Synthesis of 26,27-bisnorcastasterone analogs and analysis of conformation-activity relationship for brassinolide-like activity

Article abstract of DOI:10.1016/j.bmc.2005.10.024

Three castasterone (CS) derivatives with varied side-chain moieties, 26,27-bisnorcastasterone (20S-bisnorCS), 20-epi-26,27-bisnorcastasterone (20R-bisnorCS), and 21,26,27-trisnorcastasterone (trisnorCS), were synthesized stereoselectively from either stig

Full text of DOI:10.1016/j.bmc.2005.10.024

Bioorganic & Medicinal Chemistry 14 (2006) 1761–1770
Synthesis of 26,27-bisnorcastasterone analogs and analysis
of conformation–activity relationship for brassinolide-like activity
Shuji Yamamoto,a Bunta Watanabe,a Junko Otsuki,a Yoshiaki Nakagawa,a,*
Miki Akamatsub and Hisahi Miyagawaa
aDivision of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
bDivision of Environmental Science and Technology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Received 24 August 2005; revised 17 October 2005; accepted 17 October 2005
Available online 8 November 2005
Abstract—Three castasterone (CS) derivatives with varied side-chain moieties, 26,27-bisnorcastasterone (20S-bisnorCS), 20-epi-
26,27-bisnorcastasterone (20R-bisnorCS), and 21,26,27-trisnorcastasterone (trisnorCS), were synthesized stereoselectively from
either stigmasterol or dehydroisoandrosterone. The 50% effective doses (ED50, nmol/plant) in the concentration–response curve
for brassinolide-like activity in the rice lamina inclination assay were determined to be 0.020 nmol (pED50 = 10.7) for 20S-bisnorCS,
3.2 nmol (pED50 = 8.5) for 20R-bisnorCS, and 2.0 nmol (pED50 = 8.7) for trisnorCS. An analog containing an ester linkage between
the steroid and the side-chain moiety of 20S-bisnorCS was also synthesized and its activity was evaluated to be 3.2 nmol
(pED50 = 8.5), being equipotent to 20R-bisnorCS and trisnorCS. The activity of 20S-bisnorCS was 1/40 that of CS. The conforma-
tion analysis was conducted using a systematic search, showing that the activity decreases with an increase in the degree of freedom
of the side chain of the steroidal skeleton.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
It is well known that steroid hormones bind to nuclear
receptors and the ligand–receptor complexes act on gen-
ome in animals, but no nuclear receptor has been iden-
tified in plants to date. In plants, a transmembrane
receptor kinase, brassinosteroid-insensitive1 (BRI1),
functions as a receptor of brassinolide.6 The binding do-
main of BRI1 was recently determined in Arabidopsis
using a biotin-tagged photo-affinity CS.7 Numerous ef-
forts have also been devoted to the chemical synthesis
and structure–activity relationship (SAR) studies of
BL analogs to obtain potent compounds and good
probes for the function analysis of biosynthetic and
metabolic enzymes and receptors.8–16
Brassinolide (BL, 1; Fig. 1) is a steroid hormone, which
is essential for the growth and development of plants,
and its structure was determined by Grove and co-work-
ers a quarter century ago.1 BL has a number of interest-
ing substructures, including a seven-membered ring
lactone structure as part of the B-ring moiety and four
asymmetric carbons in the side-chain moiety. Various
physiological effects on plant growth and improvement
of stress tolerance are associated with BL structure. Lat-
er, castasterone (CS, 2; Fig. 1) was isolated in chestnut
insect gall by Yokota and co-workers,2 which is the bio-
synthetic immediate precursor of BL.3 Since the discov-
ery of BL, more than 50 BL analogs have been identified
in the plant kingdom, and these BL-like compounds are
collectively called brassinosteroids (BRs).4,5
Our group synthesized new BRs containing the ester link-
age between the steroid skeleton and the side chain such
as compound 3 (BL-ester, Fig. 1) and discussed the struc-
ture–activity relationship for the BL-activity of com-
pounds with various side-chain moieties.15 The activity
of ester analogs was drastically enhanced by the intro-
duction of an OH group at the a-position of the ester
moiety. The compound with R-configuration with re-
spect to the a-carbon was 10 times more potent than
the corresponding S-form. However, even for the most
potent compound, its activity was 1/1000 that of BL.
We assumed that the reduction in activity is possibly
Abbreviations: BL, brassinolide; CS, castasterone; ED50, 50% effective
dose; BR, brassinosteroid; TsOH, toluenesulfonic acid; BuOH, buta-
nol; HRMS, high-resolution mass spectrum; THF, tetrahydrofuran;
DMAP, 4-dimethylaminopyridine; PPTS, pyridinium p-toluenesulfo-
nate; PDC, pyridinium dichromate.
Keywords: Brassinosteroids; Castasterone; Rice lamina inclination;
Conformation analysis.
*
Corresponding author. Fax: +75 753 6123; e-mail: naka@kais.
kyoto-u.ac.jp
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.024

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S. Yamamoto et al. / Bioorg. Med. Chem. 14 (2006) 1761–1770
28
O
OH
OH
21
18
26
23
O
22
24
20
25
27
OH
OH
17
OH
D16
12
13
19
5
11
15
14
C
HO
HO
HO
HO
HO
HO
9
8
1
10
2
3
7
A
4
B
O
6
O
H
H
H
O
O
O
Brassinolide (BL,1)
Castasterone (CS, 2)
3 (BL-ester)
Figure 1. Chemical structures of brassinosteroids.
due to the lack of the substituent at the position corre-
sponding to C21 position of BRs. We also demonstrated
that the replacement of the side chain of CS to that of
ponasterone A (PonA), which is an insect molting hor-
mone agonist, eliminated the activity, but its molting
hormonal activity remained.17,18 It is also reported that
the configurations of C22 and C23 with hydroxyl groups
are both R for the high BL-like activity.19
sis for these compounds to discuss the relationship
between the conformation and the activity.
2. Materials and methods
2.1. General for chemical synthesis
Reactions requiring anhydrous conditions were con-
ducted under argon atmosphere using oven-dried glass
wares. Anhydrous solvents used in this study were either
commercially available or prepared conventionally in
this laboratory. Chemicals were purchased from Aldrich
Chemical Corp. (Milwaukee, WI, USA), Tokyo Kasei
Kogyo Co. Ltd. (Tokyo, Japan), Wako Pure Chemical
Industries, Ltd. (Osaka, Japan), and Nacalai Tesque
Inc. (Kyoto, Japan), unless noted. Starting materials in
Schemes 1–3 were synthesized previously in our labora-
tory. Column chromatography was conducted using
Kieselgel 60 (Merck, Darmstadt, Germany) as the
It is known that steroid hormones and their agonists
change the conformation of their receptor proteins, trig-
gering a series of molecular events leading to various
biological responses.20,21 It is generally accepted that li-
gands with a stable conformation are able to bind tightly
to the receptor and induce strong biological activity. To
date, the conformational flexibility of the side chain of
epimeric vitamins has been examined and the contribu-
tion of the conformers was discriminated using the ener-
gy window concept.22,23 Based on this concept,
Yamamoto and co-workers studied the relationship be-
tween the conformation and function of vitamin D to
demonstrate to clarify the conformation of vitamin D
responsible for the receptor binding.24
1
absorbent. H and 13C NMR spectra were recorded on
a Bruker AC-300 or ARX-500 NMR spectrometer in
deuteriochloroform (CDCl3) with tetramethylsilane as
the internal standard. Optical rotations were measured
on a JASCO P-1000 polarimeter. High-resolution mass
spectrum (HRMS) was recorded on a JEOL JMS 700
spectrometer (Tokyo, Japan). Elemental analyses were
performed at the Microanalytical Center of Kyoto Uni-
versity. Melting points were determined with a Yanako
melting point apparatus (Yanagimoto Seisakusho Co.
Ltd., Kyoto, Japan) and are uncorrected.
In this report, we synthesized both 20S (4) and 20R (5)
enantiomers of 26,27-bisnorcastasterone analog and
21,26,27-trisnorcastasterone (6, Fig. 2) to examine the ef-
fect of C21 methyl group on the BL-like activity. Even
though it is appropriate to use the BL- or CS-type com-
pounds to examine the effect of C21 methyl group, the
construction of the original side chain is not easy as com-
pared with that of 26,27-bisnor type compounds. In com-
pounds 4–6, the carbon at 24-position is not asymmetric
anymore, and the decrease in the number of asymmetric
carbons dramatically lower the labor of synthetic chem-
ists. The corresponding ester analog 7 with CS-type ste-
roidal skeleton was also synthesized, and its BL-like
activity was compared with those of compounds 4–6. In
the further study, we performed the conformation analy-
2.2. Synthesis of (22R,23R)-2a,3a,22,23-tetrahydroxy-
26,27-bisnor-5a-ergostan-6-one (4; Scheme 1)
2.2.1. Ethyl (22R,23S)-2a,3a-isopropylidenedioxy-22,23-
dihydroxy-6-oxo-5a-cholan-24-oate (10). Triethyl phos-
phonoacetate (11 ml, 55 mmol) was added dropwise to
a stirred suspension of NaH (60% oil suspension;
O
OH
OH
OH
O
OH
OH
OH
OH
HO
HO
HO
HO
HO
HO
HO
HO
H
H
H
H
O
O
O
O
7
6
5
4
Figure 2. Newly synthesized brassinosteroids.

S. Yamamoto et al. / Bioorg. Med. Chem. 14 (2006) 1761–1770
1763
O
OH
O
CO2Et
CHO
CO2Et
CO2Et
OH
c
b
a
O
O
O
O
O
O
O
O
H
H
H
H
O
O
O
O
11
10
9
8
O
OH
O
O
OH
O
CO2Et
OH
e
f, g, h
HO
HO
c, d
O
O
O
O
H
H
H
O
O
O
O
O
4
12
13
Scheme 1. Reagents and conditions: (a) (EtO)2P(O)CH2CO2Et, NaH, THF; (b) OsO4, (DHQD)2PHAL, K3Fe(CN)6, K2CO3, MeSO2NH2, t-BuOH–
H2O; (c) 2,2-dimethoxypropane, p-TsOH, CHCl3; (d) 2,2-dimethyl-1,3-dioxolane, p-TsOH, reflux; (e) MeLi, THF, ꢀ78 ꢁC; (f) MsCl, Et3N, DMAP,
CH2Cl2; (g) H2, Pd/C, EtOH; (h) 70% aq AcOH, reflux.
2.2 g, 55 mmol) in tetrahydrofuran (THF; 80 ml) at
0 ꢁC. After stirring the mixture for 30 min at room tem-
perature, a THF solution of 8 (7.47 g, 18.6 mmol),
which is derived from stigmasterol according to the con-
ventional method,25 was added and stirred for 2 h.
Saturated aqueous NH4Cl (200 ml) was added to the
solution and extracted with ethyl acetate (4 · 100 ml).
The combined organic layer was dried over anhydrous
MgSO4, filtered, and concentrated to give a crude yellow
solid, which was purified by flash column chromatogra-
phy (hexane/ethyl acetate = 7:2, v/v) to provide 9 as a
colorless solid (7.35 g). To a solution of 9 in a mixture
of t-BuOH (85 ml) and H2O (85 ml), dihydroquini-
dine–phthalazine ligands (DHQD)2PHAL (1.17 g,
1.5 mmol), K3Fe(CN)6 (8.69 g, 26 mmol), K2CO3
(3.65 g, 26 mmol), 10% (w/v) OsO4/t-BuOH (9 ml,
OsO4: 0.35 mmol), and CH3SO2NH2 (1.67 g,
17.6 mmol) were added successively. After stirring over-
night at room temperature, Na2SO3 (5.0 g, 40 mmol)
was added to the reaction mixture, which was then
OH
OH
OBn
OBn
a
b
c, d
AcO
AcO
HO
16
15
14
O
O
O
O
O
O
h, i
e, f, g
j, k
HO
H
18
19
17
OH
O
OH
O
O
OH
l
HO
HO
O
O
H
5
O
20
H
O
Scheme 2. Reagents and conditions: (a) (R)-2-benzyloxy-3-methylbutanal, EtAlCl2, CH2Cl2–hexane; (b) K2CO3, MeOH–H2O; (c) H2, Pd/C, EtOAc;
(d) 2,2-dimethoxypropane, p-TsOH, CHCl3; (e) MsCl, Et3N, DMAP, CH2Cl2; (f) KHCO3, acetone–H2O, reflux; (g) H2, Pd/C, EtOH; (h) CrO3,
H2SO4, acetone; (i) NaBr, p-TsOH, DMF, reflux; (j) OsO4, NMO, THF–H2O; (k) 2,2-dimethoxypropane, p-TsOH, CHCl3; (l) 80% aq AcOH, 60 ꢁC.

1764
S. Yamamoto et al. / Bioorg. Med. Chem. 14 (2006) 1761–1770
CO2Me
CHO
CO2Et
f, g, h, i
a, b, c
d, e
HO
THPO
HO
21
22
23
OH
CO2Et
O
O
CO2Et
OH
j, k, l
HO
HO
O
O
H
O
H
O
H
O
O
6
24
25
Scheme 3. Reagents and conditions: (a) 3,4-dihydro-2H-pyran, PPTS, CH2Cl2; (b) LiAlH4, THF; (c) PDC, Celite, CH2Cl2; (d) (EtO)2P(O)CH2-
CO2Et, NaH, THF; (e) PPTS, EtOH, reflux; (f) MsCl, Et3N, DMAP, CH2Cl2; (g) KHCO3, acetone–H2O, reflux; (h) CrO3, H2SO4, acetone; (i) NaBr,
p-TsOH, DMF, reflux; (j) OsO4, (DHQD)2PHAL, K3Fe(CN)6, K2CO3, MeSO2NH2, t-BuOH–H2O; (k) 2,2-dimethoxypropane, p-TsOH, CHCl3;
(l) 2,2-dimethyl-1,3-dioxolane, p-TsOH, reflux.
23
D
extracted with CHCl3 (1 · 150 ml, 3 · 50 ml). The com-
bined organic layer was washed with 2 M KOH (150 ml)
and brine (150 ml), dried over anhydrous MgSO4, and
the solvent was evaporated. The residue was purified
by flash column chromatography (CHCl3/CH3OH =
50:1, v/v) to afford 10 (3.62 g, 68% for two steps) as a
colorless solid. Mp: 220 ꢁC. NMR dH (CDCl3): 0.66
(3H, s), 0.67 (3H, s), 1.05 (3H, d, J = 6.1), 1.31 (3H, t,
J = 7.2), 1.34 (3H, s), 1.51 (3H, s), 2.13 (1H, m), 2.32
(1H, dd, J = 4.3, 13.0), 2.38 (1H, d, J = 6.7), 2.55 (1H,
dd, J = 4.0, 13.0), 3.02 (1H, d, J = 5.8), 3.80 (1H, m),
4.07–4.13 (1H, m), 4.20–4.37 (3H, m). NMR dC
(CDCl3): 11.7, 12.61, 12.64, 14.2, 21.1, 22.5, 23.9, 26.5,
27.7, 28.6, 37.6, 39.3, 39.8, 41.1, 42.5, 42.7, 46.8, 51.5,
72.8, 72.9, 76.4, 80.9, 107.5, 109.7, 110.9, 171.8. ½aꢁ
+45.9 (c 0.98, CHCl3).
2.2.3. (22R,23S)-6,6-Ethylenedioxy-2a,3a,22,23-bis(iso-
propylidenedioxy)-26,27-bisnor-5a-ergostan-24-ol (13).
To a solution of 12 (2.00 g, 3.4 mmol) in THF (30 ml)
was added dropwise 1.14 M CH3Li/Et2O (12 ml,
13.7 mmol) at ꢀ78 ꢁC under argon. After 30 min, satu-
rated aqueous NH4Cl (5 ml) was added dropwise, and
the reaction mixture was warmed to room temperature.
Saturated aqueous NH4Cl (100 ml) was added and
extracted with ethyl acetate (1 · 100 ml, 3 · 50 ml).
The combined organic layer was washed with saturated
aqueous NaCl (100 ml) and dried over anhydrous
MgSO4. After evaporating the solvent, the residue was
purified with flash column chromatography (hexane/eth-
yl acetate = 3:1) to provide 13 (1.37 g, 70%) as a color-
less amorphous solid. NMR dH (CDCl3): 0.66 (3H, s),
0.83 (3H, s), 1.00 (3H, d, J = 6.1), 1.16 (3H, s), 1.25
(3H, s), 1.33 (3H, s), 1.39 (6H, s), 1.48 (3H, s), 2.08
(1H, s), 3.61 (1H, d, J = 8.6), 3.72–3.78 (1H, m), 3.87–
3.99 (3H, m), 4.06–4.13 (2H, m), 4.27 (1H, m). NMR
dC (CDCl3): 11.8, 12.6, 13.4, 20.8, 22.0, 24.0, 24.7,
26.6, 27.3, 27.5, 27.6, 28.3, 28.6, 33.0, 36.9, 38.0, 39.5,
41.0, 42.4, 42.7, 45.5, 52.9, 53.6, 55.6, 64.2, 65.5, 69.8,
72.8, 73.0, 78.2, 83.1, 107.5, 107.9, 109.7. HR-MS m/z
52.4, 53.2, 56.4, 62.1, 72.1, 72.3, 73.1, 73.8, 107.9,
23
D
173.6, 211.4. ½aꢁ +34.0 (c 1.13, CHCl3).
2.2.2. Ethyl (22R,23S)-6,6-ethylenedioxy-2a,3a,22,23-
bis(isopropylidenedioxy)-5a-cholan-24-oate (12). To a
solution of 10 (3.61 g, 7.1 mmol) in CHCl3 (50 ml) were
added 2,2-dimethoxypropane (5 ml) and catalytic
amount of p-TsOH, and then the mixture was stirred
for 30 min at room temperature. The reaction mixture
was diluted with CHCl3 (200 ml) and washed with satu-
rated aqueous NaHCO3 (100 ml). The organic layer was
dried over anhydrous MgSO4 and the solvent was evap-
orated to afford 11. Compound 11 was dissolved in 2,2-
dimethyl-1,3-dioxolane (10 ml) with catalytic amount of
p-TsOH. After stirring for 2 h at 90 ꢁC, the mixture was
diluted with CHCl3 (200 ml), and washed with saturated
aqueous NaHCO3 (100 ml). The organic layer was dried
over anhydrous MgSO4, and the solvent was evaporat-
ed. The residue was purified by flash column chromatog-
raphy (hexane/ethyl acetate = 7:2) to provide 12 (3.02 g,
72% for two steps) as colorless solid. Mp 199 ꢁC. NMR
dH (CDCl3): 0.68 (3 H, s), 0.84 (3H, s), 0.98 (3H, d,
J = 6.6), 1.29 (3H, t, J = 7.1), 1.33 (3H, s), 1.40 (3H,
s), 1.46 (3H, s), 1.48 (3H, s), 3.72–3.79 (1H, m), 3.87–
3.99 (3H, m), 4.06–4.14 (1H, m), 4.18–4.33 (5H, m).
NMR dC (CDCl3): 11.8, 12.2, 13.4, 14.3, 20.7, 22.0,
24.1, 25.4, 26.6, 26.6, 27.7, 28.6, 32.9, 36.9, 38.0, 39.4,
41.0, 42.5, 42.7, 45.5, 52.9, 53.1, 55.7, 61.2, 64.2, 65.5,
(M+) Calcd for C34H56O7: 576.4026. Found: 576.4034.
22:6
D
½aꢁ
+41.0 (c 0.13, MeOH).
2.2.4.
(22R,23R)-2a,3a,22,23-Tetrahydroxy-26,27-bis-
nor-5a-ergostan-6-one (4). To a solution of 13 (478 mg,
0.83 mmol), triethylamine (0.58 ml, 4.16 mmol) in
CH2Cl2 (10 ml) with catalytic amount of 4-dimethylami-
nopyridine (DMAP) was added methanesulfonyl chlo-
ride (0.13 ml, 1.7 mmol) at 0 ꢁC. After stirring for 4 h
at room temperature, the reaction mixture was diluted
with CHCl3 (30 ml) and washed with saturated aqueous
NaHCO3 (30 ml). The aqueous layer was extracted with
CHCl3 (3 · 10 ml), and the combined organic layer was
washed with brine (30 ml). After being dried over
anhydrous Na2SO4, the solvent was evaporated. The
residue was purified by flash column chromatography

S. Yamamoto et al. / Bioorg. Med. Chem. 14 (2006) 1761–1770
1765
(hexane/ethyl acetate = 5:1) to afford the olefin com-
pounds (296 mg), a mixture of 23- and 24-ene com-
pounds. This olefin mixture (285 mg) was dissolved in
EtOH (2 ml) and hydrogenated with Pd/C to provide a
colorless amorphous solid, which was dissolved in 70%
aqueous acetic acid (5 ml). After stirring for 5 h at
80 ꢁC, the reaction mixture was concentrated. The resi-
due was azeotropically concentrated with toluene and
purified by flash column chromatography (CHCl3/
MeOH = 15:1–9:1) to provide 4 (70 mg, 20% for three
steps) as a colorless solid. Mp: 291–292 ꢁC (EtOH).
(lit.,26 290–295) NMR dH (CDCl3/C5D5N = 1/1): 0.69
(3H, s), 0.79 (3H, s), 0.98 (3H, d, J = 6.7), 1.06 (3H, d,
J = 6.6), 1.10 (3H, d, J = 6.8), 2.29 (1H, dd, J = 4.4,
12.9), 2.89 (1H, dd, J = 2.5, 12.3), 3.56 (1H, dd,
J = 2.4, 7.6), 3.72 (1H, d, J = 7.5), 3.86 (1H, m), 4.20
(1H, m). NMR dC (CDCl3/C5D5N = 1/1): 12.0, 12.5,
13.6, 15.2, 20.8, 21.3, 23.9, 27.2, 27.8, 29.8, 37.7, 38.2,
39.6, 40.7, 42.5, 42.8, 46.8, 51.1, 52.6, 53.8, 56.6, 68.0,
68.4, 73.4, 76.3, 212.1. HRMS m/z (M+) Calcd for
C26H44O5: 436.3189. Found: 436.3192.
sponding triol. This triol was dissolved in CHCl3
(10 ml) and then 2,2-dimethoxypropane (2 ml) and cata-
lytic amount of p-TsOH were added to this solution.
After stirring for 5 min, MeOH (5 ml) was added to
the reaction mixture and stirred for further 5 min. The
mixture was diluted with CHCl3 (100 ml) and washed
with saturated aqueous NaHCO3 (50 ml). The organic
layer was dried over anhydrous MgSO4, filtered, and
concentrated to afford crude 17 (0.89 g), which was used
for the next step without purification.
To a mixture of crude 17 (0.89 g, synthesized from
1.98 mmol of 16), Et3N (0.56 ml, 4.0 mmol), and catalyt-
ic DMAP in CH2Cl2 (10 ml) was added methanesulfonyl
chloride (0.23 ml, 3.0 mmol) at 0 ꢁC. After stirring for
30 min at room temperature, the reaction mixture was
diluted with CHCl3 (100 ml) and then washed successive-
ly with 1 M HCl (50 ml) and saturated aqueous NaHCO3
(50 ml). The organic layer was concentrated to give crude
mesylate. The mixture of this mesylate and KHCO3
(0.92 g, 9.2 mmol) in acetone/H2O (48 ml–12 ml) was
stirred for 1.5 h at 75 ꢁC and then concentrated. The pre-
cipitate was dissolved in ethyl acetate (60 ml), washed
with water (30 ml), and the aqueous layer was extracted
with ethyl acetate (3 · 15 ml). The combined organic
layer was washed with 0.5 M HCl (50 ml), saturated
aqueous NaHCO3 (50 ml), and brine (50 ml), dried over
anhydrous MgSO4, filtered, and concentrated to give
crude 3a,5-cycloalcohol. This cycloalcohol was dissolved
in EtOH (10 ml). To this solution was added 10% Pd/C
(0.25 g) and the resulting mixture was stirred overnight
at room temperature under H2 atmosphere. Pd/C was fil-
tered off and the filtrate was concentrated to give crude
18 (0.84 g) as a colorless amorphous solid, which was
used for the next step without purification.
2.3. Synthesis of (20R,22R,23R)-2a,3a,22,23-tetrahydr-
oxy-26,27-bisnor-5a-ergostan-6-one (5; Scheme 2)
2.3.1. (20R,22R,23R)-23-Benzyloxy-26,27-bisnorergost-
5,16-diene-3b,22-diol (16). To a solution of compound
14 (1.41 g, 4.1 mmol) prepared according to the conven-
tional method,27 and (R)-2-benzyloxy-3-methyl-butanal
(1.05 g, 5.5 mmol) in CH2Cl2 (30 ml) was added drop-
wise 1 M EtAlCl2/hexane (15 ml, 15 mmol) at ꢀ78 ꢁC,
and the reaction mixture was stirred for 30 min. One
molar HCl (10 ml) was added to the mixture and then
warmed to room temperature. One molar HCl
(150 ml) was added and extracted with CH2Cl2
(1 · 50 ml, 4 · 20 ml). The combined extract was washed
with brine (80 ml) and dried over anhydrous MgSO4.
The solvent was evaporated to afford a yellow oil, which
was purified by flash column chromatography (hexane/
ethyl acetate = 15:1–10:1) to provide colorless oil
(1.27 g) containing 15. This material was hydrolyzed
with K2CO3 in CH3OH/H2O at 80 ꢁC and then purified
by flash column chromatography (hexane/ethyl ace-
tate = 5/2) to afford compound 16 (1.08 g, 53% for two
steps) as a colorless amorphous solid. NMR dH (CDCl3)
d: 0.80 (3 H, s), 0.97 (3H, d, J = 6.8), 0.98 (3H, d,
J = 6.8), 1.05 (3H, s), 1.048 (3H, d, J = 6.9), 2.13 (1H,
ddd, J = 3.0, 6.6, 14.9), 2.53 (1H, d, J = 6.9), 3.29 (1H,
dd, J = 2.1, 5.8), 4.56 (1H, d, J = 11.1), 4.70 (1H, d,
J = 11.1), 5.37 (1H, m), 5.49 (1H, m), 7.36 (5H, m).
NMR dC (CDCl3): 16.5, 18.3, 18.7, 18.8, 19.3, 20.8,
30.1, 30.5, 31.51, 31.56, 31.62, 34.8, 35.9, 36.7, 37.2,
Crude compound 19 derived from 18 was converted to
20 as a colorless amorphous solid (overall yield: 9%
for nine steps from 16) according to conventional meth-
ods.28 NMR dH (CDCl3): 0.62 (3H, s), 0.68 (3H, s), 0.94
(6H, d, J = 6.9, 2· CH3), 0.98 (3H, d, J = 6.6), 1.34 (3H,
s), 1.36 (3H, s), 1.38 (3H, s), 1.50 (3H, s), 2.33 (1H, dd,
J = 4.3, 12.9), 2.55 (1H, dd, J = 4.0, 12.4), 3.54 (1H, dd,
J = 6.0, 6.5), 3.64 (1H, dd, J = 4.8, 6.6), 4.06 – 4.13 (1H,
m), 4.28 (1H, m). NMR dC (CDCl3): 12.7, 13.2, 15.8,
17.6, 20.0, 20.8, 22.4, 22.5, 24.0, 26.5, 27.4, 27.7, 28.6,
31.8, 33.8, 37.2, 37.4, 41.1, 42.5, 43.3, 46.9, 48.9, 51.5,
53.8, 55.6, 72.1, 72.3, 84.3, 84.4, 107.9, 108.0, 211.4.
25
½aꢁ +26.0 (c 0.37, CHCl3). HRMS m/z (M+) Calcd
D
for C32H52O5: 516.3815. Found: 516.3802.
2.3.3. (20R,22R,23R)-2a,3a,22,23-Tetrahydroxy-26,27-
bisnor-5a-ergostan-6-one (5). Compound 20 (44 mg,
84 lmol) was dissolved in 80% aqueous acetic acid
(5 ml) and stirred for 6 h at 60 ꢁC. The reaction mixture
was concentrated and azeotropically concentrated with
toluene. The residue was purified by flash column chro-
42.3, 47.5, 50.7, 56.5, 71.7, 73.2, 73.7, 82.0, 121.5,
123.7, 127.7, 127.8, 128.4, 138.5, 141.0, 158.2. ½aꢁ
25
D
+39.7 (c 0.56, CHCl3).
2.3.2. (20R,22R,23R)-2a,3a,22,23-Bis(isopropylidenedi-
oxy)-26,27-bisnor-5a-ergostan-6-one (20). To the solu-
tion of 16 (0.98. 1.98 mmol) in ethyl acetate (10 ml)
was added 10% Pd/C (Aldrich Co., Degussa type,
200 mg), then the mixture was stirred at room tempera-
ture for 30 min under H2 atmosphere. After filtration of
Pd/C, the solvent was evaporated to give the corre-
matography (CHCl3/MeOH = 20:1) to provide
5
(21.7 mg, 59%) as a colorless amorphous solid. NMR
dH (CDCl3): 0.65 (3H, s), 0.76 (3H, s), 0.93 (3H, d,
J = 7.5), 0.96 (3H, d, J = 7.1), 0.97 (3H, d, J = 6.7),
2.31 (1H, dd, J = 4.5, 13.6), 2.69 (1H, dd, J = 3.1,
12.5), 3.36 (1H, m), 3.43 (1H, m), 3.78 (1H, m), 4.05

1766
S. Yamamoto et al. / Bioorg. Med. Chem. 14 (2006) 1761–1770
(1H, m). NMR dC (CDCl3): 13.2, 13.6, 14.6, 17.4, 19.7,
21.0, 23.2, 24.0, 26.4, 31.4, 35.1, 37.7 (2· C), 40.1, 42.7,
43.5, 46.8, 49.9, 50.9, 54.2, 55.9, 68.1, 68.2, 74.9, 75.3,
213.1. HRMS m/z (M+) Calcd for C26H44O5: 436.3189.
Found: 436.3178.
20.8, 24.6, 28.2, 31.6, 31.9, 33.4, 36.6, 37.3, 37.7, 42.1,
42.3, 49.6, 50.4, 55.9, 60.1, 71.7, 121.3, 121.6, 140.8,
24
D
149.4, 166.8. ½aꢁ ꢀ71.7 (c 0.26, CHCl3). HRMS m/z
(M+) Calcd for C25H38O3: 386.2821. Found: 386.2827.
2.4.3. Ethyl (22E)-21-nor-5a-chol-2,22-dien-24-oate (24).
Compound 24 was synthesized according to the similar
method as shown in Scheme 2. NMR dH (CDCl3):
0.63 (3H, s), 0.72 (3H, s), 1.29 (3H, t, J = 7.1), 4.18
(2H, q, J = 7.1), 5.57 (1H, m), 5.69 (1H, m), 5.82 (1H,
dt, J = 15.6, 1.4), 6.94 (1H, dt, J = 7.4, 15.5). NMR dC
(CDCl3): 12.4, 13.5, 14.3, 20.8, 21.7, 24.2, 28.1, 33.4,
2.4. Synthesis of (22R,23R)-2a,3a,22,23-tetrahydro-
21,26,27-trisnor-5a-ergostan-6-one (6; Scheme 3)
2.4.1. 3b-Tetrahydropyranyloxypregn-5-en-21-al (22). To
a solution of 2129 (10.61 g, 30.6 mmol) in CH2Cl2 were
added 3,4-dihydro-2H-pyran (3.1 ml, 34 mmol) and cata-
lytic amount of pyridinium p-toluenesulfonate (PPTS),
and stirred overnight at room temperature. The reaction
mixture was diluted with CHCl3 (200 ml) and washed
with saturated aqueous NaHCO3 (100 ml). The organic
layer was dried over anhydrous MgSO4, filtered, and con-
centrated to afford the crude THP ether. The residue was
dissolved in THF (100 ml), and LiAlH4 (2.90 g,
76.4 mmol) was added to this solution at 0 ꢁC. After stir-
ring for 1 h at room temperature, water (2.9 ml), 10%
aqueous NaOH (2.9 ml), and water (8.7 ml) were added
dropwise at 0 ꢁC, successively. The precipitate was filtered
off, and the filtrate was concentrated to afford the crude
21-hydroxy compound. This alcohol was dissolved in
CH2Cl2, and Celiteꢂ (10 g) and pyridinium dichromate
(PDC; 17.0 g, 45 mmol) were added. After stirring for
2 h, i-PrOH (10 ml) was added, and the precipitate was fil-
tered off. The filtrate was washed with 10% aqueous
NaOH (100 ml) and brine (100 ml). The organic layer
was dried over Na2SO4, filtered, and concentrated. The
residue was purified by flash column chromatography
(hexane/ethyl acetate = 10:1) to provide 22 (4.86 g, 40%
for three steps) as a colorless solid. NMR dH (CDCl3):
0.62 (3H, s), 1.02 (3H, s), 2.24 (1H, ddd, J = 2.3, 9.6,
15.7), 2.50 (1H, ddd, J = 2.3, 4.5, 15.7), 3.45–3.58 (2H,
m), 3.88–3.94 (1H, m), 4.72 (1H, m), 5.35 (1H, m), 9.77
(1H, t, J = 2.3).
37.4, 37.7, 39.4, 40.0, 42.6, 47.0, 49.6, 53.6, 53.8, 55.9,
60.1, 121.5, 124.5, 124.9, 149.0, 166.7, 211.8. ½aꢁ
24
D
+13.2 (c 0.78, CHCl3).
2.4.4. Ethyl (22R,23R)-6,6-ethylenedioxy-2a,3a,22,23-
bis(isopropylidenedioxy)-21-nor-5a-cholan-24-oate (25).
To a mixture of 24 (2.30 g, 6.0 mmol), (DHQD)2PHAL
(0.94 g, 1.2 mmol), K3Fe(CN)6 (9.88 g, 30 mmol), and
K2CO3 (4.15 g, 30 mmol) were added 10% (w/v) OsO4/t-
BuOH (7.6 ml, OsO4: 0.30 mmol), t-BuOH (60 ml), H2O
(60 ml), and CH3SO2NH2 (1.71 g, 18 mmol), successive-
ly. After stirring for 2 days at room temperature, Na2SO3
(8.0 g, 64 mmol) was added to the reaction mixture, which
was then extracted with CHCl3 (1 · 200 ml, 3 · 100 ml).
The combined extract was washed with 2 M KOH
(150 ml) and brine (150 ml), dried over anhydrous
MgSO4, filtered, and concentrated to afford the crude
2,3,22,23-tetraol (3.29 g). This tetraol compound
(3.19 g) was dissolved in CHCl3 (30 ml), and 2,2-dimeth-
oxypropane (10 ml) and catalytic amount of p-TsOH
were added to the solution. After 3 h, the reaction mixture
was diluted with CHCl3 (100 ml) and washed with satu-
rated aqueous NaHCO3 (50 ml). The organic layer was
dried over anhydrous MgSO4, filtered, and concentrated.
The residue was purified by flash column chromatogra-
phy (hexane/ethyl acetate = 3:1) to afford the bisaceto-
nide, which was protected by 2,2-dimethyl-1,3-
dioxolane (7 ml) with catalytic amount of p-TsOH, in a
similar manner to that used for the preparation of 12 in
Scheme 1, to give the pure colorless solid 25 (0.83 g,
37% for three steps). Mp: 220 ꢁC. NMR dH (CDCl3):
0.59 (3H, s), 0.85 (3H, s), 1.30 (3H, t, J = 7.1), 1.33 (3H,
s), 1.43 (3H, s), 1.46 (3H, s), 1.48 (3H, s), 3.73–3.77 (1H,
m), 3.88–3.97 (3H, m), 4.07–4.14 (3H, m), 4.21–4.28
(3H, m). NMR dC (CDCl3): 12.5, 13.4, 14.2, 20.5, 22.0,
24.6, 25.8, 26.6, 27.3, 27.8, 28.6, 32.9, 34.3, 37.1, 38.1,
2.4.2. Ethyl (22E)-3b-hydroxy-21-norchol-5,22-diene-24-
oate (23). To a stirred suspension of NaH (60% oil sus-
pension; 0.97 g, 24 mmol) in THF was added dropwise
triethyl phosphonoacetate (4.8 ml, 24 mmol). After
10 min, a solution of 22 (4.86 g, 12.1 mmol) in THF
was added and stirred for 3 h. Saturated aqueous NH4Cl
(100 ml) was added to the solution and extracted with
ethyl acetate (5 · 50 ml). The combined extract was
washed with brine (100 ml), dried over MgSO4, filtered,
and concentrated. The residue was purified by flash col-
umn chromatography (hexane/ethyl acetate = 10:1) to
afford the 5,22-dien. This diene was dissolved in EtOH
(50 ml), and PPTS (0.30 g) was added to this solution.
After stirring for 1 h at 80 ꢁC, the reaction mixture
was concentrated to the oil, which was dissolved with
CHCl3 (200 ml) and washed with saturated aqueous
NaHCO3 (100 ml). The organic layer was dried over
MgSO4, filtered, and concentrated. The residue was
purified by flash column chromatography (hexane/Ethyl
acetate = 2:1) to provide 23 (3.93 g, 85% for two steps) as
a colorless solid. NMR dH (CDCl3): 0.63 (3H, s), 1.02
(3H, s), 1.28 (3H, t, J = 7.1), 3.52 (1H, m), 4.18 (2H, q,
J = 7.1), 5.35 (1H, m), 5.81 (1H, d, J = 15.6), 6.95 (1H,
dt, J = 15.5, 7.4). NMR dC (CDCl3): 12.4, 14.3, 19.4,
41.2, 42.1, 42.7, 45.6, 46.6, 53.4, 55.0, 61.2, 64.2, 65.5,
72.8, 73.0, 78.2, 79.5, 107.6, 109.6, 110.8, 171.0. ½aꢁ
24
D
+57.3 (c 1.04, CHCl3). Anal. Calcd for C33H52O8: C,
68.72; H, 9.09. Found: C, 68.68; H, 8.80.
2.4.5. (22R,23R)-2a,3a,22,23-Tetrahydroxy-21,26,27-tri-
snor-5a-ergostan-6-one (6). The conversion of compound
25 to compound 6 was done in a similar manner to that
used for the preparation of 4 from 13 in Scheme 1. Color-
less amorphous solid (31% for four steps). NMR dH
(CDCl3): 0.59 (3H, s, 18-H), 0.77 (3H, s, 19-H), 0.93
(3H, d, J = 6.8, 25-H or 28-H), 0.97 (3H, d, J = 6.8, 28-
H or 25-H), 1.35 (1H, dt, J = 3.5, 12.7, 11-H), 1.43 (1H,
dt, J = 3.7, 12.3, 9-H), 1.53–1.59 (2H, m, 1-H and 20-
H), 1.92 (1H, td, J = 3.4, 15.2, 16-H), 1.95–1.98 (1H, m,

S. Yamamoto et al. / Bioorg. Med. Chem. 14 (2006) 1761–1770
1767
4-H), 2.03 (1H, t, J = 12.8, 7-H), 2.26 (3H, br s, 2-OH, 22-
OH, and 23-OH), 2.32 (1H, dd, J = 4.5, 13.0), 7-H), 2.49
(1H, br s, 3-OH), 2. 70 (1H, dd, J = 3.0, 12.6, 5-OH), 3.12
(1H, m, 23-OH), 3.58 (1H, m, 22-H), 3.76 (1H, m, 2-H),
4.04 (1H, m, 3-H). NMR dC (CDCl3): 12.7, 13.6, 16.7,
19.8, 21.0, 24.4, 26.3, 27.8, 30.0, 34.3, 37.0, 37.7, 40.3,
42.5, 42.6, 46.2, 46.8, 50.8, 54.2, 55.9, 68.3, 68.4, 70.5,
80.0, 212.1. HRMS m/z (M+) Calcd for C25H42O5:
422.3032. Found: 422.3045.
reaction between steroidal 22-aldehyde 8 and triethyl
phosphonoacetate provided the geometrically pure (E)-
a,b-unsaturated ester 9. This ester compound 9 was oxi-
dized to 22R,23S-diol 10 (68% yield for two steps) by
Sharpless asymmetric dihydroxylation reaction using
(DHQD)2PHAL as a chiral catalyst.32 The 22,23-dihy-
droxy and 6-carbonyl groups were protected as aceto-
nide and ketal, respectively, giving compound 12 in
72% yield for two steps. Methylation of compound 12
with CH3Li gave compound 13 in 70% yield, which
was then transformed to 4 in three steps.
2.4.6. 2a,3a-Dihydroxy-17b-[(2R,3S)-2-hydroxy-3-meth-
ylpentanoyl]oxy-5a-androstan-6-one (7). Compound 7
was synthesized according to the previously reported
method.15 Mp: 235–237 ꢁC. NMR dH (CDCl3): 0.77
(3H, s), 0.81 (3H, s), 0.87 (3H, d, J = 6.9), 1.02 (3H, d,
J = 6.9), 2.32 (1H, dd, J = 13.0, 4.4), 2.71 (1H, dd,
J = 12.7, 3.0), 2.74 (1H, d, J = 6.1), 3.77 (1H, m), 4.03–
4.06 (2H, m), 4.71 (1H, dd, J = 9.0, 7.7). NMR dC
(CDCl3): 12.11, 13.58, 16.60, 18.69, 20.67, 23.28, 26.25,
27.36, 32.18, 36.30, 37.33, 40.15, 42.49, 43.08, 46.13,
Next we synthesized 5, the 20-epimer of 4, using carbonyl-
ene reaction reported by Watanabe et al.33 in order to
examine the effect of configuration of symmetric 20-
carbon. Even though 20-epiBL and 20-epiCS were syn-
thesized by Kametani et al.,34 their activity values have
not been reported and no discussion was made for the
effect of C21 methyl group. Steroidal (Z)-17(20)-olefin
14
and
(R)-2-benzyloxy-3-methylbutanal
were
50.71, 50.91, 53.59, 68.20, 68.29, 74.82, 83.64, 174.92,
211.36. ½aꢁ ꢀ2.0 (c 0.93, CHCl3). Anal. Calcd for
condensed in the presence of EtAlCl2 to provide 15.
The acetyl group of 15 was removed by alkaline hydro-
lysis to give 16 in 53% for two steps. Regioselective
hydrogenation of D5,16-steroidal compound with Pd/C
was reported by Hershberg et al.,35 but it failed for 16.
Instead, deprotection of 23-benzyloxy group without
hydrogenation of double bond successfully occurred to
give 3,22,23-triol compound, which was then protected
with acetonide to provide 17. Compound 17 was con-
verted to 3a,5-cycloalcohol via mesylation and alkaline
hydrolysis, and the following hydrogenation with Pd/C
effected the reduction of 16(17)-double bond to afford
18. Compound 18 was converted to 19 according to
the conventional methods in 19% yield from 16.
Dihydroxylation of 19 provided the mixture of 2a,3a-
and 2b,3b-diol, which were separated after derivatiza-
tion to acetonided by column chromatography to pro-
vide 20 in 47% yield for two steps. Deprotection of 20
under acidic condition afforded 5 in 59% yield. Hydroxy
group of the known steroid 21 was protected as THP
ether, then reduction of methoxycarbonyl moiety fol-
lowed by PDC-oxidation afforded 22-aldehyde 22 in
40% for three steps. The HWE reaction of 22 provided
the (E)-a,b-unsaturated ester, which was treated with
acid to provide 23 in 85% yield for two steps. Steroidal
part of 23 was modified by conventional methods to give
2,22-dien 24 in 62% yield for four steps. Asymmetric
dihydroxylation of 24 followed by protection of 22,23-
diol and 6-ketone provided compound 25, which was
then converted into 6 in the similar manner as that for
4 from 13.
23
D
C24H38O6: C, 68.22; H, 9.06. Found: C, 67.95; H, 8.87.
2.5. Computations
All computations such as constructions of structures,
structural optimization, and conformational analysis
were executed using SYBYL ver. 6.91 (Tripos Co., St.
Louis, MO, USA). Analog structures were constructed
by modifying the structure of (22S, 23S)-homobrassino-
lide whose X-ray crystallographic data are available in
Cambridge Database.30 The side-chain moiety of opti-
mized (22S,23S)-28-homobrassinolide was replaced with
each of compounds 4–7, and each structure was fully
optimized by the semi-empirical molecular orbital meth-
od, PM3 in MOPAC 5.0. The optimized structures of
the corresponding BL derivatives of 4–7 are shown in
Figure 3. Conformation analyses were executed by a
SYBYL module, Systematic Search. To execute the sys-
tematic search, each molecule was minimized by molec-
ular mechanics using the ÔPowell methodÕ with SYBYL
default parameter. The mobility of the side chain of syn-
thetic analogs was examined using the SYBYL system-
atic search, in which C17–C20 bond was rotated 360ꢁ
with 10ꢁ intervals, and each C20–C22, C22–C23, and
C23–C24 bond was done 360ꢁ with 30ꢁ intervals to yield
360/10 · (360/30)3 = 62,208 for each compound. From
these conformers, infeasible conformations suffering
from van der Waals repulsion were eliminated. System-
atic searches were also executed for BL, 20-epiBL, and
20-norBL, in which the C17–C20 bond was rotated
360ꢁ with 10ꢁ intervals, and each C20–C22, C22–C23,
C23–C24, and C24–C25 bond was done 360ꢁ with 30ꢁ
intervals to yield 360/10 · (360/30)4 = 746,496.
To compare the activity between the acyl side-chain
analogs and alkyl side-chain analogs, we also synthe-
sized the corresponding ester analog 7. Although we
previously prepared some analogs with acyl side chain,15
their steroid moiety and the length of side chains are dif-
ferent from those synthesized in this study.
3. Result and discussion
3.1. Chemistry
3.2. Bioassays
Compound 4 was synthesized according to Zhou et alÕs
The synthesized analogs were subjected to the rice
lamina inclination assay under the synergistic condition
method.31 First, Horner–Wadsworth–Emmons (HWE)

1768
S. Yamamoto et al. / Bioorg. Med. Chem. 14 (2006) 1761–1770
Figure 3. Initial conformations of 26,27-bisnorBL (A), 21-epi-25,26-bisnorBL (B), 21,25,26-trisnorBL (C), and compound 3 (D).
with indole-3-acetic acid (IAA). Biological activity was
evaluated quantitatively as the reciprocal logarithm of
the ED50 (ED50: 50% effective dose per plant in moles),
pED50, calculated in the same manner as reported by
Uesusuki et al.15 The pED50 values of the synthesized
compounds are listed in Table 1.
group in the bean second internode bioassay. These ana-
logs, however, also lack 22,23-diol that are critically
important for the hormonal activity, so the contribution
of C21 methyl to the activity was unclear. The low activ-
ity of ester analogs is probably due to the lack of C21
substituent like in the case of 6.
Compound 4 showed the highest activity among com-
pounds tested in this study, but it was about 1/50 of
CS (2). By removing the C21-methyl group, the activity
decreased by 100-fold (4 vs 6). This finding is consistent
with the consideration by Uesusuki et al.15 that the exis-
tence of the 21-methyl group is important to enhance the
activity based on the SAR for a series of alkoxy BL ana-
logs. Strnad and Kohout also synthesized 17-alkoxy
analogs of BL lacking C21 methyl group such as
17b-3-methylbutyloxy and 17b-2-methylbutyloxy, and
reported that their activity is 100 times less potent than
24-epiBL in the bean second internode bioassay.36 Be-
sides, a remarkable reduction of the activity has been
reported for these ether BL analogs lacking C21 methyl
The activity of 5 was 100 times lower than that of 4.
Interestingly 5, 6, and the corresponding ester analog 7
were equipotent. These facts indicate that C21 methyl
group with a proper stereochemistry is important to en-
hance the activity. It is assumed that C21 methyl group
influences on the conformational flexibility of the side
chain at C17, as reported for the conformation–function
relationship of vitamin D.37
3.3. Systematic search
We analyzed the relationship between the hormonal
activity of the analogs and the spatial area occupied
by C25. The possible conformations of each analog were
searched using ÔSystematic SearchÕ module with molecu-
lar mechanics method. Thirty four possible conforma-
tions were obtained for 4, 40 for 5, and 385 for 6. In
this search experiment, infeasible conformations suffer-
ing from van der Waals repulsion were omitted. The
positions of 25-carbon were plotted for all possible con-
formers, in which steroid moiety was overlapped. One
cross sign corresponds to one conformation of the side
chain. Figure 4 indicates that the side chains of 4 mainly
occupy a certain limited area, but that of 5 occupy two
areas. However, in the case of 6, the corresponding ter-
minal carbon of the side chain covers a wider area than
those for 4 and 5 as depicted with green crosses. The
wide area is also occupied for ester analog 7 (data not
Table 1. Brassinolide-like activity of newly synthesized compounds in
the rice lamina inclination assay
Compound
pED50 (nmol)
1 (Brassinolide)
2 (Castasterone)
3 (BL-ester)
13.6a
12.3a
10.1a
4
5
6
7
10.7 0.4b
8.5 0.2b
8.7 0.2b
8.5
a From Ref. 15.
b Means standard deviation (n = 2) for 4–6.

S. Yamamoto et al. / Bioorg. Med. Chem. 14 (2006) 1761–1770
1769
Figure 4. The mobile areas of the side chains are shown by (A) red cross for 4, (B) white cross for 5, and (C) green cross for 6. The mobile areas of the
side chain of corresponding BL analogs are also plotted by (D) red cross for BL, (E) white cross for 20-epiBL, and (F) green cross for 20-norBL.
shown). The results for the systematic searches for BL
and its analogs (20-epiBL and 21norBL) gave the similar
results as shown in Figure 4D–F, although the number
of conformers was smaller than those for 4–6 (24 for
BL, 13 for 20-epiBL, and 274 for 21-norBL). The intro-
duction of a CH3 group at the C20 position prevents any
free rotation with respect to the C17–C20 bond, result-
ing in the entire molecule having a rigid conformation.
Thus, we can assume that the activity increases with
the poor conformational flexibility of the side chain.
The replacement of side-chain moiety with a benzene
structure may decrease the flexibility of side-chain moi-
ety. For the insect steroid hormone, 20-hydroxyecdy-
sone, it is demonstrated that the side-chain moiety of
20-hydroxyecdysone corresponds to the benzoyl moiety
of non-steroidal dibenzoylhydrazine-type compounds.38
Keiji Tanaka of Sankyo Agro Co. for providing rice
weed. This investigation was supported, in part, by a
Grant-in Aid for Scientific Research from the Ministry
of Education, Culture, Sports, Science and Technology
of Japan (09660117), and by the 21st century COE pro-
gram for Innovative Food and Environmental Studies
Pioneered by Entomomimetic Sciences, from the Minis-
try of Education, Culture, Sports, Science and Technol-
ogy of Japan.
References and notes
1. Grove, M. D.; Spencer, G. F.; Rohwedder, W. K.;
Mandava, N.; Worley, J. F.; Warthen, J. D., Jr.; Steffens,
G. L.; Flippen-Anderson, J. L.; Cook, J. C., Jr. Nature
1979, 281, 216.
In conclusion, we synthesized three CS analogs 4, 5, and
6 as well as the corresponding ester analog 7. The hor-
monal activity of both 5 and 6 was 100 times lower than
that of 4 which has the same configuration at C20 as
natural brassinosteroids. The corresponding ester ana-
log 7 has similar hormonal activity as 5 and 6. Results
of a systematic search for the conformation-activity
relationship for these compounds as well as BL analogs
(BL, 20-norBL, and 20-epiBL) suggested that the hor-
monal activity decreases with the degree of freedom of
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