Synthesis of ponasterone A derivatives with various steroid skeleton moieties and evaluation of their binding to the ecdysone receptor of Kc cells

Article abstract of DOI:10.1016/j.steroids.2008.08.005

A series of ponasterone A (PNA) derivatives with various steroid moieties were synthesized to measure their binding activity to the ecdysone receptors of Drosophila Kc cells. The activity of compounds was evaluated by determining the concentration required to give the 50% inhibition (IC₅₀ in M) of the incorporation of [³H]PNA to Drosophila Kc cells. Compounds with no functional groups such as OH and C{double bond, long}O group in the steroid skeleton moiety were inactive. By the introduction of functional groups such as the OH and the C{double bond, long}O group in the steroidal structure, these compounds became active. Some compounds containing the A/B-trans ring fusion, which is different from that (A/B-cis) of ecdysteroids were also active. The oxidation of CH₂ at 6-position to C{double bond, long}O, enhanced the activity 19 times, but the activity was erased by the reduction of oxo to OH group at 6-position. The activity was enhanced about 250 times by the conversion of A/B ring configuration from trans [(20R,22R)-2β,3β,20,22-tetrahydroxy-5α-cholestan-6-one: pIC₅₀ = 4.84] to cis [(20R,22R)-2β,3β,20,22-tetrahydroxy-5β-cholestan-6-one: pIC₅₀ = 7.23]. The latter cis-type compound which is the most potent among compounds synthesized in this study was equipotent to the natural molting hormone, 20-hydroxyecdysone, even though it is 1/50 of PNA.

Full text of DOI:10.1016/j.steroids.2008.08.005

steroids 7 3 ( 2 0 0 8 ) 1452–1464
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Synthesis of ponasterone A derivatives with various steroid
skeleton moieties and evaluation of their binding to the
ecdysone receptor of Kc cells
Hirokazu Arai, Bunta Watanabe, Yoshiaki Nakagawa∗, Hisashi Miyagawa
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of ponasterone A (PNA) derivatives with various steroid moieties were synthesized
to measure their binding activity to the ecdysone receptors of Drosophila Kc cells. The activity
of compounds was evaluated by determining the concentration required to give the 50%
inhibition (IC50 in M) of the incorporation of [3H]PNA to Drosophila Kc cells. Compounds
with no functional groups such as OH and C O group in the steroid skeleton moiety were
inactive. By the introduction of functional groups such as the OH and the C O group in
the steroidal structure, these compounds became active. Some compounds containing the
A/B-trans ring fusion, which is different from that (A/B-cis) of ecdysteroids were also active.
The oxidation of CH2 at 6-position to C O, enhanced the activity 19 times, but the activity
was erased by the reduction of oxo to OH group at 6-position. The activity was enhanced
about 250 times by the conversion of A/B ring configuration from trans [(20R,22R)-2␤,3␤,20,22-
tetrahydroxy-5␣-cholestan-6-one: pIC50 = 4.84] to cis [(20R,22R)-2␤,3␤,20,22-tetrahydroxy-5␤-
cholestan-6-one: pIC50 = 7.23]. The latter cis-type compound which is the most potent among
compounds synthesized in this study was equipotent to the natural molting hormone, 20-
hydroxyecdysone, even though it is 1/50 of PNA.
Received 31 January 2008
Received in revised form
29 July 2008
Accepted 7 August 2008
Published on line 2 September 2008
Keywords:
Ecdysone agonists
Ponasterone A
Drosophila
Kc cells
Ecdysone receptor
© 2008 Elsevier Inc. All rights reserved.
1.
Introduction
catalyze the final four steps of 20-OH Ecd biosynthesis have
been identified in Drosophila [4–8].
Insect molting is regulated by the molting hormone, 20-
hydroxyecdysone (20-OH Ecd, 1 in Fig. 1). The biosynthesis
of 20-OH Ecd has been studied intensively by Gilbert and co-
workers [1,2]. It is well-known that insects have to intake
cholesterol and other related steroidal compounds from their
diet because they cannot construct the steroid skeleton. In
Drosophila, ecdysone (Ecd; Fig. 1) is synthesized from choles-
terol in the prothoracic gland and converted to 20-OH Ecd
in other peripheral tissues, such as fat body and midgut,
although 3-dehydroecdysone is secreted in Bombyx mori [3].
Recently, halloween genes coding the P450 hydroxylases that
Even though cholesterol has no receptor binding activity
[9], Ecd and 2-deoxyecdysone have weak receptor binding and
hormonal activities [10,11]. We also demonstrated that Ecd
has a weak binding activity to in vitro translated ecdysone
receptor proteins [12,13] and molting hormonal activity in tis-
sue [14]. In addition, there are many steroidal compounds
that have molting hormonal activity in plants [11] and a few
animals, which are available in the web site EcdyBase (URL:
http://ecdybase.org/). Among them, ponasterone A (PNA, 2
in Fig. 1), which is isolated from Podocarpus Nakaii, is well-
known as the most potent ecdysteroid [15,16]. To date, many
Abbreviations: Ecd, ecdysone; 20-OH-E, 20-hydroxyecdysone; PNA, ponasterone A; CS, castasterone.
Corresponding author. Tel.: +81 75 753 6117; fax: +81 75 753 6123.
∗
E-mail address: naka@kais.kyoto-u.ac.jp (Y. Nakagawa).
0039-128X/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2008.08.005

steroids 7 3 ( 2 0 0 8 ) 1452–1464
1453
Fig. 1 – Structures of ecdysteroids and PNA/CS hybrid compound.
ecdysteroids have been identified and their hormonal activ-
ity has been evaluated in the cell-based assay [17,18]. The
structure-activity relationship (SAR) of steroidal compounds
is quantitatively analyzed using the three-dimensional quan-
titative SAR (3D QSAR) method [11,19].
In addition to ecdysteroids, non-steroidal compounds
such as diacylhydrazines [20,21], acylaminoketones,
[22,23] N-benzoyltetrahydroquinolines, [24] 3,5-di-t-butyl-4-
hydroxybenzamides [25] and oxadiazolines [26] are reported
to be ecdsyone agonists. We reported that the side chain
moiety of ecdysteroids that is mimicked by the t-butyl-
aminobenzoyl moiety of dibenzoylhydrazines is important
to express the molting hormonal activity [27-29]. Moreover,
crystal structure analyses of the ecdysone receptor proteins
with ligand molecules demonstrated that steroidal and
non-steroidal ligand molecules are only partially overlap-
ping the ligand binding cavities of ecdysone receptor (EcR)
of the tobacco budworm Heliothis virescens [30,31]. Thus, a
steroid compound PNA and a nonsteroidal ecdysone agonist,
a dibenzoylhydrazine-type compound (BYI06830) [30], are
overlapped as shown in Fig. 2, which is constructed by fitting
the conserved 4 amino acid residues in the ligand binding
domain (LBD) of both the PNA- and the BYI06830-bound
receptors (http://www.ncbi.nlm.nih.gov/) using the modeling
software SYBYL 6.9 (Tripos, USA).
Fig. 3 – Structures of plant steroid hormones.
thesized the brassinosteroid/ecdysteroid hybrid compounds
and found that the PNA/castasterone (PNA/CS: Fig. 3) hybrid
compound 3 carrying the steroid moiety of a plant steroid hor-
mone, CS, and the side chain of PNA, has a binding affinity to
the EcRs and a hormonal activity against lepidopteran tissue
[14,33]. Even though the steroid mother skeleton of ecdys-
teroids is replaceable with that of the brassinosteroids to show
the ecdysone activity, the potency of the PNA/CS was 1/40 and
1/250 that of PNA against Diptera Kc and Lepidoptera Sf-9 cells,
respectively. [33] This result indicates that the modification of
the steroid mother skeleton may change the proper position-
ing of the side chain moiety of ecdysteroids in ligand binding
pocket.
In this study, we synthesized a number of PNA analogs by
modifying the mother skeleton moiety of PNA, and studied
the SAR which would be fruitful to design new chemistry.
The design of new compounds based on the structure of
ecdysteroids will reach to the development of other ecdysone
agonists possessing a broad insecticidal activity.
According to Voigt et al., few brassinosteroid/ecdysteroid
hybrid compounds have weak ecdysone activity, while some
other compounds are antagonists [32]. Previously we also syn-
2.
Experimental
2.1.
Synthesis
Chemicals were purchased from Aldrich Chemical Co., Inc.
(Milwaukee, Wisconsin, USA), Wako Pure Chemical Indus-
tries, Ltd. (Osaka, Japan), Tokyo Chemical Industry Co., Ltd.
(Tokyo, Japan), Kanto Chemical Co., Inc. (Tokyo, Japan),
and Nacalai Tesque Inc. (Kyoto, Japan). Dess–Martin peri-
odinane was also prepared according to the conventional
method [34,35]. Oven-dried glassware and positive Ar pres-
Fig. 2 – Superposition between PNA (black) and BYI06830
(gray).

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steroids 7 3 ( 2 0 0 8 ) 1452–1464
Table 1 – Binding activity of ecdysteroids against ecdysone receptor of Kc cellsa
Compound
No.b
pIC50 (M)
Compound
Structure
pIC50 (M)
Structure
No.
1c
7.34f
20
6.10 0.18
2d
8.89f
21
4.05 0.27
3e
6.49g
24
26
28
32
<3.61 (42%)
5.02 0.45
<3.61 (19%)
4.84 0.30
4
4.38 0.28
<3.61 (23%)
4.38 0.17
8
9

steroids 7 3 ( 2 0 0 8 ) 1452–1464
1455
Table 1 (Continued )
Compound
pIC50 (M)
Compound
Structure
pIC50 (M)
No.b
Structure
No.
12
<3.61 (8%)
34
7.23 0.10
a
Values are mean standard deviation for two experiments. The values in parentheses are the inhibition % at the corresponding concentration.
b
c
d
e
f
Corresponds to the compound number in the text and schemes.
20-Hydroxyecdysone (20-OH Ecd).
Ponasterone A (PNA).
PNA/CS hybrid compound.
Ref. [36].
g
Ref. [33].
sure were used to keep anhydrous conditions. Anhydrous
solvents were either commercially available or prepared con-
ventionally in the laboratory. Flash column chromatography
was conducted using Kieselgel 60 (Merck, Darmstadt, Ger-
many) as the adsorbent. NMR spectra were recorded on
a Bruker ARX-500 (500 MHz for 1H and 125 MHz for 13C)
or Bruker AVANCE-400 (400 MHz for 1H and 100 MHz for
13C) or Bruker AC-300 (300 MHz for 1H and 75 MHz for
13C) in deuteriochloroform unless otherwise noted. Melt-
ing points (mp) were measured with a Yanagimoto melting
point apparatus (Yanagimoto Seisakusho, Kyoto, Japan) and
uncorrected. Optical rotations were measured on a JASCO
P-1010 polarimeter (JASCO, Tokyo, Japan). Synthesized com-
pounds listed in Table 1 were submitted to either elemental or
high-resolution mass spectrum (HRMS) analysis. HRMS were
recorded on a JEOL JMS 700 spectrometer (Tokyo, Japan). Ele-
mental analyses were performed at the Microanalytical Center
of Kyoto University. The synthetic methods are summarized
in Schemes 1–4.
2.1.1. (20R,22R)-20,22-Dihydroxycholest-5-ene (4)
(Scheme 1)
Compound 4 was synthesized from pregnenolone according to
the procedure previously reported [33]. The NMR (500 MHz for
1H and 125 MHz for 13C) spectrum of compound 4 is follows:
ı 0.89 (3H, s), 0.90 (3H, d, J = 6.3 Hz), 0.91 (3H, d, J = 6.3 Hz), 1.02
(3H, s), 1.22 (3H, s), 3.39 (1H, m), 3.53 (1H, m), 5.36 (1H, m); 13C
NMR (125 MHz) ı 13.57, 19.38, 20.38, 20.93, 21.92, 22.36, 22.94,
28.08, 29.15, 31.27, 31.61, 31.74, 36.32, 36.48, 37.23, 40.18, 42.25,
43.17, 50.04, 54.72, 56.67, 71.74, 76.39, 77.41, 121.54, 140.76;
HRMS (FAB) m/z: C27H46O3Na [M+Na]+, calcd 441.3345, found
441.3341.
2.1.2. (20R,22R)-20,22-Isopropylidenedioxycholest-5-ene
(7) (Scheme 1)
A
mixture of compound
4
(0.50 g, 1.19 mmol), 2,2-
catalytic
dimethoxypropane (0.29 mL, 2.38 mmol), and
a
amount of p-TsOH·H2O in CHCl3 (5 mL) was stirred for 1 min
at room temperature. Since the reaction did not complete,
Scheme 1 – Reagents and conditions: (a) (MeO)2CMe2, p-TsOH·H2O, CHCl3, RT; (b) TsCl, pyridine, RT; (c) LiEt3BH, THF, RT; (d)
60% AcOH/THF, 80 ◦C; (e) H2, 10% Pd–C, EtOH, RT, 2 d.

1456
steroids 7 3 ( 2 0 0 8 ) 1452–1464
Scheme 2 – Reagents and conditions: (a) (MeO)2CMe2, p-TsOH•H2O, CHCl3, RT, 2 min; (b) BH3•SMe2, THF, RT, overnight; (c)
Dess–Martin periodinane, CH2Cl2, RT, 2 h; (d) 80% AcOH, THF, 80 ◦C, 2 h; (e) benzoic acid, PPh3, DEAD, THF, RT, overnight; (f)
NaOH, MeOH, 60 ◦C, 30 min; (g) 60% AcOH, 80 ◦C.
additional 2,2-dimethoxypropane (0.29 mL, 2.38 mmol) was
added and stirred for another 1 min. The mixture was diluted
with CHCl3 (50 mL), washed with saturated aqueous NaHCO3
(20 mL) solution, and dried over anhydrous MgSO4. The sol-
vent was removed in vacuo to afford compound 5 (0.88 g) as a
yellowish oil. This crude material was dissolved in anhydrous
pyridine (8.8 mL). p-Toluenesulfonyl chloride was added to
the solution (0.68 g, 3.57 mmol) and the reaction mixture
was stirred overnight at room temperature. The mixture was
added 3M HCl (88 mL) at 0 ◦C and extracted with EtOAc (4×
30 mL). The combined organic layer was washed successively
with brine (30 mL) and saturated aqueous NaHCO3 (30 mL)
solution, and dried over anhydrous MgSO4. The solvent was
removed in vacuo to give compound 6 (0.69 g) as a colorless
Scheme 3 – Reagents and conditions: (a) 2,3-Dihydro-2H-pyran, p-TsOH·H2O, CH2Cl2, RT, 9 h; (b), BH3·SMe2, THF, RT, 12 h; (c)
Dess–Martin periodinane, CH2Cl2, RT, 3 h; (d) 60% AcOH, EtOH, 80 ◦C; (e) NaBH4, EtOH, 0 ◦C, 15 min then RT, 1 h.

steroids 7 3 ( 2 0 0 8 ) 1452–1464
1457
Scheme 4 – Reagents and conditions: (a) MsCl, Et3N, CH2Cl2, RT, 30 min; (b) LiBr·H2O, Li2CO3, DMF, reflux, 1.5 h; (c) (i) AcOAg,
I2, AcOH, RT, 1 h, (ii) H2O, 90 ◦C, 1 h; (d) 1N NaOH, THF, RT, 1 h; (e) 60% AcOH, 80 ◦C, 4 h; (f) K2CO3, MeOH/H2O, reflux, overnight.
solid. This crude material was dissolved in anhydrous THF
(11.2 mL). LiEt3BH (1.05 M in THF, 3.20 mL, 3.36 mmol) was
added to the solution at 0 ◦C under argon and the reaction
mixture was stirred overnight at room temperature. Since the
reaction did not complete, extra amount of LiEt3BH (1.05 M in
THF, 5.33 mL, 5.60 mmol) was added at 0 ◦C and stirred for 1
day at room temperature. At this point, since 6 still remained,
more LiEt3BH (1.05 M in THF, 5.33 mL, 5.60 mmol) was added
at 0 ◦C. After stirring overnight at room temperature, 10%
NaOH (8.0 mL) and 30% H2O2 (6.0 mL) were added dropwise
successively at 0 ◦C, and stirred for 15 min at the same tem-
perature. The mixture was diluted with water (40 mL) and
extracted with EtOAc (4× 20 mL). The combined organic layer
was washed successively with 20% Na2S2O3·5H2O (30 mL)
and brine (15 mL), and dried over anhydrous MgSO4. The
solvent was evaporated and the crude product was purified by
flash column chromatography (hexane/EtOAc = 300:1–200:1)
to obtain compound 7 (0.40 g, 76% from 4) as a colorless solid;
1H NMR (500 MHz) ı 0.81 (3H, s), 0.90 (6H, d, J = 6.5 Hz), 1.00 (3H,
s), 1.15 (3H, s), 1.30 (3H, s), 1.41 (3H, s), 3.63 (1H, dd, J = 9.3 and
2.7 Hz), 5.27 (1H, m).
solvent was removed in vacuo, and the residue was sub-
jected to flash column chromatography (hexane/EtOAc = 9:1)
to afford compound 8 (43 mg, 14%) as a colorless solid.
Unreacted 7 was treated as above repeatedly and the total
yield of 8 was 80 mg (25%), mp 136–137 ◦C (EtOH); [␣]D
16
−20.9 (c 0.64, EtOH); 1H NMR (400 MHz) ı 0.89 (3H, s), 0.90
(3H, d, J = 6.4 Hz), 0.91 (3H, d, J = 6.4 Hz), 1.00 (3H, s), 1.22
(3H, s), 3.39 (1H, m), 5.27 (1H, m); 13C NMR (100 MHz) ı
13.59, 19.46, 20.37, 20.61, 21.92, 22.36, 22.51, 22.94, 23.91,
28.00, 28.08, 29.13, 31.20, 31.72, 32.84, 36.32, 37.51, 39.86,
40.25, 43.17, 50.49, 54.73, 56.77, 76.39, 77.47, 118.79, 143.74;
HRMS (FAB) m/z: C27H46O2Na [M+Na]+, calcd 425.3396, found
425.3398.
2.1.4. (20R,22R)-5˛-Cholestane-3ˇ,20,22-triol (9)
(Scheme 1)
A
mixture of compound 4 (0.94 g, 2.25 mmol) and 10%
Pd–C (0.47 g) in EtOH (20 mL) was stirred for 2 days under
hydrogen at room temperature. The catalyst was removed
by filtration through Celite®. The filtrate was concentrated
in vacuo and the residue was recrystallized from MeOH
to yield compound
9
(0.57 g, 60%) as
a
colorless solid,
2.1.3. (20R,22R)-Cholest-5-ene-20,22-diol (8) (Scheme 1)
A solution of compound 7 (0.35 g, 0.79 mmol) in 60% AcOH
(7.5 mL) and THF (13 mL) was stirred 4 days at 80 ◦C. The
mp 90–91 ◦C (MeOH); [␣]D
+18.2 (c 0.50, CHCl3); 1H NMR
27
(C5D5N, 500 MHz)
0.96 (3H, d, J = 6.3 Hz), 1.19 (3H, s), 1.54 (3H, s), 3.77 (1H,
ı 0.85 (3H, s), 0.95 (3H, d, J = 6.4 Hz),

1458
steroids 7 3 ( 2 0 0 8 ) 1452–1464
dd, J = 10.0 and 5.0 Hz), 3.88 (1H, m); 13C NMR (C5D5N,
125 MHz) ı 12.58, 14.23, 21.26, 21.59, 22.61, 23.31, 24.46, 28.40,
29.24, 30.21, 32.40, 35.18, 35.84, 37.23, 37.61, 39.37, 41.02,
43.85, 45.31, 52.55, 54.73, 55.70, 56.91, 70.65, 76.58, 76.68;
HRMS (FAB) m/z: C27H48O3Na [M+Na]+, calcd 443.3501, found
443.3496.
(400 MHz) ı 0.78 (3H, s), 0.86 (3H, s), 0.90 (3H, d, J = 6.4 Hz), 0.91
(3H, d, J = 6.4 Hz), 1.20 (3H, s), 3.38 (1H, m); 13C NMR (100 MHz) ı
12.22, 13.78, 20.30, 20.67, 21.91, 22.15, 22.36, 22.92, 23.81, 26.80,
28.07, 29.00, 32.03, 34.81, 36.21, 36.31, 38.67, 40.53, 43.46, 47.02,
54.71, 54.82, 56.56, 76.38, 77.20, 77.49. Analysis calculated for
C27H48O2: C, 80.14; H, 11.96. Found: C, 79.91; H, 11.99.
2.1.8. (20R,22R)-3ˇ-Hydroxy-20,22-isopropylidenedioxy-
5˛-cholestan-6-one (16) (Scheme 2)
2.1.5. (20R,22R)-20,22-Isopropylidenedioxy-3ˇ-
(p-toluenesulfonyloxy)-5˛-cholestane (10) (Scheme 1)
A mixture of compound 13 (3.33 g, 6.25 mmol) that was derived
from pregnenolone [33], 2,2-dimethoxypropane (1.53 mL,
12.5 mmol), and a catalytic amount of p-TsOH·H2O in CHCl3
(30 mL) was stirred for 1 min at room temperature. Since the
reaction did not complete, additional 2,2-dimethoxypropane
(0.77 mL, 6.24 mmol) was added and stirred for another 1 min.
The reaction mixture was diluted with CHCl3 (120 mL), washed
with saturated aqueous NaHCO3 solution (50 mL), and dried
over anhydrous MgSO4. The solvent was removed in vacuo to
afford a colorless solid (4.78 g). This crude product was dis-
solved in anhydrous THF (50 mL). BH3·SMe2 solution (2.0 M
in toluene, 12.5 mL, 25.0 mmol) was added to the solution at
0 ◦C under argon. The mixture was stirred overnight at room
temperature. 10% NaOH (10 mL) and 30% H2O2 (10 mL) were
added to the reaction mixture at 0 ◦C and this was stirred
for 30 min at the same temperature. The reaction mixture
was diluted with EtOAc (200 mL), washed successively with
brine (50 mL) and 20% Na2S2O3·5H2O (130 mL), and dried over
anhydrous MgSO4. The solvent was removed in vacuo to yield
compound 14 (5.23 g) as a colorless solid. This crude mate-
rial was dissolved in anhydrous CH2Cl2 (50 mL). Dess–Martin
periodinane (4.76 g, 11.2 mmol) was added to the solution at
0 ◦C, and the mixture was stirred for 1 h at room temperature.
Since the reaction was not completed, Dess–Martin period-
inane (0.53 g, 1.25 mmol) was added again at 0 ◦C, and the
mixture was stirred for another 1 h at room temperature. 20%
Na2S2O3·5H2O/saturated aqueous NaHCO3 (75 mL) solution
was added to the mixture and this stirred for 30 min at room
temperature. The mixture was diluted with CH2Cl2 (200 mL),
and the separated organic layer was dried over anhydrous
MgSO4. The solvent was removed in vacuo to give compound
15 (5.02 g) as a colorless solid. This crude material was dis-
solved in 80% AcOH (50 mL), and THF (10 mL), and the mixture
was stirred for 2 h at 80 ◦C. The solvent was removed in vacuo,
and the residue was subjected to flash column chromatogra-
phy (CHCl3/MeOH = 50:1) to yield compound 16 (2.96 g, quant.
from 13) as a colorless solid. 1H NMR (400 MHz) ı 0.77 (3H, s),
0.80 (3H, s), 0.90 (6H, d, J = 6.6 Hz), 1.17 (3H, s), 1.30 (3H, s), 1.41
(3H, s), 2.21 (1H, dd, J = 12.4 and 2.6 Hz), 2.33 (1H, dd, J = 13.1 and
4.4 Hz) 3.58 (1H, m), 3.62 (1H, dd, J = 9.2 and 2.9 Hz).
A
mixture of compound
9
(0.50 g, 1.19 mmol), 2,2-
catalytic
dimethoxypropane (0.72 mL, 5.95 mmol), and
a
amount of p-TsOH·H2O in CHCl3 (5 mL) was stirred for 1 min
at room temperature. After addition of CHCl3 (50 mL), the
organic layer was washed with saturated aqueous NaHCO3
solution (20 mL) and dried over anhydrous MgSO4. The solvent
was removed in vacuo to afford a yellowish oil (0.88 g). This
crude product was dissolved in anhydrous pyridine (7.6 mL)
and p-toluenesulfonyl chloride (0.68 g, 3.57 mmol) was added
to the mixture. After stirring overnight at room temperature,
the reaction mixture was added with 3M HCl (76 mL) at
0 ◦C and extracted with EtOAc (4× 30 mL). The combined
organic layer was washed successively with brine (30 mL) and
saturated aqueous NaHCO3 solution (30 mL), and dried over
anhydrous MgSO4. The solvent was removed in vacuo to give
compound 10 (0.54 g, 74% from 9) as a colorless solid, which
was used without further purification. 1H NMR (500 MHz) ı
0.76 (3H, s), 0.78 (3H, s), 0.89 (6H, d, J = 6.6 Hz), 1.11 (3H, s), 1.29
(3H, s), 1.40 (3H, s), 2.44 (3H, s), 3.61 (1H, dd, J = 9.3 and 2.8 Hz),
4.42 (1H, m), 7.32 (2H, d, J = 8.2 Hz), 7.79 (2H, d, J = 8.2 Hz).
2.1.6. (20R,22R)-20,22-Isopropylidenedioxy-5˛-cholestane
(11) (Scheme 1)
LiEt3BH (1.05 M in THF, 2.51 mL, 2.64 mmol) was added at 0 ◦C
to compound 10 (0.54 g, 0.88 mmol) in anhydrous THF (8.8 mL)
under argon. The reaction mixture was stirred overnight at
room temperature. Since the reaction did not complete, addi-
tional LiEt3BH (1.05 M in THF, 4.19 mL, 4.40 mmol) was added
at 0 ◦C and the mixture was stirred overnight at room temper-
ature. To the reaction mixture were added successively 10%
NaOH (8.0 mL) and 30% H2O2 (6.0 mL) dropwise at 0 ◦C, and
the mixture was stirred for 30 min at the same temperature. At
this point, water (40 mL) was added and extracted with EtOAc
(4× 20 mL). The combined organic layer was washed succes-
sively with 20% Na2S2O3·5H2O (30 mL) and brine (15 mL), then
dried over anhydrous MgSO4. The solvent was evaporated and
the crude product was purified by flash column chromatogra-
phy (hexane/EtOAc = 500:1) to obtain the compound 11 (0.38 g,
97%) as a colorless solid. 1H NMR (500MHz) ı 0.78 (6H, s), 0.90
(6H, d, J = 6.6 Hz), 1.13 (3H, s), 1.30 (3H, s), 1.41 (3H, s), 3.62 (1H,
dd, J = 9.1 and 2.7 Hz).
2.1.9. (20R,22R)-3˛-Benzoyloxy-20,22-
2.1.7. (20R,22R)-5˛-Cholestane-20,22-diol (12)
(Scheme 1)
isopropylidenedioxy-5˛-cholestan-6-one (17) (Scheme 2)
Diethyl azodicarboxylate solution (40% in toluene, 1.44 mL,
3.16) mmol was added to a mixture of compound 16 (0.75 g,
1.58 mmol), triphenylphosphine (0.83 g, 3.16 mmol), and ben-
zoic acid (0.39 g, 3.16 mmol) in anhydrous THF (10 mL) at 0 ◦C,
and the mixture was stirred overnight at room temperature.
After addition of silica gel (13 g), the solvent was removed in
vacuo and residue was subjected to flash column chromatogra-
phy (hexane/EtOAc = 10:1) to afford compound 16 (0.62 g, 68%)
A solution of compound 11 (0.23 g, 0.52 mmol) in 60% AcOH
(5.5 mL) and THF (11 mL) was stirred overnight at 80 ◦C. The
solvent was evaporated and the residue was subjected to flash
column chromatography (hexane/EtOAc = 9:1) to yield com-
pound 12 (34 mg, 16%) as a colorless solid. Unreacted 11 was
treated as above repeatedly and the total yield of 12 was 69 mg
(33%), mp 122–123 ◦C (EtOH); [␣]D24 +19.5 (c 0.48, EtOH); 1H NMR

steroids 7 3 ( 2 0 0 8 ) 1452–1464
1459
as a colorless solid.1H NMR (400 MHz) ı 0.81 (6H, s), 0.91 (6H,
d, J = 6.6 Hz), 1.15 (3H, s), 1.30 (3H, s), 1.41 (3H, s), 2.14 (1H, d,
J = 12.0 Hz), 2.35 (1H, dd, J = 13.1 and 4.4 Hz), 2.67 (1H, dd, J = 12.4
and 3.0 Hz), 3.63 (1H, dd, J = 9.0 and 2.9 Hz), 5.38 (1H, t, J = 2.7 Hz),
7.45 (2H, m), 7.54 (1H, m), 8.02 (2H, m).
(100 MHz) ı 13.58, 20.33, 20.70, 21.83, 22.34, 22.90, 23.14, 23.63,
28.03, 29.21, 29.80, 34.30, 34.81, 36.26, 36.34, 37.90, 39.98, 40.05,
42.72, 43.83, 54.67, 56.79, 59.30, 70.09, 76.38, 77.08, 213.76;
HRMS (FAB) m/z: C27H47O4 [M+H]+, calcd 435.3474, found
435.3483.
2.1.10. (20R,22R)-3˛-Hydroxy-20,22-isopropylidenedioxy-
5˛-cholestan-6-one (18) (Scheme 2)
2.1.14. (20R,22R)-20,22-Isopropylidenedioxy-3ˇ-
(tetrahydropyran-2-yloxy)cholest-5-ene (22) (Scheme 3)
A mixture of crude compound 5 (2.27 g, 96%, 4.75 mmol),
2,3-dihydro-2H-pyrane (1.0 mL), and a catalytic amount of p-
TsOH·H2O in anhydrous CH2Cl2 (5 mL) was stirred for 9 h at
room temperature. The reaction mixture was diluted with
CH2Cl2 (25 mL), washed with saturated aqueous NaHCO3
solution (10 mL), and dried over anhydrous Na2SO4. The sol-
vent was evaporated, and the residue was subjected to flash
column chromatography (hexane/EtOAc = 20:1) to afford com-
pound 22 (2.00 g, 78%) as a colorless solid.
A mixture of compound 17 (0.62 g, 1.07 mmol) and NaOH
(0.42 g, 10.5 mmol) in MeOH was stirred for 30 min at 60 ◦C. The
solvent was removed in vacuo, and the residue was dissolved in
CHCl3 (50 mL). The organic layer was washed with saturated
aqueous NH4Cl solution (50 mL), and the separated aqueous
layer was re-extracted with CHCl3 (4× 20 mL). The combined
organic layer was washed with brine (50 mL) and dried over
anhydrous MgSO4. The solvent was removed in vacuo and
the residue was subjected to flash column chromatography
(hexane/EtOAc = 2:1) to obtain compound 18 (0.30 g, 59%) as a
colorless solid. 1H NMR (400 MHz) ı 0.74 (3H, s), 0.79 (3H, s), 0.90
(6H, d, J = 6.6 Hz), 1.15 (3H, s), 1.30 (3H, s), 1.41 (3H, s), 2.12 (1H,
m), 2.31 (1H, dd, J = 13.0 and 4.5 Hz), 2.71 (1H, t, J = 7.9 Hz), 3.62
(1H, dd, J = 9.3 and 2.9 Hz), 4.17 (1H, br, s).
2.1.15. (20R,22R)-20,22-Isopropylidenedioxy-3ˇ-
(tetrahydropyran-2-yloxy)-5˛-cholestan-6˛-ol (23)
(Scheme 3)
BH3·SMe2 solution (2.0 M in toluene, 7.2 mL, 14.4 mmol) was
added to a solution of compound 22 (2.00 g, 3.68 mmol) in
anhydrous THF (23 mL) at 0 ◦C under argon, and the mixture
was stirred for 12 h at room temperature. 10% NaOH (7.2 mL)
and 30% H2O2 (7.2 mL) were added to the reaction mixture
at 0 ◦C and stirred for 15 min. The mixture was diluted with
EtOAc (160 mL), successively washed with brine (50 mL) and
20% Na2S2O3·5H2O (75 mL), and dried over anhydrous Na2SO4.
The solvent was removed in vacuo to yield compound 23 (2.11 g,
quant.) as a colorless solid, which was subsequently used
without purification.
2.1.11. (20R,22R)-3˛-Hydroxy-20,22-isopropylidenedioxy-
5ˇ-cholestan-6-one (19) (Scheme 2)
Compound 19 (0.13 g, 26%) was obtained in the course of
preparation of compound 18 as described above as a more
polar material than compound 18. 1H NMR (400 MHz) ı 0.79
(3H, s), 0.86 (3H, s), 0.91 (6H, d, J = 6.6 Hz), 1.15 (3H, s), 1.30 (3H,
s), 1.41 (3H, s), 3.63 (1H, dd, J = 9.0 and 2.7 Hz), 3.64 (1H, m).
2.1.12. (20R,22R)-3˛,20,22-Trihydroxy-5˛-cholestan-6-one
(20) (Scheme 2)
2.1.16. (20R,22R)-5˛-Cholestane-3ˇ,6˛,20,22-tetraol (24)
(Scheme 3)
A solution of compound 18 (0.30 g, 0.63 mmol) in 60% AcOH
(10 mL) was stirred for 7 h at 80 ◦C. The solvent was removed
in vacuo, and the residue was subjected to flash column
chromatography (CHCl3/MeOH = 40:1) to obtain compound 20
(0.23 g, 84%) as a colorless oil. Recrystallization from EtOAc
A solution of compound 23 (1.05 g, 98%, 1.84 mmol) in 60%
AcOH (12 mL) and EtOH (2 mL) was stirred for 6.5 h at 80 ◦C.
The solvent was removed in vacuo, and the residue was sub-
jected to flash column chromatography (2 times) to separate
compound 24 from 23. The latter was treated as above, and
the combined 24 was further purified by recrystallization
from EtOH to give a pure product (0.33 g, 42%) as a colorless
gave a colorless solid, mp 185–186 ◦C (EtOAc); [␣]D −5.3 (c
20
0.52, EtOH); 1H NMR (400 MHz) ı 0.74 (3H, s), 0.88 (3H, s), 0.90
(3H, d, J = 6.5 Hz), 0.92 (3H, d, J = 6.5 Hz), 1.21 (3H, s), 2.31 (1H,
dd, J = 13.0 and 4.5 Hz), 2.71 (1H, t, J = 7.9 Hz), 3.38 (1H, m),
4.17 (1H, br, s); 13C NMR (100 MHz) ı 12.30, 13.66, 20.34, 20.93,
21.75, 22.34, 22.93, 23.59, 27.65, 28.05, 28.15, 29.20, 31.66, 36.28,
37.24, 39.95, 41.49, 43.76, 46.68, 51.69, 53.76, 54.65, 56.71, 65.41,
76.39, 77.14, 212.53; HRMS (FAB) m/z: C27H47O4 [M+H]+, calcd
435.3474, found 435.3472. Analysis calculated for C27H46O4: C,
74.55; H, 10.74. Found: C, 74.61; H, 10.67.
solid, mp 132–134 ◦C (EtOH); [␣]D +31.9 (c 0.54, EtOH); 1H
20
NMR (C5D5N, 400 MHz) ı 0.93 (3H, s), 0.94 (3H, d, J = 6.5 Hz), 0.96
(3H, d, J = 6.4 Hz), 1.20 (3H, s), 1.56 (3H, s), 3.07 (1H, m), 3.76
(1H, m), 3.95 (1H, m), 5.09 (1H, d, J = 4.4 Hz), 5.91 (1H, s), 6.00
(1H, s), 6.13 (1H, s); 13C NMR (C5D5N, 100MHz) ı 13.57, 13.78,
13.82, 14.22, 21.27, 21.58, 22.61, 22.69, 23.32, 24.51, 28.41, 30.22,
32.45, 33.82, 34.19, 36.62, 37.24, 38.16, 40.92, 43.83, 52.91, 54.44,
56.79, 68.78, 73.10, 76.57, 83.97; HRMS (FAB) m/z: C27H48O4Na
[M+Na]+, calcd 459.3450, found 459.3462.
2.1.13. (20R,22R)-3˛,20,22-Trihydroxy-5ˇ-cholestan-6-one
(21) (Scheme 2)
A solution of compound 19 (0.13 g, 0.27 mmol) in 60% AcOH
(10 mL) was stirred for 4 h at 80 ◦C. The solvent was removed
in vacuo, and the residue was subjected to flash column
chromatography (CHCl3/MeOH = 40:1) to obtain compound 21
(85 mg, 73%) as a colorless oil. Recrystallization from EtOAc
2.1.17. (20R,22R)-20,22-Isopropylidenedioxy-3ˇ-
(tetrahydropyran-2-yloxy)-5˛-cholestan-6-one (25)
(Scheme 3)
Dess–Martin periodinane (0.86 g, 2.02 mmol) was added to a
solution of compound 23 (1.05 g, 98%, 1.84 mmol) in anhydrous
CH2Cl2 (16 mL) at 0 ◦C. After stirring for 2 h at room tempera-
ture, additional Dess–Martin periodinane (0.22 g, 0.50 mmol)
was added at 0 ◦C, and the mixture was stirred for another 1 h
gave a colorless solid, mp 104–106 ◦C (EtOAc); [␣]D −24.8
24
(c 0.44, EtOH); 1H NMR (400 MHz) ı 0.86 (3H, s), 0.87 (3H, s),
0.91 (3H, d, J = 6.5 Hz), 0.92 (3H, d, J = 6.4 Hz), 1.22 (3H, s), 3.38
(1H, m), 3.64 (1H, ddd, J = 15.4 and 10.4 and 4.9 Hz); 13C NMR

1460
steroids 7 3 ( 2 0 0 8 ) 1452–1464
at room temperature. 20% Na2S2O3·5H2O/saturated aqueous
NaHCO3 solution (10 mL) was added to the mixture and stirred
for 5 min at room temperature. The mixture was diluted with
CH2Cl2 (65 mL), and the separated organic layer was dried over
anhydrous Na2SO4. The solvent was removed in vacuo, and the
residue was subjected to flash column chromatography (hex-
the reaction mixture was stirred for 20 min at room temper-
ature. The mixture was diluted with CH2Cl2 (60 mL), washed
with saturated aqueous NaHCO3 solution (15 mL), and dried
over anhydrous MgSO4. The solvent was removed in vacuo
to obtain a pale yellowish solid (2.26 g). This crude mate-
rial was dissolved in anhydrous DMF (35 mL). LiBr·H2O (4.24 g,
40.4 mmol) and Li2CO3 (2.99 g, 40.4 mmol) were added to the
solution and the mixture was refluxed for 1.5 h. 1N HCl (50 mL)
was added to the cooled mixture and this was extracted
with CHCl3 (4× 30 mL). The combined organic layer was suc-
cessively washed with saturated aqueous NaHCO3 solution
(50 mL) and brine (50 mL), and dried over anhydrous MgSO4.
The solvent was removed in vacuo to obtain a pale yellow-
ish oil, which was purified by flash column chromatography
(hexane/EtOAc = 30:1) to yield compound 29 (0.95 g, 52%) as a
colorless solid. 1H NMR (400 MHz) ı 0.72 (3H, s), 0.81 (3H, s), 0.90
(6H, d, J = 6.6 Hz), 1.15 (3H, s), 1.31 (3H, s), 1.41 (3H, s), 3.62 (1H,
dd, J = 9.2 and 3.0 Hz), 5.57 (1H, m), 5.68 (1H, m).
ane/EtOAc = 7:1) to give compound 25 (0.43 g, 42%) as a color-
less solid, mp 150-151 ◦C (EtOH); [␣]D −14.1 (c 0.50, EtOH); 1
H
27
NMR (300 MHz) ı 0.77 (3H, s), 0.79 (3H, s), 0.90 (6H, d, J = 6.5 Hz),
1.14 (3H, s), 1.29 (3H, s), 1.41 (3H, s), 3.48 (1H, m), 3.56 (1H, m),
3.62 (1H, dd, J = 9.2 and 2.8 Hz), 3.90 (1H, m), 4.76 (1H, m).
2.1.18. (20R,22R)-3ˇ,20,22-Trihydroxy-5˛-cholestan-6-one
(26) (Scheme 3)
A solution of compound 25 (1.73 g, 3.10 mmol) in 60% AcOH
(24 mL) and EtOH (4 mL) was stirred for 8.5 h at 80 ◦C. After
evaporation, the residue was subjected to flash column chro-
matography (CHCl3/MeOH = 30:1) to separate compound 26 as
a colorless solid and unreacted compound 25. The latter was
treated as above, and the total yield of compound 26 was 1.12 g
2.1.21. (20R,22R)-2ˇ-Acetoxy-3ˇ-hydroxy-20,22-
(83%), mp 180–181 ◦C (EtOH); [␣]D −6.4 (c 0.52, EtOH); 1H NMR
isopropylidenedioxy-5˛-cholestan-6-one (30) (Scheme 4)
Iodine (0.55 g, 2.18 mmol) was added to a mixture of compound
29 (0.95 g, 2.08 mmol) and silver(I) acetate (0.78 g, 4.68 mmol)
in AcOH (35 mL) in portion under argon, and the reaction mix-
ture was stirred for 1 h at room temperature. Water (37.4 ␮L,
2.08 mmol) was added to the mixture, and stirred for 1 h at
90 ◦C. After addition of NaCl (1.3 g), the mixture was cooled
to room temperature. The insoluble materials were filtered
off, and the filtrate was concentrated in vacuo to obtain a
pale yellowish solid, which was purified by flash column chro-
matography (CHCl3/MeOH = 50:1) to yield compound 30 (1.01 g,
91%) as a colorless solid. 1H NMR (400 MHz) ı 0.79 (3H, s), 0.89
(3H, s), 0.90 (6H, d, J = 6.6 Hz), 1.14 (3H, s), 1.29 (3H, s), 1.41 (3H,
s), 2.09 (3H, s), 2.17 (1H, dd, J = 12.1 and 2.8 Hz), 2.26 (1H, dd,
J = 11.7 and 2.0 Hz), 2.33 (1H, dd, J = 13.2 and 4.5 Hz), 3.61 (1H,
dd, J = 9.2 and 2.9 Hz), 3.67 (1H, m), 5.15 (1H, m).
20
(400 MHz) ı 0.76 (3H, s), 0.88 (3H, s), 0.90 (3H, d, J = 6.5 Hz), 0.90
(3H, d, J = 6.6 Hz), 1.21 (3H, s), 2.33 (1H, dd, J = 13.0 and 4.3 Hz),
3.38 (1H, m), 3.58 (1H, m); 13C NMR (100 MHz) ı 13.65, 20.34,
21.38, 21.76, 22.34, 22.50, 22.90, 23.62, 28.05, 28.16, 29.21, 30.00,
30.66, 36.26, 36.64, 37.16, 39.92, 40.86, 43.77, 46.51, 53.86, 54.67,
56.78, 70.64, 76.36, 83.35, 210.66; HRMS (FAB) m/z: C27H47O4
[M+H]+, calcd 435.3474, found 435.3473.
2.1.19. (20R,22R)-5˛-Cholestan-3ˇ,6ˇ,20,22-tetraol (28)
(Scheme 3)
NaBH4 (0.69 g, 18.3 mmol) was added to a solution of com-
pound 25 (1.02 g, 1.83 mmol) in EtOH (20 mL) at 0 ◦C. After
stirring for 15 min at the same temperature, the reaction mix-
ture was stirred for 1 h at room temperature. 1M HCl (20 mL)
was added to the mixture at 0 ◦C, and extracted with CHCl3
(4× 10 mL). The combined organic layer was washed with
saturated aqueous NaHCO3 solution (20 mL) and dried over
anhydrous MgSO4. The solvent was removed under reduced
pressure to give compound 27 (1.10 g) as a colorless solid.
This crude material was dissolved in 60% AcOH (12 mL) and
EtOH (2 mL), and the mixture was stirred for 9 h at 80 ◦C. After
evaporation, the residue was subjected to flash column chro-
matography (CHCl3/MeOH = 15:1) to separate compound 28
and unreacted 27. The latter was treated as above, and the
total yield of 28 was 0.31 g (39%) as a colorless solid, mp 112-
2.1.22. (20R,22R)-2ˇ,3ˇ-Dihydroxy-20,22-
isopropylidenedioxy-5˛-cholestan-6-one (31) (Scheme 4)
A mixture of compound 30 (0.53 g, 1.0 mmol) in 1M NaOH
(15 mL) and THF (8 mL) was stirred for 1 h at room temperature.
After addition of saturated aqueous NH4Cl solution (40 mL),
the aqueous layer was extracted with CHCl3 (4× 15 mL). The
combined organic layer was washed successively with sat-
urated aqueous NH4Cl solution (30 mL) and brine (30 mL),
and dried over anhydrous MgSO4. The solvent was removed
in vacuo to obtain a pale yellowish solid, which was puri-
fied by flash column chromatography (CHCl3/MeOH = 40:1) to
yield compound 31 (0.27 g, 55%) as a colorless solid. 1H NMR
(400 MHz) ı 0.80 (3H, s), 0.90 (6H, d, J = 6.6 Hz), 0.99 (3H, s), 1.14
(3H, s), 1.29 (3H, s), 1.41 (3H, s), 2.21 (1H, dd, J = 12.1 and 2.6 Hz),
2.33 (1H, dd, J = 13.1 and 4.5 Hz), 3.62 (1H, dd, J = 9.1 and 2.9 Hz),
3.63 (1H, m), 4.03 (1H, m).
113 ◦C (EtOAc); [␣]D +12.1 (c 0.51, EtOH); 1H NMR (300 MHz) ı
20
0.90 (3H, d, J = 6.3 Hz), 0.91 (3H, s), 0.91 (3H, d, J = 6.3 Hz), 1.04 (3H,
s), 1.21 (3H, s), 3.38 (1H, m), 3.66 (1H, m), 3.82 (1H, d, J = 2.1 Hz);
13C NMR (C5D5N, 100 MHz) ı 13.82, 14.23, 16.34, 21.28, 21.57,
22.61, 23.32, 24.61, 28.41, 30.23, 30.57, 36.13, 37.24, 39.29, 40.90,
43.95, 48.58, 53.16, 54.96, 55.75, 56.83, 71.22, 71.41, 76.58, 76.69;
HRMS (FAB) m/z: C27H48O4Na [M+Na]+, calcd 459.3450, found
459.3462.
2.1.23. (20R,22R)-2ˇ,3ˇ,20,22-Tetrahydroxy-5˛-cholestan-
6-one (32) (Scheme 4)
A solution of compound 31 (0.30 g, 0.61 mmol) in 60% AcOH
was stirred for 4 h at 80 ◦C. The solvent was removed in vacuo,
and the residue was purified by flash column chromatogra-
phy (CHCl3/MeOH = 20:1) to obtain a compound 32 (0.20 g, 71%)
2.1.20. (20R,22R)-20,22-Isopropylidenedioxy-5˛-cholest-2-
en-6-one (29) (Scheme 4)
Methanesulfonyl chloride (0.63 mL, 8.4 mmol) was added to a
mixture of compound 16 (1.90 g, 4.0 mmol) and triethylamine
(1.5 mL, 10.0 mmol) in anhydrous CH2Cl2 (15 mL) at 0 ◦C, and

steroids 7 3 ( 2 0 0 8 ) 1452–1464
1461
as a colorless solid, mp 226–227 ◦C (hexane/EtOH); [␣]D +3.5
(c 0.52, EtOH); 1H NMR (400 MHz) ı 0.88 (3H, s), 0.90 (3H, d,
J = 6.5 Hz), 0.91 (3H, d, J = 6.5 Hz), 0.99 (3H, s), 1.21 (3H, s), 2.20
(1H, dd, J = 12.4 and 2.9 Hz), 2.32 (1H, dd, J = 13.1 and 4.2 Hz),
3.38 (1H, m), 3.64 (1H, m), 4.03 (1H, br, s); 13C NMR (C5D5N,
100 MHz) ı 14.11, 15.81, 21.26, 21.89, 22.45, 22.62, 23.33, 24.13,
25.57, 28.41, 30.24, 36.85, 37.23, 40.55, 41.04, 43.59, 44.18, 46.70,
54.84, 55.49, 56.90, 57.93, 70.02, 72.15, 76.54, 210.53; HRMS (FAB)
m/z: C27H47O5 [M+H]+, calcd 451.3423, found 451.3422.
21
rithm of IC50, pIC50, was used as the index of the binding
activity.
3.
Results and discussion
3.1.
Chemistry
The analogs with non-oxygenated steroidal mother skeleton
moiety (8 and 12) were prepared according to the procedure
of Scheme 1. Since there are three OH groups in the starting
material (4), protections of OH are sometimes needed for cer-
tain reactions. The 20,22-diol moiety of compound 4 [33] was
protected as acetonide and 3␤-hydroxyl group was tosylated
to give compound 6. Even though LiEt3BH was used to remove
the tosyl group of 6, large amounts of LiEt3BH (13 equiv.) and a
long reaction time (6 days) were required to obtain compound
7 in a favorable yield. The hydrolysis of compound 7 by 60%
acetic acid/THF was quite sluggish, and the yield of compound
8 was low (25%) in spite of the repetition of the hydrolytic reac-
tion. The double bond existing in the B-ring of compound 4
was hydrogenated to lead compound 9 using 10% Pd–C, which
was then converted to 12 using a similar procedure to that
used for the preparation of compound 8 from compound 5.
Other acids such as BiCl3, trifluoroacetic acid, HBr, and HCl
were used to hydrolyze acetonide, but the satisfactory result
was not obtained.
The syntheses of 3␣-OH analogs (20 and 21) are summa-
rized in Scheme 2. The diol moiety of compound 13 was
protected using dimethoxypropane, and the acetonide was
submitted to hydroboration of the double bond at C-5 with
BH3·SMe2 obtaining the 6␣-alcohol 14. Since the addition of
BH3·SMe2 to double bond is sterically hindered by the presence
of the methyl group (C19) at C-10 position, the stereochem-
istry of OH at C6 of 24 should be a alpha configuration having
6␤-H[38,39]. Compound 28 with 6␣-H is derived from the cor-
responding ketone 25 by reducing the carbonyl group carrying
minor byproduct 24.
The 6␣-OH group of 14 was oxidized to a ketone with
Dess–Martin periodinane, and the TBDMS group, which is
used to protect the 3-OH group was selectively removed
without hydrolyzing acetonide under the moderate acid
condition (80% AcOH, THF, 80 ◦C). The yield of compound
16 from compound 13 was quantitative. The stereochemistry
inversion of the 3␤-OH group was achieved by a Mitsunobu
reaction affording compound 17 in 68% yield. By hydrolyz-
ing compound 17 under basic condition A/B trans (18) and
A/B cis (19) were obtained in 59% and 26% yield, respectively.
The stereochemistry of the A/B-ring fusion was determined
by NMR. Even though the corresponding compounds have not
been reported to date, the chemical shifts of H-5␣ of 32 and H-
5␤ of 34 are thought to be similar to the analogous compounds
such as brassinosteroids shown in Fig. 4. [40] The chemical
shift of 5␤-H of 35 (A/B cis) is 2.41 ppm (dd, J = 10 and 5 Hz),
and those of 5␣-H of 36 and 37 (A/B trans) are 2.21 ppm (dd,
J = 11.7 and 3 Hz) and 2.22 ppm (dd, J = 11.7 and 3 Hz), respec-
tively. Thus, the A/B ring fusion of 32 and 34 are assigned to be
trans and cis, respectively. The similar shifts of H-NMR for H-
5 are also reported for 22,23-epoxyecdysteroids (i.e. 2.42 ppm;
J = 12 and 3.5 Hz for 5␣-H; 3.02 ppm, J = 12 and 3 Hz for 5␤-H). [41]
2.1.24. (20R,22R)-2ˇ,3ˇ-Dihydroxy-20,22-
isopropylidenedioxy-5ˇ-cholestan-6-one (33) (Scheme 4)
A mixture of compound 30 (0.24 g, 0.45 mmol) and K2CO3 (2.0 g)
in MeOH (60 mL) and water (10 mL) was refluxed overnight.
The solvent was evaporated and water (50 mL) was added
to the residue. The aqueous layer was extracted with CHCl3
(1× 50 mL, 4× 20 mL), and the combined organic layer was
washed with brine (50 mL), and dried over anhydrous MgSO4.
The solvent was removed in vacuo to obtain a pale yellowish
solid, which was purified by flash column chromatography
(CHCl3/MeOH = 40:1) to yield compound 33 (95 mg, 43%) as a
colorless solid. 1H NMR (400MHz) ı 0.79 (3H, s), 0.90 (6H, d,
J = 6.6 Hz), 0.91 (3H, s), 1.15 (3H, s), 1.29 (3H, s), 1.41 (3H, s), 2.43
(1H, dd, J = 12.9 and 4.7 Hz), 3.62 (1H, dd, J = 9.1 and 2.9 Hz), 3.78
(1H, m), 4.05 (1H, m).
2.1.25. (20R,22R)-2ˇ,3ˇ,20,22-Tetrahydroxy-5ˇ-cholestan-
6-one (34) (Scheme 4)
A mixture of compound 33 (0.14 g, 0.29 mmol) in 60% AcOH
was stirred for 4 h at 80 ◦C. The solvent was removed in vacuo,
and the residue was subjected to flash column chromatogra-
phy (CHCl3/MeOH = 20:1) to obtain compound 34 (0.12 g, 88%)
as a colorless solid, mp 130–132 ◦C (EtOAc); [␣]D −46.1 (c
21
0.52, EtOH); 1H NMR (400 MHz) ı 0.88 (3H, s), 0.90 (3H, d,
J = 6.5 Hz), 0.91 (3H, d, J = 6.2 Hz), 0.92 (3H, s), 1.22 (3H, s), 2.44
(1H, dd, J = 12.9 and 4.5 Hz), 3.37 (1H, m), 3.78 (1H, m), 4.04
(1H, m); 13C NMR (C5D5N, 100 MHz) ı 13.98, 21.18, 21.62, 22.46,
22.61, 23.21, 23.98, 28.40, 30.21, 33.31, 36.58, 37.15, 38.24, 40.54,
40.66, 40.75, 43.12, 44.10, 54.72, 55.42, 56.85, 67.52, 68.78, 76.49,
214.43; HRMS (FAB) m/z: C27H47O5 [M+H]+, calcd 451.3423,
found 451.3422.
2.1.26. Binding assay
The inhibition of the binding of [3H]PNA (85.2 Ci/mmol; ARC
Inc., Carlsbad, CA, USA) to Kc cells was examined accord-
ing to previously reported methods [9,36]. In brief, 400 ␮L
of cell suspension (4 × 106 cells/ml) containing 1 ␮L of DMSO
solution of the test compound and 2 ␮L of the 70% ethanol
solution of [3H] PNA (0.5 ␮M, ca. 60,000 dpm) was incubated
for 30 min at 25 ◦C. The reaction mixture was immediately
filtered through a glass filter (GF/F) and washed three times
with water (1 mL) which was used to rinse the test tube.
The filter was dried under infrared light and transferred to
the vial for a liquid scintillation counter (LSC). The radioac-
tivity collected in the filter was counted with LSC in 3 mL
of Aquasol-2 (Packard Instrument Co., Meriden, CT, USA).
The concentration-response curve for the inhibition of the
[3H]PNA binding was drawn for each compound. The con-
centration required to give 50% inhibition (IC50 in M) was
determined by probit analysis [37] and the reciprocal loga-

1462
steroids 7 3 ( 2 0 0 8 ) 1452–1464
Fig. 4 – H NMR chemical shifts (ı) for H␣ or H␤ at C5 for A/B cis and trans compounds. See Ref. [40].
The compounds 18 and 19 were hydrolyzed to compounds 20
and 21 in fairly good yields (84% and 73%) under the condition
with 60% acetic acid in THF.
uration to the cis enhanced the activity 250 times (34 vs. 32);
the activity of 34 was equivalent or slightly higher than that
of the insect molting hormone, 20-OH Ecd (pIC50 = 7.34).
In the above structure-activity relationship (SAR), the effect
of the configuration of the 3-OH group on the activity was
somewhat curious. Even though the configurations of the 3-
OH group of 20-OH Ecd and PNA are both ␤, the activity of
the compound 26 with 3␤-OH group was 10 times lower than
its 3␣-epimer (compound 20). This was also the case for the
compounds carrying the cis-2,3-dihydroxy groups, where com-
pound 32 carrying the 3␤-OH group was about 50 times less
potent than the (2␣, 3␣)-compound (3). The apparent incon-
sistency in these data is likely explained by the difference in
the configuration of the A/B ring fusion between the series of
tested compounds in this study and the natural ecdysteroids.
As illustrated in Fig. 5, the 3-OH groups of compounds 20 and
3 are axially positioned to be located in a space close to that
occupied by the 2,3-diOH groups of ecdysteroids, such as 20E
and PNA. By contrast, the 3␤-OH groups of compounds 26 and
32 are away from the 2,3-diOH groups of ecdysteroids. Con-
versely, it is suggested that the location of a hydrogen bond
forming group in this space is very important for the molecular
interaction between the steroid compound and the EcR.
As the compounds without functionality in the steroid
skeleton were inactive, it is obvious that the properly located
functional groups play a very important role in the binding
of the steroidal ligand to its receptor. However, as indicated
by the difference in the potency between the 3-OH epimers,
Compound 5 was used as starting material to synthesize
3␤-OH analogs (26 and 28), as shown in Scheme 3. The 3␤-
OH group of compound 5 was protected by the THP group
(compound 22) with a 78% yield. Since the protection of OH
by THP is not strong, both THP and isopropylidene groups
compound 23 were easily removed using 60% acetic acid with
a 42% yield. Compound 22 was converted to compound 23
quantitatively by the hydroboration of the double bond of the
B-ring. Compound 23 was oxidized to compound 25 using
Dess–Martin periodinane with a 42% yield, and compound 25
was hydrolyzed to compound 26 with a 83% yield. Compound
25 was also converted to compound 28, in which the 6-keto
group was reduced to the 6␤-OH group with NaBH4. The acid
hydrolysis of compound 27 afforded compound 28.
The analogs with the 2␤,3␤-diol moiety were prepared from
compound 16, as shown in Scheme 4. After mesylating the
3␤-OH group of compound 16, MsOH was eliminated to afford
compound 29 with a 52% yield, by refluxing in DMF with a
combination of LiBr·H2O and Li2CO3. Woodward oxidation of
compound 29 gave compound 30 with a 91% yield. By the brief
treatment of compound 30 with 1 M NaOH in THF, the A/B
trans steroid 31 was obtained in 55% yield, and hydrolyzed
to compound 32 in 71% yield under acidic condition. On the
other hand, by hydrolyzing compound 30 with K2CO3 in boil-
ing aqueous MeOH overnight, the A/B cis compound 33 was
obtained in 43% yield. This was then hydrolyzed to compound
34 under acidic condition.
3.2.
Receptor binding activity
The binding activity of newly synthesized steroidal com-
pounds is listed in Table 1. Compounds 8 and 12, without
functional groups such as OH and C O group in the steroid
skeleton were inactive. Introduction of the OH group at the
3-position of the steroidal skeleton elevated the activity to a
measurable level (pIC50 = 4.38 for 4 and 9), even though the
configuration of the A/B ring fusion is different from that of the
ecdysteroids. The additional modification of the B-ring moiety
where CH2 was oxidized to C O at the C6 position increased
the activity about 10 times (26 vs. 9). Interestingly, the activity
was lost by the reduction of the oxo to the hydroxyl group (26
vs. 24 or 28). Further hydroxylation at 2-position had no effect
on the activity (32 vs. 26). The conversion of A/B trans config-
Fig. 5 – Superposition of A/B ring moieties of ecdysteroids
on the corresponding moiety of 20-OH Ecd (broken line).
The sign “>” indicates the difference of the activity. (A)
3-Hydroxy analogs (20 vs. 26) (B) 2,3-dihydroxyanalogs (3
vs. 32).

steroids 7 3 ( 2 0 0 8 ) 1452–1464
1463
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functionalities are important not only for the high affinity
of the ligand-receptor interaction, but also for the capacity
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Acknowledgements
This study was supported, in part, by the 21st Century
COE program for Innovative Food and Environmental Stud-
ies Pioneered by Entomomimetic Sciences, from Ministry of
Education, Culture, Sports, Science and Technology of Japan.
A part of experiments was performed in the RI center of Kyoto
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