Asymmetric synthesis and effect of absolute stereochemistry of YCZ-2013, a brassinosteroid biosynthesis inhibitor

Article abstract of DOI:10.1016/j.bmcl.2013.09.056

The four stereoisomers of 2RS,4RS-1-[[2-(2,4-dichlorophenyl)-4-(2-(2- propenyloxy)phenoxymethyl)-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole (YCZ-2013), a novel brassinosteroid biosynthesis inhibitor, were prepared. The diastereomers of 2RS,4R-5 and 2RS,4S-5 were prepared by using the corresponding optically pure R and S toluene-4-sulfonic acid 2,3-dihydroxypropyl ester (R-4,S-4). The enatiomerically and diastereomerically pure acetonide (5) was obtained by a method involving diastereoselective crystallisation of the tosylate salt, followed by re-equilibration with the mother liquor and chromatography. The optical purity of four target compounds (YCZ-2013) was confirmed by chiral high-performance liquid chromatography (HPLC) and NMR. The effects of these stereoisomers on Arabidopsis stem elongation indicated that the cis isomers of 2S,4R-YCZ-2013 and 2R,4S-YCZ-2013 exhibited potent inhibitory activity with IC₅₀ values of approximately 24 ± 3 and 24 ± 2 nM, respectively. The IC₅₀ values of the trans isomers of 2S,4S-YCZ-2013 and 2R,4R-YCZ-2013 are approximately 1510 ± 50 and 3900 ± 332 nM, respectively. Co-application of brassinolide (10 nM), the most potent BR, and GA₃ (1 μM) to Arabidopsis seedlings grown in the dark with 2R,4S-YCZ-2013 and 2S,4R-YCZ-2013 revealed that brassinolide recovered the induced dwarfism of Arabidopsis seedlings, whereas GA₃ showed no effect.

Full text of DOI:10.1016/j.bmcl.2013.09.056

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6915–6919
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Asymmetric synthesis and effect of absolute stereochemistry
of YCZ-2013, a brassinosteroid biosynthesis inhibitor
⇑
Keimei Oh , Kazuhiro Yamada, Yuko Yoshizawa
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:
The four stereoisomers of 2RS,4RS-1-[[2-(2,4-dichlorophenyl)-4-(2-(2-propenyloxy)phenoxymethyl)-1,3-
dioxolan-2-yl]methyl]-1H-1,2,4-triazole (YCZ-2013), a novel brassinosteroid biosynthesis inhibitor, were
prepared. The diastereomers of 2RS,4R-5 and 2RS,4S-5 were prepared by using the corresponding opti-
cally pure R and S toluene-4-sulfonic acid 2,3-dihydroxypropyl ester (R-4,S-4). The enatiomerically and
diastereomerically pure acetonide (5) was obtained by a method involving diastereoselective crystallisa-
tion of the tosylate salt, followed by re-equilibration with the mother liquor and chromatography. The
optical purity of four target compounds (YCZ-2013) was confirmed by chiral high-performance liquid
chromatography (HPLC) and NMR. The effects of these stereoisomers on Arabidopsis stem elongation indi-
cated that the cis isomers of 2S,4R-YCZ-2013 and 2R,4S-YCZ-2013 exhibited potent inhibitory activity
with IC50 values of approximately 24 3 and 24 2 nM, respectively. The IC50 values of the trans isomers
of 2S,4S-YCZ-2013 and 2R,4R-YCZ-2013 are approximately 1510 50 and 3900 332 nM, respectively.
Received 10 August 2013
Revised 11 September 2013
Accepted 21 September 2013
Available online 29 September 2013
Keywords:
Brassinosteroid biosynthesis inhibitor
Plant hormone
Azole derivatives
Co-application of brassinolide (10 nM), the most potent BR, and GA3 (1 lM) to Arabidopsis seedlings
grown in the dark with 2R,4S-YCZ-2013 and 2S,4R-YCZ-2013 revealed that brassinolide recovered the
induced dwarfism of Arabidopsis seedlings, whereas GA3 showed no effect.
Ó 2013 Elsevier Ltd. All rights reserved.
Brassinosteroids (BRs) are a group of naturally occurring steroi-
dal plant hormones that serve as important signal mediators with
well-defined functions, including stem elongation, pollen tube
growth, leaf bending, leaf unrolling, root inhibition, proton pump
activation.1–3 Because BRs control several important agronomic
traits such as flowering, plant architecture, seed yield, and stress
tolerance,2,3 efforts have been made to control BR levels in plant
tissues using genetic approaches. Overexpression of DWARF4, an
enzyme that catalyses a rate-limiting step in BR biosynthesis, en-
hances plant growth and seed yield in Arabidopsis.4 Similarly,
transgenic rice plants overexpressing a sterol C-22 hydroxylase
that catalyses a key step in BR biosynthesis displayed increased
biomass and seed yields,5 and available evidence indicates that
mutations in BR biosynthesis may be a means to improve biomass
production.6
Specific inhibitors targeting BR biosynthesis are quite useful for
manipulating BR levels in plant tissues. In fact, it is thought that
the use of inhibitors to control BR levels has advantages over ge-
netic mutants because inhibitors can be used at different stages
of plant growth and development.7 Moreover, they can be applied
to different plant species with great ease. Consequently, we carried
out a systematic search for specific inhibitors of BR biosynthesis.
Recently, we discovered a new class of highly potent and selective
BR biosynthesis inhibitors (YCZ, the general structure is shown in
Fig. 1) of the triazole type.8–10 Our previous effort was limited to
the synthesis and determination of the inhibition activity of Ara-
bidopsis stem elongation of epimeric mixtures of YCZ. Because
these compounds contain two chiral centres on the 1,3-dioxolane
ring (C-2 and C-4, see Fig. 1), each compound gives rise to a total
of four stereoisomers. In some cases, stereoisomers of azole com-
pounds have different biological activity.11,12 Thus, the study of
the effect of absolute stereochemistry at each chiral centre of
YCZ on BR biosynthesis inhibition represents a straightforward ap-
proach to determine 3D structural information of the inhibitor of
BR biosynthesis inhibition.
To explore the stereochemistry-activity relationships of YCZ on
BR biosynthesis, we chose 2RS,4RS-1-[[2-(2,4-dichlorophenyl)-4-
(2-(2-propenyloxy)phenoxymethyl)-1,3-dioxolan-2-yl]methyl]-1H-
1,2,4-triazole (YCZ-2013, the chemical structure is shown in Fig. 1)
as the target compound in this study. This choice was based on
the following reasons: First of all, we investigated the instrumen-
tal analysis data of related compounds in the literature and found
that the stereochemical properties of the itraconazole intermediate
(see Fig. 1), which shares a common chemical structure with an
intermediate of the target compound YCZ-2013 in the process of
chemical synthesis, have been studied in considerable detail.13
These instrumental analysis data are quite useful regarding the
identification of the absolute stereochemical configuration of
each isomer of YCZ-2013. Secondly, our previous work has also
⇑
Corresponding author. Tel.: +81 18 872 1590; fax: +81 18 872 1676.
E-mail address: jmwang@akita-pu.ac.jp (K. Oh).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.09.056

6916
K. Oh et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6915–6919
Figure 1. Chemical structures of YCZs and their related compounds.
demonstrated that an analogue with an allyloxy substitution at po-
sition 2 of ring B (see general structure of YCZ in Fig. 1) exhibited
excellent inhibitory activity on BR biosynthesis.10
The development of a synthetic route for the preparation of dia-
stereomeric and enantiomeric pure key intermediates 2S,4R-5 and
2R,4R-5 are outlined in Scheme 1A. Compound 1 was prepared by
coupling 1,2,4-triazole with 2,4-dichlorophenacyl bromide using a
method previously described.14 The key intermediate of the diaste-
reomer mixture of 2RS,4R-5 was prepared based on the following
steps: Tosylation of commercially available optically pure isopro-
pylidene glycerol S-2 was achieved by a standard protocol (tosyl
chloride in pyridine at 0 °C) to produce the corresponding R-3.
Scheme 1. General route for chemical synthesis. Reagents and conditions: (a) TsCl, pyridine, 0 °C, acetone; (b) HCl, Reflux, 6 h; (c) 3 equiv TfOH, toluene, rt, 60 h; (d)
recrystallisation and PTLC purification; (e) phenol, KOH, DMF, 50 °C, 12 h.

K. Oh et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6915–6919
6917
Hydrolysis of R-3 with 1 M HCl in MeOH yielded the corresponding
toluene-4-sulfonic acid 2,3-dihydroxypropyl ester (R-4). Then, a
diastereoselective ketalisation of 1 with optically pure R-4 was car-
ried out using 3 equivalents of trifluoromethanesulfonic acid
(TfOH) in toluene at room temperature for 60 h to generate the cor-
responding crude diastereomeric mixture of 2RS,4R-5 as we previ-
ously described.8 At this point, the stereochemistry at C-4 in
2RS,4R-5 emanates from the chiral starting material R-4, while C-
2 is a new chiral centre that was created during ketalisation. After
neutralisation and work-up of the reaction mixture, the crude
product was dissolved in iso-butyl methyl ketone and treated with
tosic acid. Crystals from the crude solution are mainly tosylate salts
of 2R,4R-5. The diastereomeric highly pure 2R,4R-5 was obtained
by recrystallisation from acetonitrile using a method described
previously.13,15 On the other hand, the 2S,4R-5 predominantly re-
mained in the mother liquid during the purification process. To iso-
late the diastereomerically pure 2S,4R-5, the mother liquid was
concentrated under reduced pressure. The remaining light yellow
oil was subjected to PTLC and column chromatography (3:7, ethyl
acetate:hexane). With these purification processes, 2S,4R-5 was
obtained at a high purity that was sufficient for NMR characterisa-
tion. The 1H NMR spectra illustrate the obvious differences not
only in the aromatic region (7–8.5 ppm) but also in the aliphatic
region (3.4–4.8 ppm), indicating different 1,3-dioxolane ring con-
figurations in the cis and trans diastereomers (data not shown).
Our 1H NMR data for each isomer were in good agreement with
those of literature.13,15 Preparation of diastereomerically pure
2S,4S-5 and 2R,4S-5 was carried out in a similar way by using R-
2 as the starting material (Scheme 1B). In this case, diastereomer-
ically pure 2S,4S-5 was obtained in a similar way to 2R,4R-5. Mean-
while, diastereomerically pure 2R,4S-5 was obtained from the
mother liquid in the processes of the recrystallisation of 2S,4S-5
followed by PTLC and column chromatography as described above.
The optical purity of stereoisomers of 2S,4R-5, 2R,4R-5, 2S,4S-5
and 2R,4S-5 were confirmed by chiral HPLC (Daicel Chem. Ltd,
CHIRALPAK OJ, /4.6 mm, 250 mm) under previously reported con-
ditions.16 The retention times of each stereoisomers are listed in
Table 1. We found that the optical purities of each purified stereo-
isomer were greater than 99% (data not shown). Thus, we have suc-
cessfully purified the four stereo-isomers by separating the
diastereomer mixture of 2RS,4R-5 to afford pure 2R,4R-5, and
2S,4R-5. By the same strategy, diastereomerically pure 2R,4S-5
and 2S,4S-5 were obtained. At this stage, although the diastereo-
meric purity of these stereoisomers has been well established,
their enantiomeric purity is still undetermined. To confirm the
enantiomeric purity of these stereoisomers, we determined the
retention time of the enantiomeric mixture by mixing 2S,4S-5 with
2R,4R-5, and 2S,4R-5 with 2R,4S-5. The mixture was subjected to
chiral HPLC analysis. As shown in Table 1, both mixtures were
separated by chiral HPLC. This result clearly established the
enantiomeric purity of all the stereoisomers. Next, we compared
the rotation of the prepared stereoisomers with the data previously
reported. As shown in Table 1, we found that the properties of
2R,4R-5 and 2S,4S-5 are in good agreement with the data previ-
ously reported.13,15 However, to the best of our knowledge, the
specific rotations of 2R,4S-5 and 2S,4R-5 have not been reported
in the literature.
With the building blocks of key intermediates in hand, the dis-
placement of the O-tosyl group in compounds 5 with the phenolic
oxygen in 2-allyloxyphenol under basic conditions afforded the fi-
nal products YCZ-2013.8,17 The optical purity of each purified ste-
reoisomer were determined by using chiral HPLC, and their specific
rotations were determined. As shown in Table 1, the retention
times of the four stereoisomers of YCZ-2013 are 11.57, 19.16,
13.40, and 13.79 min. Enantiomers prepared by mixing 2R,4S-
YCZ-2013 with 2S,4R-YCZ-2013 and 2S,4S-YCZ-2013 with 2R,4R-
YCZ-2013 can be separated by chiral HPLC (Table 1). This result
clearly established the stereochemical purity of the four stereoiso-
mers of YCZ-2013.
To evaluate the biological activity of the synthesised com-
pounds on inhibition of BR biosynthesis, an assay system using
Arabidopsis seedlings grown in the dark was used.11 In the dark,
the hypocotyls and roots of Arabidopsis seedlings grow signifi-
cantly, and the apical hook of the cotyledon is maintained without
opening. 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.18 This unique phenotype
of de-etiolation in the dark has been used in screens for BR biosyn-
thesis inhibitors.8 In the present study, we adopted this assay to
determine the effects of test compounds on the hypocotyl elonga-
tion of Arabidopsis seedlings grown in the dark.
The chemical structures of the compounds used for biological
studies are shown in Table 2. The test compounds were used at
concentrations of 0, 0.001, 0.005, 0.01, 0.05, 1, 10, and 100 lM,
and the IC50 values were calculated accordingly. As shown in
Table 2, the cis isomers of 2R,4S-YCZ-2013 and 2S,4R-YCZ-2013
exhibited potent inhibitory activity, retarding the hypocotyl elon-
gation of Arabidopsis seedling grown in the dark, with IC50 values
of 24 3 and 24 2 nM, respectively. In contrast, the trans isomers
of 2S,4S-YCZ-2013 and 2R,4R-YCZ-2013 exhibited relatively weak
inhibitory activity with IC50 values of 1510 50 and
3900 332 nM, respectively. This result clearly indicated that the
inhibitory activity of the cis-isomers (2R,4S-YCZ-2013 and 2S,4R-
YCZ-2013) is more potent those that of the trans-isomers (2S,4S-
YCZ-2013 and 2R,4R-YCZ-2013). Sekimata and his co-workers re-
ported the stereochemical structure–activity relationship of
Brz220 on the retardation of Arabidopsis hypocotyls growth.11 Pre-
viously reported data for Brz220 are listed in Table 2 as a reference
for discussion. The IC50 values of the four stereoisomers of
YCZ-2013 range from 24 to 3900 nM, while isomers of Brz220
Table 1
Specific rotations and retention times of 5s and YCZ-2013 in chiral HPLC
Compound
[
a]
D
[a]
D
(reference)
rt (min)
2S,4R-5
2R,4R-5
2S,4S-5
2R,4S-5
À6.84 0.19 (c = 1, 25.0 °C, CHCl3)
—
18.61
15.69
16.28
12.99
15.34, 16.70
12.65, 18.66
19.16
11.57
13.40
+15.42 0.24 (c = 1, 24.6 °C, CHCl3)
+16.44 (c = 1%, EtOH)⁄
À16.30 0.25 (c = 1, 24.6 °C, CHCl3)
À16.37 (c = 1%, MeOH)⁄
+4.66 0.39 (c = 1, 25.3 °C, CHCl3)
—
—
—
—
—
—
—
—
—
2S,4S-5+2R,4R-5
2S,4R-5+2R,4S-5
2S,4S-YCZ-2013
2R,4S-YCZ-2013
2S,4R-YCZ-2013
2R,4R-YCZ-2013
2S,4S-YCZ-2013+2R,4R-YCZ-2013
2S,4R-YCZ-2013+2R,4S-YCZ-2013
—
—
+10.47 0.51 (c = 1, 22.2 °C, CHCl3)
+10.93 0.64 (c = 1, 22.1 °C, CHCl3)
À14.60 1.80 (c = 1, 22.3 °C, CHCl3)
À17.58 0.61 (c = 1, 22.0 °C, CHCl3)
13.79
13.53, 18.81
11.54, 13.16
—
—

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K. Oh et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6915–6919
Table 2
IC50 Values obtained from Arabidopsis hypocotyl growth
Compound
Structure
IC50 (nM)
1510 50
Compound
Structure
IC50 (nM)
2S,4S-YCZ-2013
Brz22022 (2S,4S)
2330
2R,4S-YCZ-2013
2S,4R-YCZ-2013
2R,4R-YCZ-2013
24
24
2
3
Brz22011 (2R,4S)
Brz22012 (2S,4R)
Brz22021 (2R,4R)
6950
1210
3900 332
18700
Data for Brz220 are adopted from Ref. 11.
range from 1210 to 18700 nM. This result clearly indicates that the
inhibitory potency of YCZ-2013 is greater than Brz220. The rate of
the IC50 values of the active form of YCZ-2013 with Brz220 is
approximately 1/50, which is in agreement with our previous
observations when using epimeric mixtures.10
Additionally, the IC50 of the inactive form of Brz220 (2R,4R-
Brz220, IC50 = 18700 nM) which is about 15 times to that of active
form (2S,4R-Brz220, IC50 = 1210 nM). In contrast, the IC50 value of
the inactive form of YCZ-2013 (2R,4R-YCZ-2013) is 3900 nM which
is approximately 60 times to that of active form (2R,4S-YCZ-2013,
IC50 = 24 nM; 2S,4R-YCZ-2013, IC50 = 24 nM). This observation
indicated that the propyl moiety of Brz220 may not fit well to
the binding pocket which allows Brz220 to be more flexible.
Another factor that greatly influences the usage of YCZ in
manipulating the BR level in plants is its selectivity. The assay
method used in the present work is based on the determination
of the inhibitory activity of YCZ-2013 on the retardation of stem
elongation of Arabidopsis seedlings. To determine the selectivity
of YCZ-2013 on BR biosynthesis inhibition, other factors involved
in stem elongation of Arabidopsis need to be determined. GA bio-
synthesis inhibitors such as paclobutrazol retard the stem elonga-
tion of many plant species by blocking ent-kaurene oxidation and
also mildly affect other cytochrome P450 monooxygenases.25 This
retardation can be rescued by the application of GA. To rule out the
possibility of GA biosynthesis inhibition by our analogues, we
tested the effects of brassinolide (BL), the most biologically active
BR, and GA3 on the recovery of chemically induced dwarfism of
Arabidopsis seedlings grown in the dark. The cis isomers of 2R,4S-
YCZ-2013 and 2S,4R-YCZ-2013 were subjected to the bioassay at
The establishment of the biosynthetic pathways of BRs and
functional analysis of BR biosynthesis enzymes provided evidence
that several steps in BR biosynthesis are catalysed by P450
enzymes.19 CYP90s are involved in C22 and C23 side-chain hydrox-
ylation of BRs.20 CYP92A6 catalyses the biochemical conversion of
castasterone from typhasterol,21 and CYP85A2 catalyses the lac-
tonisation of castasterone to brassinolide.22 Furthermore, the inhi-
bition mechanisms of cytochrome P450 have been studied in
considerable detail.23 It has been demonstrated that triazole deriv-
atives have a widespread ability to inhibit P450s due to the intrin-
sic affinity of the nitrogen electron pair in heterocyclic molecules
for the prosthetic heme iron. The triazoles thus bind not only to
lipophilic regions of the protein but also simultaneously to the
prosthetic heme iron.24 In this context, one factor that affects the
binding affinity of the inhibitor to the target enzyme is the struc-
ture of the lipophilic moiety of the inhibitor. The cis and/or trans
isomer of the inhibitor is the most significant factor involved in
the 3D configuration. Data obtained in the present work provide
evidence that the cis isoforms of the YCZ-2013 are more potent
than those of trans isoforms, indicating that the structural require-
ments for the lipophilic binding of the inhibitor with the target en-
zyme are at the opposite site of the 1,3-dioxolane moiety. It is
worth mentioning that the main difference in chemical structures
of YCZ-2013 and Brz220 are 2-(2-propenyloxy)phenoxymethyl
moiety (for YCZ-2013) and propyl moiety (for Brz-220). Regarding
the inhibitory potency of YCZ-2013 is greater than Brz220, it is rea-
sonable to estimate that the binding pocket is relatively fit well to
2-(2-propenyloxy)phenoxymethyl moiety than propyl moiety.
a concentration of 0.5
lM, and Arabidopsis seedlings were grown
in the presence or absence of BL (10 nM) or GA3 (1
lM) for 5 days
in the dark. The data shown in Table 3 are expressed as a percent-
age relative to the untreated control. As shown in Table 3, in the
presence of BL (10 nM) or GA3 (1 lM), the average hypocotyl
length of Arabidopsis seedlings was approximately 108.4 8.1%
and 105.2 6.8%, respectively. This result indicates that BL and
GA3 stimulate the hypocotyl elongation of Arabidopsis seedlings.
We found that compounds 2R,4S-YCZ-2013 and 2S,4R-YCZ-2013

K. Oh et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6915–6919
6919
Table 3
4. Choe, S.; Fujioka, S.; Noguchi, T.; Takatsuto, S.; Yoshida, S.; Feldmann, K. A. Plant
J. 2001, 26, 573.
Retardation of Arabidopsis seedling growth by YCZ-2013s and rescue of growth with
5. Wu, C. Y.; Trieu, A.; Radhakrishnan, P.; Kwok, S. F.; Harris, S.; Zhang, K.; Wang,
J.; Wan, J.; Zhai, H.; Takatsuto, S.; Matsumoto, S.; Fujioka, S.; Feldmann, K. A.;
Pennell, R. Plant Cell 2008, 20, 2130.
BL and GA3
No.
Hypocotyl length relative to untreated Arabidopsis
6. Sakamoto, T.; Morinaka, Y.; Ohnishi, T.; Sunohara, H.; Fujioka, S.; Ueguchi-
Tanaka, M.; Mizutani, M.; Sakata, K.; Takatsuto, S.; Yoshida, S.; Tanaka, H.;
Kitano, H.; Matsuoka, M. Nat. Biotechnol. 2006, 24, 105.
seedlings (%)
Chem.*
Chem. + BL (10 nM)
Chem. + GA3 (1 lM)
7. Blackwell, H. E.; Zhao, Y. Plant Physiol. 2003, 133, 448.
8. Oh, K.; Yamada, K.; Asami, T.; Yoshizawa, Y. Bioorg. Med. Chem. Lett. 2013, 22,
1625.
9. Yamada, K.; Yoshizawa, Y.; Oh, K. Molecules 2013, 17, 4460.
10. Yamada, K.; Yajima, O.; Yoshizawa, Y.; Oh, K. Bioorg. Med. Chem. 2013, 21, 2451.
11. Sekimata, K.; Ohnishi, T.; Mizutani, M.; Todoroki, Y.; Han, S. Y.; Uzawa, J.;
Fujioka, S.; Yoneyama, K.; Takeuchi, Y.; Takatsuto, S.; Sakata, K.; Yoshida, S.;
Asami, T. Biosci. Biotechnol. Biochem. 2008, 72, 7.
Control
2R,4S-YCZ-2013
2S,4R-YCZ-2013
100
21.8 0.7
22.6 0.8
108.4 8.1
100.5 2.2
97.4 1.7
102.6 2.9
105.2 6.8
24.1 0.4
25.0 0.1
25.4 1.0
2RS,4RS-YCZ-2013 24.9 1.8
*
The final concentration of the chemicals was 0.5 lM.
exhibited high inhibitory activity on Arabidopsis seedling elonga-
tion. The hypocotyl length of the chemically treated or untreated
Arabidopsis seedlings was approximately 21.8 0.7% and
22.6 0.8%, respectively. The racemic mixture of YCZ-2013
12. Oh, K.; Shimura, Y.; Ishikawa, K.; Ito, Y.; Asami, T.; Murofushi, N.; Yoshizawa, Y.
Bioorg. Med. Chem. 2008, 1090, 16.
13. Shi, W.; Nacev, B. A.; Bhat, S.; Liu, J. O. ACS Med. Chem. Lett. 2010, 1, 155.
14. Oh, K.; Nakai, K.; Yamada, K.; Yoshizawa, Y. J. Pestic. Sci. 2012, 37, 80.
15. Tanoury, G. J.; Hett, R.; Wilkinson, H. S.; Wald, S. A.; Senanayake, C. H.
Tetrahedron: Asymmetry 2003, 14, 3487.
(0.5 lM) retarded the stem elongation of Arabidopsis seedlings in
16. Kunze, K. L.; Nelson, W. L.; Kharasch, E. D.; Thummel, K. E. Drug Metab. Dispos.
2006, 34, 583.
the dark by approximately 24.9 1.8%. Co-application of BL
(10 nM) showed significant recovery; 2R,4S-YCZ-2013 co-applied
with BL reversed the hypocotyl lengths to 100.5 2.2%, and in
the case of 2S,4R-YCZ-2013 and BL treatment, it was reversed to
97.4 1.7%. A similar recovery rate can be observed with the treat-
ment of a racemic mixture of YCZ-2013 (Table 3). However, co-
17. 2R,4S-1-[4-(2-Allyloxyphenoxymethyl)-2-(2,4-dichlorophenyl)-[1,3]-dioxolan-2-
ylmethyl]-1H-[1,2,4]-triazole (2R,4S-YCZ-2013): Yield: 22%, mp: 71.8–73.3 °C.
1H NMR (400 MHz, CDCl3) d: 3.68 (dd, J = 6.3, 10.0 Hz, 1H), 3.95–4.00 (m, 3H),
4.43–4.48 (m, 1H), 4.53–4.56 (m, 2H), 4.80–4.91 (m, 2H), 5.22–5.26 (m, 1H),
5.35–5.40 (m, 1H), 5.98–6.07 (m, 1H), 6.86–6.98 (m, 4H), 7.22 (dd, J = 2.3,
8.5 Hz, 1H), 7.47 (d, J = 2.1 Hz, 1H), 7.50 (d, J = 8.5 Hz, 1H), 7.85 (s, 1H), 8.18 (s,
1H). The HRMS-ESI calcd for C22H21Cl2N3O4Na [M+Na]+ was 484.0801, with
484.0808 determined experimentally.
application of GA3 (1 lM) instead of BL did not show a significant
recovery of the chemical induced dwarfism of Arabidopsis seedlings
(see Table 3). These results clearly indicate that the dwarfism of
Arabidopsis seedlings induced by 2R,4S-YCZ-2013 and 2S,4R-YCZ-
2013 can be recovered by the application of BL (10 nM) but not
2S,4R-1-[4-(2-Allyloxyphenoxymethyl)-2-(2,4-dichlorophenyl)-[1,3]-dioxolan-2-
ylmethyl]-1H-[1,2,4]-triazole (2S,4R-YCZ-2013): Yield: 26%, mp: 71.7–72.7 °C 1
H
NMR (400 MHz, CDCl3) d: 3.68 (dd, J = 6.3, 10.0 Hz, 1H), 3.95–4.00 (m, 3H),
4.43–4.48 (m, 1H), 4.53–4.56 (m, 2H), 4.80–4.91 (m, 2H), 5.22–5.26 (m, 1H),
5.35–5.40 (m, 1H), 5.98–6.07 (m, 1H), 6.86–6.98 (m, 4H), 7.22 (dd, J = 2.3,
8.5 Hz, 1H), 7.47 (d, J = 2.1 Hz, 1H), 7.50 (d, J = 8.5 Hz, 1H), 7.85 (s, 1H), 8.18 (s,
1H). The HRMS-ESI calcd for C22H21Cl2N3O4Na [M+Na]+ was 484.0801, with
484.0805 determined experimentally.
2S,4S-1-[4-(2-Allyloxyphenoxymethyl)-2-(2,4-dichlorophenyl)-[1,3]-dioxolan-2-
ylmethyl]-1H-[1,2,4]-triazole: Yield: 9% (2S,4S-YCZ-2013), mp: 112.9–113.9 °C
1H NMR (400 MHz, CDCl3) d: 3.84–3.88 (m, 2H), 4.01–4.06 (m, 2H), 4.23–4.29
(m, 1H), 4.46–4.48 (m, 2H), 4.71–4.80 (m, 2H), 5.23–5.26 (m, 1H), 5.30–5.36
(m, 1H), 5.93–6.02 (m, 1H), 6.79–6.95 (m, 4H), 7.17 (dd, J = 2.3, 8.5 Hz, 1H),
7.43 (d, J = 2.1 Hz, 1H), 7.58 (d, J = 8.5 Hz, 1H), 7.91 (s, 1H), 8.18 (s, 1H). The
HRMS-ESI calcd for C22H21Cl2N3O4Na [M+Na]+ was 484.0801, with 484.0808
determined experimentally.
2R,4R-1-[4-(2-Allyloxyphenoxymethyl)-2-(2,4-dichlorophenyl)-[1,3]-dioxolan-2-
ylmethyl]-1H-[1,2,4]-triazole: Yield: 6% (2R,4R-YCZ-2013), mp: 116.1–117.9 °C
1H NMR (400 MHz, CDCl3) d: 3.84–3.88 (m, 2H), 4.01–4.06 (m, 2H), 4.23–4.29
(m, 1H), 4.46–4.48 (m, 2H), 4.71–4.80 (m, 2H), 5.23–5.26 (m, 1H), 5.30–5.36
(m, 1H), 5.93–6.02 (m, 1H), 6.79–6.95 (m, 4H), 7.17 (dd, J = 2.3, 8.5 Hz, 1H),
7.43 (d, J = 2.1 Hz, 1H), 7.58 (d, J = 8.5 Hz, 1H), 7.91 (s, 1H), 8.18 (s, 1H). The
HRMS-ESI calcd for C22H21Cl2N3O4Na [M+Na]+ was 484.0801, with 484.0809
determined experimentally.
by GA (1 lM).
In conclusion, we conducted stereochemically restricted syn-
thesis of four stereo-isomers of YCZ-2013. Stereochemical struc-
ture–activity relationship studies indicate that the cis isomers
(2R,4S-YCZ-2013 and 2S,4R-YCZ-2013) exhibited potent inhibition,
retarding the stem elongation of Arabidopsis seedlings grown in the
dark. The IC50 values of the cis isomers are smaller than 1/60 that of
trans isomers, indicating that the configuration of the cis isomers
may fit to the binding site of the target enzymes better than the
trans. 2R,4S-YCZ-2013 and 2S,4R-YCZ-2013 are the most potent
inhibitors of BR biosynthesis at present. We expect that further
application studies of this synthetic series may lead to the estab-
lishment of new technology to regulate plant growth and
development.
18. Choe, S.; Dilkes, B. P.; Gregory, B. D.; Ross, A. S.; Yuan, H.; Noguchi, T.; Fujioka,
S.; Takatsuto, S.; Tanaka, A.; Yoshida, S.; Tax, F. E.; Feldmann, K. A. Plant Physiol.
1999, 119, 897.
Acknowledgments
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This study was supported in part by the Akita Prefectural Uni-
versity President’s Research Project to K. Oh. We thank Professor
Emeritus of Akita Prefectural University and Tokyo University No-
boru Murofushi for his thoughtful suggestions on preparing this
manuscript.
22. Kim, T. W.; Hwang, J. Y.; Kim, Y. S.; Joo, S. H.; Chang, S. C.; Lee, J. S.; Takatsuto,
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