Synthesis and biological evaluation of novel azole derivatives as selective potent inhibitors of brassinosteroid biosynthesis

Article abstract of DOI:10.1016/j.bmc.2013.03.006

Brassinosteroids (BRs) are phytohormones that control several important agronomic traits, such as flowering, plant architecture, seed yield, and stress tolerance. To manipulate the BR levels in plant tissues using specific inhibitors of BR biosynthesis, a series of novel azole derivatives were synthesized and their inhibitory activity on BR biosynthesis was investigated. Structure-activity relationship studies revealed that 2RS, 4RS-1-[4-(2- allyloxyphenoxymethyl)-2-(4-chlorophenyl)-[1,3]dioxolan-2-ylmethyl]-1H-[1,2,4] triazole (G₂) is a highly selective inhibitor of BR biosynthesis, with an IC₅₀ value of approximately 46 ± 2 nM, which is the most potent BR biosynthesis inhibitor observed to date. Use of gibberellin (GA) biosynthesis mutants and BR signaling mutants to analyze the mechanism of action of this synthetic series indicated that the primary site of action is BR biosynthesis. Experiments feeding BR biosynthesis intermediates to chemically treated Arabidopsis seedlings suggested that the target sites of this synthetic series are CYP90s, which are responsible for the C-22 and/or C-23 hydroxylation of campesterol.

Full text of DOI:10.1016/j.bmc.2013.03.006

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel azole derivatives as
selective potent inhibitors of brassinosteroid biosynthesis
⇑
Kazuhiro Yamada, Osamu Yajima, Yuko Yoshizawa, Keimei Oh
Department of Biotechnology, Faculty of Bioresource Sciences, Akita Prefectural University, 241-438, Shimoshinjo Nakano, Akita 010-0195, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Brassinosteroids (BRs) are phytohormones that control several important agronomic traits, such as
flowering, plant architecture, seed yield, and stress tolerance. To manipulate the BR levels in plant tis-
sues using specific inhibitors of BR biosynthesis, a series of novel azole derivatives were synthesized and
their inhibitory activity on BR biosynthesis was investigated. Structure–activity relationship studies
revealed that 2RS, 4RS-1-[4-(2-allyloxyphenoxymethyl)-2-(4-chlorophenyl)-[1,3]dioxolan-2-ylmethyl]-
Received 30 January 2013
Revised 5 March 2013
Accepted 7 March 2013
Available online xxxx
1H-[1,2,4]triazole (G2) is
a highly selective inhibitor of BR biosynthesis, with an IC50 value of
Keywords:
Brassinosteroid biosynthesis inhibitor
Plant hormone
approximately 46 2 nM, which is the most potent BR biosynthesis inhibitor observed to date. Use of
gibberellin (GA) biosynthesis mutants and BR signaling mutants to analyze the mechanism of action
of this synthetic series indicated that the primary site of action is BR biosynthesis. Experiments feeding
BR biosynthesis intermediates to chemically treated Arabidopsis seedlings suggested that the target sites
of this synthetic series are CYP90s, which are responsible for the C-22 and/or C-23 hydroxylation of
campesterol.
Azole derivatives
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
increased biomass and seed yields,8 and available evidence indi-
cates that mutations in BR biosynthesis may be a means to im-
Plant responses to internal and external stimuli through the
activation of gene expression are regulated by signal transduction
pathways. Brassinosteroids (BRs) serve as important signal media-
tors that are involved in plant growth and development. Molecular
genetic analysis established the role of BRs as endogenous plant
hormones with well-defined functions including extreme dwarf-
ism, delayed senescence, male sterility, and constitutive photo-
morphogenesis in the dark.1–3 Exogenous application of BR has
been shown to promote cell expansion and division, regulating leaf
senescence, pollen development, fruit ripening, and to modulate
the plant response to numerous environmental cues.4 Because
BRs control several important agronomic traits such as flowering,
plant architecture, seed yield, and stress tolerance,5,6 various ef-
forts have been made to control BR levels in plant tissues using ge-
netic approaches. Using transgenic techniques to manipulate
endogenous BR content has a remarkable effect on plant growth.
Overexpression of DWARF4, an enzyme that catalyzes a rate-limit-
ing step in BR biosynthesis, enhances plant growth and seed yield
in Arabidopsis.7 Similarly, transgenic rice plants over expressing a
sterol C-22 hydroxylase that catalyzes a key step in BR biosynthesis
prove biomass production.9
An alternative method to manipulate the BR levels in plant tis-
sues is the use of specific inhibitors that target the enzymes in-
volved in BR biosynthesis. Asami and Yoshida reported the
discovery of brassinazole (Brz, chemical structure shown in
Fig. 1), the first class of synthetic BR biosynthesis inhibitors.10,11
Studies on the mode of actions of Brz have shown that the target
site(s) of Brz is DWARF4.12 Brz has been used not only to reveal
the role of BR in various plant species,13 but also for the isolation
and characterization of genes that function in BR signaling.14,15
The mutants, BZR1 (Brassinazole Resistant 1)16 and BZR2 (Brassi-
nazole Resistant 2),17 also known as BES1, were identified as nucle-
ar transcription factors that are dominant gain-of-function
mutants resistant to Brz. BZR1 and BZR2 are components located
downstream of the BR receptors in BR signal transduction path-
ways. The signal transduction of BR is thought to function through
BR binding to the extracellular domain of the leucine-rich-repeat
receptor-like kinase (LRR–RLK) BRI1 (Brassinosteroid Insensitive
1)18 and BAK1, a coreceptor of BRI1,19 which convey the BR signal
to downstream components including BIN2 (BR-Insensitive 2)20
and BSU1 (bri1 Suppressor 1).21 Subsequent phosphorylation of
BZR1 and BZR2/BES1 inhibits their activity through multiple mech-
anisms.22 BR-induced dephosphorylation activates the BZR1 and
BZR2/BES1 proteins, which directly regulate the transcription of
Abbreviation: BR, brassinosteroid; BL, brassinolide; GA, gibberellin; Brz,
brassinazole; DMF, dimethylformamide; mp, melting point; rt, room temperature.
⇑
Corresponding author. Tel.: +81 18 872 1590; fax: +81 18 872 1676.
E-mail address: jmwang@akita-pu.ac.jp (K. Oh).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.03.006
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2
K. Yamada et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
studied in detail.31 Azole derivatives have been demonstrated to
have widespread ability as inhibitors of P450s, due to the intrinsic
affinity of the nitrogen electron pair in heterocyclic molecules for
the prosthetic heme iron. The azoles bind not only to lipophilic re-
gions of the protein but also simultaneously to the prosthetic heme
iron. Therefore, we carried out a biorational approach using keto-
conazole as a molecular scaffold, which lead to the discovery of a
new series of BR biosynthesis inhibitors (YCZ series, general chem-
ical structure shown in Fig. 1).32
Initial structure–activity relationship studies indicated that the
introduction of a mono substitution of a chlorine atom at position
4 or a double substitution of chlorine atoms at positions 2 and 4 on
ring A (Fig. 1) exhibited potent inhibition of BR biosynthesis.33 We
also found that substitutions on ring D (as shown in Fig. 1) dramat-
ically affected the inhibitory potency against BR biosynthesis of
this synthetic series.32 To study further the structure–activity
relationships of this synthetic series and determine their mode of
action, we synthesized new compounds by the chemical optimiza-
tion of ring D and ring C. Subsequently, we used GA-deficient mu-
tants, BR signaling mutants and BR biosynthetic intermediates to
determine the target site of the inhibitors.
Figure 1. Chemical structures of brassinosteroid biosynthesis inhibitors and
general structure of chemicals in this study.
2. Results and discussion
2.1. Chemistry
BR-responsive genes. Consequently, inhibitors of BR biosynthesis
are useful tool for searching for components of the BR signal trans-
duction pathway. This method has advantages over mutant screens
using BR-deficient mutants as a background because it can be used
at different stages of plant growth and development.16 Moreover, it
can be applied to different plant species with great ease. In this
context, searching for selective and potent inhibitors of BR biosyn-
thesis represents a useful approach to dissecting the functions of
BRs as well as their signal transduction pathways.
Our research interest is the design and synthesis of novel plant
hormone biosynthesis inhibitors.23–27 In the course of this work,
we carried out a systemic search for novel BR biosynthesis inhibi-
tors based on the following observations. First, molecular analysis
of enzymes involved in BR biosynthesis provided a broad hint for
the design of new inhibitors. Several steps in BR biosynthesis pro-
ceed through the action of P450 enzymes. CYP90s are involved in
C-22 and C-23 side-chain hydroxylation of BR,28 CYP92A6 cata-
lyzes the biochemical conversion of castasterone from typhaster-
ol,29 and CYP85A2 catalyzes the lactonization of castasterone to
brassinolide.30 Accordingly, strategies for designing P450 inhibi-
tors can be attributed to the identification of BR synthesis inhibi-
tors. Second, cytochrome P450 inhibition mechanisms have been
Preparation of target compounds G–Gc were carried out by a
method described previously (Scheme 1).32 This method consisted
of the following four steps: (1) formation of 1-(4-chlorophenyl)-2-
azoleyl ethanone B–Bc; (2) tosylation of isopropylideneglycerol C;
(3) deprotection of isopropylidene ketal D; and (4) ketal formation
to generate F–Fc. Compounds B–Bc were prepared by reacting a-
bromoketone A with different azoles in DMF using a method that
we described previously.34 When using 1,2,4-triazole as a reagent
to prepare compound B, compound Ba is obtained as a by-product
and separated by column chromatography. The tosylation of iso-
propylidene glycerol C was achieved using a standard protocol (to-
syl chloride in pyridine at 0 °C), and hydrolysis of C with 1 M HCl in
MeOH yielded glyceryl tosylate E. Ketal formation to generate F–Fc
was carried out using 3 equiv of trifluoromethanesulfonic acid
(TfOH) in toluene at room temperature for 60 h, according to a pre-
viously described method.35 To generate compounds Ga–Gc, com-
pounds Fa–Fc were reacted with 2-ethoxyphenol in a basic
condition, as described previously.36 Other compounds were pre-
pared by reacting compound F with corresponding phenols or
naphthols by the same method. All of the compounds synthesized
Scheme 1. Reagents and conditions: (a) azoles, triethylamine, DMF, À10 °C, 1 h, rt, 3 h; (b) TsCl, pyridine, 0 °C; (c) MeOH, HCl, reflux, 6 h; (d) 3 equiv TfOH, toluene, rt, 60 h;
and (e) phenols, KOH, DMF, 50 °C, 12 h.
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K. Yamada et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
in this work consist of four stereoisomers, and they were subjected
to biological studies without further purification.
To investigate the site of action of this synthetic series on BR bio-
synthesis, intermediates of BR biosynthesis were applied to inhib-
itor-treated Arabidopsis seedlings for the biological evaluation of
their effects and/or response.
2.2. Bioassay systems
To evaluate the biological activity of the synthesized com-
pounds on BR biosynthesis inhibition, an assay system using Ara-
bidopsis seedlings grown in the dark was used.37 In the dark, the
hypocotyls and roots of Arabidopsis seedlings grow significantly,
and the apical hook of the cotyledon is maintained without open-
ing. However, BR synthesis-deficient Arabidopsis mutants such as
dwarf 1 show remarkable dwarfism and the opening of the apical
hook of cotyledons in the dark.38 This unique phenotype with de-
etiolation in the dark has been used in screening for BR biosynthe-
sis inhibitors.32 In the present study, we adapted this assay to
determine the effects of test compounds on the hypocotyl elonga-
tion of Arabidopsis seedlings grown in the dark, and we co-applied
BL and GA with the test compounds to determine the reversibility
of their effects. With this assay system, we evaluated the biological
activities of the synthesized compounds. To investigate the selec-
tivity of this synthetic series, the GA biosynthesis mutant (ga4-1)
and BR signaling mutants Bes1-D (obtained from Arabidopsis Infor-
mation Resource, Stock No. CS65988) and bzr1-1D (obtained from
Arabidopsis Information Resource, Stock No. CS65987) were used.
2.3. Inhibitory effects of azole derivatives on hypocotyl
elongation of Arabidopsis seedlings
In a previous work, we reported the discovery of a new BR bio-
synthesis inhibitor based on ketoconazole as a molecular scaffold,
the potent inhibitor YCZ-18 (structure shown in Fig. 1).32 In the
course of that research, we found that an alkyloxy substituent at
position 2 of the phenyl ring (ring D) significantly promoted the
biological activity of this triazole derivative in retarding the hypo-
cotyl elongation of Arabidopsis seedlings grown in the dark.33 To
further determine the structure–activity relationships of this syn-
thetic series, 15 analogues with different substituent on the phenyl
and/or aromatic rings (ring D) were prepared. The chemical struc-
tures of the newly synthesized triazole analogs used for biological
studies are shown in Table 1. To identify the chemical structure of
ring D that was responsible for the retardation of Arabidopsis stem
elongation, YCZ-18, which has an ethoxy substituent at the posi-
tion 2 of the phenyl ring, was used as a baseline reference, and
Brz220 (chemical structure shown in Fig. 1), a selective BR biosyn-
Table 1
Inhibitory activity of triazole derivatives on Arabidopsis seedling growth
a
No.
R
Inhibition of Arabidopsis stem elongation (IC50
)
(l
M)
G1
2-Propoxyphenyl
2-Allyloxyphenyl
0.06 0.01
G2
G3
0.05 0.01
0.28 0.01
2-Butoxyphenyl
G4
G5
2-But-3-enyloxyphenyl
2-Allylphenyl
0.13 0.02
0.21 0.05
G6
G7
G8
2-Isobutoxyphenyl
0.23 0.04
0.32 0.02
0.33 0.06
2-tert-Butoxyphenyl
2-(3-Methyl-but-2-enyloxy)phenyl
(continued on next page)
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K. Yamada et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Table 1 (continued)
a
No.
R
Inhibition of Arabidopsis stem elongation (IC50) (lM)
G9
2-(3-Methylbutoxy)phenyl
2-Cyclopentyloxyphenyl
2-Phenylphenyl
0.85 0.03
G10
0.19 0.02
0.48 0.12
G11
G12
G13
G14
2-Benzylphenyl
4-Phenylphenyl
Naphthalen-1-yl
2.43 0.20
0.42 0.08
0.71 0.09
G15
Naphthalen-2-yl
0.41 0.08
0.13 0.01
0.61 0.14
YCZ-18
Brz220
a
The IC50 values of the test compounds for the inhibition of Arabidopsis stem elongation were calculated by defining the hypocotyl length of untreated Arabidopsis seedlings
as 0% inhibition, the hypocotyl length of 0 mm as 100% inhibition. Data were collected from 15 to 20 seedlings. All of the experiments were performed at least in duplicate to
establish repeatability.
thesis inhibitor,37 was used as a positive control for structure–
activity relationship discussions. The concentrations of all of the
position 2 or a phenyl group at position 4 of the phenyl ring (G11
–G13) greatly reduced the biological activity, with an IC50 value be-
test compounds were 0, 0.1, 0.5, 2, 10 and 50
lM, and the IC50 val-
tween 0.41 0.08 and 2.43 0.20 lM, respectively. These results
ues were calculated accordingly. As shown in Table 1, introduction
of a propoxy or allyloxy group to the position 2 of the phenyl ring
(G1, G2) enhanced the retardation of hypocotyl elongation in Ara-
bidopsis seedlings grown in the dark, with an IC50 value of
indicate that the presence of a bulky biphenyl group weakens the
biological activity of this synthetic series. The introduction of a
naphthalene moiety instead of a 2-ethoxyphenyl substitution
(YCZ-18) either at position 1 or 2 of the naphthalenyl (G14–G15
also reduced the inhibitory activity, with IC50 values of
0.71 0.09 and 0.41 0.08 M, respectively. The data obtained in
)
0.06 0.01 and 0.05 0.01
was found to be 0.61 0.14
l
l
M, respectively. The IC50 of Brz220
M in our assay system. Introduction
l
of a butoxy (G3) or a but-3-enyloxy (G4) at position 2 of phenyl ring
(ring D) caused a negative effect on retarding the hypocotyl elon-
gation of Arabidopsis seedling, with IC50 values of 0.28 0.01 and
this work provided information about the chemical structure of
ring D and its relationship to the biological activity of this synthetic
series.
0.13 0.02
l
M, respectively. This result indicates that the length
Another factor that may affect the biological activity of this syn-
thetic series is the chemical structure of ring C (Fig. 1). To explore
the structure–activity relationships of ring C, three analogs with
different azole moieties were prepared, and their biological activity
was investigated. As shown in Table 2, the introduction of a 1,3,4-
triazole (Ga) and pyrazole (Gc) moiety to the YCZ-18 skeleton in-
stead of the 1,2,4-triazole moiety greatly reduced the inhibitory
activity of retardation of hypocotyl elongation of Arabidopsis seed-
of the alkyl chain at this position is sensitive to the biological activ-
ity. A propyloxy or an allyloxy group at position 2 of the phenyl
(ring D) gave the best result. Interestingly, the introduction of an
allyl group at position 2 of the phenyl ring (G5) reduced the biolog-
ical activity, with an IC50 value of 0.21 0.05 lM. This result sug-
gests that an oxygen atom is important for promoting biological
activity. Next, we determined the effect of branching structure of
the alkyl substituent at position 2 of the phenyl (ring D) on retard-
ing the hypocotyl elongation of Arabidopsis seedlings. As shown in
Table 1, the IC50 values of analogs with branching structures (G6–
lings, with IC50 values of 11.2 2.68 and 11.8 4.84 lM, respec-
tively. This result clearly indicates that the position of the
nitrogen atom in the azole moiety is important for BR biosynthesis
inhibition. The analog with an imidazole moiety (Gb) exhibited
inhibitory activity on the retardation of hypocotyl elongation of
Arabidopsis seedlings, with IC50 values of 0.74 0.08 lM (Table 2).
We thus discovered compounds with highly potent inhibitory
activity. For the analogs synthesized in this work, we found that
G9) are between 0.23 0.04 and 0.85 0.03 lM. This result indi-
cates that the branching structures have a negative effect on the
biological activity. The inhibitory potency of the analog with a
cyclopentane moiety (G10) is, however, slightly higher than those
with branching structure analogs, with an IC50 value of
0.19 0.02
lM. Introduction of a phenyl group or benzyl group at
at a concentration of 0.5 lM, G1–G4 and G8–G10 inhibited the stem
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K. Yamada et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
Table 2
seedlings were grown in the presence of BL (10 nM) or GA3
(1 M) for 5 days in the dark. Consequently, analogs G12, Ga and
Inhibitory activity of azole derivatives on Arabidopsis seedling growth
l
Gc were not applied for this biological evaluation.
Data shown in Table 3 are expressed as a percentage relative to
the untreated control. In the presence of BL (10 nM) or GA3 (1 lM),
the average hypocotyls length of Arabidopsis seedlings was approx-
imately 121 10% and 116 8% of that of the control, respectively.
This result indicates that BL and GA3 stimulate hypocotyl elonga-
tion of Arabidopsis seedlings. We found that at a concentration of
No.
X
Y
Z
IC50 (lM)
0.5 lM, the hypocotyls length of YCZ-18 treated Arabidopsis seed-
Ga
Gb
Gc
YCZ-18
Brz220
C
C
N
N
N
C
C
C
N
N
C
11.2 2.68
0.74 0.08
11.8 4.84
0.13 0.01
0.61 0.14
lings was approximately 27 3%, whereas the positive control
Brz220 was approximately 50 4% of that of the control. Co-appli-
cation of BL (10 nM) on the YCZ-18 and Brz220 treated seedlings
showed excellent recoveries; the hypocotyl lengths were
101 6% and 102 7%, respectively. When co-application of GA3
N
(1 lM) to the chemical treated seedlings did not show significant
recoveries on stem elongation, which were 26 2% and 52 5%
of that of the control, respectively. On the other hand, at a concen-
elongation of Arabidopsis seedlings less than 30% of that of the con-
trol. We also found that the inhibitory potency of most of the ana-
logs is higher than that of Brz220.
tration of 1
bidopsis seedlings were 38 5% of that of the control. When co-
application of BL (10 nM) or GA3 (1 M) to the chemical treated
lM of trinexapac-ethyl, the hypocotyl lengths of Ara-
l
2.4. Rescue hypocotyl elongation of Arabidopsis seedlings by BL
and GA
seedlings, the hypocotyl lengths of Arabidopsis seedlings were
53 4% and 90 4%, respectively. Our data clearly indicates that
both YCZ-18 and Brz220 are highly selective inhibitors of BR bio-
synthesis rather than GA biosynthesis, while trinexapac-ethyl is a
selective inhibitor of GA biosynthesis.
Next, we determined the selectivity of analogs synthesized in
this work by a method as described above. As shown in Table 3,
the chemically induced dwarfism of Arabidopsis seedlings caused
by all the analogs (G1–G11, G13–G15) underwent an excellent recov-
ery with BL (10 nM) application; the hypocotyl lengths were re-
versed to control levels (from 92 7% to 124 6%). This result
suggests that the activity of the analogs on retardation of stem elon-
gation of Arabidopsis may be primarily due to the inhibition of BR
biosynthesis. To verify this hypothesis, we determined the effect
of GA on the recovery of the chemically induced dwarf phenotype.
GA biosynthesis inhibitors such as uniconazole retard the stem
elongation of many plant species by blocking ent-kaurene oxida-
tion and also mildly affect other cytochrome P450 monooxygena-
ses.10 Stem elongation can be rescued by the application of GA.
To rule out the possibility of GA biosynthesis inhibition by our ana-
logs, we tested the effects of brassinolide (BL), the most biologi-
cally active BR, and GA3 on the recovery of the chemically
induced dwarfism of Arabidopsis seedlings grown in the dark.
Brz220 was used as a positive control for BR biosynthesis inhibi-
tors. Trinexapac-ethyl, an none azole type GA biosynthesis inhibi-
tor, was used as positive control for GA biosynthesis inhibitors.39
Compounds with IC50 values less than 1
lM were subjected to
the bioassay at
a concentration of 0.5 lM, and Arabidopsis
Co-application of GA3 (1 lM) resulted in different recoveries for dif-
Table 3
Retardation of Arabidopsis seedling growth by triazole derivatives and rescue of growth by BL and GA
No.
R
Hypocotyl length, relative to untreated Arabidopsis seedlings (%)
Chem.
100
Chem. + BL (10 nM)
121 10
Chem. + GA (1 lM)
Cont.
G1
G2
G3
G4
G5
G6
G7
G8
G9
G10
G11
G13
G14
G15
YCZ-18
—
116
26
25
42
26
51
36
53
40
88
39
71
72
74
60
26
90
52
8
2
2
5
2-Propoxyphenyl
2-Allyloxyphenyl
2-Butoxyphenyl
2-But-3-enyloxyphenyl
2-Allylphenyl
2-Isobutoxyphenyl
2-tert-Butoxyphenyl
2-(3-Methyl-but-2-enyloxy)phenyl
2-(3-Methyl-butoxy)phenyl
2-Cyclopentyloxyphenyl
2-Phenylphenyl
21
26
29
25
34
34
36
29
29
25
53
54
62
44
27
38
50
2
111
106
114
118
95
114
120
113
120
124
101
98
5
8
6
3
8
9
6
5
7
6
7
7
2
2
2
3
3
7
3
3
3
7
9
7
7
3
5
4
3
5
5
5
6
8
4
9
9
7
9
2
4-Phenylphenyl
Naphthalen-1-yl
Naphthalen-2-yl
2-Ethoxyphenyl
93 10
92
101
53
7
6
5
7
Trinexapac-ethyla
Brz220
4
5
—
102
a
The concentration used was 1 lM.
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K. Yamada et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Table 4
Retardation of Arabidopsis seedling growth by azole derivatives and rescue of growth by BL and GA
No.
X
Y
Z
Hypocotyl length, relative to untreated Arabidopsis seedlings (%)
Chem.
100
Chem. + BL (10 nM)
Chem. + GA (1 lM)
Control
Gb
YCZ-18
Trinexapac-ethyl
Brz220
110
101
101
53
8
8
6
5
7
107
77
26
90
6
6
2
C
N
C
C
N
N
75
27
38
50
6
3
5
4
4
—
102
52
5
ferent analogs. G1, G2 and G4 did not show significant recovery with
GA application; values shifted from 21 2% to 26 2%, 26 2% to
25 2% and 25 2% to 26 3%, respectively. Interestingly, the ana-
log G5 recovered partially with the co-application of GA3 (51 5%)
while G9 was remarkably reversed by application of GA3
(88 8%). This result suggests that G9 may inhibit GA biosynthesis
as a side effect. Other analogs exhibited slightly recovered stem
elongation of Arabidopsis seedling after application of GA.
Another factor that may affect the biological activity is the azole
moiety. As shown in Table 4, analog Gb exhibited good recovery
from BL (from 75 6% to 101 8%) but did not recover after appli-
cation of GA3. Although imidazole derivatives have been used as
P450 inhibitors,26,31 our data suggests that 1,2,4-triazole moiety
is important for BR biosynthesis inhibition.
nexapac-ethyl inhibited the stem elongation of bes1-D (Fig. 1 of
Supplementary data). Therefore, the bes1-D and bzr1-1D mutants
are sensitive indicators of BR biosynthesis inhibitors. We then
choose these two mutants for investigation of the inhibitory prop-
erties of YCZ-18 on the induction of dwarfism in Arabidopsis seed-
lings. As shown in Figure 2A3 (right) and 2B (orange bar), when in
the presence of YCZ-18 (0.5 lM), the hypocotyl length of the bes1-
D mutant is approximately 15.4 0.3 mm, whereas in the absence
of YCZ-18, the hypocotyl length of bes1-D mutant is approximately
18.3 0.8 mm (Fig. 2A3 left, and 2B red bar). Similarly, the brz-1-
1D mutant also exhibited resistance to YCZ-18 treatment. As
shown in Figure 2A4 (right, and Fig. 2B yellow bar), in the presence
of YCZ-18 (0.5 lM), the hypocotyl length of brz-1-1D was approx-
imately 14.6 1.2 mm, while in the absence of YCZ-18, the hypo-
cotyls length of brz-1-1D are approximately 15.2 0.8 mm
(Fig. 2A4 left, and 2B blue bar). These data indicate that the inhibi-
tion of stem elongation of Arabidopsis seedlings by YCZ-18 is due to
the inhibition of BR biosynthesis.
2.5. Characterization of YCZ-18 as a selective inhibitor of BR
biosynthesis
2.5.1. YCZ-18 inhibits hypocotyls elongation of ga4-1 mutant in
the presence of GA3
2.5.3. Determination of the target site(s) of YCZ-18 on inhibition
of BR biosynthesis
Mutants of GA biosynthesis are useful tool for the study of the
mechanisms of action of YCZ because they provide a specific selec-
tion background to rule out GA biosynthesis inhibitors from the
chemicals that induce dwarfism of Arabidopsis. The ga4-1 mutant
(obtained from the Arabidopsis Information Resource, Stock No.
CS61) has an absolute requirement for GA for germination and suc-
cessive elongation growth.40 Thus, we choose YCZ-18 as an analog
to determine its effects on the dark growth of wild type and ga4-1
mutant seedlings. As shown in Figure 2A1 (right) and 2B (white
To determine the target site(s) of YCZ inhibition of BR biosyn-
thesis, we used the straightforward approach of feeding of BR bio-
synthesis intermediates to BR biosynthesis inhibitor treated
Arabidopsis seedlings. Thus, we used commercially available BR
biosynthesis intermediates to determine which intermediate could
rescue the YCZ-induced dwarfism of Arabidopsis seedlings grown
in the dark. We choose YCZ-18 (0.5 lM) as an analog and campes-
terol (CR), campestanol (CN), teasterone (TE), castasterone (CS) as
BR biosynthesis intermediates for this study (Fig. 3A). CN was pre-
pared by the reduction of CR by a method described previously.42
bar), in the presence of YCZ-18 (0.5 lM), the hypocotyl lengths
of Arabidopsis seedlings are approximately 3.4 0.4 mm, whereas
the controls are approximately 15.6 0.4 mm (Fig. 2A1 left, and
2B black bar). In contrast, for the ga4-1 mutant, when germinating
In the presence of 0.5
opsis seedlings was approximately 2.8 0.5 mm (Fig. 3B left plant
and 3C blue bar). When TE (100 M, Fig. 3C, green bar), CS
lM YCZ-18, the hypocotyl length of Arabid-
in the presence of 1
mately 19.6 0.6 mm (Fig. 2A2 left, and 2B dark green bar). In
the presence of GA3 (1 M) and YCZ-18 (0.5 M), the hypocotyl
lM GA3, the hypocotyl lengths are approxi-
l
(100 nM, Fig. 3C, orange bar) and BL (10 nM, Fig. 3C, black bar)
were applied to the growth media, the hypocotyl length of Arabid-
opsis seedlings was restored from 2.8 0.5 to 10.5 0.7, 9.2 0.6
and 11.7 0.7 mm (Fig. 3B and C), respectively. This result indi-
cates that TE, CS and BL reversed the YCZ-18 induced dwarfism
of Arabidopsis seedlings grown in the dark, implying that the en-
zymes downstream of TE did not inhibited by YCZ-18 (Fig. 3A).
l
l
lengths of Arabidopsis seedlings are approximately 3.5 0.3 mm
(Fig. 2A2 right, and 2B light green bar). This result clearly indicates
that the YCZ-18 induced dwarfism of Arabidopsis seedlings is not
due to the inhibition of GA biosynthesis.
2.5.2. Brz-resistant mutants bes1-D (bzr-2) and bzr-1-1D are
resistant to YCZ-18 treatment
The application of CN (100
lM, Fig. 3C, yellow bar) or CR
(100 M, Fig. 3C, red bar) did not reverse the YCZ-18 induced
l
Brassinazole-resistant Arabidopsis mutants are useful tools to
determine BR biosynthesis inhibitors.41 bes1-D (also known as
bzr-2) and brassinazole resistant1-1D (bzr1-1D) mutants carry a
dominant gain of function mutation in transcription factor function
as a major component in BR signaling. Both mutants exhibit a
constitutive BR response even in the absence of BR.16,17 Indeed, tri-
dwarfism as the hypocotyl length shifted from 2.8 0.2 to
2.6 0.3 mm and from 2.8 0.2 to 2.5 0.2 mm, respectively, sug-
gesting that YCZ-18 inhibits enzymes downstream of CN. Taking
these results together, at least one of the target sites of YCZ-18
in BR biosynthesis is the oxidation step from CN to TE, which is
performed by CYP90B1, CYP90C1 and CYP90D1 (Fig. 3A).28,43 As
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K. Yamada et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
A
B
A1
A2
A3
A4
3 mm
WT WT
ga4-1
ga4-1
bes1-D
bes1-D
bzr1-1D
bzr1-1D
Cont
YCZ-18
GA3
GA3+YCZ-18
YCZ-18
YCZ-18
Figure 2. Sensitivity of Arabidopsis wild type, GA biosynthetic mutant ga4-1, and BR signaling mutants bes1-D and bzr1-1D to YCZ-18. (A1) Seedlings of wild-type(Col-0)
grown on ½ MS media in the dark for 5 days, either with 0.5 M YCZ-18 or DMSO (n >15). (A2) The GA biosynthetic mutant ga4-1 (Ler-0), grown on ½ MS media containing
M GA3 in the dark for 7 days, with either with 0.5 M YCZ-18 or DMSO (n P5). (A3–A4) The BR signaling mutants bes1-D (En) and bzr1-1D (Col) grown on ½ MS media
with either 0.5 M YCZ-18 or DMSO. (B) Average hypocotyl length of Arabidopsis seedlings at 5 days (for ga4-1, 7 days) of treatment (as shown in A1–A4). Error bars represent
l
1
l
l
l
standard deviation (n P5). All the experiments were duplicated to establish repeatability.
in a similar experiment performed by Fujioka et al., both cathaster-
one and TE rescued the defective hypocotyl growth of the dark-
grown det2 mutant.44 Additionally, Asami et al., used the same
method to determine the target site of Brz.45 Nevertheless, to char-
acterize the target site of YCZ in the inhibition of BR biosynthesis,
careful experiments should be conducted to determine the binding
YCZ indicate that this synthetic series is composed of selective
inhibitors of BR biosynthesis over GA biosynthesis. Feeding exper-
iments of BR biosynthesis intermediates suggests that the target
sites are the steps responsible for side-chain hydroxylation in BR
biosynthesis.
affinity of these enzymes to YCZ.
4. Experimental
4.1. General
Although the compounds used in the biological assays consisted
of four stereoisomers, the asymmetric synthesis of these stereoiso-
mers has been well established.35 We expect that further studies
on the stereochemical structure–activity relationship of this syn-
thetic series may provide insights regarding the binding site of this
synthetic series.
Chemicals for synthesis were purchased from Kanto Chemicals
Co., Ltd (Tokyo, Japan) and Tokyo Kasei Co., Ltd (Tokyo, Japan).
Brassinolide, castaserone, teasterone were purchased from Burash-
ino K.K. (Toyama, Japan), GA3 and campesterol were purchased
from Wako Pure Chemical Industries, Ltd (Tokyo, Japan). Melting
points (mp) were determined using a Yanako melting point appa-
ratus. 1H NMR spectra were recorded with a JEOL ECP-400 spec-
trometer, with chemical shifts expressed in ppm downfield from
tetramethylsilane, an internal standard. High resolution mass spec-
tra by electrospray ionization Fourier transform ion cyclotron res-
onance (ESI-FTICR) was taken using an Exactive MS System
(Thermo Fisher Scientific, USA).
3. Conclusions
We discovered and tested novel selective and specific inhibitors
of BR biosynthesis. Structure–activity relationship studies revealed
that analog 2RS, 4RS-1-[4-(2-allyloxyphenoxymethyl)-2-(4-chloro-
phenyl)-[1,3]dioxolan-2-ylmethyl]-1H-[1,2,4]triazole (G2) is
highly selective inhibitor of BR biosynthesis, with an IC50 value of
approximately 46 2 nM. Studies of the mechanism of action of
a
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8
K. Yamada et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
A
B
C
Figure 3. Responses of Arabidopsis seedlings to inhibitor treatments and BL and BR biosynthesis intermediates complementation. (A) Outline of the biosynthetic pathway of
brassinosteroid (BR) and chemical structure of the biosynthesis intermediates used in the experiment. (B) Col-0 grown on ½ MS media containing 0.5 M YCZ-18 in the dark
for 5 days. From left: without co-application of BR biosynthesis intermediates or BL; co-application of 100 M CR; 100 M CN; 100 M TE; 100 nM CS and 10 nM BL. (C)
Average hypocotyl length at day 5 of treatment (n >15). Error bars represent standard deviation. Experiments were duplicated to establish repeatability.
l
l
l
l
4.2. Chemical synthesis
pension of 1H-1,2,4-triazole (5.52 g, 0.08 mol) and phenacyl
bromide A (9.95 g, 0.05 mol) in 30 mL acetone, was added triethyl-
amine (8.1 g, 0.05 mol) drop-wise over a period of 1 h with the
temperature below 0 °C, and the reaction mixture was stirred for
another 30 min at room temperature. The mixture was filtered to
remove triethylamine hydrobromide salt precipitates. The precipi-
4.2.1. Preparation of 1-(4-chlorophenyl)-2-[1,2,4]-triazol-1-yl-
ethanone (B)
Compound B was prepared using phenacyl bromide as the start-
ing material, as described previously.17 To a vigorously stirred sus-
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K. Yamada et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
9
tate was washed with 3 Â 10 mL acetone. The filtrate was evapo-
rated under reduced pressure, and the residues were dissolved in
50 mL chloroform and then washed with 2 Â 25 mL water. After
evaporation of chloroform, the yellow solid was recrystallized with
2-propanol, and target compound B was obtained as a white solid
(yield: 22.7%). 1H NMR (400 MHz, CDCl3) d: 5.69 (s, 2H), 7,55 (t,
J = 7.8 Hz, 2H), 7.68 (t, J = 7.5 Hz, 1H), 8.00 (d, J = 7.8 Hz, 2H), 8.03
(s, 1H), 8.26 (s, 1H).
reaction mixture was stirred at room temperature for 60 h. The
reaction was quenched by adding saturated sodium bicarbonate
solution (25 mL), extracted with EtOAc (3 Â 30 mL), washed with
brine, and dried over anhydrous sodium sulfate and concentrated
under reduced pressure. The mixture was purified by flash chro-
matography on silica gel (EtOAc/hexanes = 1:1) affording target
compound F (yield: 49.5%). 1H NMR (400 MHz, CDCl3) d: 2.48 (s,
3H), 3.55 (dd, J = 6.1, 10.4 Hz, 1H), 3.70 (dd, J = 4.5, 8.8 Hz, 1H),
3.77–3.84 (m, 2H), 4.20–4.26 (m, 1H), 4.44 (s, 2H), 7.34 (s, 4H),
7.38–7.40 (m, 2H), 7.77–7.79 (m, 2H), 7.80 (s, 1H), 8.06 (s, 1H).
Other compounds (Fa–Fc) were prepared in a similar way by
the reaction of corresponding azole with the corresponding ketone
(Ba–Bc).
Other azole derivatives (Ba–Bc) were prepared in a similar way
by reacting of corresponding azoles with the 4-chlorophenacyl
bromide.
4.2.1.1.
1-(4-Chlorophenyl)-2-[1,2,4]-triazol-4-yl-ethanone
(Ba). 1H NMR (400 MHz, acetone-d6) d: 5.93 (s, 2H), 7.64–7.68
(m, 2H), 8.10–8.14 (m, 2H), 8.35 (s, 2H).
4.2.4.1. Toluene-4-sulfonic acid 2-(4-chlorophenyl)-2-[1,2,4]-
triazol-4-ylmethyl-[1,3]-dioxolan-4-ylmethyl ester (Fa).
NMR (400 MHz, acetone-d6) d: 2.49 (s, 3H), 3.63 (dd, J = 5.3, 8.7 Hz,
1H), 3.78–3.82 (m, 1H), 3.85–3.91 (m, 2H), 4.30–3.34 (m, 1H), 4.50
(s, 1H), 7.42–7.47 (m, 4H), 7.52–7.55 (m, 2H), 7.83–7.86 (m, 2H),
8.16 (s, 2H).
1H
1
4.2.1.2. 1-(4-Chlorophenyl)-2-imidazol-1-yl-ethanone (Bb). H NMR
(400 MHz, CDCl3) d: 5.44 (s, 2H), 6.97–6.98 (m, 1H), 7.17–7.18 (m, 1H),
7.50–7.54 (m, 2H), 7.71 (s, 1H), 7.91–7.95 (m, 2H).
4.2.1.3. 1-(4-Chlorophenyl)-2-pyrazol-1-yl-ethanone (Bc). 1H NMR
(400 MHz, CDCl3)d: 5.56 (s, 2H), 6.38 (t, J = 2.2 Hz, 1H),7.46–7.50 (m,
3H), 7.59 (d, J = 1.6 Hz, 1H), 7.91–7.94 (m, 2H).
4.2.4.2. Toluene-4-sulfonic acid 2-(4-chlorophenyl)-2-imidazol-
1-ylmethyl-[1,3]-dioxolan-4-ylmethyl ester (Fb).
1H NMR
(400 MHz, CDCl3) d: 2.40 (s, 3H), 3.38 (dd, J = 6.3, 10.4 Hz, 1H),
3.51–3.54 (m, 1H), 3.63–3.69 (m, 2H), 3.97–4.07 (m, 2H), 4.11–
4.16 (m, 1H), 6.76–6.81 (m, 2H), 7.20–7.32 (m, 7H), 7.67–7.71
(m, 2H).
4.2.2. Preparation of 2,2-dimethyl-4-(4-
methylbenzenesulfonate)-1,3-dioxolane-4-methanol (D)
A dry 50 mL round-bottomed flask was charged with p-toluene-
sulfonyl chloride (7.1 g, 37 mmol), to which pyridine (15 mL) was
added while stirring under nitrogen with a magnetic stir bar. The
reaction flask was placed inside a container; an ice/water mixture
could be added in the event that the reaction becomes too exother-
mic. 2,2-Dimethyl-1,3-dioxolane-4-methanol C (3.29 g, 25 mmol)
was added slowly and stirred for 1.5 h. The mixture was slowly
4.2.4.3. Toluene-4-sulfonic acid 2-(4-chlorophenyl)-2-pyrazol-
1-ylmethyl-[1,3]-dioxolan-4-ylmethyl ester (Fc).
1H NMR
(400 MHz, CDCl3) d: 2.46 (s, 3H), 3.54 (dd, J = 6.5, 8.6 Hz, 1H),
3.71–3.79 (m, 2H), 3.86 (dd, J = 5.5, 10.5 Hz, 1H), 4.05–4.13 (m,
2H), 4.35 (s, 2H), 6.23 (t, J = 2.1 Hz, 1H), 7.22–7.25 (m, 2H), 7.27–
7.33 (m, 3H), 7.34–7.41 (m, 2H), 7.48 (d, J = 8.2 Hz, 1H), 7.62–
7.66 (m, 2H).
added to
a vigorously stirred mixture of de-ionized water
(70 mL) and crushed ice (70 g) in a 300 mL Erlenmeyer flask and al-
lowed to stir for an additional 0.5 h. The white precipitate was col-
lected on filter paper and washed with cold water (H2O). The
product was dried under high vacuum and/or anhydrous sodium
sulfate to obtain target compound D (yield: 58.0%). 1H NMR
(400 MHz, CDCl3) d: 1.32 (s, 3H), 1.34 (s, 3H), 2.46 (s, 3H), 3.77
(dd, J = 4.9, 9.0 Hz, 1H), 3.95–4.06 (m, 3H), 4.25–4.31 (m, 1H),
7.35 (d, J = 8.1 Hz, 2H), 7.80 (d, J = 8.4 Hz, 2H).
4.2.5. Preparation of 1-[2-(4-chlorophenyl)-4-(2-
propoxyphenoxymethyl)-[1,3]-dioxolan-2-ylmethyl]-1H-
[1,2,4]-triazole (G1)
Potassium hydroxide (160 mg, 2.8 mmol) was added to a solu-
tion of compound F (485 mg, 0.78 mmol) and 2-propokyphenol
(110 mg, 0.72 mmol) in dry DMF (5 mL), and the reaction mixture
was heated at 50 °C overnight. After cooling to room temperature,
the reaction mixture was diluted with water (20 mL) and EtOAc
(20 mL), and the organic phase was separated. The aqueous phase
was extracted with EtOAc (3 Â 20 mL). All of the organic layers
were combined, washed with brine (20 mL), dried over anhydrous
sodium sulfate, filtered, concentrated, and purified by flash chro-
matography on silica gel (EtOAc/hexanes = 1:1), affording target
compound G1 (yield: 20.0%), mp: 103.2–104.5 °C. 1H NMR
(400 MHz, CDCl3) d: 1.00 (t, J = 7.3 Hz, 3H), 1.74–1.80 (m, 2H),
3.61 (dd, J = 6.6, 9.9 Hz, 1H), 3.90–3.96 (m, 5H), 4.41–4.44 (m,
1H), 4.51–4.60 (m, 2H), 6.84–6.98 (m, 4H), 7.34–7.42 (m, 4H),
7.87 (s, 1H), 8.17 (s, 1H). The HRMS-ESI calcd for C22H24ClN3O4Na
[M+Na]+ was 452.1348, with 452.1352 found experimentally.
Other compounds (G2–G15, Ga–Gc) were prepared in a similar
method, by reacting F with the corresponding phenols.
4.2.3. Preparation of 1-tosyloxy-2, 3-propanediol (E)
Compound D (8.21 g, 30 mmol) was dissolved in methanol
(300 mL). Then, 0.5 N hydrochloric acid (30 mL) was added, and
the resulting mixture was heated to reflux. Acetone and methanol
were slowly distilled off. Additional methanol (50 mL) and 0.5 N
hydrochloric acid (20 mL) was added, and the mixture were kept
at room temperature until ketal hydrolysis was completed. The
mixture was diluted with saturated sodium bicarbonate solution
and extracted with EtOAc (3 Â 80 mL). The EtOAc extracts were
combined, washed with brine (20 mL), dried over anhydrous so-
dium sulfate, filtered, concentrated, and purified by flash chroma-
tography on silica gel (EtOAc/hexanes = 7:3), affording target
compound E (yield: 64.8%). 1H NMR (400 MHz, CDCl3) d: 2.46 (s,
3H), 3.63 (dd, J = 5.1, 11.4 Hz, 1H), 3.71 (dd, J = 4.2, 11.5 Hz, 1H),
3.92–3.99 (m, 1H), 4.06–4.13 (m, 2H), 7.37 (d, J = 6.6 Hz, 2H), and
7.81 (d, J = 6.6 Hz, 2H).
4.2.5.1.
1-[4-(2-Allyloxyphenoxymethyl)-2-(4-chlorophenyl)-
Yield:
[1,3]-dioxolan-2-ylmethyl]-1H-[1,2,4]-triazole (G2).
4.2.4. Preparation of toluene-4-sulfonic acid 2-(4-
chlorophenyl)-2-[1,2,4]-triazol-1-ylmethyl-[1,3]-dioxolan-4-
ylmethyl ester (F)
Trifluoromethanesulfonic acid (1.5 mL, 16 mmol) was added to
a solution of 1-tosyloxy-2, 3-propanediol E (1 g, 4.0 mmol) and ke-
tone B (1 g, 3.9 mmol) in toluene (10 mL) under nitrogen. Then, the
13.0%; mp: 76.6–78.9 °C. 1H NMR (400 MHz, CDCl3) d: 3.69 (dd,
1H, J = 6.2, 9.9 Hz, 1H), 3.90–3.99 (m, 3H), 3.97–3.99 (m, 2H),
4.42–4.48 (m, 1H), 4.53–4.64 (m, 4H), 5.22–5.24 (m, 1H), 5.34–
5.38 (m, 1H), 5.95–6.05 (m, 1H), 6.84–6.98 (m, 4H), 7.34–7.41
(m, 4H), 7.93 (s, 1H), 8.29 (s, 1H). The HRMS-ESI calcd for
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10
K. Yamada et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
C22H22ClN3O4Na [M+Na]+ was 450.1191, with 450.1195 found
experimentally.
(s, 1H). The HRMS-ESI calcd for C24H26ClN3O4Na [M+Na]+ was
478.1504, with 478.1510 found experimentally.
4.2.5.2.
1-[4-(2-Butoxyphenoxymethyl)-2-(4-chlorophenyl)-
Yield:
4.2.5.9. 1-[4-(2-Allylphenoxymethyl)-2-(4-chlorophenyl)-[1,3]-
[1,3]-dioxolan-2-ylmethyl]-1H-[1,2,4]-triazole (G3).
dioxolan-2-ylmethyl]-1H-[1,2,4]-triazole
(G10).
Yield:
40.4%; mp: 109.5–110.8 °C. 1H NMR (400 MHz, CDCl3) d: 0.93 (t,
J = 7.5 Hz, 3H), 1.39–1.48 (m, 2H), 1.65–1.73 (m, 2H), 3.75 (dd,
J = 5.7, 10.1 Hz, 1H), 3.90–4.04 (m, 5H), 4.42–4.47 (m, 1H), 4.54–
4.70 (m, 2H), 6.84–6.98 (m, 4H), 7.33–7.40 (m, 4H), 7.95 (s, 1H),
8.42 (s, 1H). The HRMS-ESI calcd for C23H26ClN3O4Na [M+Na]+
was 466.1504, with 466.1509 found experimentally.
45.7%; mp: 109.0–110.7 °C. 1H NMR (400 MHz, CDCl3) d: 3.31 (d,
J = 6.7 Hz, 2H), 3.50 (dd, J = 6.7, 9.8 Hz, 1H), 3.85–3.96 (m, 3H),
4.37–4.40 (m, 1H), 4.53 (s, 2H), 4.96–5.02 (m, 2H), 5.87–5.97 (m,
1H), 6.74 (d, J = 6.7 Hz, 1H), 6.91–6.95 (m, 1H), 7.12–7.22 (m,
1H), 7.36–7.39 (m, 2H), 7.42–7.45 (m, 2H), 7.93 (s, 1H), 8.24 (s,
1H). The HRMS-ESI calcd for
434.1242, with 434.1248 found experimentally.
C
22H22ClN3O3Na [M+Na]+ was
4.2.5.3.
phenyl)-[1,3]-dioxolan-2-ylmethyl]-1H-[1,2,4]-triazole
(G4).
Yield: 45.3%; mp: 102.0–104.0 °C. 1H NMR (400 MHz,
1-[4-(2-But-3-enyloxyphenoxymethyl)-2-(4-chloro-
4.2.5.10.
1-[4-(Biphenyl-2-yloxymethyl)-2-(4-chlorophenyl)-
[1,3]-dioxolan-2-ylmethyl]-1H-[1,2,4]-triazole (G11).
Yield:
CDCl3) d: 2.49 (q, J = 6.5 Hz, 2H), 3.90 (d, J = 4.0 Hz, 2H), 4.00–
4.13 (m, 4H), 4.45–4.48 (m, 1H), 4.58–4.76 (m, 2H), 5.05–5.16
(m, 2H), 5.81–5.91 (m, 1H), 6.87–7.01 (m, 4H), 7.35–7.41 (m,
4H), 8.04 (s, 1H), 8.71 (s, 1H). The HRMS-ESI calcd for
7.3%, oil. 1H NMR (400 MHz, CDCl3) d: 3.57–3.61 (m, 1H), 3.72–
3.75 (m, 1H), 3.80–3.83 (m, 1H), 3.88–3.92 (m, 1H), 4.25–4.28
(m, 1H), 4.34 (s, 2H), 6.91 (d, J = 8.3 Hz, 1H), 7.06–7.10 (m, 1H),
7.23–7.38 (m, 9H), 7.44–7.47 (m, 2H), 7.86 (s, 1H), 8.08 (s, 1H).
The HRMS-ESI calcd for C25H22ClN3O3Na [M+Na]+ was 470.1242,
with 470.1250 found experimentally.
C
23H24ClN3O4Na [M+Na]+ was 464.1348, with 464.1351 found
experimentally.
4.2.5.4. 1-[2-(4-Chlorophenyl)-4-(2-isobutoxyphenoxymethyl)-
4.2.5.11.
1-[4-(2-Benzylphenoxymethyl)-2-(4-chlorophenyl)-
Yield:
[1,3]-dioxolan-2-ylmethyl]-1H-[1,2,4]-triazole (G5).
Yield:
[1,3]-dioxolan-2-ylmethyl]-1H-[1,2,4]-triazole (G12).
52.4%; mp: 77.5–79.9 °C. 1H NMR (400 MHz, CDCl3) d: 1.00 (dd,
J = 4.6, 6.6 Hz, 6H), 2.00–2.10 (m, 1H), 3.66 (dd, J = 6.3, 10.0 Hz,
1H), 3.72 (d, J = 6.6 Hz, 2H), 3.92–3.97 (m, 3H), 4.39–4.44 (m,
1H), 4.51–4.60 (m, 2H), 6.84–7.00 (m, 4H), 7.33–7.36 (m, 2H),
7.39–7.42 (m, 2H), 7.89 (s, 1H), 8.23 (s, 1H). The HRMS-ESI calcd
for C23H26ClN3O4Na [M+Na]+ was 466.1504, with 466.1509 found
experimentally.
42.4%; mp: 93.0–94.8 °C. 1H NMR (400 MHz, CDCl3) d: 3.40 (dd,
J = 6.9, 9.4 Hz, 1H), 3.56–3.59 (m, 1H), 3.71–3.81 (m, 2H), 3.87–
3.96 (m, 2H), 4.23–4.29 (m, 1H), 4.45 (d, J = 3.7 Hz, 2H), 6.73 (d,
J = 8.0 Hz, 1H), 6.91–6.95 (m, 1H), 7.10–7.15 (m, 4H), 7.19–7.23
(m, 3H), 7.35–7.42 (m, 4H), 7.91 (s, 1H), 8.10 (s, 1H). The HRMS-
ESI calcd for
484.1404 found experimentally.
C
26H24ClN3O3Na [M+Na]+ was 484.1398, with
4.2.5.5.
phenyl)-[1,3]-dioxolan-2-ylmethyl]-1H-[1,2,4]-triazole
(G6).
Yield: 14.5%; mp: 134.7–135.4 °C. 1H NMR (400 MHz,
CDCl3) d: 1.32 (s, 9H), 3.60 (dd, J = 6.4, 9.7 Hz, 1H), 3.86–3.97 (m,
3H), 4.39–4.41 (m, 1H), 4.51–4.61 (m, 2H), 6.82–7.06 (m, 4H),
7.35–7.43 (m, 4H), 7.97 (s, 1H), 8.32 (s, 1H). The HRMS-ESI calcd
for C23H26ClN3O4Na [M+Na]+ was 466.1504, with 466.1509 found
experimentally.
1-[4-(2-tert-Butoxyphenoxymethyl)-2-(4-chloro-
4.2.5.12.
1-[4-(Biphenyl-4-yloxymethyl)-2-(4-chlorophenyl)-
[1,3]-dioxolan-2-ylmethyl]-1H-[1,2,4]-triazole (G13).
Yield:
20.8%; mp: 168.7–170.1 °C. 1H NMR (400 MHz, CDCl3) d: 3.56
(dd, J = 6.2, 9.8 Hz, 1H), 3.82–3.86 (m, 2H), 3.90–3.94 (m, 1H),
4.36–4.41 (m, 1H), 4.53 (s, 2H), 6.89–6.92 (m, 2H), 7.29–7.33 (m,
1H), 7.37–7.44 (m, 4H), 7.46–7.56 (m, 6H), 7.92 (s, 1H), 8.21 (s,
1H). The HRMS-ESI calcd for
470.1242, with 470.1249 found experimentally.
C
25H22ClN3O3Na [M+Na]+ was
4.2.5.6. 1-{2-(4-Chlorophenyl)-4-[2-(3-methyl-but-2-enyloxy)-
4.2.5.13. 1-[2-(4-Chlorophenyl)-4-(naphthalen-1-yloxymethyl)-
phenoxymethyl]-[1,3]-dioxolan-2-ylmethyl}-1H-[1,2,4]-triazole
[1,3]-dioxolan-2-ylmethyl]-1H-[1,2,4]-triazole (G14).
Yield:
Yield: 47.9%; mp: 95.7–97.3 °C. 1H NMR (400 MHz,
13.9%; mp: 105.6–107.2 °C. 1H NMR (400 MHz, CDCl3) d: 3.76
(dd, J = 6.2, 9.6 Hz, 1H), 3.98–4.06 (m, 3H), 4.51–4.57 (m, 3H),
6.73–6.75 (m, 1H), 7.36–7.42 (m, 3H), 7.45–7.52 (m, 5H), 7.78–
7.82 (m, 1H), 7.94 (s, 1H), 8.11–8.13 (m, 1H), 8.31 (s, 1H). The
HRMS-ESI calcd for C23H20ClN3O3Na [M+Na]+ was 444.1085, with
444.1091 found experimentally.
(G7).
CDCl3) d: 1.67 (s, 3H), 1.71 (s, 3H), 3.68–3.73 (m, 1H), 3.92–4.00
(m, 3H), 4.42–4.65 (m, 5H), 5.38–5.41 (m, 1H), 6.85–6.95 (m,
4H), 7.32–7.39 (m, 4H), 7.88 (s, 1H), 8.25 (s, 1H). The HRMS-ESI
calcd for C24H26ClN3O4Na [M+Na]+ was 478.1504, with 478.1510
found experimentally
4.2.5.7. 1-{2-(4-Chlorophenyl)-4-[2-(3-methylbutoxy)-phenoxy-
4.2.5.14. 1-[2-(4-Chlorophenyl)-4-(naphthalen-2-yloxymethyl)-
methyl]-[1,3]-dioxolan-2-ylmethyl}-1H-[1,2,4]-triazole
[1,3]-dioxolan-2-ylmethyl]-1H-[1,2,4]-triazole (G15).
Yield:
(G8).
Yield: 37.2%; mp: 80.4–81.8 °C. 1H NMR (400 MHz,
42.5%; mp: 168.2–170.4 °C. 1H NMR (400 MHz, CDCl3) d: 3.72
(dd, J = 5.8, 10.0 Hz, 1H), 3.85–3.97 (m, 3H), 4.42–4.45 (m, 1H),
4.56 (s, 2H), 7.05 (d, J = 2.5 Hz, 1H), 7.12 (dd, J = 2.5, 9.0 Hz, 1H),
7.33–7.41 (m, 3H), 7.43–7.50 (m, 3H), 7.74–7.78 (m, 3H), 7.95 (s,
CDCl3) d: 0.92 (d, J = 3.3 Hz, 3H), 0.94 (d, J = 3.3 Hz, 3H), 1.54–
1.65 (m, 2H), 1.73–1.80 (m, 1H), 3.84 (dd, J = 3.8, 10.1 Hz, 1H),
3.91–3.95 (m, 1H), 3.98–4.09 (m, 4H), 4.44–4.50 (m, 1H), 4.57–
4.76 (m, 2H), 6.86–7.00 (m, 4H), 7.35–7.40 (m, 4H), 8.01 (s, 1H),
8.60 (s, 1H). The HRMS-ESI calcd for C24H28ClN3O4Na [M+Na]+
was 480.1661, with 480.1665 found experimentally.
1H), 8.31 (s, 1H). The HRMS-ESI calcd for
C23H20ClN3O3Na
[M+Na]+ was 444.1085, with 444.1092 found experimentally.
4.2.5.15.
1-[2-(4-Chlorophenyl)-4-(2-ethoxyphenoxymethyl)-
4.2.5.8. 1-[2-(4-Chlorophenyl)-4-(2-cyclopentyloxyphenoxym-
[1,3]-dioxolan-2-ylmethyl]-4H-[1,2,4]-triazole (Ga).
Yield:
ethyl)-[1,3]-dioxolan-2-ylmethyl]-1H-[1,2,4]-triazole
16.9%; mp: 128.2–130.7 °C. 1H NMR (400 MHz, CDCl3) d: 1.37 (t,
J = 8.5 Hz, 3H), 3.66 (dd, J = 6.0, 10.1 Hz, 1H), 3.87–3.92 (m, 2H),
3.96–4.06 (m, 3H), 4.23–4.38 (m, 2H), 4.41–4.47 (m, 1H), 6.85–
6.98 (m, 4H), 7.35–7.40 (m, 4H), 8.10 (s, 2H). The HRMS-ESI calcd
(G9).
Yield: 37.2%; mp: 97.3–99.0 °C. 1H NMR (400 MHz,
CDCl3) d: 1.56–1.89 (m, 8H), 3.78 (dd, J = 5.7, 10.1 Hz, 1H), 3.90–
3.93 (m, 1H), 3.99–4.07 (m, 2H), 4.42–4.47 (m, 1H), 4.55–4.77
(m, 3H), 6.88–6.98 (m, 4H), 7.34–7.40 (m, 4H), 7.97 (s, 1H), 8.45
Please cite this article in press as: Yamada, K.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.03.006

K. Yamada et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
11
for C21H22ClN3O4Na [M+Na]+ was 43.1191, with 438.1193 found
experimentally.
4. Mandava, N. B. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1988, 39, 23.
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4.2.5.16.
1-[2-(4-Chlorophenyl)-4-(2-ethoxyphenoxymethyl)-
[1,3]-dioxolan-2-ylmethyl]-1H-imidazole
(Gb).
Yield:
16.6%; mp: 104.2–105.6 °C. 1H NMR (400 MHz, CDCl3) d: 1.38 (t,
J = 7.0 Hz, 3H), 3.52 (dd, J = 6.5, 10.0 Hz, 1H), 3.84–3.93 (m, 3H),
4.00–4.06 (m, 2H), 4.14–4.25 (m, 2H), 4.38–4.44 (m, 1H), 6.83–
6.97 (m, 6H), 7.29–7.42 (m, 5H). The HRMS-ESI calcd for
C
22H23ClN2O4Na [M+Na]+ was 437.1239, with 437.1238 found
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4.2.5.17.
1-[2-(4-Chlorophenyl)-4-(2-ethoxyphenoxymethyl)-
[1,3]-dioxolan-2-ylmethyl]-1H-pyrazole (Gc).
Yield: 69.0%;
mp: 84.5–85.6 °C. 1H NMR (400 MHz, CDCl3) d: 1.34 (t, J = 7.0 Hz,
3H), 3.71–3.79 (m, 2H), 3.88–4.02 (m, 4H), 4.23–4.29 (m, 1H),
4.41 (s, 2H), 6.25 (t, J = 2.1 Hz, 1H), 6.76–6.93 (m, 4H), 7.27–7.30
(m, 2H), 7.39–7.42 (m, 2H), 7.46–7.50 (m, 2H). The HRMS-ESI calcd
for C22H23ClN2O4Na [M+Na]+ was 437.1239, with 437.1237 found
experimentally.
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Acknowledgment
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We thank Professor Noboru Murofushi for his thoughtful sug-
gestions on preparing the manuscript.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.03.006.
References and notes
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Please cite this article in press as: Yamada, K.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.03.006