Design and chemical synthesis of root gravitropism inhibitors: Bridged analogues of ku-76 have more potent activity

Article abstract of DOI:10.1016/j.phytochem.2020.112508

Previously, we found (2Z,4E)-5-phenylpenta-2,4-dienoic acid (ku-76) to be a selective inhibitor of root gravitropic bending of lettuce radicles at 5 μM, with no concomitant growth inhibition, and revealed the structure–activity relationship in this inhibitory activity. The conformation of ku-76 is flexible owing to the open-chain structure of pentan-2,4-dienoic acid with freely rotating single bonds, and the (2Z)-alkene moiety may be isomerized by external factors. To develop more potent inhibitors and obtain insight into the target biomolecules, various analogues of ku-76, fixed through conformation and/or configuration, were synthesized and evaluated. Stereochemical fixation was effective in improving the potency of gravitropic bending inhibition. Finally, we found highly potent conformational and/or configurational analogues (ku-257, ku-294 and ku-308), that did not inhibit root growth. The inhibition of root curvature by these analogues was comparable to that of naptalam.

Full text of DOI:10.1016/j.phytochem.2020.112508

Phytochemistry 179 (2020) 112508
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Design and chemical synthesis of root gravitropism inhibitors: Bridged
analogues of ku-76 have more potent activity
,
Mitsuru Shindoa *, Saki Makigawab, Kozue Kodamaa, Hiromi Sugiyamaa, Kenji Matsumotoa,
Takayuki Iwataa, Naoya Wasanoc, Arihiro Kanoa, Miyo Terao Moritad, Yoshiharu Fujiic
a Institute for Materials Chemistry and Engineering, Kyushu University, Kasuga-koen, Kasuga, 816-8580, Japan
b Interdisciplinary Graduate School of Engineering Sciences, Kyushu University, Kasuga-koen, Kasuga, 816-8580, Japan
c International Environmental and Agricultural Sciences, Tokyo University of Agriculture and Technology, 3-5-8, Saiwai-cho, Fuchu, Tokyo, 183-8509, Japan
d Division of Plant Environmental Responses, National Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki, 444-8585, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Previously, we found (2Z,4E)-5-phenylpenta-2,4-dienoic acid (ku-76) to be a selective inhibitor of root gravi-
Lactuca sativa
Compositae
tropic bending of lettuce radicles at 5 μM, with no concomitant growth inhibition, and revealed the structur-
e–activity relationship in this inhibitory activity. The conformation of ku-76 is flexible owing to the open-chain
structure of pentan-2,4-dienoic acid with freely rotating single bonds, and the (2Z)-alkene moiety may be iso-
merized by external factors. To develop more potent inhibitors and obtain insight into the target biomolecules,
various analogues of ku-76, fixed through conformation and/or configuration, were synthesized and evaluated.
Stereochemical fixation was effective in improving the potency of gravitropic bending inhibition. Finally, we
found highly potent conformational and/or configurational analogues (ku-257, ku-294 and ku-308), that did
not inhibit root growth. The inhibition of root curvature by these analogues was comparable to that of naptalam.
Gravitropism
Inhibitor
Organic synthesis
Structure modification
1. Introduction
2017) (Fig. 1). While these chemicals can also be regarded as gravi-
tropism inhibitors, they tend to inhibit growth. Selective gravitropism
Gravitropism is a fundamental property of the plants, consisting of
the curvature that develops in the growing organs of plants in response
to gravity. Generally, roots grow downward (positive gravitropism) via
several sequential processes (Morita and Tasaka, 2004; Morita, 2010). In
roots, gravity-sensing cells are columella cells in root caps at the root tip
(Blancaflor et al., 1998), where amyloplast sedimentation is thought to
elicit signal transduction (the starch-statolith hypothesis) (Kiss et al.,
1989, 1997; Sack, 1997), leading to a change in the direction of auxin
transport (Friml et al., 2002) (Harrison and Masson, 2008). Auxins play
a crucial role in the process from signal transmission to asymmetric
organ growth (Su et al., 2017; Sato et al., 2015; Nakamura et al., 2019).
In order to identify plant growth regulators, gravitropism has been
used to screen chemicals (Surpin et al., 2005; Nishimura et al., 2012,
2014). Using this methodology, several organic compounds have been
found to be auxin transport inhibitors, such as 1-N-naphthylphthalamic
acid (naptalam, NPA) (Mentzer et al., 1950), gravacin (Surpin et al.,
2005; Rojas-Pierce et al., 2007), alkoxy-auxins (Tsuda et al., 2011), and
auxin biosynthesis inhibitors, such as yucasin DF (Tsugafune et al.,
inhibitors, which suppress only gravitropic bending while not affecting
other plant growth events, are valuable tools for research on the
mechanism of gravitropism. Furthermore, inhibitors would be helpful
for developing a new class of agrochemicals, with potential uses as plant
growth regulators and weed suppressors.
During the study of allelochemicals (Nishikawa et al., 2013a, 2013b;
Okuda et al., 2014; Fukuda et al., 2016), we found that cis-cinnamic acid
(cis-CA) shows a weak gravitropic bending inhibition of lettuce roots as
well as growth inhibition. Furthermore, we developed ku-76, a syn-
thetic analogue of cis-CA, which inhibits gravitropic bending of lettuce
roots at a concentration of 5 μM (Fujii et al., 2016). Since ku-76 did not
show a significant suppression of growth (elongation) at this concen-
tration, we recognized it as a new lead compound for a potent selective
gravitropic bending inhibitor. In a previous paper, we revealed the
structure–activity relationship, which designated the essential structural
features to be (2Z,4E)-configuration, an aromatic ring, and a carboxylic
acid for inhibitory activity (Shindo et al., 2020) (Fig. 2).
The conformation of ku-76 would not be fixed, owing to the open-
* Corresponding author
E-mail address: shindo@cm.kyushu-u.ac.jp (M. Shindo).
https://doi.org/10.1016/j.phytochem.2020.112508
Received 1 April 2020; Received in revised form 22 August 2020; Accepted 25 August 2020
Available online 6 September 2020
0031-9422/© 2020 Elsevier Ltd. All rights reserved.

M. Shindo et al.
Phytochemistry 179 (2020) 112508
Fig. 3. Design of stereochemically fixed analogues of ku-76.
followed by hydrolysis (Fig. 7(b)).
Fig. 1. Examples of non-selective gravitropism inhibitors.
2.2. Inhibitory activity test for root gravitropic bending
chain structure of penta-2,4-dienoic acid with its freely rotating single
bonds (C3–C4, C5-benzene), in spite of the conjugation between the
aromatic ring and double bonds. Moreover, the (2Z)-alkene moiety may
be isomerized into a more stable (E)-form by external factors such as UV
light. To develop more potent inhibitors and to obtain insight into their
pharmacophores, analogues of ku-76, fixed through conformation and/
or configuration, will be useful. Herein, we describe the synthesis and
evaluation of various stereochemically fixed synthetic analogues of ku-
76.
The inhibitory activity test for root gravitropic bending was con-
ducted according to the method reported in our previous paper (Shindo
et al., 2020): 2-day lettuce seedlings (Lactuca sativa L. cv. Great Lakes,
Compositae) were transferred to agar plates containing test compounds
(0.05 μM–50 μM) and arranged so that the roots were parallel to the
gravity vector. The seedlings were preincubated vertically for 1 h and
were gravistimulated by turning at 90◦. After 24 h, root images were
captured by camera, and the angles (θ, degree) of gravitropic curvature
of the root as well as the extent of root growth during the 24 h period
were measured (Fig. 8).
2. Results and discussion
2.3. Evaluation and discussion
2.1. Design and synthesis of various bridged analogues of ku-76
2.3.1. Naphthyl analogues (1)
Ring-closure by bridging between C4 and the aromatic ortho-posi-
tion, designated in Fig. 3 by a black dashed curve line, constrains the C5-
benzene single bond rotation.
The α- and β-naphthyl analogue (1a and 1b) showed inhibitory ac-
tivity at 0.05 μM–50 μM as well as ku-76 for comparison, although
significant elongation inhibition was also observed (Fig. 9). Therefore,
naphthyl-type analogues were judged to be much less potent for selective
root-bending inhibition. Thus, we did not perform further research on 1.
The conformationally constrained analogues were designed as
shown in Fig. 4. Naphthalene analogues 1a and 1b, five, six, and seven-
membered carbocyclic analogues 2a-c, heterocyclic five membered-
analogues 3a-d, quinoline, and quinoxaline analogues (nitrogen-con-
taining six membered-heterocycles: 4a-d) were synthesized.
2.3.2. Benzocarbocyclic analogues (2)
Benzocarbocyclic analogues with a styryl skeleton 2 inhibited grav-
Furthermore, we planned to synthesize ortho-benzoic acid analogues
5a-d (Fig. 5) with a benzene ring in place of (2Z)-alkene, indicated in
Fig. 3 by a gray dashed curve line. While (Z)-alkenes are easily iso-
merized by UV or radical sources, the (2Z)-alkene in benzene is
configurationally fixed to the external factors.
itropic bending at 50 μM and tended to inhibit elongation, except for 2b,
which was also potent for gravitropic bending inhibition at 5
μM
(Fig. 10). Consequently, the five-membered ring was thought to be a
better bridging unit than the six- and seven-membered ones.
The naphthalene analogues 1 were synthesized according to our
previous report (Nishikawa et al., 2013a). Other analogues having
(Z)-alkene 2–4 were prepared by cis-selective olefination of the corre-
sponding aldehydes, followed by hydrolysis (Fig. 6) (Fig S1; Table S1).
Some of the aldehydes were prepared according to the literature.
The styryl analogues (5a and 5b) were prepared by Heck reaction of
vinyl arenes with ethyl o-bromobenzoate, followed by hydrolysis (Fig. 7
(a)) (Shahzad et al., 2010). o-Arylbenzoates (5c and 5d) were synthe-
sized by the Suzuki coupling of arylboronic acids and o-bromobenzoate,
2.3.3. Benzoheterocyclic analogues (3)
As a bridging unit, five-membered heteroaromatics containing sul-
fur, oxygen, and nitrogen atoms were examined in a dose–response
relationship study at 0.2–10 μM. Benzothiophene, benzofuran, indole,
and benzoxazoline analogues (3a, 3b, 3c, and 3d) were found to be
potent at a concentration of at least 5
μM (Fig. 11). Benzoxazoline
analogue (3d) did not exhibit elongation inhibitory activity, but rather
elongated the roots, while the others somewhat inhibited elongation at
5 μM. Among these analogues, 3a (ku-257) exhibited the most potent
inhibitory activity, which was highly effective even at 0.2
μM for
gravitropic bending inhibition. In total, the introduction of a heteroatom
in the five-membered ring was linked with more potent inhibitory ac-
tivity along with much less inhibition of elongation.
2.3.4. Quinoline and quinoxaline analogues (4)
Quinoline and quinoxaline analogues 4 with six-membered hetero-
cycles tended to be less potent than any of the five-membered hetero-
aromatics (3) tested at 50 μM (Fig. 12). Quinoline analogue 4b was
stronger than the other analogues 4a, 4c and 4d, indicating that the
penta-2,4-dienoic carbon skeleton, not involving heteroatoms, is highly
important for potency. Compared with the naphthalene analogue 1a, 4b
Fig. 2. Lead compound, ku-76 and its SAR.
2

M. Shindo et al.
Phytochemistry 179 (2020) 112508
Fig. 4. Conformationally fixed analogues.
Fig. 5. Configurationally fixed (stilbene type) analogues.
Fig. 6. Synthesis of bridged carboxylate analogues from aldehydes. (a) ethyl 2-[bis(2-isopropylphenoxy)phosphoryl]acetate, Triton B, THF, ꢀ 78 ◦C, (b) 10% NaOH
aq., EtOH, rt.
showed less elongation inhibitory activity.
concentrations. Consequently, these series of analogues were stronger
inhibitors than ku-76, suggesting that their conformational and/or
configurational fixation is effective for gravitropic bending inhibition.
These structures, especially 5b, reminded us of the structure of NPA with
an amide bond instead of E-alkene in 5b (Fig. 1). From the standpoint of
structural similarity, NPA was employed in the inhibitory activity test
for comparison (Fig. 13(e)). As a result, the bending inhibitory activity
of NPA was stronger than that of 5b, although it was virtually compa-
rable to that of 5a and 5c; however, NPA also suppressed elongation. In
terms of selective inhibitory activity without suppression of elongation,
5a and 5c seem to be equal or slightly more potent inhibitors than NPA.
2.3.5. o-Styrylbenzoic acid analogues (5)
o-Styrylbenzoic acid 5a (ku-294) inhibited gravitropic bending even
at 0.05 μM, while elongation was not inhibited. The bending inhibitory
activity of (E)-2-(2-(naphthalen-1-yl)vinyl)benzoic acid 5b was strong
as well; however, inhibition of elongation was also detected. Benzoic
acid analogues having a formal cis-alkene moiety seem to be more potent
than ku-76. The double-bridged analogue 5c (ku-308), having the fused
structure of 1a and 5a, was found to be somewhat more potent with no
concomitant elongation inhibition at 0.05 μM although the results were
variable. Similar analogue 5d, which can be regarded as a hybrid
compound of 3b and 5a, also exhibited strong inhibitory activity against
bending, although elongation was also slightly inhibited at lower
3

M. Shindo et al.
Phytochemistry 179 (2020) 112508
Fig. 7. Synthesis of configurationally fixed analogues 5.
Fig. 8. Inhibitory activity test for gravitropic bending. Gravitropic vectors before (g1) and after (g2) reorientation are indicated. Length (L, cm) of the root and the
angle (θ, degree) of the curvature after reorientation were measured. This figure is representative of a control experiment where no treatment with chemicals
was performed.
3. Experimental section
3.2. Synthesis
3.1. Materials
3.2.1. Synthesis of (Z)-3-(3,4-dihydronaphthalen-2-yl)acrylic acid (2a)
To a solution of 3,4-dihydronaphthalene-2-carbaldehyde (7a, 379
mg, 2.39 mmol) in THF (8.0 mL) was added Triton B (40% MeOH so-
lution, 1.32 mL, 3.35 mmol) at ꢀ 78 ◦C under argon atmosphere. After
stirring for 20 min, 2-[bis(2-isopropylphenoxy)phosphoryl] acetate
(220 mg, 0.543 mmol) in THF (5.0 mL) was added to the mixture. The
resulting mixture was stirred for 2.5 h at ꢀ 78 ◦C, and the reaction was
quenched with saturated aqueous NH4Cl. The mixture was extracted
with EtOAc, and the combined organic layer was washed with brine,
dried over MgSO4, filtered and concentrated in vacuo. The crude product
was purified by silica gel column chromatography (hexane/EtOAc = 97/
3 to 95/5) to give ethyl ester of 2a (466.9 mg, 86%, Z:E = >99:1) as a
pale yellow oil; 1H-NMR (270 MHz, CDCl3) δ: 1.30 (t, J = 7.2 Hz, 3H),
2.60 (t, J = 7.8 Hz, 2H), 2.83 (t, J = 7.8 Hz, 2H), 4.20 (q, J = 7.2 Hz, 2H),
5.75 (d, J = 12.7 Hz, 1H), 6.56 (d, J = 12.7 Hz, 1H), 6.71 (s, 1H),
7.07–7.19 (m, 4H); 13C-NMR (100 MHz, CDCl3) δ: 14.2 (q), 25.8 (t), 28.1
(t), 60.3 (t), 117.9 (d), 126.5 (d), 127.1 (d), 127.2 (d), 128.1 (d), 133.8
(2C, apparent d), 136.1 (s), 136.3 (s), 142.9 (d), 166.7 (s); IR (NaCl)
1715 cmꢀ 1; EIMS m/z 228 (M+), 153 (100%); Elem. Anal. calcd for
The 1H and 13C NMR spectra were recorded on a JNM EX-270 (270 and
67.5 MHz), AL-400 (400 and 100 MHz), and a JNM ECA-600 spectrometer
(600 and 150 MHz). Chemical shifts were reported in ppm downfield from
Me4Si (TMS) used as the internal standard or relative to the residual pro-
tonated solvent peaks (acetone-d6: 2.05 and 29.8, TFA-d: 11.5 and 116.6).
Splitting patterns are designed as “br, s, d, t, q, and m,” indicating “broad,
singlet, doublet, triplet, quartet, andmultiplet,” respectively. TheIRspectra
were recorded on a SHIMADZU IRPrestige-21 FT-IR spectrophotometer
using a KBr disk or a NaCl cell. Mass spectra were obtained on a JEOL JMS-
700oraJEOLJMS-T100CS. High-resolutionmassspectrawereobtainedon
a JEOL JMS-700 or a JEOL JMS-T100CS. Column chromatography was
performed on silica gel (Kanto Chemical Co.). Thin-layer chromatography
was performed on pre-coated plates (0.25 mm, silica gel Merck 60 F254).
Reaction mixtures were stirred magnetically. Preparation of ku-76 (Abe
et al., 2012), 1a and 1b (Nishikawa et al., 2013b) were reported previously.
3,4-Dihydronaphthalene-2-carbaldehyde, 1H-indene-2-carbaldehyde, 6,
7-dihydro-5H-benzo[7]annulene-8-carbaldehyde and benzoxazole-2-
carbaldehyde were synthesized according to the literature procedure (See
supplemental information). 2-Benzofurancarboxaldehyde was purchased
from Sigma-Aldrich Japan. Benzo[b]thiophene-2-carboxaldehyde, indo-
le-2-carboxaldehyde, 2-quinolinecarboxaldehyde, 3-quinolinecarbox-
aldehyde and 4-quinolinecarboxaldehyde were purchased from Tokyo
Chemical Industry. Quinoxaline-2-carbaldehyde was purchased from
Apollo Scientific.
C15H16O2: C, 78.92; H, 7.06, found: C, 78.68; H, 7.07.
To a solution of the ester above (356 mg, 1.56 mmol) in EtOH (6.0
mL) was added 10% NaOH aq (8.0 mL) at room temperature. After being
stirred for 1 h, the mixture was washed with hexane. After the water
layer was acidified with 3M HCl, the mixture was extracted with EtOAc.
The combined organic layer was washed with brine, dried over MgSO4,
filtered and concentrated in vacuo. The crude product (219.0 mg, 70%,
Z:E = >99:1) was pure enough. The compound was recrystallized from
hexane/EtOAc = 95/5 before used for the inhibitory test for gravitropic
4

M. Shindo et al.
Phytochemistry 179 (2020) 112508
Fig. 9. Inhibitory activity tests of gravitropic bending (left) and elongation (right) for naphthyl analogues (a) ku-76, (b) 1a and (c) 1b. 1a completely inhibited
elongation at 5 and 50 M. Data for gravitropic bending and elongation represent mean ± SD. Asterisk indicates statistically significant differences between
treatments and controls at p < 0.05 (Dunnett’s test, n = 7).
μ
5

M. Shindo et al.
Phytochemistry 179 (2020) 112508
Fig. 10. Inhibitory activity tests of gravitropic bending and elongation for benzocarbocyclic analogues 2 (50
μ
M, unless otherwise noted). Data for gravitropic
bending (left) and elongation (right) represent mean ± SD. Asterisk indicates statistically significant differences between treatments and controls at p < 0.05
(Dunnett’s test, n = 7).
bending.
12.2 Hz, 1H), 6.71 (d, J = 12.2 Hz, 1H), 6.75 (s, 1H), 7.14–7.21 (m, 4H);
13C-NMR (150 MHz, CDCl3) δ: 29.9 (t), 31.6 (t), 34.7 (t), 116.8 (d),
125.9 (d), 127.6 (d), 129.2 (d), 131.1 (d), 136.2 (s), 137.1 (d), 139.1 (s),
142.1 (s), 148.9 (d), 171.6 (s); IR (KBr) 1686 cmꢀ 1; EIMS m/z 214 (M+,
100%); Elem. Anal. calcd for C14H14O2: C, 78.48; H, 6.59. found: C,
78.28; H, 6.71.
2a: colorless prism (219.0 mg, 70%, Z:E = >99:1), mp. 104–105 ◦C
(hexane/EtOAc); 1H-NMR (270 MHz, CDCl3) δ: 2.65 (t, J = 7.8 Hz, 2H),
2.84 (t, J = 7.8 Hz, 2H), 5.78 (d, J = 12.5 Hz, 1H), 6.69 (d, J = 12.5 Hz,
1H), 6.76 (s, 1H), 7.08–7.19 (m, 4H); 13C-NMR (100 MHz, CDCl3) δ:
26.0 (t), 28.1 (t), 116.6 (d), 126.6 (d), 127.24 (d), 127.26 (d), 128.3 (d),
133.6 (s), 135.1 (d), 136.26 (s), 136.28 (s), 146.2 (d), 171.8 (s); IR (KBr)
1690 cmꢀ 1; EIMS m/z 200 (M+, 100%); Elem. Anal. calcd for C13H12O2:
C, 77.98; H, 6.04, found: C, 77.86; H, 6.00.
3.2.4. Synthesis of (Z)-3-(benzo[b]thiophen-2-yl)acrylic acid (3a)
The title compound was synthesized according to the procedure of
4.2.1. from benzo[b]thiophene-2-carboxaldehyde.
3.2.2. Synthesis of (Z)-3-(1H-inden-2-yl)acrylic acid (2b)
3a: starting from 877 mg of the aldehyde; colorless needles (87% for
1
◦
The title compound was synthesized according to the procedure of
4.2.1. from 1H-indene-2-carbaldehyde.
2 steps, Z:E = >99:1), mp. 154–156 C (CH3CN); H-NMR (400 MHz,
CDCl3) δ: 5.92 (d, J = 12.6 Hz, 1H), 7.17 (d, J = 12.6 Hz, 1H), 7.33–7.40
(m, 2H), 7.67 (s, 1H), 7.78–7.83 (m, 2H); 13C-NMR (150 MHz, Acetone-
d6) δ: 117.5 (d), 122.9 (d), 125.1 (d), 125.3 (d), 126.8 (d), 133.3 (d),
Ethyl ester of 2b: starting from 22 mg of the aldehyde: yellow oil
(22%, Z:E = >99:1); 1H-NMR (600 MHz, CDCl3) δ: 1.34 (t, J = 7.0 Hz,
3H), 3.94 (s, 2H), 4.24 (q, J = 7.0 Hz, 2H), 5.75 (d, J = 12.4 Hz, 1H),
6.86 (d, J = 12.4 Hz, 1H), 7.22 (s, 1H), 7.42 (d, J = 6.9 Hz, 1H), 7.47 (d,
J = 6.9 Hz, 1H); 13C-NMR (150 MHz, CDCl3) δ: 14.3 (q), 40.9 (t), 60.1
(t), 116.7 (d), 122.0 (d), 123.9 (d), 126.5 (2C, d), 138.0 (d), 140.7 (d),
143.2 (s), 143.5 (s), 145.4 (s), 166.3 (s); IR (NaCl) 1721 cmꢀ 1; EIMS m/z
214 (M+), 168 (100%); HRMS (FAB) Calcd for C14H14O2 (M+):
214.0994, found: 214.0993.
137.6 (d), 138.8 (s), 139.2 (s), 143.9 (s), 167.2 (s); IR (KBr) 1680 cmꢀ 1
;
EIMS m/z 204 (M+, 100%); Elem. Anal. calcd for C11H8O2S: C, 64.69; H,
3.95, found: C, 64.58; H, 3.87.
3.2.5. Synthesis of (Z)-3-(benzofuran-2-yl)acrylic acid (3b)
The title compound was synthesized according to the procedure of
4.2.1. from 2-benzofurancarboxaldehyde.
2b: starting from 399 mg of the ester; pale yellow prism (50%, Z:E =
Ethyl ester of 3b: starting from 438 mg of the aldehyde; colorless oil
(97%, Z:E = >99:1); 1H-NMR (400 MHz, CDCl3) δ: 1.35 (t, J = 7.1 Hz,
3H), 4.28 (q, J = 7.1 Hz, 2H), 5.96 (d, J = 13.0 Hz, 1H), 6.88 (d, J = 13.0
Hz, 1H), 7.21–7.25 (m, 1H), 7.31–7.35 (m, 1H), 7.44 (d, J = 8.2 Hz, 1H),
7.62 (d, J = 7.7 Hz, 1H), 7.95 (s, 1H); 13C-NMR (100 MHz, CDCl3) δ:
14.3 (q), 60.5 (t), 111.2 (d), 112.5 (d), 118.6 (d), 122.2 (d), 123.1 (d),
126.1 (d), 128.8 (s), 130.2 (d), 151.6 (s), 154.8 (s), 165.8 (s); IR (NaCl)
1718 cmꢀ 1; EIMS m/z 216 (M+, 100%); Elem. Anal. calcd for C13H12O3:
C, 72.21; H, 5.59, found: C, 72.30; H, 5.51.
1
◦
>99:1); mp. 121–123 C (hexane: AcOEt = 9:1); H-NMR (400 MHz,
CDCl3) δ: 3.97 (s, 2H), 5.80 (d, J = 12.6 Hz, 1H), 6.99 (d, J = 12.6 Hz,
1H), 7.26–7.32 (m, 3H), 7.44–7.51 (m, 2H); 13C-NMR (150 MHz, CDCl3)
δ: 40.9 (t), 115.2 (d), 122.2 (d), 124.0 (d), 126.6 (d), 126.9 (d), 140.5
(d), 142.3 (d), 143.0 (s), 143.3 (s), 145.7 (s), 171.0 (s); IR (KBr) 1686
cmꢀ 1; EIMS m/z 186 (M+), 168 (100%); HRMS (FAB) Calcd for
C
12H10O2 (M+): 186.0681, found: 168.0685.
3.2.3. Synthesis of (Z)-3-(6,7-dihydro-5H-benzo[7]annulen-8-yl)acrylic
acid (2c)
3b: starting from 500 mg of the ester; colorless prism (93%, Z:E =
>99:1), mp. 147–149 ◦C (CH3CN); 1H-NMR (400 MHz, CDCl3) δ: 5.99
(d, J = 13.0 Hz, 1H), 6.91 (d, J = 13.2 Hz, 1H), 7.24 (t, J = 7.6, 7.6 Hz,
1H), 7.34 (dd, J = 7.6, 7.6 Hz, 1H), 7.46 (d, J = 7.6 Hz, 1H), 7.62 (d, J =
7.6 Hz, 1H), 7.89 (s, 1H); 13C-NMR (100 MHz, acetone-d6) δ: 111.9 (d),
112.9 (d), 120.2 (d), 123.1 (d), 124.1 (d), 127.1 (d), 129.7 (s), 130.7 (d),
152.8 (s), 155.5 (s), 167.0 (s); IR (KBr) 1701 cmꢀ 1; EIMS m/z 188 (M+,
100%); Elem. Anal. calcd for C11H8O3: C, 70.21; H, 4.29, found: C,
69.91; H, 4.24.
The title compound was synthesized according to the procedure of
4.2.1. from 6,7-dihydro-5H-benzo[7]annulene-8-carbaldehyde.
Ethyl ester of 2c: starting from 399 mg of the aldehyde; pale yellow
oil (29%, Z:E = >99:1); 1H-NMR (270 MHz, CDCl3) δ: 1.27 (t, J = 7.2 Hz,
4H), 2.11–2.16 (m, 2H), 2.47 (t, J = 6.6 Hz, 2H), 2.79–2.83 (m, 2H),
4.18 (q, J = 7.2 Hz, 2H), 5.73 (d, J = 12.6 Hz, 1H), 6.56 (d, J = 12.6 Hz,
1H), 6.70 (s, 1H), 7.09–7.19 (m, 4H).
2c: starting from 159 mg of the ester; colorless needles (94%, Z:E =
>99:1); mp. 58–59 ◦C (hexane); 1H-NMR (400 MHz, CDCl3) δ: 2.11–2.18
(m, 2H), 2.49 (t, J = 6.5 Hz, 2H), 2.80 (t, J = 6.0 Hz, 2H), 5.76 (d, J =
3.2.6. Synthesis of (Z)-3-(1H-indol-2-yl) acrylic acid (3c)
The title compound was synthesized according to the procedure of
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Phytochemistry 179 (2020) 112508
Fig. 11. Inhibitory activity tests of gravitropic bending and elongation for benzoheterocyclic analogues. Dose–response relationship study for (a) 3a, (b) 3b, (c) 3c,
and (d) 3d are shown. Data for gravitropic bending and elongation represent the mean ± SD. Asterisk indicates statistically significant differences between treatments
and controls at p < 0.05 (Dunnett’s test, n = 7).
4.2.1. from indole-2-carboxaldehyde.
oil (79%, Z:E = >99:1); 1H-NMR (600 MHz, CDCl3) δ: 1.35 (t, J = 7.2 Hz,
3H), 4.37 (q, J = 7.2 Hz, 2H), 6.44 (d, J = 12.7 Hz, 1H), 6.78 (d, J = 12.7
Hz, 1H), 7.34–7.39 (m, 2H), 7.51 (d, J = 7.7 Hz, 1H), 7.75 (d, J = 7.7 Hz,
1H); 13C-NMR (150 MHz, CDCl3) δ: 14.0 (q), 61.3 (t), 110.6 (d), 120.6
(d), 122.4 (d), 124.7 (d), 126.1 (d), 129.0 (d), 141.4 (s), 150.4 (s), 159.3
(s), 165.6 (s); IR (KBr) 1719 cmꢀ 1; EIMS m/z 217 (M+, 100%); Elem.
Anal. calcd for C12H11NO3: C, 66.35; H, 5.10; N, 6.45, found: C, 66.20;
H, 5.08; N, 6.24.
Ethyl ester of 3c: starting from 145 mg of the aldehyde; pale yellow
solid (77%, Z:E = >99:1); 1H-NMR (270 MHz, CDCl3) δ: 1.36 (t, J = 7.0
Hz, 3H), 4.29 (q, J = 7.0 Hz, 2H), 5.79 (d, J = 12.7 Hz, 1H), 6.77 (s, 1H),
6.94 (d, J = 12.7 Hz, 1H), 7.06–7.12 (m, 1H), 7.22–7.29 (m, 1H), 7.44
(d, J = 8.2 Hz, 1H), 7.62 (d, J = 8.2 Hz, 1H), 11.87 (s, 1H).
3c: starting from 166 mg of the ester; yellow prisms (98%, Z:E =
>99:1); mp. 133–135 ◦C (benzene); 1H-NMR (400 MHz, CDCl3) δ: 5.85
(d, J = 12.6 Hz, 1H), 6.84 (s, 1H), 7.07 (d, J = 12.6 Hz, 1H), 7.11
(apparent t, J = 7.7 Hz, 1H), 7.27–7.31 (m, 1H), 7.45 (d, J = 8.2 Hz, 1H),
7.64 (d, 8.2 Hz, 1H), 11.6 (s, 1H); 13C-NMR (100 MHz, CD3OD) δ: 112.2
(d), 112.7 (d), 114.4 (d), 121.0 (d), 122.3 (d), 125.5 (d), 129.0 (s), 135.1
(s), 136.1 (d), 138.9 (s), 171.2 (s); IR (KBr) 1676, 3329 cmꢀ 1. EIMS m/z
187 (M+), 169 (100%); Anal. calcd for C11H9NO2: C, 70.58; H, 4.85; N,
7.48, found: C, 70.38; H, 4.79; N, 7.39.
3d: starting from 240 mg of the ester; pale brown needles (77%, Z:E
= >99:1); mp. 92–94 ◦C (hexane, EtOAc); 1H-NMR (400 MHz, CDCl3) δ:
6.59 (d, J = 13.5 Hz, 1H), 6.97 (d, J = 13.5 Hz, 1H), 7.48–7.56 (m, 2H),
7.65 (d, J = 7.6 Hz, 1H), 7.81 (dd, J = 7.6 Hz, 1H); 13C-NMR (100 MHz,
CDCl3) δ: 111.5 (d), 120.0 (d), 121.7 (d), 126.4 (d), 127.8 (d), 133.3 (d),
138.3 (s), 149.6 (s), 159.8 (s), 164.3 (s); IR (KBr) 1719 cmꢀ 1; EIMS m/z
189 (M+), 145 (100%); Elem. Anal. calcd for C10H7NO3: C, 63.49; H,
3.73; N, 7.40, found: C, 63.56; H, 3.74; N, 7.45.
3.2.7. Synthesis of (Z)-3-(benzo[d]oxazol-2-yl)acrylic acid (3d)
The title compound was synthesized according to the procedure of
4.2.1. from benzoxazole-2-carbaldehyde.
3.2.8. Synthesis of (Z)-3-(quinolin-2-yl)acrylic acid (4a)
The title compound was synthesized according to the procedure of
4.2.1. from 2-quinolinecarboxaldehyde.
Ethyl ester of 3d: starting from 292 mg of the aldehyde; pale yellow
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Phytochemistry 179 (2020) 112508
Fig. 11. (continued).
4.2.1. from 3-quinolinecarboxaldehyde.
Ethyl ester of 4a: starting from 451 mg of the aldehyde; pale orange
oil (quant, Z:E = >99:1); 1H-NMR (400 MHz, CDCl3) δ: 1.23 (t, J = 7.6
Hz, 3H), 4.23 (q, J = 7.6 Hz, 2H), 6.26 (d, J = 12.4 Hz, 1H), 7.16 (d, J =
12.4 Hz, 1H), 7.54 (dd, J = 7.6, 7.6 Hz, 1H), 7.68–7.72 (m, 2H), 7.81 (d,
J = 7.6 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H), 8.13 (d, J = 8.7 Hz, 1H); 13C-
NMR (100 MHz, CDCl3) δ: 14.0 (q), 60.5 (t), 121.9 (d), 124.0 (d), 126.8
(d), 127.3 (s), 127.4 (d), 129.4 (d), 129.5 (d), 135.6 (d), 140.3 (d), 147.6
(s), 154.1 (s), 166.4 (s); IR (NaCl) 1721 cmꢀ 1. EIMS m/z 227 (M+), 182
(100%); Elem. Anal. calcd for C14H13NO2: C, 73.99; H, 5.77; N, 6.16,
found: C, 73.74; H, 5.83; N, 6.03.
Ethyl ester of 4b: starting from 471 mg of the aldehyde; pale yellow
oil (96%, Z:E = 17:1), 1H-NMR (270 MHz, CDCl3) δ: 1.25 (t, J = 7.1 Hz,
4H), 4.21 (q, J = 7.1 Hz, 2H), 6.15 (d, J = 12.6 Hz, 1H), 7.10 (d, J = 12.6
Hz, 1H), 7.53–7.59 (m, 1H), 7.70–7.76 (m, 1H), 7.85 (d, J = 8.1 Hz, 1H),
8.09 (d, J = 8.4 Hz, 1H), 8.58 (d, J = 2.1 Hz, 1H), 8.96 (d, J = 2.1 Hz,
1H).
4b: starting from 654 mg of the ester; colorless prisms (88%, Z:E =
>99:1); mp. 175–177 ◦C (EtOH); 1H-NMR (270 MHz, CD3OD) δ: 6.21 (d,
J = 12.5 Hz, 1H), 7.19 (d, J = 12.5 Hz, 1H), 7.63 (ddd, J = 8.2, 6.9, 1.2
Hz, 1H), 7.79 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.94 (d, J = 8.2 Hz, 1H),
8.01 (d, J = 8.4 Hz, 1H), 8.58 (s, 1H), 9.01 (d, J = 2.3 Hz, 1H); 13C-NMR
(150 MHz, acetone-d6) δ: 122.8 (d), 127.7 (d), 128.2 (s), 129.3 (d),
129.9 (d), 130.8 (d), 137.2 (d), 140.7 (d), 145.5 (s), 148.6 (s), 152.3 (d),
167.2 (s); IR (KBr) 1686, 1637 cmꢀ 1; FAB-MS m/z 200 (M + H+); Elem.
Anal. calcd for C12H9NO2: C, 72.35; H, 4.55, N; 7.03, found: C, 71.93; H,
4.51; N; 7.01.
4a: starting from 500 mg of the ester; colorless needles (23%, Z:E =
>99:1); mp. 72–74 ◦C (toluene); 1H-NMR (400 MHz, CDCl3) δ: 6.48 (d, J
= 13.0 Hz, 1H), 7.01 (d, J = 13.0 Hz, 1H), 7.55 (d, J = 8.2 Hz, 1H), 7.70
(ddd, J = 7.6, 7.6, 1.1 Hz, 1H), 7.89 (m, 1H), 8.11 (d, J = 7.6 Hz, 1H),
8.42 (d, J = 8.2 Hz, 1H); 13C-NMR (150 MHz, CD3OD) δ: 125.4 (d),
126.4 (d), 129.4 (s), 129.5 (d), 130.1 (d), 132.0 (d), 133.6 (d), 137.6 (d),
142.1 (d), 144.3 (s), 152.7 (s), 170.0 (s); IR (KBr) 1707 cmꢀ 1. EIMS m/z
199 (M+), 155 (100%); Elem. Anal. calcd for C12H9NO2: C, 72.35; H,
4.55, N; 7.03, found: C, 72.46; H, 4.49; N; 6.92.
3.2.10. Synthesis of (Z)-3-(quinolin-4-yl)acrylic acid (4c)
The title compound was synthesized according to the procedure of
4.2.1. from 4-quinolinecarboxaldehyde.
3.2.9. Synthesis of (Z)-3-(quinolin-3-yl)acrylic acid (4b)
The title compound was synthesized according to the procedure of
Ethyl ester of 4c: starting from 471 mg of the aldehyde; pale pink oil
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Phytochemistry 179 (2020) 112508
Fig. 12. Inhibitory activity tests of gravitropic bending and elongation for quinoline and quinoxaline analogues 4: (a) 50 μM for 4a-4d, (b) 0.05–50 μM for 4a, (c)
0.05–50 μM for 4b. Data for gravitropic bending (left) and elongation (right) represent the mean ± SD. Asterisk indicates statistically significant differences between
treatments and controls at p < 0.05 (Dunnett’s test, n = 7).
9

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Phytochemistry 179 (2020) 112508
Fig. 13. Inhibitory activity tests of gravitropic bending and elongation for o-styrylbenzoic acid analogues 5 and NPA (0.05–5
μ
M). Data for gravitropic bending and
elongation represent the mean ± SD. Asterisk indicates statistically significant differences between treatments and controls at p < 0.05 (Dunnett’s test, n = 7).
10

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Phytochemistry 179 (2020) 112508
Fig. 13. (continued).
4.2.1. from quinoxaline-2-carbaldehyde.
(quant, Z:E = 20:1); 1H-NMR (400 MHz, CDCl3) δ: 0.98 (t, J = 7.0 Hz,
3H), 3.99 (q, J = 7.0 Hz, 2H), 6.36 (d, J = 12.1 Hz, 1H), 7.35 (d, J = 4.3
Hz, 1H), 7.45 (d, J = 12.1 Hz, 1H), 7.56 (apparent t, J = 7.6 Hz, 1H),
7.73 (apparent t, J = 7.6 Hz, 1H), 7.88 (d, J = 8.2 Hz, 1H), 8.14 (d, J =
8.7 Hz, 1H), 8.90 (d, J = 4.3 Hz, 1H); 13C-NMR (150 MHz, CDCl3) δ: 13.6
(q), 60.5 (t), 120.1 (d), 124.4 (d), 125.1 (d), 125.9 (s), 126.7 (d), 129.4
(d), 130.0 (d), 139.0 (d), 142.2 (s), 148.0 (s), 149.6 (d), 165.0 (s); IR
(KBr) 1722 cmꢀ 1; EIMS m/z 227 (M+), 154 (100%); HRMS (FAB) Calcd
for C14H14O2N (M + H+): 228.1025, found: 228.1027.
Ethyl ester of 4d: starting from 474 mg of the aldehyde; pale purple
oil (92%, Z:E = >99:1); 1H-NMR (400 MHz, CDCl3) δ: 1.23 (t, J = 7.0 Hz,
3H), 4.23 (q, J = 7.0 Hz, 2H), 6.39 (d, J = 12.1 Hz, 1H), 7.16 (d, J = 12.1
Hz, 1H), 7.75–7.79 (m, 2H), 8.04 (dd, J = 6.3, 3.4 Hz, 1H), 8.10 (dd, J =
6.3, 3.4 Hz, 1H), 9.03 (s, 1H); 13C-NMR (100 MHz, CDCl3) δ: 14.0 (q),
60.9 (t), 125.9 (d), 129.2 (d), 129.3 (d), 130.1 (d), 130.2 (d), 137.5 (d),
141.4 (s), 141.7 (s), 145.6 (d), 149.5 (s), 165.9 (s); IR (KBr) 1721 cmꢀ 1
;
EIMS m/z 228 (M+), 199 (100%); HRMS (FAB) Calcd for C13H13O2N2
(M + H+): 229.0977, found: 229.0973.
4c: starting from 500 mg of the ester; colorless prism (45%, Z:E =
20:1); mp. 210–212 ◦C (EtOH); 1H-NMR (600 MHz, TFA-d) δ: 6.65 (d, J
= 12.4 Hz, 1H), 7.69 (d, J = 12.4 Hz, 1H), 7.86 (d, J = 5.8 Hz, 1H), 7.93
(apparent t, J = 7.6 Hz, 1H), 8.13 (apparent t, J = 7.6 Hz, 1H), 8.18 (d, J
= 8.4 Hz, 1H), 8.20 (d, J = 8.2 Hz, 1H), 8.91 (d, J = 5.8 Hz, 1H); 13C-
NMR (150 MHz, TFA-d) δ: 122.6 (d), 122.7 (d), 128.2 (d), 128.4 (d),
129.4 (s), 133.4 (d), 138.6 (d), 139.6 (s), 142.1 (d), 144.6 (d), 159.3 (s),
171.7 (s); IR (KBr) 1697 cmꢀ 1; FAB-MS m/z 200 (M + H+); Elem. Anal.
calcd for C12H9NO2: C, 72.35; H, 4.55; N, 7.03, found: C, 72.14; H, 4.33;
N, 6.98.
4d: starting from 400 mg of the ester; pale pink needles (85%, Z:E =
>99:1); mp. 105 ◦C (decomposed, EtOH); 1H-NMR (400 MHz, CDCl3) δ:
6.59 (d, J = 13.3 Hz, 1H), 7.15 (d, J = 13.3 Hz, 1H), 7.93–7.97 (m, 2H),
8.10 (dd, J = 6.2, 3.5 Hz, 1H), 8.21 (dd, J = 6.2, 3.5 Hz, 1H), 9.04 (s,
1H); 13C-NMR (100 MHz, CDCl3) δ: 127.1 (d), 129.6 (d), 132.2 (d),
132.3 (d), 132.5 (d), 132.9 (d), 137.6 (s), 142.7 (s), 145.7 (s), 147.4 (d),
165.6 (s); IR (KBr) 1678 cmꢀ 1; FAB-MS m/z 201 (M + H+); Anal. calcd
for C11H8N2O2: C, 66.00; H, 4.03; N, 13.99, found: C, 65.87; H, 3.97; N,
13.98.
3.2.11. Synthesis of (Z)-3-(quinoxalin-2-yl)acrylic acid (4d)
3.2.12. Synthesis of (E)-2-styrylbenzoic acid (5a)
The title compound was synthesized according to the procedure of
The title compound was synthesized according to the literature
11

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Phytochemistry 179 (2020) 112508
procedure (Shahzad et al., 2010): 1H-NMR (270 MHz, CDCl3) δ: 7.03 (d,
J = 16.3 Hz, 1H), 7.24–7.31 (m, 2H), 7.34–7.40 (m, 3H), 7.55–7.61 (m,
3H), 7.74–7.76 (m, 1H), 8.05–8.13 (m, 2H); The spectral data were
consistent with those reported in the literature.
configurational fixed analogues 5a (ku-294) and 5c (ku-308) as well as
3a (ku-257) were the most potent inhibitors without inhibition of
growth. The inhibitory activity of the potent analogues was comparable
to that of NPA.
These highly potent analogues would be very useful tools in the
elucidation of the mechanism of gravitropic inhibitory activity and yield
important clues for the design of new types of agrochemicals.
3.2.13. Synthesis of (E)-2-(2-(naphthalen-1-yl)vinyl)benzoic acid (5b)
The title compound was synthesized according to the literature
procedure (Shahzad et al., 2010): 1H-NMR (400 MHz, CDCl3) δ: 7.41 (t,
J = 7.7 Hz, 1H), 7.52 (m, 3H), 7.64 (dd, J = 7.7, 7.7 Hz, 1H), 7.78 (d, J =
16.4 Hz, 1H), 7.82 (d, J = 7.7 Hz, 2H), 7.87 (d, J = 8.7 Hz, 2H), 8.08 (d,
J = 16.4 Hz, 1H), 8.12 (dd, J = 7.7, 1.9 Hz, 1H), 8.26 (d, J = 7.7 Hz, 1H);
The spectral data were consistent with those reported in the literature.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
3.2.14. Synthesis of 2-(naphthalen-2-yl)benzoic acid (5c)
The title compound was synthesized according to the literature
procedure (Wang et al., 2013): 1H-NMR (600 MHz, CDCl3) δ: 7.45–7.51
(m, 5H), 7.61 (td, J = 7.6, 1.4 Hz, 1H), 7.81–7.87 (m, 4H), 7.99 (dd, J =
8.2, 1.4 Hz, 1H); The spectral data were consistent with those reported
in the literature.
Acknowledgment
This research was financially supported by JSPS KAKENHI Grant
Number (No. JP22390002, JP24106731, JP16H01157, JP26293004,
JP17K14449, JP18H04418, JP18H04624, and JP18H02557), Science
and Technology Research Promotion Program for Agriculture, Forestry,
Fisheries and Food industry (M.S.). This work was performed under the
Cooperative Research Program “Network Joint Research Center for
Materials and Devices.”
3.2.15. Synthesis of 2-(benzofuran-2-yl)benzoic acid (5d)
The title compound was synthesized according to the literature
procedure (Yamaguchi et al., 1995): 1H-NMR (600 MHz, CDCl3) δ: 6.99
(s, 1H), 7.23–7.30 (m, 2H), 7.48–7.51 (m, 2H), 7.62 (apparent t, J = 8.6
Hz, 2H), 7.76 (d, J = 7.6 Hz, 1H), 7.91 (d, J = 7.6 Hz, 1H); The spectral
data were consistent with those reported in the literature.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.phytochem.2020.112508.
3.3. Inhibitory test for gravitropic bending
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