Synthesis of brassinosteroids of varying acyl side chains and evaluation of their brassinolide-like activity

Article abstract of DOI:10.1271/bbb.68.1097

Brassinosteroids containing various side chain moieties were synthesized and their activity was determined as the reciprocal logarithm of the ED ₅₀ (50% effective dose per plant in moles) in the rice lamina inclination assay using synergist indole-3-acetic acid (IAA). The introduction of a hydroxyl group in the α-position to the carbonyl group of the ester structure significantly enhanced the activity. 2α,3α-Dihydroxy- 17β-[(2R,3S)-2-hydroxy-3-methylpentanoyl]oxy-B-homo-7-oxa-5α- androstan-6-one showed the highest activity, for which the pED₅₀ was determined to be 10.5 under synergistic conditions with IAA. Under identical conditions, the pED₅₀ values of brassinolide and castasterone were determined to be 13.6 and 12.3 respectively. With respect to the α-carbon of the acyl moiety, the R-form was 10 times more potent than the corresponding S-form. Substituting the terminal structure (Et) of the side chain to that of the most potent compound, brassinolide (i-Pr), did not increase the activity.

Full text of DOI:10.1271/bbb.68.1097

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Synthesis of Brassinosteroids of Varying Acyl Side
Chains and Evaluation of Their Brassinolide-like
Activity
Shinya UESUSUKIa, Bunta WATANABEa, Shuji YAMAMOTOa, Junko OTSUKIa, Yoshiaki
NAKAGAWAa & Hisashi MIYAGAWAa
a Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University
Published online: 22 May 2014.
To cite this article: Shinya UESUSUKI, Bunta WATANABE, Shuji YAMAMOTO, Junko OTSUKI, Yoshiaki NAKAGAWA & Hisashi
MIYAGAWA (2004) Synthesis of Brassinosteroids of Varying Acyl Side Chains and Evaluation of Their Brassinolide-like
Activity, Bioscience, Biotechnology, and Biochemistry, 68:5, 1097-1105, DOI: 10.1271/bbb.68.1097
To link to this article: http://dx.doi.org/10.1271/bbb.68.1097
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Biosci. Biotechnol. Biochem., 68 (5), 1097–1105, 2004
Synthesis of Brassinosteroids of Varying Acyl Side Chains
and Evaluation of Their Brassinolide-like Activity
Shinya UESUSUKI, Bunta WATANABE, Shuji YAMAMOTO, Junko OTSUKI,
Yoshiaki NAKAGAWA,y and Hisashi MIYAGAWA
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Received December 8, 2003; Accepted February 17, 2004
Brassinosteroids containing various side chain moi-
eties were synthesized and their activity was determined
as the reciprocal logarithm of the ED50 (50% effective
dose per plant in moles) in the rice lamina inclination
assay using synergist indole-3-acetic acid (IAA). The
introduction of a hydroxyl group in the ꢀ-position to the
carbonyl group of the ester structure significantly
enhanced the activity. 2ꢀ,3ꢀ-Dihydroxy-17ꢁ-[(2R,3S)-2-
hydroxy-3-methylpentanoyl]oxy-B-homo-7-oxa-5ꢀ-an-
drostan-6-one showed the highest activity, for which the
pED50 was determined to be 10.5 under synergistic
conditions with IAA. Under identical conditions, the
pED50 values of brassinolide and castasterone were
determined to be 13.6 and 12.3 respectively. With
respect to the ꢀ-carbon of the acyl moiety, the R-form
was 10 times more potent than the corresponding S-
form. Substituting the terminal structure (Et) of the side
chain to that of the most potent compound, brassinolide
(i-Pr), did not increase the activity.
oxa, was originally determined by Grove et al.1) Later,
castasterone (2, CS in Fig. 1) containing a 6-keto 6-
member B-ring instead of a 7-member ring was also
identified in chestnut insect gall.2) In the last two
decades, more than 50 BL analogs have been identified
in plants and these brassinolide-like compounds are
collectively called brassinosteroids (BRs).3–5) Since they
have excellent activity in evoking cell elongation and
cell division, which are essential for plant growth and
various forms of stress-resistance, they are expected to
be used in the future as versatile plant growth regu-
lators.6–9)
To date a number of BRs including BL have been
synthesized, and the structure-activity relationship
(SAR) of BRs has been extensively analyzed.10–21) The
cumulative results of these studies indicate that the
structure requirements for high activity are: (1) a 2ꢀ,3ꢀ-
diol, (2) a trans A/B ring system, (3) a 6-keto or 7-oxa-
6-keto moiety in the B-ring, (4) hydroxyl groups at C-22
and C-23, and (5) a methyl or ethyl substituent at C-24.
The stereochemistry of the steroid ring system of BL
(2ꢀ,3ꢀ-dihydroxy, trans configuration of A/B-ring
fusion, configurations of 18 and 19-CH3, C-17 config-
uration) is easily constructed from steroid compounds
such as pregnenolone and stigmasterol using conven-
tional methods.4,22) But, the construction of the alkyl
chain moiety with defined stereochemistry is not easy,
because four asymmetric carbons exist consecutively in
the side chain. Kohout and his coworkers combined
2ꢀ,3ꢀ,17ꢁ-trihydroxy-7-oxa-B-homo-5ꢀ-androstan-6-
one containing the steroid moiety of BL with amino
acids and found weak activity for some compounds
using a bean hypocotyls elongation bioassay.23) These
compounds have an ester bond in the side chain moiety.
Synthesis of the ester analogs is attractive, because the
ester bond is easily constructed from alcohols and
carboxylic acids to derive various analogs.
Key words: brassinolide; castasterone; brassinosteroids;
plant hormone; rice lamina inclination assay
Brassinolide (1, BL in Fig. 1) is a known steroidal
plant hormone and its structure, which characteristically
has a 7-member B-ring moiety containing a 6-keto-7-
Fig. 1. Structures of Brassinolide (1) and Castasterone (2).
In this study, we synthesized a number of ester
y
To whom correspondence should be addressed. Fax: +81-75-753-6123; E-mail: naka@kais.kyoto-u.ac.jp
Abbreviations: BR, brassinosteroids; BL, brassinolide; SAR, structure-activity relationship; RLI, rice lamina inclination; DMAP, dimethylamino-
pyridine; NMO, N-methylmorpholine N-oxide; PPTS, pyridinium p-toluenesulfonate; MsCl, methanesulfonyl chloride; p-TsOH, p-toluenesulfonic
acid; TBDPS, tert-butyldiphenylsilyl; TBAF, tetra-n-butylammonium fluoride; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; BnBr, benzyl bromide;
DEAD, diethyl azodicarboxylate; PDC, pyridinium dichromate; DCC, dicyclohexylcarbodiimide; IAA, indole-3-acetic acid

1098
S. UESUSUKI et al.
Scheme 1.
analogs and measured their BL-like activity in a rice
lamina inclination (RLI) bioassay24) using intact rice
seedlings under the synergistic condition with indole-3-
acetic acid (IAA). BRs were chemically prepared from
the 17-hydroxy compound (20; key intermediate in
Scheme 1), which compound was derived from pregne-
nolone (15 in Scheme 1) and various carboxylic acids.
apparatus (Yanagimoto Seisakusho Co., Ltd., Kyoto,
Japan) and are uncorrected. The structures of the final
compounds and their melting points and spectral data
are shown in Table 1. HRMS data are shown for
compounds 12–14 instead of their elemental analysis
data. Spectral and elemental analysis data for inter-
mediates are shown in the text.
Materials and Methods
Construction of the key intermediate (20) (Scheme 1).
3ꢁ-Bromo-5ꢀ-pregnan-6,20-dione (17). Synthetic
procedures are basically the same as those reported
previously.25) Acetic acid (150 ml) and 48% HBr
(7.5 ml) were successively added to compound 16
(13.49 g, 42.9 mmol) and the mixture was dissolved
following ultrasonification. After adding water (750 ml)
to the mixture, the aqueous layer was extracted with
toluene (150 ml ꢀ 4). Saturated aqueous NaHCO3
(900 ml) was added to the combined organic layers
and the mixture was stirred for 5 min. The aqueous layer
was then extracted with toluene (300 ml ꢀ 2). The
combined organic layers were washed with saturated
aqueous NaHCO3 solution and brine and dried over
anhydrous MgSO4. The solvent was evaporated in vacuo
to afford a colorless solid compound 17 (16.71 g, 99%),
which was used without purification. NMR ꢂH (CDCl3):
0.63 (3H, s), 0.80 (3H, s), 1.23 (1H, qd, J ¼ 12:1,
6.2 Hz), 1.26–1.39 (4H, m), 1.48 (1H, td, J ¼ 12:5,
3.9 Hz), 1.61–1.74 (3H, m), 1.78–1.83 (2H, m), 1.90–
2.04 (3H, m), 2.09 (1H, dt, J ¼ 12:2, 3.2 Hz), 2.12 (3H,
s), 2.14–2.19 (2H, m), 2.21–2.27 (2H, m), 2.34 (1H, dd,
J ¼ 13:3, 4.5 Hz), 2.54 (1H, t, J ¼ 9:1 Hz), 3.93 (1H, tt,
J ¼ 12:3, 4.5 Hz). NMR ꢂC (CDCl3): 13.11, 13.37,
General. Reactions requiring anhydrous conditions
were performed using oven-dried glassware and con-
ducted under positive pressure using argon. Anhydrous
solvents were either commercially available or prepared
conventionally in the laboratory. Chemicals were pur-
chased from Aldrich Chemical Co., Inc. (Milwaukee,
Wisconsin, U.S.A.), Tokyo Kasei Kogyo Co., Ltd.
(Tokyo, Japan), Wako Pure Chemical Industries, Ltd.
(Osaka, Japan), and Nacalai Tesque Inc. (Kyoto, Japan),
1
unless otherwise noted. H- and 13C-NMR spectra were
recorded on a Bruker AC-300 or ARX-500 NMR
spectrometer in deuteriochloroform (CDCl3) with tetra-
methylsilane as the internal standard. Chemical shifts for
carboxylic acids were not evident, because the signals
are broad and small. IR spectra were recorded on a
Shimadzu IR-420 spectrometer (Shimadzu, Kyoto,
Japan). Optical rotations were measured on a JASCO
DIP-1000 or P-1000 polarimeter. High-resolution mass
spectra (HRMS) were measured on JMS600H (JEOL,
Tokyo, Japan). Elemental analyses were performed at
the Microanalytical Center at Kyoto University. Melting
points were determined on a Yanako melting point

Structure-activity Relationship of Brassinosteroids
1099
Table 1. Structures of Newly Synthesized Compounds and Their Physical Properties
R
HO
O
HO
H
O
No.
R
mp/Optical rotation
192
Chemical shifts (ppm) for 1H-NMRaÞ
Elemental analysis or HRMS
0.82 (3H, s), 0.93 (3H, s), 1.14 (3H, t,
J ¼ 7:6 Hz), 2.33 (2H, q, J ¼ 7:6 Hz), 3.13
(1H, dd, J ¼ 12:1, 4.6 Hz), 3.71 (1H, ddd,
J ¼ 12:0, 4.7, 2.8 Hz), 4.02–4.13 (3H, m),
4.62 (1H, dd, J ¼ 9:1, 8.0 Hz).
O
Found: C, 66.69%; H, 8.99%
Calcd: C, 66.98%; H, 8.69%
3
29
O
O
½ꢀꢁD þ 25:3 (c 0.54, CHCl3)
0.82 (3H, s), 0.89 (3H, t, J ¼ 7:4 Hz), 0.93
(3H, s), 0.93 (3H, d, J ¼ 6:4 Hz), 3.12 (1H,
dd, J ¼ 12:0, 4.5 Hz), 3.69–3.73 (1H, m),
4.02–4.09 (3H, m), 4.62 (1H, t, J ¼ 8:4).
175
½ꢀꢁD þ 36:8 (c 0.56, CHCl3)
Found: C, 68.53%; H, 8.96%
Calcd: C, 68.78%; H, 9.23%
4
5
6
29
O
0.82 (3H, s), 0.93 (3H, s), 3.12 (1H, dd,
J ¼ 12:1, 4.6 Hz), 4.02–4.08 (3H, m), 4.61
(1H, t, J ¼ 7:9 Hz).
O
200
½ꢀꢁD þ 39:0 (c 0.56, CHCl3)
Found: C, 69.35%; H, 9.17%
Calcd: C, 69.61%; H, 8.99%
29
O
0.94 (3H, s), 0.97 (3H, s), 3.14 (1H, dd,
J ¼ 12:1, 4.6 Hz), 4.03–4.12 (3H, m), 4.87
(1H, t, J ¼ 8:9 Hz), 7.42–7.47 (2H, m),
7.54–7.57 (1H, m), 8.01–8.04 (2H, m).
O
O
256
½ꢀꢁD þ 81:1 (c 0.38, CHCl3)
Found: C, 70.46%; H, 7.66%
Calcd: C, 70.56%; H, 7.74%
29
O
O
0.84 (3H, s), 0.88 (3H, d, J ¼ 6:9 Hz), 0.93
(3H, s), 1.04 (3H, d, J ¼ 6:9 Hz), 2.73 (1H,
d, J ¼ 6:0 Hz), 3.12 (1H, dd, J ¼ 12:0,
4.6 Hz), 3.69–3.74 (1H, m), 4.02–4.09 (4H,
m), 4.74 (1H, dd, J ¼ 9:0, 7.9 Hz).
176
½ꢀꢁD þ 34:8 (c 0.94, CHCl3)
Found: C, 65.45%; H, 8.58%
Calcd: C, 65.73%; H, 8.73%
7
8
27
OH
0.84 (3H, s), 0.87 (3H, d, J ¼ 6:9 Hz), 0.93
(3H, s), 1.02 (3H, d, J ¼ 6:9 Hz), 2.74 (1H,
d, J ¼ 6:1 Hz), 3.12 (1H, dd, J ¼ 12:0,
4.6 Hz), 3.68–3.76 (1H, m), 4.03–4.10 (4H,
m), 4.67 (1H, dd, J ¼ 9:0, 7.7 Hz).
O
198
½ꢀꢁD þ 47:8 (c 1.04, CHCl3)
Found: C, 65.73%; H, 8.43%
Calcd: C, 65.73%; H, 8.73%
31
O
OH
0.84 (3H, s), 0.91 (3H, t, J ¼ 7:5 Hz), 0.93
(3H, s), 1.00 (3H, d, J ¼ 6:9 Hz), 3.13 (1H,
dd, J ¼ 12:1, 4.6 Hz), 3.69–3.75 (1H, m),
4.02–4.09 (4H, m), 4.72 (1H, dd, J ¼ 8:9,
7.8 Hz).
O
181
½ꢀꢁD þ 39:0 (c 0.95, CHCl3)
Found: C, 66.17%; H, 8.71%
Calcd: C, 66.35%; H, 8.91%
9
31
O
O
OH
0.82 (3H, d, J ¼ 6:8 Hz), 0.84 (3H, s), 0.94
(3H, s), 0.96 (3H, t, J ¼ 7:4 Hz), 3.13 (1H,
dd, J ¼ 12:1, 4.6 Hz), 3.70–3.76 (1H, m),
4.02–4.09 (1H, m), 4.18 (1H, d, 2.7 Hz),
4.72 (1H, dd, J ¼ 8:9, 7.8 Hz).
O
202
½ꢀꢁD þ 45:1 (c 1.05, CHCl3)
Found: C, 66.05%; H, 8.97%
Calcd: C, 66.35%; H, 8.91%
10
31
OH
OH
0.80 (3H, d, J ¼ 6:8 Hz), 0.83 (3H, s), 0.93
(3H, s), 0.95 (3H, d, J ¼ 7:1 Hz), 1.00 (3H,
d, J ¼ 6:6 Hz), 2.72 (1H, d, J ¼ 5:7 Hz),
3.13 (1H, dd, J ¼ 12:1, 4.5 Hz), 3.70–3.73
(1H, m), 4.02–4.14 (3H, m), 4.32 (1H, dd,
J ¼ 5:7, 2.9 Hz), 4.67 (1H, dd, J ¼ 8:7,
7.8 Hz).
O
181
½ꢀꢁD þ 43:3 (c 1.08, CHCl3)
Found: C, 66.74%; H, 9.03%
Calcd: C, 66.93%; H, 9.07%
11
26
O
Continued on next page

1100
S. UESUSUKI et al.
0.90 (3H, s), 0.91 (3H, s), 1.22 (3H, s), 1.25
(3H, s), 3.21 (1H, dd, J ¼ 12:2, 4.4 Hz),
3.60 (1H, ddd, J ¼ 12:1, 4.5, 2.7 Hz), 3.93
(1H, m), 3.94 (1H, s), 4.05 (1H, dd,
J ¼ 11:6 Hz), 4.21 (1H, dd, J ¼ 12:5,
9.4 Hz), 4.70 (1H, dd, J ¼ 9:0, 7.9 Hz).bÞ
O
OH
127
½ꢀꢁD þ 38:0 (c 0.40, EtOH)
Calculated: 455.2645 (C24H39O6)
Found: 455.2635
12
27
O
OH
0.76 (3H, s), 0.92 (3H, s), 2.80 (1H, d,
J ¼ 6:3 Hz), 2.98 (1H, dd, J ¼ 14:0,
6.3 Hz), 3.10 (1H, dd, J ¼ 14:0, 4.9 Hz),
3.11 (1H, dd, J ¼ 12:0, 4.4 Hz), 3.70–3.73
(1H, m), 4.01–4.08 (3H, m), 4.46 (1H, dt,
J ¼ 5:1, 6.2 Hz), 4.62 (1H, dd, J ¼ 8:8,
7.6 Hz), 7.20–7.29 (5H, m).
O
204
½ꢀꢁD þ 49:8 (c 0.50, CHCl3)
Calculated: 487.2695 (C28H39O7)
Found: 487.2708
13
14
24
O
OH
0.84 (3H, s), 0.89–0.94 (9H, m), 3.13 (1H,
dd, J ¼ 12:1, 4.5 Hz), 3.37 (3H, s), 3.64
(1H, d, J ¼ 4:4 Hz), 3.69–3.74 (1H, m),
4.02–4.14 (3H, m), 4.69 (1H, dd, J ¼ 8:7,
8.0 Hz).
O
77
½ꢀꢁD þ 61:8 (c 1.09, CHCl3)
Calculated: 467.3008 (C26H43O7)
Found: 467.3010
25
O
OCH3
a) Measured in CDCl3 except for compound 12.
b) Measured in CD3OD.
21.28, 22.79, 24.12, 31.41, 32.31, 33.35, 37.59, 38.44,
39.16, 40.57, 44.33, 46.35, 50.30, 53.72, 56.74, 58.97,
63.35, 208.91, 209.00. IR ꢃmax (nujor) cmꢂ1: 1690,
1350, 1270, 1230, 1180, 1150, 710.
(1H, dd, J ¼ 10:2, 4.4 Hz), 3.67 (1H, td, J ¼ 8:5,
5.6 Hz), 4.03–4.09 (2H, m), 4.35–4.40 (2H, m). NMR
ꢂC (CDCl3): 11.20, 19.71, 22.57, 23.62, 23.82, 26.56,
27.70, 30.41, 33.53, 36.07, 36.51, 39.36, 40.27, 43.47,
46.68, 54.92, 70.60, 72.47, 73.08, 81.34, 107.60, 176.44.
IR ꢃmax (nujor) cmꢂ1: 3500, 1710. Found: C, 69.53; H,
9.18. Calcd. for C22H34O5: C, 69.81; H, 9.05.
Synthesis of hydroxycarboxylic acids (Scheme 2). ꢀ-
Hydroxycarboxylic acids and their O-protected analogs
were synthesized according to the conventional methods
summarized in Scheme 2. Chiral isoleucine and valine
were used as starting materials to prepare hydroxycar-
boxylic acids for compounds 7–10, 14.
5ꢀ-Pregn-2-en-6,20-dione (18). Compound 17 (17.42
g, 44.1 mmol), LiBr H O (23.22 g, 221 mmol), and
ꢃ
2
Li2CO3 (24.61 g, 333 mmol) were suspended in anhy-
drous DMF and stirred for 2 h at 110 ꢄC under an argon
atmosphere. After cooling the mixture to room temper-
ature, EtOAc (1 l) was added. The precipitate was
removed by filtration and the filtrate was washed with
water and brine. The organic layer was dried over
anhydrous MgSO4 and the solvent was evaporated in
vacuo to yield a pale yellow solid compound 18
(14.17 g), which was used without purification. NMR
ꢂH (CDCl3): 0.64 (3H, s), 0.72 (3H, s), 1.23–1.26 (1H,
m), 1.33–1.38 (2H, m), 1.42–1.53 (2H, m), 1.63–1.80
(5H, m), 1.99–2.04 (4H, m), 2.09–2.13 (1H, m), 2.13
(3H, s), 2.15–2.20 (1H, m), 2.22–2.29 (1H, m), 2.36 (1H,
dd, J ¼ 9:4, 4.8 Hz), 2.38 (1H, dd, J ¼ 13:2, 4.1 Hz),
2.55 (1H, t, J ¼ 8:9 Hz), 5.56–5.58 (1H, m), 5.69 (1H,
m). NMR ꢂC (CDCl3): 13.32, 13.53, 21.12, 21.74, 22.80,
24.15, 31.44, 37.52, 38.58, 39.39, 39.94, 44.26, 46.80,
53.32, 53.87, 56.85, 63.46, 124.38, 125.03, 209.02,
211.27. IR ꢃmax (nujor) cmꢂ1: 1690, 1650, 1350, 1230,
1190, 1160, 680.
(2S,3S)-2-(tert-Butyldiphenylsilyl)oxy-3-methylpenta-
30
noic acid (25). Colorless oil: ½ꢀꢁD ꢂ 1:2ꢄ (c 1.20,
CHCl3). NMR ꢂH (CDCl3): 0.68 (3H, t, J ¼ 7:4 Hz),
0.90 (3H, d, J ¼ 6:9 Hz), 1.12 (9H, s), 1.13–1.30 (1H,
m), 1.31–1.45 (1H, m), 1.53–1.66 (1H, m), 4.21 (1H, d,
J ¼ 3:8 Hz), 7.34–7.48 (6H, m), 7.60–7.69 (4H, m).
NMR ꢂC (CDCl3): 11.61, 14.44, 19.36, 24.84, 26.96,
40.20, 76.26, 127.82, 127.85, 130.20, 132.16, 132.58,
135.79, 135.82, 174.02. Found: C, 71.23; H, 8.26. Calcd.
for C22H30O3Si: C, 71.31; H, 8.16.
(2R,3S)-2-(tert-Butyldiphenylsilyl)oxy-3-methylpenta-
28
noic acid (28). Colorless oil: ½ꢀꢁD þ 7:0ꢄ (c 1.22,
CHCl3). NMR ꢂH (CDCl3): 0.75 (3H, t, J ¼ 7:4 Hz),
0.90 (3H, d, J ¼ 6:9 Hz), 1.01–1.16 (1H, m), 1.16 (9H,
s), 1.39–1.53 (1H, m), 1.56–1.69 (1H, m), 4.21 (1H, d,
J ¼ 3:2 Hz), 7.34–7.43 (6H, m), 7.61–7.66 (4H, m).
NMR ꢂC (CDCl3): 11.85, 14.22, 19.50, 24.88, 27.01,
40.01, 76.38, 127.79, 130.14, 132.71, 135.34, 135.87,
135.89, 174.69. Found: C, 71.28; H, 8.21. Calcd for
C22H30O3Si: C, 71.31; H, 8.16.
17ꢁ-Hydroxy-2ꢀ,3ꢀ-isopropylidenedioxy-B-homo-7-
oxa-5ꢀ-androstan-6-one (20). Compound 19, which was
derived from compound 18 by osmium oxidation, was
converted to the key intermediate 20 using conventional
methods,25) and is summarized in Scheme 1. Colorless
31
crystal: m.p. 233 ꢄC. ½ꢀꢁD þ 23:3ꢄ (c 0.98, CHCl3).
NMR ꢂH (CDCl3): 0.79 (3H, s), 0.90 (3H, s), 1.08–1.19
(3H, m), 1.32 (3H, s), 1.35–1.44 (3H, m), 1.46–1.54 (1H,
m), 1.52 (3H, s), 1.59–1.65 (1H, m), 1.71–1.76 (1H, m),
1.78–1.86 (4H, m), 1.91 (1H, dq, J ¼ 13:6, 3.3 Hz),
2.05–2.21 (1H, m), 2.34 (1H, J ¼ dd, 16.0, 3.6 Hz), 3.29
(2R,3S)-2-Methoxy-3-methylpentanoic acid (30). Col-
25
orless oil: ½ꢀꢁD þ 43:5ꢄ (c 1.15, CHCl3). NMR ꢂH
(CDCl3): 0.94 (3H, d, J ¼ 6:9 Hz), 0.94 (3H, t,
J ¼ 7:4 Hz), 1.28–1.37 (1H, m), 1.49–1.56 (1H, m),

Structure-activity Relationship of Brassinosteroids
1101
Scheme 2.
1.87 (1H, m), 3.44 (3H, s), 3.73 (1H, d, J ¼ 3:7 Hz).
NMR ꢂC (CDCl3): 11.73, 14.05, 25.86, 38.05, 59.06,
83.42, 177.99. Found: C, 57.34; H, 9.58. Calcd for
C7H14O3: C, 57.51; H, 9.65.
CHCl3). NMR ꢂH (CDCl3): 0.88 (3H, d, J ¼ 6:9 Hz),
0.90 (3H, d, J ¼ 6:9 Hz), 1.13 (9H, s), 1.86–1.96 (1H,
m), 4.13 (1H, d, J ¼ 3:6 Hz), 7.26–7.45 (6H, m), 7.61–
7.67 (4H, m). Found: C, 70.47; H, 7.74. Calcd. for
C21H28O3Si: C, 70.74; H, 7.92.
(2R)-2,3-isopropylidenedioxy-3-methylbutanoic acid
(32). Compound 31, which was prepared according to
the conventional method,26) was used as the starting
(R)-2-(tert-Butyldiphenylsilyl)oxy-3-methylbutanoic
acid. To derive the hydroxycarboxylic acid moiety of
compound 8, D-valine was used instead of L-isoleucine
20
material. Colorless solid: ½ꢀꢁD þ 20:7ꢄ (c 0.96, EtOH).
23
NMR ꢂH (CDCl3): 1.24 (3H, s), 1.40 (3H, s), 1.51 (3H,
s), 1.54 (3H, s), 4.39 (1H, s).
in Scheme 2. Colorless oil: ½ꢀꢁD þ 13:8ꢄ (c 0.93,
CHCl3). NMR ꢂH (CDCl3): 0.87 (3H, d, J ¼ 6:9 Hz),
0.89 (3H, d, J ¼ 6:9 Hz), 1.11 (9H, s), 1.31–1.93 (1H,
m), 4.15 (1H, d, J ¼ 3:6 Hz), 7.36–7.59 (6H, m), 7.64–
7.67 (4H, m). Found: C, 71.02; H, 7.76. Calcd for
C21H28O3Si: C, 70.74; H, 7.92.
(2R,3S)-2-Benzyloxy-3,4-dimethylpentanoic acid (34).
Compound 33, which was derived from crotyl alcohol in
our laboratory, was used as the starting material. Brown
27
oil: ½ꢀꢁD þ 40:3ꢄ (c 1.30, CHCl3). NMR ꢂH (CDCl3):
0.87 (1H, d, J ¼ 6:1 Hz), 0.92 (1H, d, J ¼ 6:2 Hz), 0.97
(1H, d, J ¼ 6:2 Hz), 1.72 (1H, m), 4.09 (1H, d,
J ¼ 2:5 Hz), 4.43 (1H, d, J ¼ 11:3 Hz), 4.75 (1H, d,
J ¼ 11:3 Hz), 7.32–7.37 (5H, m). NMR ꢂC (CDCl3):
11.57, 19.76, 21.05, 29.81, 42.84, 73.02, 79.94, 128.00,
128.06, 128.43, 137.19, 177.91.
Synthesis of BL analogs (Scheme 3). Condensation of
a key intermediate with the corresponding carboxylic
acid was performed using dicyclohexylcarbodiimide
(DCC) and dimethylaminopyridine (DMAP) in CH2Cl2,
and protective groups were removed as summarized in
Scheme 3.
(R)-2-(tert-Butyldiphenylsilyl)oxy-3-phenylpropanoic
acid. The corresponding hydroxycarboxylic acid for
compound 13 was derived from D-phenylalanine. Color-
Bioassay. BL activity was measured in a dwarf rice
lamina inclination assay developed by Fujioka et al.24)
Briefly, the seeds of dwarf rice Oryza sativa cv. Tan-
ginbozu were soaked in aqueous 0.2% Benlate-T solu-
tion for 2 d at 30 ꢄC under light. Germinated seeds were
planted on 20 ml of 1% agar medium (ca. 1 cm depth) in
a beaker (50 ml volume), then incubated 3 d under the
conditions mentioned above to obtain the seedlings (ca.
3 cm). A 0.5 ꢄl ethanol solution of indole-3-acetic acid
(IAA, 50 mM) was applied to the top portion of the
20
less oil: ½ꢀꢁD ꢂ 5:6ꢄ (c 1.14, CHCl3). NMR ꢂH
(CDCl3): 1.06 (9H, s), 2.87 (1H, dd, J ¼ 13:7, 5.2 Hz),
2.95 (1H, dd, J ¼ 13:7, 6.1 Hz), 4.47 (1H, t,
J ¼ 5:5 Hz), 7.11–7.58 (15H, m). Found: C, 74.08; H,
7.08. Calcd. for C25H28O3Si: C, 74.22; H, 6.98.
(S)-2-(tert-Butyldiphenylsilyl)oxy-3-methylbutanoic
acid. To derive the hydroxycarboxylic acid moiety of
compound 7, L-valine was used instead of L-isoleucine
24
in Scheme 2. Colorless oil: ½ꢀꢁD ꢂ 12:3ꢄ (c 1.51,

1102
S. UESUSUKI et al.
Scheme 3.
lamina by micro-syringe, then various doses of test
compounds were applied in ethanol solution (0.5 ꢄl)
with a microsyringe to the same part for the IAA
treatment. After maintaining the plants for 2 d under
identical growth conditions, the external angle between
the lamina and its leaf sheath was measured using a
circular protractor. Seven seedlings were planted in each
beaker, and three sets were used for each dose, yielding
21 observations. Both a negative (solvent) and a positive
control (1 nmol of BL) were used in each experiment.
hydrolysis and protection of 2ꢀ,3ꢀ-diol to afford 7-
oxalactone 20 in 43% yield from 19. The total yield of
20 from starting material 15 was 17% in 10 steps.
Hydroxycarboxylic acid derivatives were derived
from ꢀ-amino acid according to the method reported
by Irie et al.,30) as summarized in Scheme 2. L-
Isoleucine 21 was treated with NaNO2 in AcOH
followed by hydrolysis with K2CO3 to give 22. Selective
benzylation of 22 afforded the benzyl ester 23. After
protecting the hydroxyl group of compound 23 by tert-
butyldiphenylsilyl (TBDPS), the benzyl group was
removed to afford compound 25. (2R)-2-Hydroxycar-
boxylic acid derivative 28 was derived from compound
23 using the Mitsunobu reaction.31) 2-Methoxycarbox-
ylic acid 30 was converted from 26 using conventional
methods. The synthesis of 2-benzyloxycarboxylic acid
34 was performed by treatment of alcohol 33 with
pyridinium dichromate (PDC) in DMF (Scheme 2).32)
BL analogs having various ester substructures at the
C-17 position of the steroid skeleton were synthesized
by condensing the key intermediate 20 with the
corresponding carboxylic acids (Scheme 3) using DCC
and DMAP. Deprotection of the hydroxyl groups was
performed using hydrogen and palladium-carbon. Yields
in the condensation and deprotection steps were between
45–88%. Although compounds 4 and 6 were already
synthesized and reported to be active in the bean
hypocotyls elongation assay, the evaluated activity was
qualitative.23)
Results and Discussion
Synthetic study
Pregnenolone 15 was converted to its methanesulfo-
nate, then solvolyzed with KHCO3 in aqueous acetone.
Subsequent Jones oxidation yielded 3ꢀ,5-cyclo ketone
16 in 77% yield from 15. Treatment of 16 with HBr in
AcOH gave 17 quantitatively. This ring-opening reac-
tion proceeded instantaneously, and the prolonged
reaction time caused epimerization with respect to C-
17. Although the treatment of 16 with p-toluenesulfonic
acid (p-TsOH) in sulfolane27) directly afforded 18, this
was unfavorable in that the products contained 30% of
the undesired epimer of 18 with respect to C-17. The
low yield of 18 (28% from 15) previously reported25) is
probably due to the epimerization at C-17. This
epimerization could not be avoided, even though multi-
ple reaction conditions were attempted (p-TsOH and
NaBr in DMF).28)
Dehydrobromination of 17 was performed with LiBr/
Li2CO3 in DMF29) to give compound 18 in high yield as
well as its regioisomer. Without Li2CO3, C-17 epime-
rization occurred. Crude 18 was then submitted to
further dihydroxylation using OsO4/N-methylmorpho-
line N-oxide (NMO) without purification. The re-
gioisomer was inert under these conditions and was
easily separated from 19 by column chromatography.
After recrystallization, the 2ꢀ,3ꢀ-diol analog 19 was
obtained in 51% yield from 16. Compound 19 was
subjected to Baeyer-Villiger oxidation followed by basic
Bioassay
The synthesized compounds were subjected to the rice
lamina inclination bioassay.24) The dose-response curves
for BL 1 and compounds 9 and 10 are shown in Fig. 2.
In each curve, the inclination angles caused by BL
treatment and control were set as 100% and 0%
respectively. From these dose-response curves, a 50%
effective dose (ED50; mol) was evaluated by probit
transformation33) and the reciprocal logarithmic value of
ED50, pED50, was used as an index of BL-like activity.
The pED50 values of the newly synthesized compounds

Structure-activity Relationship of Brassinosteroids
1103
group (14) caused a 25-fold reduction in activity.
Previously Luo and his coworkers measured the BL-
activity of BL and its O-methylated analogs in the
presence and absence of IAA. They found that under
assay conditions similar to those in our studies, the
conversion of 22-OH of BL to 22-OCH3 resulted in a
10-fold decrease in activity. Methylation of the 23-OH,
which is presumed to be the ꢀ-OH group of our ester
compounds (7–13), was detrimental to activity.35) This
observation is not consistent with the fact that the
activity was maintained by the methylation of the ꢀ-OH
group of the ester side chain. These contradictory results
may be due to different physicochemical properties such
as hydrophobicity and steric effect differences between
compounds containing an ester versus an alkyl moiety.
In fact, inactive 23-OMe BL becomes active upon
methylation of the 22-OH group corresponding to a
similar increase in the molecular hydrophobicity.35)
Considering these results, we concluded that the oxygen
of the 22-OH group works as a hydrogen bond acceptor
similar to carbonyl oxygen.
Fig. 2. Dose-response Relationships for BL (1, ), Compound 9 (
and 10 ( ).
)
Table 2. Brassinolide-like Activity of Brassinosteroids in the Rice
Lamina Inclination Assay
Introduction of the hydroxyl group at the terminal of
the ester side chain decreased activity by 20 times (8 vs
12). Pharis and his coworkers synthesized the 25- and
26-hydroxy derivatives of BL and found that these
hydroxylated compounds showed lower activity than
BL.20) Generally, if compounds are hydroxylated they
are easily removed from the target tissues, even though
the potent BL has four OH groups. The hydroxylation at
C2 of typhasterol enhanced activity by 10 times (2 vs
typhasterol).6) Examination of the activity of more
hydroxylated compounds will be interesting from the
viewpoint of BL metabolism in the regulation of
hormonal activity.
Compound
pED50 (mol)aÞ
Compound
pED50 (mol)
1bÞ
2dÞ
3
13:6 ꢅ 0:4 (n ¼ 4)cÞ
12:3 ꢅ 0:1 (n ¼ 3)cÞ
<8.0 (13%)
<8.0 (41%)
<8.0 (48%)
<8.0 (44%)
8.6
8
9
9.6
9.3
10
11
12
13
14
10.5
10.1
4
5
8.3
<8.7 (16%)
9.1
6
7
a) Percent values in parentheses are the inclination % at the corresponding
dose. 100% for BL treatment and 0% for control.
b) Brassinolide.
c) Mean ꢅ standard deviation for repetition (n).
d) Castasterone.
When the side chain of compounds 7 and 8 was
elongated, activity increased 10 times (7 vs 9, and 8 vs
10). But further addition of a methyl group at the ꢅ
position of compound 10, which makes the terminal
alkyl structure similar to that of BL, was not favorable to
activity (10 vs 11). Modification of the terminal i-Pr
moiety (8) to benzyl (13), caused a subsequent loss of
activity. As explained above, the hydrophobic and steric
effects of the side chain moiety must correlate with the
activity. In order to delineate the essential physicochem-
ical property, a three-dimensional structure-activity
relationship study is in progress for the expanded set
of compounds.
are listed in Table 2. A activity in terms of pED50 was
not obtained for compounds 3–6, but three compounds
(4–6) gave a significant response (>40%) at the highest
doses.
Structure-activity relationship study
Since most of the BRs contain both 22- and 23-
hydroxyl groups in the side chain moiety, we assumed
that the carbonyl oxygen of these ester compounds plays
the role of the 22-hydroxyl oxygen. Based on this
hypothesis we introduced a hydroxyl group at the alpha
position relative to the carbonyl of the ester moiety to
mimic the 23-hydroxyl group of BL. The activity was
enhanced dramatically by introduction of the hydroxyl
group next to the carbonyl group of compound 4 (vs 9 or
10). As shown in Table 2, the R-form was 10 times more
potent than the corresponding S-isomer (7 vs 8, and 9 vs
10), which is consistent with the fact that the R
configuration at C-23 is favored for BR activity.34)
Thus, we concluded that the ꢀ-OH group of ester
compounds corresponds to the 23-OH group of BL.
In a further study, we found that conversion of the ꢀ-
hydroxyl group of the side chain of 10 to a methoxy
Acknowledgments
We are thankful to Dr. Craig Wheelock of the
University of California at Davis for reviewing this
manuscript. We are also indebted to Dr. Kazuhiro Irie of
the Graduate School of Agriculture of Kyoto University
for measurement of the high resolution mass spectra,
and to Dr. Keiji Tanaka of Sankyo Agro Co. for
providing rice seeds. This investigation was supported,
in part, by a Grant-in-Aid for Scientific Research from

1104
S. UESUSUKI et al.
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2441 (1983).
17) Back, T. G., Janzen, L., Pharis, R. P., and Yan, Z.,
Synthesis and bioactivity of C-2 and C-3 methyl ether
derivatives of brassinolide. Phytochem., 59, 627–634
(2002).
the Ministry of Education, Culture, Sports, Science and
Technology of Japan (09660117), and by the Fujisawa
Foundation.
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