A specific and potent inhibitor of brassinosteroid biosynthesis possessing a dioxolane ring.

Article abstract of DOI:10.1021/jf011716w

Screening for brassinosteroid biosynthesis inhibitors was performed to find azole derivatives that induced dwarfism, to resemble brassinosteroid-deficient mutants in Arabidopsis, and which could be rescued by brassinosteroid. Through this screening experiment, propiconazole fungicide was selected as a likely inhibitor of brassinosteroid biosynthesis and, thus, propiconazole derivatives with optimized activity and selectivity were synthesized. The biological activity of these compounds was evaluated by examining cress stem elongation. Among the compounds tested, 2RS,4RS-1-[2-(4-trifluoromethylphenyl)-4-n-propyl-1,3-dioxolan-2-ylmethyl]-1H-1,2,4-triazole (12) showed the most potent capability to retard cress stem elongation in the light. The compound-induced hypocotyl dwarfism was restored by the coapplication of 10 nM brassinolide but not by 1 microM gibberellin. These results suggest that 12 should affect brassinosteroid biosynthesis. The potency and specificity of 12 were greater than those of brassinazole, a previously reported brassinosteroid biosynthesis inhibitor.

Full text of DOI:10.1021/jf011716w

3486 J. Agric. Food Chem. 2002, 50, 3486−3490
A Specific and Potent Inhibitor of Brassinosteroid Biosynthesis
Possessing a Dioxolane Ring
KATSUHIKO SEKIMATA,†,‡ SUN-YOUNG HAN,†,‡ KOICHI YONEYAMA,§
YASUTOMO TAKEUCHI,§ SHIGEO YOSHIDA,‡ AND TADAO ASAMI*,‡
Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan;
RIKEN, Hirosawa 2-1, Wako, Saitama 351-0198, Japan; and Center for Research on Wild Plants,
Utsunomiya University, Minemachi, Utsunomiya, Tochigi 350, Japan
Screening for brassinosteroid biosynthesis inhibitors was performed to find azole derivatives that
induced dwarfism, to resemble brassinosteroid-deficient mutants in Arabidopsis, and which could be
rescued by brassinosteroid. Through this screening experiment, propiconazole fungicide was selected
as a likely inhibitor of brassinosteroid biosynthesis and, thus, propiconazole derivatives with optimized
activity and selectivity were synthesized. The biological activity of these compounds was evaluated
by examining cress stem elongation. Among the compounds tested, 2RS,4RS-1-[2-(4-trifluorometh-
ylphenyl)-4-n-propyl-1,3-dioxolan-2-ylmethyl]-1H-1,2,4-triazole (12) showed the most potent capability
to retard cress stem elongation in the light. The compound-induced hypocotyl dwarfism was restored
by the coapplication of 10 nM brassinolide but not by 1 µM gibberellin. These results suggest that 12
should affect brassinosteroid biosynthesis. The potency and specificity of 12 were greater than those
of brassinazole, a previously reported brassinosteroid biosynthesis inhibitor.
KEYWORDS: Brassinosteroid; inhibitor; brassinosteroid biosynthesis; gibberellin biosynthesis; bras-
sinazole
INTRODUCTION
inhibitor, brassinazole, and showed that this compound was a
useful tool for investigating the functions of brassinosteroids
(14-16). Thus, the development of more specific and potent
brassinosteroid biosynthesis inhibitors is worthwhile.
The application of biologically active brassinosteroid homo-
logues causes remarkable growth responses in plants, including
stem elongation, pollen tube growth, leaf bending, leaf unrolling,
root inhibition, proton pump activation (1), initiation of ethylene
synthesis (2), tracheary element differentiation (3, 4), and cell
elongation (5). The functions of endogenous brassinosteroids
have been revealed by identifying several brassinosteroid-
deficient mutants of Arabidopsis, pea, tomato, and rice (6) and
have revealed that brassinosteroids are essential for normal plant
growth and development. Over 40 brassinosteroid analogues
have been isolated from various plant species so far (7-9).
Consequently, brassinosteroids have been recognized as a new
class of phytohormone (8, 10).
Specific biosynthesis inhibitors are useful in determining the
physiological functions of endogenous substances. As shown
in mode of action studies of gibberellins, gibberellin-deficient
mutants and gibberellin biosynthesis inhibitors are both quite
effective (11, 12). Similarly, a specific inhibitor of brassino-
steroid biosynthesis should provide a new and complementary
approach to understanding the functions of brassinosteroids (13).
Asami and Yoshida reported a brassinosteroid biosynthesis
Many steps of brassinosteroid biosynthesis are thought to be
catalyzed by cytochrome P450 enzymes. These steps include
the production of 6R-hydroxycampestanol from campestanol,
cathasterone from 6-oxocampestanol(17), teasterone from ca-
thasterone (18), castasterone from typhasterole, and brassinolide
from castasterone (8). Thus, the biosynthetic pathway of
brassinosteroids includes several potential active sites for
cytochrome P450 inhibitors. An important criterion for assessing
the inhibition of cytochrome P450 in brassinosteroid biosyn-
thesis by any compound is the nature of inhibition of other
cytochrome P450 enzymes of physiological importance. Specific
brassinosteroid biosynthesis inhibitors were developed from
uniconazole, which is a triazole-type inhibitor targeting a P450
that catalyzes an early step of gibberellin biosynthesis and partly
inhibits brassinosteroid biosynthesis (19). Chemical modification
of uniconazole led to the isolation of brassinazole (20) and
Brz2001 (21) (Figure 1). Pyrimidine derivatives of a P450-
inhibiting fungicide were also found to inhibit brassinosteroid
biosynthesis (22). Therefore, we have begun to screen known
cytochrome P450 inhibitors to search for new potent inhibitors
of brassinosteroid biosynthesis (Table 1). Among the tested
compounds, propiconazole and some triazole derivatives inhib-
ited brassinosteroid biosynthesis. We chose propiconazole as a
* Corresponding author (telephone +81-48-467-9526; fax +81-48-462-
4674; e-mail tasami@postman.riken.go.jp).
† Saitama University.
‡ RIKEN.
§ Utsunomiya University.
10.1021/jf011716w CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/04/2002

Novel Brassinosteroid Biosynthesis Inhibitor
J. Agric. Food Chem., Vol. 50, No. 12, 2002 3487
Table 3. 1,3-Dioxanes Tested in This Study
no.
R
1
R
2
R
3
18
19
4′-Cl
4′-Cl
H
CH
H
CH
Figure 1. Structures of brassinazole and Brz2001.
3
3
Table 1. Retardation of Growth of Cress Seedlings by 1 µM
Concentrations of Various Cytochrome P450 Inhibitors and Rescue
from Their Effect by Brassinolidea
hypocotyl length (cm)
compd
control
brassinazole
bitertanol
pacrobutrazol
propiconazole
tebconazole
triadimefon
triadimenol
without brassinolide
with brassiolide (10 nM)
2.55 ± 0.22
1.10 ± 0.15
2.60 ± 0.17
0.40 ± 0.10
0.80 ± 0.10
0.85 ± 0.17
1.35 ± 0.10
2.35 ± 0.18
3.01 ± 0.23
2.20 ± 0.25
3.30 ± 0.27
0.90 ± 0.20
2.45 ± 0.30
1.70 ± 0.20
2.30 ± 0.28
3.15 ± 0.36
Figure 2. Ketone and allyl derivatives tested in this study.
a Data are means ± SE obtained from 20 seedlings.
Table 2. Mono- and Disubstituted Dioxolanes Tested in This Study
no.
R
1
R
2
R
3
no.
R
1
R
2
R
3
a
P
2′-Cl, 4′-Cl n-C H
H
9
4′-Cl
C H
2 5
H
H
H
H
H
H
H
H
H
3
7
Figure 3. Synthetic pathway of triazole derivatives: (a) AlCl3, Br2, ether;
(b) diol, p-toluenesulfonic acid, toluene; (c) 1,2,4-triazole, K CO , acetone;
1
2
3
4
5
6
7
8
3′-Cl, 4′-Cl n-C H
H
H
H
H
H
H
10 4′-Cl
11 4′-Cl
12 4′-CF3
13 4′-Br
14 4′-C H
15 4′-CH
16 4′-NO
17 4′-OCH
n-C H
3
7
4
9
4′-Cl
2′-Cl
3′-Cl
H
n-C H
CH OCH
3
7
2
3
2
3
n-C H
n-C H
3
7
3
7
(d) allyl-MgBr, THF.
n-C H
n-C H
3
7
3
7
n-C H
n-C H
3
7
6
5
3
7
3 (20, 23). Bromine was introduced to the R-position of a ketone to
give an R-bromoketone. Ketalization of the R-bromoketone with the
diols in toluene, in the presence of p-toluenesulfonic acid, gave
diastereomixtures of the dioxolanes, which were converted to the
corresponding diastereomixtures of triazoles by treating them with 1,2,4-
triazole in DMSO. For the synthesis of compound 21, R-bromoketone
was coupled with triazole under basic conditions; subsequent reaction
with allylmagnesium bromide resulted in a target compound.
2-Bromo-1-(4-trifluoromethylphenyl)ethanone. A 10 mM solution
of 1.88 g of 4′-(trifluoromethyl)acetophenone in 5 mL of pure anhydrous
ether was cooled in an ice bath, 0.1 g of anhydrous aluminum chloride
was introduced, and 1.59 g of 10 mM bromine was added gradually
through a separatory funnel with continuous stirring. After the bromine
had been added, the solvent and dissolved hydrogen bromide were
removed at once under reduced pressure with a slight current of air.
4-(Trifluoromethyl)phenacyl bromide remained as a solid mass of
brownish yellow crystals. The color was removed by shaking with a
mixture of 10 mL of water and 10 mL of petroleum ether. The organic
layer was dried with anhydrous sodium sulfate and concentrated at
reduced pressure; the resulting residues were recrystallized from
methanol. White crystals were obtained in 81% yield: mp 50-53 °C;
1H NMR (CDCl3) δ 4.48 (2H, s), 7.80 (2H, d, J ) 8.2 Hz), 8.1 (2H,
d, J ) 8.2 Hz).
4′-Cl
4′-Cl
4′-Cl
H
CH
CH
n-C H
3
3
7
CH
H
n-C H
3
3
2
3
7
n-C H
3
3
3 7
a P, propiconazole.
lead compound and have begun the development of new
brassinosteroid biosynthesis inhibitors. The objective of this
study was to synthesize and evaluate the biological activity of
a novel group of potent brassinosteroid biosynthesis inhibitors.
MATERIALS AND METHODS
Chemicals. Chemicals for synthesis were purchased from Kanto
Chemicals Co. Ltd. (Tokyo, Japan) and Tokyo Kasei Co. Ltd. (Tokyo,
Japan). Triazole derivatives were purchased from Kanto Chemicals Co.
Ltd. Melting points (mp) were determined on a Yanagimoto micromelt-
ing point apparatus and are uncorrected. 1H NMR spectra were recorded
with tetramethylsilane (TMS) as an internal standard, using a Bruker
300 spectrometer operating at 300 MHz. Chromatographic separations
were performed on a flash chromatograph (silica gel FL-60D, Fuji
Silysia Chemical Ltd.).
Synthesis. The compounds in Tables 2 and 3 and Figure 2 were
synthesized using previously described methods, as illustrated in Figure
2RS,4RS-2-Bromomethyl-2-(4-trifluoromethylphenyl)-1,3-di-
oxolane. A solution of 2-bromo-1-(4-trifluoromethylphenyl)ethanone

3488 J. Agric. Food Chem., Vol. 50, No. 12, 2002
Sekimata et al.
(1.34 g, 5 mM), 1,2-pentanediol (0.60 mg, 5.8 mM), and p-toluene-
sulfonic acid (0.17 mg, 1 mM) in toluene (100 mL) was heated under
reflux for 16 h. After cooling, diethyl ether (200 mL) was added. The
resulting solution was washed with water, dried with sodium sulfate,
filtered, and evaporated at reduced pressure. The residue was purified
by chromatography over silica gel, using n-hexane as the eluent. A
yellow oil was obtained in a 60% yield. 1H NMR (CDCl3) data of each
diastereomer (a and b, tentatively designated) of 2RS,4RS-2-bromom-
ethyl-2-(4-trifluoromethylphenyl)-1,3-dioxolane: (a) δ 0.98 (3H, t, J
) 7.3 Hz), 1.29-1.89 (4H, m), 3.63 (2H, s) 3.73-3.81 (1H, m), 3.96-
4.04 (2H, m), 7.66 (4H, s); (b) δ 0.93 (3H, t, J ) 7.0 Hz), 1.29-1.89
(4H, m), 3.43 (1H, t, J ) 8.1 Hz), 3.61 (1H, d, J ) -14.8 Hz) 3.64
(1H, d, J ) -14.8 Hz), 4.32 (1H, dd, J ) 5.7, 8.1 Hz), 4.40-4.49
(1H, m), 7.66 (4H, s).
2RS,4RS-1-[2-(4-Trifluoromethylphenyl)-4-n-propyl-1,3-dioxolan-2-
ylmethyl]-1H-1,2,4-triazole (12). To a solution of (0.44 g, 6.4 mM)
1,2,4-triazole in 10 mL of DMSO was added (0.41 g, 6 mM) 85%
pure potassium hydroxide, and the mixture was stirred at 45 °C until
it became a clear colorless solution. Then (1.37 g, 4 mM) 2RS,4RS-
2-bromomethyl-2-(4-trifluoromethylphenyl)[1,3]dioxolane was added
in 2 mL of DMSO. The mixture was stirred at 140 °C overnight. The
yellow solution was cooled, and 100 mL of ice-water and 120 mL of
diethyl ether were added. The water phase was separated, and the
organic solution was washed with water, dried with sodium sulfate,
filtered, and evaporated. The residue was purified by chromatography
over silica gel, using ethyl acetate/n-hexane (7:3) as the eluent. White
crystals were obtained in 40% yield: mp 62-65 °C; 1H NMR (CDCl3)
data of each diastereomer (a and b, tentatively designated) of 12 (a) δ
0.92 (3H, t, J ) 7.0 Hz), 1.26-1.45 (4H, m), 3.24 (1H, t, J ) 6.6 Hz),
3.91 (2H, m), 4.50 (2H, s), 7.66 (4H, s), 7.94 (1H, s), 8.23 (1H, s); (b)
δ 0.89 (3H, t, J ) 7.0 Hz), 1.25-1.53 (4H, m), 3.34 (1H, t, J ) 8.3
Hz), 3.76-3.82 (1H, m), 4.04 (1H, dd, J ) 5.7, 8.3 Hz), 4.49 (1H, d,
J ) -14.6 Hz), 4.53 (1H, d, J ) -14.6 Hz), 7.67 (4H, s), 8.04 (1H,
s), 8.51 (1H, s). Anal. Calcd for C16H18F3N3O2: C, 56.30; H, 5.32; N,
12.31; F, 16.70. Found: C, 56.14; H, 5.31; N, 11.95; F, 16.98.
Triazole derivatives 1-11 and 13-19 were prepared in a similar
way.
sucrose (w/v) in Agripots (Kirin Brewery Co., Ltd., Tokyo, Japan) with
or without chemicals. These chemicals were dissolved and diluted with
DMSO. DMSO alone was added in a control experiment. Cress seeds
were grown in 16-h light (240 meinstein m-2 s-1) and 8-h dark
conditions in a growth chamber at 25 °C for 8 days. Hypocotyl length
of 20 seedlings of cress was measured with a rular and each experiment
was repeated three times.
RESULTS AND DISCUSSION
It has been demonstrated that cress (L. satiVum L.) is very
sensitive to an internal deficiency of brassinosteroids and is
therefore a useful species for evaluating brassinosteroid bio-
synthesis inhibitors. For example, cress hypocotyl growth was
retarded by the treatment of brassinazole or Brz2001, and this
retardation was canceled by the coapplication of the most potent
brassinosteroid, brassinolide, but not by other plant hormones
(20, 21).
Evaluation of Cytochrome P450 Inhibitors as New Lead
Compounds for Brassinosteroid Biosynthesis Inhibitors.
From studies using cytochrome P450 inhibitors, the azole moiety
of the inhibitors is believed to act as a ligand to bind to the
iron atom of the heme prosthetic group of the cytochrome P450
enzyme, forming a coordinated complex. In some cases, azole
derivatives have multiple inhibition sites. For example, pa-
clobutrazol, a gibberellin-biosynthesis inhibitor, retards the stem
elongation of many plant species by blocking ent-kaurene
oxidation and can also mildly affect other cytochrome P450
mono-oxygenases, such as the inhibition of sterol formation by
blocking 14R-demethylation (23). Uniconazole, also a giberellin-
biosynthesis inhibitor, was reported to be effective for reducing
the level of brassinosteroids (19). Thus, chemical modification
of azole derivatives with brassinosteroid biosynthesis inhibitory
activity may produce specific brassinosteroid biosynthesis
inhibitors. Brassinazole, the first specific brassinosteroid bio-
synthesis inhibitor, was developed on the basis of the chemical
structures of both uniconazole and paclobutrazol. To find new
lead compounds for brassinosteroid biosynthesis inhibitors,
which target cytochrome P450 existing in the brassinosteroid
biosynthesis pathway, we investigated the inhibitory potency
of several azole derivatives that are available on the market.
Among the azole derivatives tested, propiconazole was selected
as the best lead compound (Table 1). Propiconazole is a
fungicide that targets lanosterol 14R-demethylase in the ergos-
terol biosynthesis pathway (23). Moreover, this triazole is
reported to show plant growth regulation activity by the
inhibition of obtusifoliol 14R-demethylase (24, 25). In our
experiment, propiconazole-treated cress showed dwarfism that
could be rescued considerably by brassinolide treatment. This
implies that the morphological alteration of cress seedlings
treated with propiconazole should be mainly due to the
deficiency of brassinosteroid.
Biological Activity of the Propiconazole Derivatives.
Although the synthesized compounds consist of four isomers,
these compounds were subjected to biological testing without
further purification. The results obtained are shown in Figure
4. To investigate which chemical substituent on the aromatic
ring was responsible for the retardation of cress stem elongation,
various substituents were introduced onto the aromatic ring of
propiconazole (Table 2). The results were as follows: Among
chloro-substituted phenyl compounds (1-4), the 4′-chloro
derivative (2) exhibited activity as high as that of propiconazole
(P), the 3′,4′-dichloro derivative (1) was the second highest,
whereas the 2′-chloro and 3′-chloro analogues (3, 4) showed
little activity against the retardation of cress stem elongation.
Considering that propiconazole has a chlorine atom on the 2′-
1-(4-Chlorophenyl)-2-(1,2,4-triazol-1-yl)ethanone (20). A solution
of (4.43 g, 20 mM) 4′-chlorophenacyl bromide, (1.38 g, 20 mM) 1,2,4-
triazole, and (1.38 g, 10 mM) potassium carbonate in 100 mL of acetone
was stirred for 12 h at room temperature. After removal of the potassium
bromide by filtration, the solution was concentrated and 200 mL of
water was added. The solution was extracted with ethyl acetate, then
dried with sodium sulfate, filtered, and evaporated at reduced pressure.
The residue was purified by chromatography over silica gel using ethyl
acetate/n-hexane (1:1) as the eluent. White crystals were obtained in
1
81% yield: mp 149-150 °C; H NMR (DMSO) δ 6.02 (2H, s), 7.71
(2H, d, J ) 8.5 Hz), 8.06 (2H, d, J ) 8.5 Hz), 8.10 (1H, s), 8.53 (1H,
s).
2RS-2-(4-Chlorophenyl)-1-(1,2,4-triazol-1-yl)pent-4-en-2-ol (21). To
solution of (0.22 g, 1 mM) 1-(4-chlorophenyl)-2-(1,2,4-triazol-1-yl)-
ethanone (20) in dry THF (2.0 mL) under N2 at -78 °C was added
dropwise a 1 M solution of magnesium propenyl bromide (1.0 mL,
1.0 mM). The mixture was stirred for 1 h at -78 °C and for 1 h at
room temperature. The solution was diluted with saturated aqueous
ammonium chloride (10 mL), the phases were separated, and the
aqueous phase was extracted with ethyl acetate (2 × 10 mL). The
combined organic phases were dried (MgSO4), filtered, and evaporated
at reduced pressure. The residue was purified by chromatography over
silica gel using ethyl acetate/n-hexane (4:1) as the eluent. A yellow oil
1
was obtained in 30% yield: mp 104-107 °C; H NMR (CDCl3) δ
2.48 (1H, dd, J ) 8.1, 14.0 Hz), 2.79 (1H, dd, J ) 6.3, 14.0 Hz), 4.54
(1H, d, J ) -14.0 Hz), 4.63 (1H, d, J ) -14.0 Hz), 5.13 (1H, d, J )
6.3 Hz), 5.17 (1H, s) 5.61 (2H, d, J ) 6.3 Hz), 7.32 (4H, s), 8.01 (1H,
s), 8.71 (1H, s). Anal. Calcd for C13H14ClN3O: C, 59.21; H, 5.35; N,
15.93; Cl, 13.44. Found: C, 58.85; H, 5.41; N, 15.15; Cl, 14.38.
Plant Materials and Growth Conditions. Cress seeds (Lepidium
satiVum L.) were purchased locally. The seeds used for the assay were
sterilized in a 1% NaOCl solution for 20 min and washed with sterile
distilled water five times. Cress seeds were sown in 0.8% agar-solidified
medium containing half-strength Murashige and Skoog salts and 1.5%

Novel Brassinosteroid Biosynthesis Inhibitor
J. Agric. Food Chem., Vol. 50, No. 12, 2002 3489
Figure 5. Hypocotyl length of light-grown, 8-day-old cress seedlings treated
with various concentrations of 2, 12, and 13 from 0.1 to 10 µM: BL,
brassinolide. Data are means ± SE obtained from 20 seedlings.
Figure 4. Retardation of growth of cress seedlings by triazole derivatives
and rescue by BL and GA : C, control; B, brassinazole; P, propiconazole;
3
BL, brassinolide; GA , gibberellic acid. Data are means ± SE obtained
3
from 20 seedlings.
position of the phenyl ring but shows a good activity, the low
activity of 3 or 4 can be due to the absence of a chlorine atom
on the 4′-position of the phenyl ring. Further evidence comes
from the fact that derivative 5, lacking a substituent on the
aromatic ring, exhibited little activity. Chemical variations of
the substituents on the phenyl ring moiety in the synthesized
series showed that the 4′-substitution of the phenyl ring is
necessary. The key observation is the good recovery of cress
growth from the P-, 1-, and 2-induced hypocotyl dwarfism by
coapplication of brassinolide with these compounds, whereas
coapplication of gibberellic acid was less effective for recovery.
Compound 6, which lacks the side chain at the 1,3-dioxolane
moiety of the propiconazole derivatives, scarcely affected cress
stem elongation. Consequently, the side-chain effects at 1,3-
dioxolane, when the substituents on the phenyl group were fixed
to the 4′-chlorine atom, were investigated. Compound 7,
methylated at both C4 and C5 of the 1,3-dioxolane moiety of
the propiconazole derivatives, exhibited high activity. Compound
7-induced hypocotyl dwarfism was noticeably rescued by
coapplication of gibberellic acid, suggesting that the morpho-
logical changes in cress seedlings treated with 7 are at least
partly due to gibberellin deficiency; similarly, uniconazole (a
gibberellin-biosynthesis inhibitor) is known to reduce the level
of gibberellin and induce dwarfism, which is almost completely
rescued by the coapplication of gibberellin acid. For the optimal
retardation of cress stem elongation, the C4-alkyl substituent
n-propyl is required for 2. Other alkyl groups are too short
(methyl and ethyl; 8, 9) or too long (n-butyl; 10). In this series,
the degree of recovery from chemical-induced dwarfism of cress
by brassinolide treatment appeared to increase with increasing
inhibitory activity. Thus, the presence of an n-propyl group on
the 1,3-dioxolane moiety of propiconazole derivatives is es-
sential for the inhibitory activity of brassinosteroid biosynthesis.
Introduction of an oxygen atom in the alkyl side chain on the
1,3-dioxolane caused a drastic loss of inhibition of cress
hypocotyl elongation, which may indicate the presence of a
subpocket in the inhibitor-binding site, which is lipophilic and
has a limited size.
Figure 6. Hypocotyl length of light-grown, 8-day-old cress seedlings treated
with various concentrations of brassinazole, Brz2001, and 12 from 0.1 to
10 µM. Data are means ± SE obtained from 20 seedlings.
chlorine (2) ) bromine (13) > phenyl (14) > methyl (15) >
nitro (16) > methoxy (17). These results suggest the electron-
withdrawing lipophilic substituents at the 4′-position of the
phenyl group may be important for potent activity. The activities
of 2, 12, and 13 were directly compared in a dose-dependent
manner as in Figure 5. Among these chemicals, 12 was the
most potent and 12-treated cress showed the best recovery by
the coapplication of brassinolide. As a result, it was suggested
that the substituent at the 4′-position of phenyl rings should
play an important role in the inhibition of brassinosteroid
biosynthesis. A trifluoromethyl group at the 4′-position was the
best substituent for the phenyl ring. The potency of inhibitory
activity of stem elongation and degree of recovery by brassi-
nolide treatment were well correlated.
The dioxane-type compounds with a 4′-chlorophenyl ring
showed weaker activity than 12 in the hypocotyl elongation test
(Table 3). Although compound 19, with a dimethyl group on
the dioxane ring, exhibited clear inhibition, 19-treated dwarf
cress showed good recovery by coapplication of 1 µM gibber-
ellic acid and slight recovery by coapplication of 10 nM
brassinolide, indicating that 19 affects both brassinosteroid and
gibberellin biosynthesis. In Figure 3, chemicals that have no
dioxolane ring are shown. As a result, ketone derivative (20)
exhibited almost no activity. The hypocotyl length of cress
seedlings treated with 1 µM allyl derivative (21) was 230% of
that of 12. Thus, it is demonstrated that the presence of the
dioxolane moiety should be critical for the inhibition of cress
hypocotyl elongation. As an overall result, 12 is the most potent
inhibitor among the compounds in this study. The inhibitory
activities of 12, brassinazole, and Brz2001 were directly
compared in a dose-response manner as is shown in Figure 6.
At 0.1 µM or higher, 12 retarded the hypocotyl growth at the
As the above results suggest that the chlorine atom at the
4′-position of the phenyl group was probably essential for the
activity, other functional groups were then introduced into this
position to investigate the factor for enhancing the activity. Some
of the triazole derivatives having substituents other than chlorine
(12-17) also maintained potent inhibitory activity of cress
hypocotyl growth. The order of inhibitory activity of cress
hypocotyl elongation was as follows: trifluoromethyl (12) >

3490 J. Agric. Food Chem., Vol. 50, No. 12, 2002
Sekimata et al.
(13) Yokota, T. Brassinosteroids. In Biochemistry and Molecular
Biology of Plant Hormones; Hooykaas, P. J. J., Hall, M. A.,
Libbenga, K. R., Eds.; Elsevier Science: London, U.K., 1999;
pp 277-293.
level of half of the control, whereas at 0.1 µM Brz2001 or
brassinazole exhibited little activity.
In conclusion, we have discovered a novel group of potential
brassinosteroid biosynthesis inhibitors that are comparable to
Brz2001 or brassinazole in their in vivo efficacy on cress.
Inhibition of brassinosteroid biosynthesis by this new chemical
series appears to be linked to the combination of the triazole
moiety, the 4-n-propyl-1,3-dioxolane moiety, and the 4′-
trifluoromethyphenyl moiety. Compound 12 consists of four
isomers; current research will reveal the stereostructure-activity
relationship of 12, both in vivo and in vitro. This novel
brassinosteroid biosynthesis inhibitor will play an important role
in the investigation of the function of brassinosteroids, not only
in other plants but also in tissues, organs, and biochemical
processes. Moreover, this inhibitor will provide a way to reveal
new brassinosteroid pathways or other novel mutants. In addition
to its use in basic science, it may be possible to develop 12 as
a commercial plant growth regulator.
(14) Asami, T.; Yoshida, S. Brassinosteroid biosynthesis inhibitors.
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ACKNOWLEDGMENT
We thank Dr. Keisuke Sekino (SDS Biotech K.K.) for helpful
discussions about syntheses.
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