
IC50 values of the (22R)- and (22S)-isomers were determined to be 0.30 and 38.9 ␮M against Kc cells, respectively, indicating that the
(22R)-isomer is about 100 times more potent than the corresponding (22S)-isomer. IC50 values of these compounds against lepidopteran
Sf-9 cells were determined to be 0.36 and 12.9 ␮M, respectively. The molting hormonal effect was examined in a Chilo suppressalis integu-
ment system and the 50% effective concentration for the stimulation of N-acetylglucosamine incorporation into the cultured integument
was determined to be 2.7 ␮M for the (22R)-isomer, while the (22S)-isomer was inactive. On the other hand, both isomers did not show
brassinolide-like activity in the rice lamina inclination assay.
© 2004 Elsevier Inc. All rights reserved.
Keywords: Castasterone; Ponasterone A; Molting hormone activity; Chilo suppressalis; Insect cells
1. Introduction
significant BR-like activity. On the other hand, two BR
compounds, 24-epibrassinolide and 24-epicastasterone, dis-
placed specifically-bound tritiated ponasterone A (PonA; 4)
in S. littoralis imaginal disc assays [7], whereas CS did not
significantly inhibit tritiated PonA binding at the highest
concentration tested (4% inhibition at 5 ␮M) against Sf-9
cell lines [8]. It is also reported that a few BR/ES hybrid com-
pounds, such as compound 5 (Fig. 1), showed weak molting
hormonal activity [9]. Previously, we reported that diben-
zoylhydrazines and diacylhydrazines analogs have ES-like
activity and the two carbonyl oxygens of diacylhydrazines
correspond to the 20- and 22-hydroxy groups of 20E [10].
Recently, the crystal structure of EcR with bound ligand was
solved and the superposition between 20E and one of dia-
cylhydrazines was shown [11]. As we proposed, the steroid
skeleton was not necessary for strong binding to Lepidoptera
ecdysteroid receptors.
Insect molting is regulated by 20-hydroxyecdysone (20E;
1 in Fig. 1), and a number of ecdysone analogs, which are
together termed ecdysteroids (ES), have been identified in
plants, animals and microorganisms (http://ecdybase.org).
In plants, steroid hormones which regulate growth and
development are also present and are referred to as brassi-
nosteroids (BR) [1–3]. The first identified BR was brassi-
nolide (BL; 2 in Fig. 1) [4], which has a characteristic
7-membered B-ring containing a 7-oxa-6-keto system. To
date, more than 50 BRs have been characterized [5]. Cas-
tasterone (CS; 3 in Fig. 1) identified from insect galls
of the chestnut, has a six-membered ring structure in-
stead of seven-membered lactone structure at the B-ring of
BL [6].
Although ES-containing plants contain a wide variety
of structural analogs, these endogenous ES do not have
Recently, we synthesized a hybrid compound containing
the steroidal moiety of CS and the side-chain of PonA,
and showed that this hybrid compound is not BS-like but
ES-like [12]. This hybrid compound was a mixture of
∗
Corresponding author. Tel.: +81 75 753 6116; fax: +81 75 753 6123.
E-mail address: naka@kais.kyoto-u.ac.jp (Y. Nakagawa).
0039-128X/$ – see front matter © 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2004.04.005

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B. Watanabe et al. / Steroids 69 (2004) 483–493
OH
(R)
(R)
OH
(R)
OH
(S)
OH
(R)
R
HO
HO
HO
HO
OH
O
H
O
H
O
20-Hydroxyecdysone
(1; R = OH)
Brassinolide (2)
Ponasterone A (4; R = H)
OH
(R)
OH
(S)
(R)
(S)
OH
(S)
(R)
OH
HO
HO
OH
HO
H
H
O
O
Castasterone (3)
5
Fig. 1. Structures of ecdysteroids, brassinosteroids, and related compounds.
epimers with respect to C-22 of the side-chain. In this
study, we stereoselectively synthesized both the (22R)-
and (22S)-isomers (Schemes 1 and 2) and evaluated their
ES-like activity. The receptor binding activity was evaluated
by performing competitive binding assays with tritiated
PonA with insect Kc and Sf-9 cells [8,13], and the molting
hormonal activity was determined in cultured integument
of Chilo suppressalis by observing the effect on chitin syn-
thesis [14,15]. BR-like activity was also measured using
the rice lamina inclination assay [16].
OH
OH
O
c
b
6: R = H
7: R = TBDMS
a
8
9
RO
RO
RO
OH
OH
OH
O
e
12: R = TBDMS
13: R = H
f
10
RO
RO
d
+
OH
OH
OH
O
e
14: R = TBDMS
15: R = H
f
11 (more polar)
RO
RO
Scheme 1. Reagents and conditions: (a) TBDMSCl, imidazole, DMF, r.t., 5 days (b) 4-methyl-1-pentyne, n-BuLi, THF, −78 ◦C, 10 min (c) H2 Lindlar
catalyst, quinoline, EtOAc, r.t., 1.5 h, then H2, Lindlar catalyst, EtOAc, r.t., 20 min (d) VO(acac)2, TBHP, CH2Cl2, r.t., overnight, then chromatographic
separation (e) LiAlH4, THF r.t., overnight (f) TBAF, THF, r.t., 4 days.

B. Watanabe et al. / Steroids 69 (2004) 483–493
485
O
O
22
a
c
e
d
13 or 15
TsO
16: (22R); R = H
17: (22R); R = Ts
H
H
b
18: (22R)
26: (22S)
19: (22R)
27: (22S)
OH
OH
RO
24: (22S); R = H
25: (22S); R = Ts
b
OH
OH
HO
HO
HO
HO
O
O
g
f
h or i
H
H
H
H
20: (22R)
28: (22S)
21: (22R)
29: (22S)
22: (22R)
30: (22S)
23: (22R)
31: (22S)
O
O
O
O
Scheme 2. Reagents and conditions: (a) (MeO)2CMe2, p-TsOH, CHCl3, r.t., 40 min, then MeOH, overnight (b) TsCl, pyridine, r.t., 2 days (c) BH3·SMe2,
THF, r.t., overnight then 10% NaOH and 30% H2O2, 0 ◦C, 20 min (d) LiBr, Li2CO3, DMF, 120 ◦C, 1 h (e) CrO3, H2SO4, acetone, r.t., 50 min (f) OsO4,
NMO, acetone, r.t., overnight (g) (MeO)2CMe2, p-TsOH, r.t., 1 h, then chromatographic separation (h) for 22, 60% AcOH, EtOH, 80 ◦C, 6 h (i) for 30,
80% AcOH, 80 ◦C, 1 h.
75 ml) and brine (50 ml). After drying the organic layer over
anhydrous Na2SO4, the solvent was evaporated in vacuo
2. Experimental
to yield compound 7 (13.20 g) as a white solid, which was
subsequently used without purification.
2.1. Synthesis
2.1.1. General
Melting points (Mp) were measured with a Yanagimoto
2.1.2.2. (20R)-3β-(tert-Butyldimethylsilyl)oxycholesta-5-
en-22-yn-20-ol (8). 4-Methyl-1-pentyne (9.0 ml, 76.7
mmol) in anhydrous THF (30 ml) was cooled to −78 ◦C
under Ar, and 48 ml (75.8 mmol) of 1.58 M n-BuLi/hexane
was added dropwise within 25 min. After stirring the mix-
ture for 15 min, compound 7 (13.2 g, 30.0 mmol), dissolved
in anhydrous THF (90 ml), was added dropwise within
10 min. Saturated aqueous NH4Cl (5.0 ml) was added drop-
wise to the reaction mixture, which was then warmed to
room temperature. After addition of the saturated aqueous
NH4Cl (100 ml), the mixture was extracted with EtOAc (4
× 30 ml). The combined organic layers were washed with
brine (50 ml). After drying the organic layer over anhydrous
Na2SO4, the solvent was evaporated in vacuo to yield com-
pound 8 (15.72 g) as a white solid, which was used without
further purification.
melting point apparatus (Yanaco, Kyoto, Japan) and are un-
corrected. Optical rotations were measured on a JASCO
P-1010 polarimeter. HRMS was recorded on a JOEL JMS
700 spectrometer operating in the 70 eV EI mode. NMR
spectra were recorded on a Bruker ARX-500 (500 MHz for
1H and 125 MHz for 13C) in CDCl3 unless indicated oth-
erwise. Complete assignment of 13C signals were carried
1
out using H-1H COSY, HMQC and HMBC spectra. Flash
column chromatography was conducted using Kieselgel 60
(Merck, Darmstadt, Germany) as the adsorbent. Lindlar cat-
alyst was purchased from Wako Pure Chemical Industries
Ltd., Osaka, Japan and used without further treatment. An-
hydrous tert-butyl hydroperoxide (TBHP)/CH2Cl2 solution
was prepared as described in the literature [17]. Elemental
analyses were performed at the Microanalytical Center of
Kyoto University.
2.1.2.3. (20R,22Z)-3β-(tert-Butyldimethylsilyl)oxycholesta-
5,22-dien-20-ol (9). Lindlar catalyst (2.50 g) was added
to a mixture of compound 8 (15.72 g, 30 mmol), EtOAc
(160 ml) and quinoline (0.13 ml). After stirring the mixture
for 1.5 h under hydrogen at room temperature, the catalyst
was filtered out and washed with EtOAc (140 ml). The com-
bined EtOAc solutions were washed with 1N HCl (50 ml)
and saturated aqueous NaHCO3 (50 ml) successively. The
organic layer was dried over anhydrous Na2SO4 and con-
centrated in vacuo to yield a white solid (15.61 g). Since the
2.1.2. Chemical synthesis
2.1.2.1. 3β-(tert-Butyldimethylsilyl)oxypregn-5-en-20-one
(7). Imidazole (6.13 g, 90.0 mmol) and tert-butyldimethyl-
silyl chloride (TBDMSCl; 6.78 g, 45.0 mmol) were added
to pregnenolone (6; 9.49 g, 30.0 mmol) in anhydrous DMF
(50 ml), successively. After stirring for 5 days at room
temperature, toluene (200 ml) was added to the reaction
mixture, and the organic layer was washed with water (3 ×

486
B. Watanabe et al. / Steroids 69 (2004) 483–493
reaction was not complete, the products were dissolved in
EtOAc (160 ml) and stirred for a further 20 min in the pres-
ence of the Lindlar catalyst (2.50 g) under hydrogen. The
mixture was filtered through Celite®, and the solvent was
evaporated to yield compound 9 (15.38 g) as a white solid,
which was used without further purification. An analytical
sample was purified by recrystallization from acetone. Mp
152–153 ◦C; [␣]D26 −48.4◦ (c 0.76, CHCl3); 1H NMR δ
0.06 (6H, s, TBDMS), 0.84 (3H, s, 18-H3), 0.89 (9H, s, TB-
DMS), 0.89–0.95 (1H, m, 9␣-H), 0.91 (6H, d, J = 6.6 Hz,
26-H3 and 27-H3), 0.96–1.02 (1H, m, 14␣-H), 1.00 (3H,
s, 19-H3), 1.04 (1H, td, J = 13.7 and 3.7 Hz, 1␣-H), 1.15
(1H, m, 15-H), 1.24 (1H, td, J = 12.2 and 5.2 Hz, 12␣-H),
1.40 (3H, s, 21-H3), 1.43–1.73 (11H, m), 1.80 (1H, dt, J
= 13.2 and 3.4 Hz, 1␤-H), 1.97 (1H, dtd, J = 17.0, 4.9 and
2.7 Hz, 7␤-H), 2.11 (1H, ddd, J = 12.4, 3.4 and 3.1 Hz,
12␤-H), 2.16 (1H, ddd, J = 13.3, 4.9 and 2.2 Hz, 4␣-H),
2.24 (2H, m, 24-H2), 2.27 (1H, m, 4␤-H), 3.48 (1H, tt, J
= 10.9 and 4.7 Hz, 3␣-H), 5.22 (1H, dt, J = 12.1 and 7.4 Hz,
23-H), 5.31 (1H, m, 6-H), 5.44 (1H, dt, J = 12.1 and 1.5 Hz,
22-H); 13C NMR δ −4.59 (TBDMS), 13.58 (C-18), 18.26
(TBDMS), 19.43 (C-19), 20.90 (C-11), 22.40 (C-26/C-27),
22.52 (C-26/C-27), 23.24 (C-16), 23.89 (C-15), 25.94 (TB-
DMS), 29.10 (C-25), 29.56 (C-21), 31.30 (C-8), 31.81
(C-7), 32.08 (C-2), 36.60 (C-10), 37.04 (C-24), 37.38 (C-1),
40.27 (C-12), 42.81 (C-4), 42.81 (C-13), 50.16 (C-9), 56.78
(C-14), 60.64 (C-17), 72.61 (C-3), 76.92 (C-20), 121.02
(C-6), 128.29 (C-23), 137.39 (C-22), 141.61 (C-5); analysis
calculated for C33H58O2Si: C, 76.98; H, 11.35. Found: C,
76.74; H, 11.45.
18.25 (TBDMS), 19.42 (C-19), 20.89 (C-11), 22.46 (C-16),
22.56 (C-26/C-27), 22.72 (C-26/C-27), 23.50 (C-21), 23.91
(C-15), 25.92 (TBDMS), 27.50 (C-25), 31.25 (C-8), 31.77
(C-7), 32.05 (C-2), 36.09 (C-24), 36.57 (C-10), 37.36 (C-1),
40.24 (C-12), 42.78 (C-4), 42.88 (C-13), 50.19 (C-9), 56.68
(C-14), 57.01 (C-23), 61.52 (C-17), 62.25 (C-22), 71.25
(C-20), 72.59 (C-3), 121.01 (C-6), 141.57 (C-5); analysis
calculated for C33H58O3Si: C, 74.66; H, 11.01. Found: C,
74.79; H, 10.73.
2.1.2.5. (20R,22S,23S)-22,23-Epoxy-3β-(tert-butyldimeth-
ylsilyl)oxycholest-5-en-20-ol (11). Compound 11 (7.18 g,
45% from 6), derived from compound 9 as described
above, was more polar than compound 10. Mp 200–201 ◦C
(acetone/CHCl3); [␣]2D9 −80.1◦ (c 1.55, CHCl3); H NMR
1
δ 0.06 (6H, s, TBDMS), 0.85 (3H, s, 18-H3), 0.89 (9H,
s, TBDMS), 0.93 (1H, ddd, J = 11.5, 11.1 and 5.2 Hz,
9␣-H), 0.99 (3H, d, J = 6.3 Hz, 26-H3/27-H3), 1.00 (3H,
d, J = 6.6 Hz, 26-H3/27-H3), 1.01 (3H, s, 19-H3), 1.04
(2H, m), 1.17 (1H, m, 15-H), 1.24 (1H, td, J = 12.6 and
4.7 Hz, 12␣-H), 1.43 (3H, s, 21-H3), 1.46–1.58 (5H, m),
1.60–1.73 (5H, m), 1.77–1.84 (4H, m), 1.98 (1H, dtd, J
= 17.0, 4.8 and 2.6 Hz, 7␤-H), 2.07 (1H, dt, J = 12.3 and
3.2 Hz, 12␤-H), 2.17 (1H, ddd, J = 13.3, 4.8 and 2.1 Hz,
4␣-H), 2.27 (1H, m, 4␤-H), 2.75 (1H, d, J = 4.3 Hz, 22-H),
3.01 (1H, ddd, J = 9.4, 4.2 and 2.5 Hz, 23-H), 3.48 (1H, tt,
J = 11.0 and 4.7 Hz, 3␣-H), 5.32 (1H, m, 6-H); 13C NMR
δ -4.61 (TBDMS), 13.60 (C-18), 18.24 (TBDMS), 19.42
(C-19), 20.89 (C-11), 22.58 (C-26/C-27), 22.65 (C-16),
22.80 (C-26/C-27), 23.83 (C-15), 25.92 (TBDMS), 27.23
(C-21), 27.71 (C-25), 31.44 (C-8), 31.73 (C-7), 32.04 (C-2),
36.57 (C-10), 36.91 (C-24), 37.35 (C-1), 39.90 (C-12),
42.64 (C-13), 42.77 (C-4), 50.15 (C-9), 56.47 (C-17), 56.82
(C-14), 59.93 (C-23), 63.34 (C-22), 72.57 (C-3), 72.81
(C-20), 120.98 (C-6), 141.54 (C-5); analysis calculated for
C33H58O3Si: C, 74.66; H, 11.01. Found: C, 74.46; H, 10.71.
2.1.2.4. (20R,22R,23R)-22,23-Epoxy-3β-(tert-butyldimeth-
ylsilyl)oxycholest-5-en-20-ol (10). Anhydrous CH2Cl2
(60 ml) was added to a mixture of compound 9 (15.38 g,
30 mmol) and vanadyl acetylacetonate (80 mg, 0.30 mmol)
under Ar. After adding 4.43 M TBHP/CH2Cl2 (10 ml,
44.3 mmol), the mixture was stirred overnight at room tem-
perature. Dimethyl sulfide (1.5 ml, 20.4 mmol) was added
and the mixture was stirred for 30 min at room tempera-
ture followed by addition of silica gel (80 g). The solvent
was removed in vacuo and the residue was subjected to
repeated flash column chromatography (hexane/EtOAc
= 25:1, three times) to afford the product 10 (5.13 g, 32%
from 6). Mp 203 ◦C (acetone/CHCl3); [␣]2D7−24.8◦ (c 1.33,
2.1.2.6. (20R,22R)-3β-(tert-Butyldimethylsilyl)oxycholest-
5-ene-20,22-diol (12). Lithium aluminum hydride (1.98 g,
52.2 mmol) was added to an ice-cold solution of compound
10 (2.77 g, 5.22 mmol) in anhydrous THF (50 ml) and the
mixture was stirred for 30 min at 0 ◦C, then further stirred
overnight at room temperature. After cooling the mixture
with an ice-water bath, water (2.0 ml) and 15% (w/v) NaOH
(2.0 ml) were added dropwise, followed by the addition of
water (5.9 ml). After stirring for 10 min at room tempera-
ture, the precipitate was removed by filtration and the or-
ganic layer was dried over anhydrous MgSO4. The solvent
was evaporated to yield compound 12 (2.49 g) as a white
solid, which was subsequently used without purification.
1
CHCl3); H NMR δ 0.06 (6H, s, TBDMS), 0.87 (3H, s,
18-H3), 0.89 (9H, s, TBDMS), 0.92 (1H, td, J = 10.9 and
6.3 Hz, 9␣-H), 0.97–1.07 (2H, m), 0.98 (3H, d, J = 6.4 Hz,
26-H3/27-H3), 0.99 (3H, d, J = 6.4 Hz, 26-H3/27-H3), 1.00
(3H, s, 19-H3), 1.17 (1H, m, 15-H), 1.27 (1H, td, J = 11.9
and 6.1 Hz, 12␣-H), 1.34 (3H, s, 21-H3), 1.44–1.58 (6H,
m), 1.64–1.74 (5H, m), 1.75–1.84 (3H, m), 1.97 (1H, m,
7␤-H), 2.10 (1H, dt, J = 12.3 and 3.2 Hz, 12␤-H), 2.17 (1H,
ddd, J = 13.1, 5.0 and 2.1 Hz, 4␣-H), 2.27 (1H, m, 4␤-H),
2.83 (1H, d, J = 4.3 Hz, 22-H), 2.94 (1H, dt, J = 8.5 and
4.3 Hz, 23-H), 3.48 (1H, tt, J = 10.9 and 4.7 Hz, 3␣-H), 5.32
(1H, m, 6-H); 13C NMR δ -4.60 (TBDMS), 13.01 (C-18),
2.1.2.7. (20R,22R)-Cholest-5-ene-3β,20,22-triol (13).
A
1.0 M solution of tetra-n-butylammonium fluoride (TBAF)/
THF (5.7 ml, 5.7 mmol) was added to the crude compound
12 (2.49 g) dissolved in anhydrous THF (11 ml) and the
mixture was stirred for 3 days at room temperature. To

B. Watanabe et al. / Steroids 69 (2004) 483–493
487
complete the reaction, additional TBAF solution (10.5 ml,
10.5 mmol) was added and the reaction mixture was further
stirred overnight. After addition of CHCl3 (150 ml), the mix-
ture was washed with saturated aqueous NH4Cl (50 ml) and
dried over anhydrous MgSO4. The solvent was evaporated
to yield a yellowish oil (11.22 g), which was purified by
flash column chromatography (hexane/EtOAc = 2:1−1:1)
to obtain the compound 13 (2.13 g, 98%) as a white solid.
Mp 183 ◦C (EtOAc) {literature [18] 178–180 ◦C (MeOH),
literature [19] 176–178 ◦C (acetone/hexane)}; [␣]2D8 −42.5◦
(c 0.76, CHCl3); 1H NMR δ 0.89 (3H, s, 18-H3), 0.90
(3H, d, J = 6.1 Hz, 26-H3/27-H3), 0.91 (3H, d, J = 6.3 Hz,
26-H3/27-H3), 1.02 (3H, s, 19-H3), 1.22 (3H, s, 21-H3),
3.39 (1H, m, 22-H), 3.52 (1H, m, 3␣-H), 5.35 (1H, m,
6-H); 13C NMR δ 13.55, 19.37, 20.37, 20.93, 21.92, 22.35,
22.93, 23.92, 28.06, 29.15, 31.27, 31.60, 31.73, 36.32,
36.47, 37.23, 40.18, 42.24, 43.17, 50.04, 54.73, 56.67,
71.71, 76.39, 77.39, 121.52, 140.76; analysis calculated
for C27H46O3: C, 77.46; H, 10.07. Found: C, 77.24; H,
11.21.
CHCl3 (50 ml), the organic layer was washed with satu-
rated aqueous NaHCO3 (20 ml) and dried over anhydrous
MgSO4. The solvent was removed in vacuo to afford com-
pound 16 (1.76 g) as a white solid, which was used without
further purification.
2.1.2.10. (20R,22R)-20,22-Isopropylidenedioxy-5α-cholest-
2-en-6-one (20). p-Toluenesulfonyl chloride (3.00 g,
15.7 mmol) was added to compound 16 (1.76 g) in anhy-
drous pyridine (15 ml) and the mixture was stirred for 2
days at room temperature. The mixture was poured into
ice-cold 3N HCl (75 ml) and extracted with EtOAc (4 ×
30 ml). The combined organic layer was washed succes-
sively with brine (30 ml) and saturated aqueous NaHCO3
(30 ml), then dried over anhydrous MgSO4. The solvent was
removed in vacuo to give compound 17 (2.21 g) as a yellow-
ish solid, which was dissolved in anhydrous THF (16 ml).
To the mixture was added 2.0 M borane–methyl sulfide
complex/toluene (7.9 ml, 15.8 mmol) at 0 ◦C. The mixture
was stirred overnight under Ar at room temperature. After
cooling in an ice-water bath, 10% NaOH (8.0 ml) and 30%
H2O2 (8.0 ml) were added to the mixture and stirred for
20 min at 0 ◦C. After dilution with water (50 ml), the aque-
ous layer was extracted with EtOAc (4 × 30 ml). The com-
bined organic layer was washed with 20% Na2S2O3 (30 ml)
and brine (20 ml), then dried over anhydrous MgSO4. The
solvent was removed in vacuo to give compound 18 (2.29 g)
as a white solid, which was dissolved in anhydrous DMF
(35 ml) and stirred at 120 ◦C for 1 h in the presence of LiBr
(2.07 g, 19.7 mmol) and Li2CO3 (2.19 g, 29.6 mmol) under
Ar. After cooling to room temperature, insoluble materials
were filtered out and the filtrate was diluted with EtOAc
(200 ml). The organic layer was washed successively with
water (2 × 50 ml) and brine (50 ml), and then dried over
anhydrous MgSO4. The solvent was removed in vacuo to
give compound 19 (2.13 g) as a brown solid, which was
dissolved in acetone (40 ml). Jones reagent (2.0 ml) was
slowly added to this solution at 0 ◦C, and the mixture was
stirred for 50 min at the same temperature followed by ad-
dition of i-PrOH (1.0 ml). After the solvent was evaporated,
water (60 ml) was added to the residue, and the aqueous
layer was extracted with EtOAc (4 × 30 ml). The combined
organic layer was washed successively with brine (30 ml)
and saturated aqueous NaHCO3 (30 ml), then dried over
anhydrous MgSO4. The solvent was removed in vacuo to
yield a pale yellowish solid (1.67 g), which was purified
by flash column chromatography (hexane/EtOAc = 25:1)
to obtain pure compound 20 (0.66 g, 37% from 13). Mp
2.1.2.8. (20R,22S)-Cholest-5-ene-3β,20,22-triol (15). Com-
pound 11 (5.20 g, 9.80 mmol) was treated in the same way
as compound 10 to yield compound 15 (3.43 g, 84%) as
a white solid and recrystallized from acetone/CHCl3. The
melting point was not sharp (181–190 ◦C) {literature [20]
187–189 ◦C (MeOH)} and elemental analysis data was not
acceptable. This may be due to the fact that the compound
is hygroscopic. [␣]D27 −64.9◦ (c 0.89, CHCl3); H NMR δ
1
0.88 (3H, s, 18-H3), 0.90 (3H, d, J = 6.6 Hz, 26-H3/27-H3),
0.91 (3H, d, J = 6.6 Hz, 26-H3/27-H3), 0.94 (1H, td, J
= 11.2 and 5.6 Hz, 9␣-H), 1.01 (3H, s, 19-H3), 1.01 (1H,
m, 14␣-H), 1.08 (1H, m, 1␣-H), 1.15 (1H, m, 15␤-H), 1.20
(1H, m, 24-H), 1.24 (2H, m, 12␣-H and 23-H), 1.27 (3H,
s, 21-H3), 1.42–1.62 (8H, m), 1.65–1.75 (2H, m, 15␣-H
and 16-H), 1.67 (1H, m, 17␣-H), 1.78 (1H, m, 16-H), 1.84
(2H, m, 1␤-H and 2␣-H), 1.98 (1H, m, 7␤-H), 2.10 (1H,
dt, J = 12.4 and 3.2 Hz, 12␤-H), 2.23 (1H m, 4␤-H), 2.30
(1H, m, 4␣-H), 3.25 (1H, dd, J = 9.3 and 5.3 Hz, 22-H),
3.53 (1H, m, 3␣-H), 5.35 (1H, m, 6-H); 13C NMR δ 13.49
(C-18), 19.37 (C-19), 20.97 (C-11), 22.47 (C-26 / C-27),
22.60 (C-16), 22.76 (C-26 / C-27), 23.48 (C-21), 24.12
(C-15), 28.14 (C-25), 29.38 (C-23), 31.33 (C-9), 31.61
(C-1), 31.75 (C-7), 36.44 (C-24), 36.47 (C-10), 37.23 (C-1),
40.10 (C-12), 42.25 (C-4), 43.11 (C-13), 50.04 (C-9), 54.04
(C-17), 56.35 (C-14), 71.74 (C-3), 76.47 (C-20), 80.53
(C-22), 121.59 (C-6), 140.74 (C-5); HRMS m/z calculated
for C27H46O3: 418.3447. Found: 418.3457.
20
1
175 ◦C (EtOH); [␣]D +25.1◦ (c 0.25, CHCl3); H NMR
δ 0.72 (3H, s, 19-H3), 0.80 (3H, s, 18-H3), 0.90 (6H, d,
J = 6.6 Hz, 26-H3 and 27-H3), 1.11–1.19 (2H, m), 1.15
(3H, s, 21-H3), 1.20–1.35 (4H, m), 1.30 (3H, s, acetonide),
1.40–1.49 (4H, m), 1.41 (3H, s, acetonide), 1.52–1.68 (4H,
m), 1.75 (1H, dtd, J = 12.3, 10.7 and 4.1 Hz, 8␤-H), 1.93
(1H, m, 16-H), 1.98 (1H, t, J = 13.3 Hz, 7␣-H), 2.00 (2H,
m, 1-H2), 2.02 (1H, m, 4␣-H), 2.14 (1H, dt, J = 12.5 and
2.1.2.9. (20R,22R)-20,22-Isopropylidenedioxycholest-5-en-
3β-ol (16). Compound 13 (1.65 g, 3.94 mmol), 2,2-dimeth-
oxypropane (4.5 ml) and catalytic amount of p-TsOH
in CHCl3 were stirred for 40 min at room temperature,
then MeOH (3.0 ml) was added to hydrolyze undesired
3␤-(1-methyl-1-methoxyethyl)ether resulting from excess
2,2-dimethoxypropane stirring overnight. After addition of

488
B. Watanabe et al. / Steroids 69 (2004) 483–493
3.2 Hz, 12␤-H), 2.26 (1H, m, 4␤-H), 2.35 (1H, dd, J = 10.0
and 4.7 Hz, 5␣-H), 2.36 (1H, dd, J = 13.1 and 4.2 Hz, 7␤-H),
3.62 (1H, dd, J = 9.2 and 2.8 Hz, 22-H), 5.57 (1H, m, 2-H),
5.69 (1H, ddd, J = 9.9, 4.6 and 2.3 Hz, 3-H); 13C NMR
δ 13.04 (C-18), 13.51 (C-19), 21.05 (C-11), 21.70 (C-4),
21.72 (C-21), 22.49 (C-26/C-27), 22.52 (C-26/C-27), 22.58
(C-16), 23.58 (C-15), 26.75 (acetonide), 26.85 (C-23), 28.22
(C-25), 29.03 (acetonide), 36.38 (C-24), 37.32 (C-8), 39.35
(C-1), 39.78 (C-12), 39.99 (C-10), 43.40 (C-13), 46.84
(C-7), 53.38 (C-9), 53.84 (C-5), 54.73 (C-17), 56.95 (C-14),
81.34 (C-22), 84.14 (C-20), 106.61 (acetonide), 124.48
(C-2), 124.96 (C-3), 211.89 (C-6); analysis calculated for
C30H48O3: C, 78.90; H, 10.59. Found: C, 78.67; H, 10.63.
to yield compound 22 (379 mg, 59%) as a white solid. Mp
92–95 ◦C; [␣]D27 +33.9◦ (c 0.38, CHCl3); H NMR δ 0.68
1
(3H, s, 19-H3), 0.78 (3H, s, 18-H3), 0.90 (6H, d, J = 6.6 Hz,
26-H3 and 27-H3), 1.10–1.18 (2H, m), 1.14 (3H, s, 21-H3),
1.20–1.37 (6H, m), 1.29 (3H, s, 20,22-acetonide), 1.34
(3H, s, 2␣,3␣-acetonide), 1.40–1.50 (3H, m), 1.41 (3H, s,
20,22-acetonide), 1.50 (3H, s, 2␣,3␣-acetonide), 1.53–1.60
(2H, m), 1.62–1.69 (2H, m), 1.74 (1H, m, 8␤-H), 1.92 (1H,
m, 16-H), 1.97–2.05 (2H, m), 2.00 (1H, t, J = 13.3 Hz,
7␣-H), 2.12 (2H, m), 2.33 (1H, dd, J = 13.0 and 4.3 Hz,
7␤-H), 2.53 (1H, dd, J = 12.6 and 3.8 Hz, 5␣-H), 3.62 (1H,
dd, J = 9.1 and 2.7 Hz, 22-H), 4.11 (1H, m, 2␤-H), 4.28
(1H, m, 3␤-H); 13C NMR δ 12.67 (C-19), 13.03 (C-18),
21.03 (C-11), 21.70 (C-21), 22.51 (C-26 and C-27), 22.55
(C-4), 22.55 (C-16), 23.59 (C-15), 26.51 (2␣,3␣-acetonide),
26.73 (20,22-acetonide), 26.83 (C-23), 28.21 (C-25), 28.59
(2␣,3␣-acetonide), 29.02 (20,22-acetonide), 36.35 (C-24),
37.15 (C-8), 39.60 (C-12), 41.12 (C-1), 42.45 (C-10),
43.40 (C-13), 46.73 (C-7), 51.46 (C-5), 53.31 (C-9), 54.65
(C-17), 56.85 (C-14), 72.12 (C-3), 72.29 (C-2), 81.31
(C-22), 84.08 (C-20), 106.61 (20,22-acetonide), 107.84
(2␣,3␣-acetonide), 211.40 (C-6); analysis calculated for
C33H54O5: C, 74.67; H, 10.25. Found: C, 74.44; H, 10.00.
2.1.2.11. (20R,22R)-2,3-Dihydroxy-20,22-isopropylidene-
dioxy-5α-cholestan-6-one (21). To compound 20 (700 mg,
1.52 mmol) in acetone (24 ml) was added 5 mg/ml
OsO4/t-BuOH solution (0.80 ml, 0.016 mmol), 50% aqueous
N-methylmorpholine N-oxide (NMO; 0.90 ml, 3.84 mmol)
and water (1.5 ml) successively. To dissolve the starting
material completely, acetone (18 ml) was added and the
reaction mixture was stirred overnight at room tempera-
ture, then 50% Na2S2O4 (0.25 ml) was added with 5 min
stirring. To the mixture were added Celite® (0.2 g) and
activated charcoal (0.2 g) with 5 min stirring. Insoluble ma-
terials were filtered off and the filtrate was concentrated in
vacuo. To the residue was added 1N HCl (60 ml) and the
aqueous layer was extracted with CHCl3 (4 × 30 ml). The
combined organic layer was washed with saturated aqueous
NaHCO3 (30 ml), dried over anhydrous MgSO4 and con-
centrated under reduced pressure. The residue was purified
by flash column chromatography (CHCl3:MeOH = 30:1) to
give 21 (723 mg, 97%), which contained both the 2␣,3␣-
and 2␤,3␤-dihydroxy isomers in a ratio of 82/18 based on
1H NMR analysis. For the 2␣,3␣-dihydroxyisomer: δ 0.76
(3H, s, 19-H3), 0.79 (3H, s, 18-H3), 0.90 (6H, d, J = 6.6 Hz,
26-H3 and 27-H3), 1.15 (3H, s, 21-H3), 1.30 (3H, s, ace-
tonide), 1.41 (3H, s, acetonide), 2.68 (1H, dd, J = 12.6 and
2.8 Hz, 5␣-H), 3.62 (1H, m, 22-H), 3.78 (1H, m, 2␤-H),
4.05 (1H, m, 3␤-H). For the 2␤,3␤-dihydroxyisomer: δ 0.79
(3H, s, 18-H3), 0.90 (6H, d, J = 6.6 Hz, 26-H3 and 27-H3),
0.99 (3H, s, 19-H3), 1.15 (3H, s, 21-H3), 1.30 (3H, s, ace-
tonide), 1.41 (3H, s, acetonide), 3.62 (1H, m, 22-H), 3.63
(1H, m, 3␣-H), 4.04 (1H, m, 2␣-H). Since the two isomers
could not be separated by recrystallization, the mixture was
subjected to the next step.
2.1.2.13. (20R,22R)-2α,3α,20,22-Tetrahydroxy-5α-choles-
tan-6-one (23). A mixture of compound 22 (317 mg,
0.58 mmol), EtOH (1.7 ml) and 60% AcOH (10 ml) was
stirred for 6 h at 80 ◦C. After cooling the mixture to room
temperature, the solvent was evaporated and further concen-
trated azeotropically with toluene (2 × 5 ml). The residue
was purified by column chromatography (CHCl3/MeOH
= 30:1) to give compound 23 (218 mg, 81%) as a white
solid. Mp 227–230 ◦C (MeOH); [␣]2D9 +1.4◦ (c 0.49, EtOH);
1H NMR δ 0.76 (3H, s, 19-H3), 0.87 (3H, s, 18-H3), 0.90
(3H, d, J = 6.5 Hz, 26-H3/27-H3), 0.91 (3H, d, J = 6.5 Hz,
26-H3/27-H3), 1.12–1.34 (5H, m), 1.21 (3H, s, 21-H3),
1.38 (2H, m), 1.45 (2H, m), 1.55 (4H, m), 1.67–1.77 (4H,
m), 1.80–1.87 (2H, m), 1.92 (1H, m, 4␣-H), 2.00 (1H, t,
J = 12.7 Hz, 7␣-H), 2.16 (1H, m, 12␤-H), 2.30 (1H, dd,
J = 13.1 and 4.4 Hz, 7␤-H), 2.67 (1H, dd, J = 12.5 and
2.6 Hz, 5␣-H), 3.38 (1H, br.d, J = 9.0 Hz, 22-H), 3.77 (1H,
m, 2␤-H), 4.05 (1H, br.s, 3␤-H); 13C NMR δ 13.55 (C-19),
13.65 (C-18), 20.35 (C-21), 21.05 (C-11), 21.75 (C-16),
22.34 (C-26/C-27), 22.92 (C-26/C-27), 23.61 (C-15), 26.25
(C-4), 28.04 (C-25), 29.22 (C-23), 36.27 (C-24), 36.95
(C-8), 39.83 (C-12), 40.15 (C-1), 42.51 (C-10), 43.74
(C-13), 46.56 (C-7), 50.72 (C-5), 53.66 (C-9), 54.62 (C-17),
56.58 (C-14), 68.24 (C-2), 68.35 (C-3), 76.41 (C-22), 77.09
(C-20), 211.90 (C-6); analysis calculated for C27H46O5: C,
71.96; H, 10.29. Found: C, 71.69; H, 10.04.
2.1.2.12. (20R,22R)-2α,3α,20,22-Bis(isopropylidenedioxy)-
5α-cholestan-6-one (22). To mixture 21 (600 mg, 1.22
mmol) was added 2,2-dimethoxypropane (10 ml) and a cat-
alytic amount of p-TsOH, and the mixture was stirred for 1 h
at room temperature. After addition of CHCl3 (100 ml), the
organic layer was washed with saturated aqueous NaHCO3
(20 ml) and dried over anhydrous MgSO4. The solvent
was removed in vacuo and the residue was purified by
flash column chromatography (hexane/EtOAc = 7:1–5:1)
2.1.2.14. (20R,22S)-20,22-Isopropylidenedioxy-5α-cholest-
2-en-6-one (28). Compound 15 (2.56 g, 6.11 mmol) was
treated in the same way as that for compound 13 via com-
pounds 24, 25, 26, and 27 to yield compound 28 (1.45 g,
52%) as a white solid. Melting point 138 ◦C (EtOH); [␣]D19

B. Watanabe et al. / Steroids 69 (2004) 483–493
489
+31.7◦ (c 0.76, CHCl3); H NMR δ 0.71 (3H, s, 19-H3),
4.10 (1H, m, 2␤-H), 4.28 (1H, m, 3␤-H); 13C NMR δ 12.66
(C-19), 12.91 (C-18), 21.09 (C-11), 22.49 (C-26/C-27),
22.49 (C-4), 22.58 (C-26/C-27), 23.96 (C-16), 24.12
(C-15), 26.30 (20,22-acetonide), 26.44 (20,22-acetonide),
26.50 (2␣,3␣-acetonide), 27.47 (C-23), 27.77 (C-20), 28.18
(C-25), 28.58 (2␣,3␣-acetonide), 36.77 (C-24), 36.97 (C-8),
40.01 (C-12), 41.13 (C-1), 42.40 (C-10), 43.89 (C-13), 46.77
(C-7), 51.45 (C-5), 53.35 (C-9), 54.89 (C-17), 56.34 (C-14),
72.13 (C-3), 72.30 (C-2), 83.79 (C-20), 87.82 (C-22), 106.70
(20,22-acetonide), 107.83 (2␣,3␣-acetonide), 211.42 (C-6);
analysis calculated for C33H54O5: C, 74.67; H, 10.25.
Found: C, 74.40; H, 10.18.
1
0.81 (3H, s, 18-H3), 0.92 (6H, d, J = 6.6 Hz, 26-H3 and
27-H3), 1.13 (1H, m, 15-H), 1.16 (1H, m, 14␣-H), 1.19 (1H,
m, 24-H), 1.22 (1H, m, 12␣-H), 1.29 (1H, m, 9␣-H), 1.38
(1H, m, 17␣-H), 1.37 (3H, s, acetonide), 1.39 (3H, s, 21-H3),
1.41–1.52 (3H, m), 1.49 (3H, s, acetonide), 1.53–1.67 (5H,
m), 1.75 (1H, dtd, J = 12.4, 10.5 and 4.1 Hz, 8␤-H), 1.89
(1H, m, 16-H), 1.98 (1H, t, J = 12.7 Hz, 7␣-H), 1.99 (2H,
m, 1-H2), 2.02 (1H, m, 4␣-H), 2.15 (1H, dt, J = 12.5 and
3.2 Hz, 12␤-H), 2.26 (1H, m, 4␤-H), 2.34 (1H, m, 5␣-H),
2.37 (1H, dd, J = 13.2 and 4.1 Hz, 7␤-H), 3.70 (1H, dd, J
= 9.1 and 3.9 Hz, 22-H), 5.57 (1H, m, 2-H), 5.69 (1H, m,
3-H); 13C NMR δ 12.92 (C-18), 13.50 (C-19), 21.11 (C-11),
21.71 (C-4), 22.51 (C-26/C-27), 22.58 (C-26/C-27), 24.00
(C-16), 24.11 (C-15), 26.33 (acetonide), 26.46 (acetonide),
27.49 (C-23), 27.79 (C-21), 28.19 (C-25), 36.81 (C-24),
37.15 (C-8), 39.36 (C-1), 39.94 (C-10), 40.19 (C-12), 43.91
(C-13), 46.88 (C-7), 53.43 (C-9), 53.83 (C-5), 54.96 (C-17),
56.45 (C-14), 83.85 (C-20), 87.88 (C-22), 106.71 (ace-
tonide), 124.47 (C-2), 124.98 (C-3), 211.91 (C-6). Analysis
calculated for C30H48O3: C, 78.90; H, 10.59. Found: C,
78.61; H, 10.33.
2.1.2.17. (20R,22S)-2α,3α,20,22-Tetrahydroxy-5α-choles-
tan-6-one (31). A solution of compound 30 (580 mg,
1.09 mmol) in 80% AcOH (10 ml) was stirred for 1 h at
80 ◦C. After cooling to room temperature, the solvent was
evaporated and further concentrated azeotropically with
toluene (2 × 5 ml). The residue was purified by recrystal-
lization from CHCl3/MeOH to give compound 31 (284 mg,
58%) as a white solid. Mp 258 ◦C; [␣]D30 −24.6◦ (c 0.95,
EtOH); 1H NMR [C5D5N/CDCl3 = 9:1 (approximately)]; δ
0.83 (3H, s, 19-H3), 0.89 (3H, d, J = 6.6 Hz, 26-H3/27-H3),
0.90 (3H, d, J = 6.6 Hz, 26-H3/27-H3), 1.09 (3H, s, 18-H3),
1.09–1.16 (1H, m, 15-H), 1.19 (1H, m, 14␣-H), 1.27–1.37
(4H, m), 1.51 (1H, m, 15-H), 1.58 (3H, s, 21-H3), 1.58–1.66
(3H, m), 1.76–1.86 (2H, m), 1.93–2.04 (5H, m), 2.09 (1H,
td, J = 11.9 and 5.5 Hz, 23-H), 2.19–2.31 (4H, m), 2.36 (1H,
dd, J = 12.9 and 4.5 Hz, 7␤-H), 3.07 (1H, dd, J = 12.5 and
2.8 Hz, 5␣-H), 3.68 (1H, br.d, J = 9.9 Hz, 22-H), 4.00 (1H,
m, 2␤-H), 4.36 (1H, m 3␤-H); 13C NMR [C5D5N/CDCl3
= 9:1 (approximately)]; δ 13.78 (C-19), 13.92 (C-18), 21.44
(C-11), 21.93 (C-21), 22.31 (C-16), 22.76 (C-26/C-27),
23.04 (C-26/C-27), 24.00 (C-15), 27.78 (C-4), 28.58 (C-25),
29.24 (C-23), 37.18 (C-8), 37.27 (C-24), 40.37 (C-12),
41.15 (C-1), 42.60 (C-10), 43.65 (C-13), 46.90 (C-7), 51.44
(C-5), 54.00 (C-9), 54.84 (C-17), 56.97 (C-14), 68.33 (C-2),
68.84 (C-3), 76.38 (C-20), 78.33 (C-22), 211.80 (C-6); anal-
ysis calculated for C27H46O5: C, 71.96; H, 10.29. Found:
C, 71.76; H, 10.30.
2.1.2.15. (20R,22S)-2,3-Dihydroxy-20,22-isopropylidenedi-
oxy-5α-cholestan-6-one (29). Compound 28 (1.80 g,
3.94 mmol) was treated as described for compound 20 to
give compound 29 (1.80 g, 93%) as a white solid. The
ratio of 2␣,3␣- and 2␤,3␤-isomer was determined to be
83/17 based on 1H NMR analysis. For the 2␣,3␣-dihydroxy
isomer: δ 0.76 (3H, s, 19-H3), 0.80 (3H, s, 18-H3), 0.92
(6H, m, 26-H3 and 27-H3), 1.36 (3H, s, acetonide), 1.39
(3H, s, 21-H3), 1.48 (3H, s, acetonide), 2.67 (1H, dd, J
= 12.5 and 2.8 Hz, 5␣-H), 3.70 (1H, dd, J = 9.1 and 3.8 Hz,
22-H), 3.77 (1H, m, 2␤-H), 4.05 (1H, m, 3␤-H). For the
2␤,3␤-dihydroxy isomer: δ 0.80 (3H, s, 18-H3), 0.92 (6H,
m, 26-H3 and 27-H3), 0.98 (3H, s, 19-H3), 1.36 (3H, s, ace-
tonide), 1.39 (3H, s, 21-H3), 1.48 (3H, s, acetonide), 3.62
(1H, m, 3␣-H), 3.70 (1H, dd, J = 9.1and 3.8 Hz, 22-H),
4.03 (1H, m, 2␣-H). Since the two isomers could not be
separated by recrystallization, the mixture was subjected to
the next step.
2.2. Bioassay
2.1.2.16. (20R,22S)-2α,3α,20,22-Bis(isopropylidenedioxy)-
5α-cholestan-6-one (30). Compound 29 (1.60 g, 3.26
mmol) was treated as described for compound 21 to give
compound 30 (0.98 g, 58%) as a white solid. Mp 229
◦C (EtOH/CHCl3); [␣]27 +35.9◦ (c 0.79, CHCl3); 1H
NMR δ 0.68 (3H, s, 1D9-H3), 0.79 (3H, s, 18-H3), 0.91
(6H, d, J = 6.6 Hz, 26-H3 and 27-H3), 1.08–1.20 (3H, m),
1.22–1.42 (5H, m), 1.34 (3H, s, 2␣,3␣-acetonide), 1.36
(3H, s, 20,22-acetonide), 1.39 (3H, s, 21-H3), 1.43–1.67
(7H, m), 1.48 (3H, s, 20,22-acetonide), 1.50 (3H, s,
2␣,3␣-acetonide), 1.74 (1H, m, 8␤-H), 1.90 (1H, m, 16-H),
1.96–2.03 (2H, m), 2.00 (1H, t, J = 12.7 Hz, 7␣-H), 2.33
(1H, dd, J = 13.0 and 4.3 Hz, 7␤-H), 2.53 (1H, dd, J = 12.6
and 3.8 Hz, 5␣-H), 3.70 (1H, dd, J = 9.0 and 3.9 Hz, 22-H),
2.2.1. Ecdysteroid receptor binding activity
The inhibition of the incorporation of [3H]PonA
(150 Ci/mmol; ARC Inc., St. Louis, MO, USA) into Kc or
Sf-9 cells was examined according to previously reported
methods [8,13]. In brief, 400 ␮l of cell suspension (4 ×
106 cells/ml) was incubated with 1 ␮l of DMSO solution
of the test compound and 2 ␮l of the 70% ethanol solution
of [3H]PonA (0.5 nM, ca. 60000 dpm) for 30 min at 25 ◦C.
The reaction mixture was immediately filtered through a
glass filter (GF/F) and washed three times with water. The
radioactivity collected in the filter was counted with a liquid
scintillation counter (LSC) in 3 ml of Aquasol-2 (Packard
Instrument Co., Meriden, CT, USA). The concentration

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B. Watanabe et al. / Steroids 69 (2004) 483–493
curve for the inhibition of the incorporation of [3H]PonA
was derived, and the concentration required to give 50%
inhibition (IC50) was determined by probit analysis [21].
The reciprocal logarithm of IC50, pIC50, was used as the
index of the binding activity.
hydroxy-5␣-cholestan-6-one, were synthesized stereoselec-
tively from pregnenolone in good yield. In our previous
study, the alkoxylithium reagent derived from ( )-1-(ben-
zyloxymethyl)oxy-4-methyl-1-(tri-n-butylstannyl)pentane
was added to pregnenolone to introduce the side-chain [12].
However, the product was a mixture of two diastereomers
with respect to C-22 and the yield was low. We there-
fore, developed a new synthetic method to stereoselectively
construct the side-chain (Scheme 1).
2.2.2. Molting hormonal activity
Molting hormonal activity was measured using cul-
tured integument of C. suppressalis according to pre-
viously reported methods [14,15]. Briefly, integument
excised from diapause larvae of C. suppressalis was in-
cubated in Grace’s medium containing a test compound
for 24 h, then transferred to fresh medium containing
N-acetyl-[14C]glucosamine ([14C]GlcNAc: 56 mCi/mmol;
Moravek Biochemical Inc., Brea, CA, USA ) and incubated
for 3 days. The radioactivity incorporated into the cultured
integument was measured by LSC in Aquasol-2 as described
above. The 50% effective concentration (EC50 in M) was
determined from the concentration-response curve for the
incorporation of [14C]GlcNAc to the integuments by probit
analysis [21]. The reciprocal logarithm of EC50, pEC50,
was used as the index of the molting hormonal activity.
Pregnenolone 6 was converted to its TBDMS ether 7 fol-
lowed by the stereoselective addition of 4-methylpentynyl-
lithum to the C-20 carbonyl group to give the propargylic
alcohol 8. The triple bond of 8 was semihydrogenated to
afford the Z-allylic alcohol 9 in the presence of the Lindlar
catalyst, then the C-22 double bond was regioselectively
epoxidized under VO(acac)2/TBHP system [22] to give a
mixture of two compounds. They were separated using sil-
ica gel column chromatography to give the product 10 and a
more polar product 11 in 32% and 45% yield from 6, respec-
tively. The oxirane ring of 10 was cleaved by LiAlH4 [23] to
give 12. Since a part of the TBDMS ether was also cleaved
under these conditions, the crude product was treated di-
rectly with TBAF to give the triol 13 in 98% yield from 10.
1H and 13C NMR spectra of 13 were identical with those
reported for (20R,22R)-dihydroxycholesterol [19], thus the
configuration of 10 was determined to be (20R,22R,23R).
In the same way, the epimeric triol 15 was derived from
2.2.3. Brassinolide activity
The brassinolide activity was measured using the dwarf
rice lamina inclination assay reported by Fujioka et al.
[16] under synergistic condition with indole-3-acetic acid
(IAA). Briefly, the seeds of dwarf rice (Oryza sativa cv.
Tan-ginbozu, provided by Sankyo Agro, Ltd., Tokyo, Japan)
were soaked in 0.25% aqueous Benlate-T solution for 2 days
at 30 ◦C under light. Uniformly germinated seeds were se-
lected by coleoptile length (ca. 1–2 mm) and planted in 20 ml
of 1% agar medium (ca. 10 mm thickness) in a beaker (50 ml
volume), then incubated for 3 days under the same condi-
tions described above. A 0.5 ␮l (25 nmol/plant) of EtOH
solution of IAA (50 mM) was applied by micro-syringe
to the top portion of the lamina before the application of
each test compound. Various concentrations of test samples
(0.5 ␮l) in EtOH were applied to the same part of the plant.
After 2 days incubation under identical growth condition,
the external angle between the lamina and its leaf sheath
was measured using a circular protractor. Seven plants were
planted in each beaker, and three sets were used for each
concentration. Thus, each point is the average of 21 obser-
vations. In each assay, both negative (EtOH) and positive
control (brassinolide, 1 nmol/plant) were tested. To normal-
ize the measured angle, the angles obtained for positive and
negative controls were set as 0 and 100%, respectively.
1
11 in 84% yield. In this case, the H NMR spectrum for
the side-chain of 15 was identical with that reported for
(20R,22S)-3␤-acetoxycholest-5-ene-20,22-diol, [24] and
the configuration of the parent epoxide 11 was further con-
firmed to be (20R,22S,23S). This epoxide opening reaction
with LiAlH4 proved to be quite regioselective and to pro-
ceed in high yield. Application of other reagents such as
(i-Bu)2AlH [25], LiEt3BH [26], LiBH4/Ti(O-i-Pr)4 [27] and
LiBH4/Et3B [28] were unsuccessful in this transformation.
The subsequent functionalization was executed in a con-
ventional way [29] (Scheme 2). The diol moiety of 13 was
selectively protected as an acetonide followed by tosylation
of the 3␤-hydroxyl group. Hydroboration of the double
bond at C-5 with BH3·SMe2 followed by oxidative workup
gave the 6␣-alcohol 18. Introduction of a new double bond
at C-2 was accomplished by elimination of p-TsOH with
LiBr/Li2CO3 in DMF. Oxidation of the 6␣-hydroxy group
with the Jones reagent gave the 6-keto compound 20 in
37% yield from 13. Catalytic dihydroxylation of the C-2
double bond of 20 using NMO as co-oxidant [30] gave a
mixture of 2␣,3␣- and the undesired 2␤,3␤-diol in a ra-
1
tio of 82/18 as determined from H NMR analysis. These
isomers could not be separated by column chromatography
or recrystallization. However, their acetonide derivatives
could be separated by column chromatography to give
pure 2␣,3␣-acetonide 22 in 57% yield from 20. The two
acetonide groups at the A-ring and the side-chain were re-
moved by acidic hydrolysis (60% AcOH, EtOH, 80 ◦C, 6 h)
to give the desired (22R)-CS/PonA hybrid compound 23
3. Results and discussion
3.1. Synthesis
Two stereoisomers of a CS/PonA hybrid compound,
(20R,22R)- and (20R,22S)-isomers of 2␣,3␣,20,22-tetra-

B. Watanabe et al. / Steroids 69 (2004) 483–493
491
Fig. 2. Concentration–response curves of synthetic compounds (23 and
31) for the inhibition of [3H]PonA binding in intact Kc cells. Data are
Fig. 3. Concentration-response curves of synthetic compounds (23 and
31) for the molting hormonal response in the cultured integument. Data
shown as the average
S.D. (n = 3).
are shown as the average
S.D. (n = 3).
in 81% yield. The (20R,22S)-dihydroxycholesterol 15 was
treated in the same way as 13 to give the (22S)-CS/PonA
hybrid compound. The overall yields of the (22R)-isomer
and the (22S)-isomer from the starting material 6 were
5.4% and 6.1% in 14 steps, respectively. Interestingly, the
(20R,22S)-acetonide was hydrolyzed easier (80% AcOH,
80 ◦C, 1 h) than the (20R,22R)-isomer. Under these con-
ditions, the (20R,22R)-acetonide of 22 was stable, while
the 2␣,3␣-acetonide was completely removed. Similar dif-
ferences in the susceptibility to hydrolysis between two
epimeric acetonide compounds have been previously re-
ported [31].
23 was 100 times more potent than (22S)-isomer with Kc
cells, whereas the difference of the activity between the two
isomers in Sf-9 cells was 35 times.
3.3. Molting hormonal activity
The concentration-response curves for the molting hor-
monal activity of these newly synthesized compounds are
shown in Fig. 3, indicating that the (22R)-isomer evidenced
ES-like activity, but the (22S)-isomer was not effective even
at the highest concentration tested (100 ␮M). Molting hor-
monal activity in terms of pEC50 is listed in Table 1. Even
though the activity of the (22R)-isomer is 1/100 that of
PonA, it is three times higher than that of ecdysone (E). Voigt
et al. [9] synthesized twenty-four BR/ES hybrid compounds
and measured their ecdysteroid agonist activity and found
that the compounds with (22S)-configuration positively re-
sponded as ecdysteroids. Among these ecdysteroid-active
compounds, an EC50 value could only be determined
for (22S,23S,24R)-3␤,14␣,22,23-tetrahydroxy-5␣-ergost-7-
en-6-one (5; Fig. 1), even though its potency was 1/13
that of E. Our CS/PonA compounds contain a 20-hydroxy
group, but compounds synthesized by Voigt et al. lack
this 20-hydroxy group [9]. According to Voigt et al., the
3.2. Inhibition of [3H] PonA binding in Kc and Sf-9 cells
The concentration curves of the newly synthesized com-
pounds for the competitive inhibition of [3H]PonA incor-
poration to Kc cells are shown in Fig. 2. The experiment
was repeated, and the average of the pIC50 values together
with their standard deviations is listed in Table 1. A sim-
ilar binding assay was performed using the Sf-9 cell line
and the pIC50 was also determined. As shown in Table 1,
compounds 23 and 31 were active in both cell lines, with
pIC50 values ranging from 4.41 to 6.49. The (22R)-isomer
Table 1
Biological activity of newly synthesized CS/PonA hybrid compounds and ecdysteroids
Compounds
Binding activity (pIC50; M)
Kc cells
Hormonal activity (pEC50; M)
Sf-9 cells
C. suppressalis
23 (20R,22R)
31 (20R,22S)
Ecdysone
20E
6.49
4.41
5.59a
7.34a
8.89a
0.11 (5)
0.14 (2)
6.44
4.89
5.63b
6.78b
8.05b
0.06 (2)
0.24 (2)
5.57
0.08 (2)
< 4.00 (11.5%) (2)
5.05c
6.75d
7.53d
PonA
a Reference [13].
b Reference [8].
c Reference [12].
d Reference [15,32].

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B. Watanabe et al. / Steroids 69 (2004) 483–493
eggs, and to Sankyo Agro, Ltd. for the Tan-ginbozu seeds.
A part of this study was performed in the RI center of Ky-
oto University. This investigation was supported, in part, by
a Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology (for-
mally Ministry of Education, Science, and Culture) of Japan
(09660117, 10161207), and by the Fujisawa Foundation.
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