cis-Cinnamic acid selective suppressors distinct from auxin inhibitors

Article abstract of DOI:10.1248/cpb.c14-00169

The activity of cis-cinnamic acid (cis-CA), one of the allelochemicals, in plants is very similar to that of indole-3-acetic acid (IAA), a natural auxin, and thus cis-CA has long been believed to be an analog of auxin. We have reported some structure-acti

Full text of DOI:10.1248/cpb.c14-00169

600
Regular Article
Chem. Pharm. Bull. 62(6) 600–607 (2014)
Vol. 62, No. 6
cis-Cinnamic Acid Selective Suppressors Distinct from Auxin Inhibitors
Katsuhiro Okuda,a Keisuke Nishikawa,a Hiroshi Fukuda,b Yoshiharu Fujii,c and
,a
Mitsuru Shindo*
b
a Institute for Materials Chemistry and Engineering, Kyushu University; Interdisciplinary Graduate School
c
of Engineering Sciences, Kyushu University; 6–1 Kasuga-koen, Kasuga 816–8580, Japan: and International
Environmental and Agricultural Sciences, Tokyo University of Agriculture and Technology; 3–5–8 Saiwai-cho,
Fuchu, Tokyo 183–8509, Japan.
Received February 20, 2014; accepted March 10, 2014
The activity of cis-cinnamic acid (cis-CA), one of the allelochemicals, in plants is very similar to that of
indole-3-acetic acid (IAA), a natural auxin, and thus cis-CA has long been believed to be an analog of auxin.
We have reported some structure–activity relationships studies by synthesizing over 250 cis-CA derivatives
and estimating their inhibitory activities on root growth inhibition in lettuce. In this study, the compounds
that showed low- or no-activity on root growth inhibition were recruited as candidates suppressors against
cis-CA and/or auxin and tested for their activity. In the presence of cis-CA, lettuce root growth was inhib-
ited; however, the addition of some cis-CA derivatives restored control-level root growth. Four compounds,
(Z)-3-(4-isopropylphenyl)acrylic acid, (Z)-3-(3-butoxyphenyl)acrylic acid, (Z)-3-[3-(pentyloxy)phenyl]acrylic
acid, and (Z)-3-(naphthalen-1-yl)acrylic acid were selected as candidates for a cis-CA selective suppressor
they allowed the recovery of root growth from inhibition by cis-CA treatment without any effects on the
IAA-induced effect or elongating activity by themselves. Three candidates significantly ameliorated the root
shortening by the potent inhibitor derived from cis-CA. In brief, we have found some cis-CA selective sup-
pressors which have never been reported from inactive cis-CA derivatives for root growth inhibition. cis-CA
selective suppressors will play an important role in elucidating the mechanism of plant growth regulation.
Key words cis-cinnamic acid; selective suppressor; auxin; growth regulation
cis-Cinnamic acid (cis-CA; Fig. 1) is known to inhibit 6-O-(4′-hydroxy-2′-methylene-butyroyl)-1-O-cis-cinnamoyl-β-
root growth of an Arabidopsis, Avena, wheat and flax, and D-glucopyranose, as allelochemicals in Spiraea thunbergii.11)
to cause epinastic curvature in pea and tomato seedlings as While cis-CA has an inhibitory activity on root growth, it
do the plant hormones auxin and ethylene.1–5) Naturally oc- showed a growth-promoting activity on aerial moieties such
curring CA has been found in both trans- and cis-forms. as pea stem and avena coleoptile like auxin.1) The most rep-
trans-CA is generally considered to be a weak antagonist of resentative natural auxin is indole-3-acetic acid (IAA, Fig. 1).
auxin in higher plants.2,6–8) The biosynthesis of trans-CA from Because of the similar activities for plants, cis-CA has long
L-phenylalanine is catalyzed by phenylalanine ammonia-lyase been believed to be an analog of auxin.
(PAL; EC 4.3.1.5). It is then converted to 4-hydroxycinnamic
Further studies using an ethylene perception-deficient or an
acid by cinnamic acid 4-hydroxylase (EC 1.14.13.11), which ethylene biosynthesis-deficient tomato plant,4) and two auxin-
serves as the precursor for the biosynthesis of many phenolic insensitive Arabidopsis mutants5) showed that cis-CA acts on
compounds such as flavonoids, phytoalexin, salicylic acid and plant cells through both ethylene- and auxin-independent sig-
lignin.9) cis-CA can be an ultraviolet light-mediated isomeriza- naling pathways. Recently, cis-CA-upregulating Arabidopsis
tion product of trans-CA.4,5) The presence of natural cis-CA genes, MLPL1 (AT2G01520) and MLPL2 (AT2G01530), were
in plants has been reported in Brassica parachinensis10) and identified and the up-regulation occurred in auxin perception-
its glycosides, 1-O-cis-cinnamoyl-β-D-glucopyranose and deficient mutants.12) Furthermore, the treatment of cis-CA in-
Fig. 1. Structures of Cinnamic Acids (CAs), Auxins, and Anti-auxins
CA has two isoforms as cis-CA and trans-CA. Indole-3-acetic acid (IAA) is a natural auxin while 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthalene acetic acid
(NAA) are artificial auxins. p-Chlorophenoxyisobutyric acid (PICB) and L-2-aminooxy-3-phenylpropionic acid (AOPP) are recognized as anti-auxins.
The authors declare no conflict of interest.
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: shindo@cm.kyushu-u.ac.jp

June 2014
601
Fig. 2. Structures of Cinnamic Acid (CA) Derivatives
The concentrations in the brackets represent IC50 values of root growth inhibition.
duced some early auxin responsive genes only in roots without was eliminated by the anti-auxins. AOPP might decrease the
affecting the SMALL AUXIN-UP RNA (SAUR) families.13) Al- amount of intrinsic auxin because of its inhibitory activity
though these studies suggested that cis-CA was not a member of auxin biosynthesis. Compounds 4, 5, and 11 also showed
of the auxins, little is known about its physiological functions promoting activity; however, the activity of 4 appeared weaker
and mechanism of action for cis-CA.
than that of the others because only a high-concentration
We have been taking particular note of cis-CA bioactivity (50 µM) was effective.
and reported some structure–activity relationships studies.14,15)
In the presence of cis-CA, root growth was inhibited about
During these investigations, we synthesized more than 250 50% and was restored by the addition of anti-auxins and cis-
cis-CA derivatives and evaluated their inhibitory effects on CA derivatives 2, 5, 6, 7, 8, 10, 11, and 12. Using compounds
root growth in lettuce. In this study, the compounds that 2, 5, 7, 11, PCIB and AOPP, the recovery was complete and
showed low- or no-activity on root growth inhibition were control-level growth was achieved. Compounds 10 and 12
recruited as candidates for a suppressor against cis-CA and/or exhibited weak suppressive activity, with 90% growth re-
auxin (Fig. 2). Suppressors of cis-CA would aid in elucidating stored at high concentrations. These root length recovery by
the inhibitory mechanism as well as the unique compounds compounds 5, 11, PCIB and AOPP, which were also found
having the inhibitory activity against cis-CA. It would also to possess elongating activity, could not be differentiated
become evident that cis-CA and auxin regulate plant growth whether this recovery was the result of elongating activity or
through distinct signaling pathways.
suppressive activity. The chemical structure of 11 resembles
not only that of cis-CA but also abscisic acid, one of plant
hormones, however abscisic acid did not have an elongating
Results and Discussion
The assay for suppressive activity was conducted by adding activity (data not shown).
a test compound and a root growth inhibitor simultaneously.
When IAA was used as an inhibitor, it strongly inhibited
cis-CA and IAA were nominated as representative inhibitors, the root growth even at one-tenth of the concentration used
and 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphtha- for the screening assay. As shown in Table 1, only two anti-
leneacetic acid (NAA) were also used as authentic auxins. We auxins PCIB and AOPP were able to reduce the effect of IAA
initially screened for effects on the inhibitory activities of cis- significantly and the restoration levels were at most 84% and
CA, IAA, 2,4-D, and NAA (Table 1). The concentration of the 86%, respectively. Compound 12 seemed to promote inhibito-
inhibitors was set according to preliminary experiments to get ry activity of IAA. In the case of the authentic auxins, 2,4-D
the maximum response from a suppressor.
and NAA were used as inhibitors, a few compounds affected
In the absence of growth inhibitors, there was no tested their inhibitory activities. Among all of the tested compounds,
compounds that made roots shorter because the selected only PCIB, not AOPP, ameliorated the effect of 2,4-D. Com-
compounds previously showed low or no inhibitory activity. pounds 2, 10 and 11 showed recovery against NAA induced
p-Chlorophenoxyisobutyric acid (PCIB) and L-2-aminooxy- inhibition, but neither PCIB nor AOPP did.
3-phenylpropionic acid (AOPP), well known as anti-auxins,
trans-CA and compounds 1, 3, 9, and 13 did not have any
promoted lettuce root growth as much as 140% compared activity on the lettuce root regardless of the presence or ab-
with the vehicle controls because the intrinsic auxin function sence of the inhibitor. According to the results shown in Table

602
Vol. 62, No. 6
Table 1. Effects of cis-Cinnamic Acid (cis-CA) Derivatives on cis-CA and Auxins in Lettucea) (Lactuca sativa cv. Great Lakes 366)
Growth inhibitors
Suppressor candidates
µM
Control
cis-CA 5µM
IAAb) 0.5µM
2,4-Dc) 0.5µM
NAAd) 0.5µM
trans-CA
0
25
50
0
100 12
109 23
102 16
100 15
93 27
59 17
61 10
64 13
57 11
43
46
47
41
44
43
41
4
4
4
6
4
5
6
46
52
50
44
54
55
50
7
6
6
6
5
8
6
63
9
53 10
1
47
55
55
51
7
6
9
6
50
52 12
54
48 10
5
25
50
0
99 16
8
3
100 16
111 37
118 53
100 19
84 28
25
50
0
61 20
79 23
41
40
8
8
40 17
73 13
76 16
55 11
65
46
41
7
9
6
13
57
9
61 11
64 15
64 15
25
50
0
61 10
77 19
52
58
72
59
65
47
63
7
8
8
7
6
6
5
105 44
100 19
109 31
113 32
100 22
138 28*
142 26*
100 19
135 28*
133 31*
100 21
114 17
106 19
100 18
97 18
54 11
9
64
7
63
54
60
9
9
9
54
41
40
38
49
9
6
6
6
9
25
50
0
77 14
69 13
PCIBe)
56
9
55 11
25
50
0
89 15*
99 19*
56 10
81 11*
84 14*
71 12*
71 10
55
AOPPf)
56
7
45
51
59
49
54
4
6
8
6
8
5
25
50
0
93 13*
105 17*
59 14
85 14*
86 15*
61 14
51 16
41 10
71 11
74 15
54 10
80 15*
72 16
2
6
25
50
0
105 16*
93 17*
56 16
59 13
50
37
6
8
40
38
41
50
57
58
48
49
6
4
8
6
9
9
5
9
43
35
6
6
25
50
0
85 16*
93 8*
102 16
100 20
107 16
104 19
100 19
110 24
108 18
100 26
97 14
45 12
62 13
51 18
45 15
50 13
54 10
60 12
71 15
7
59 9
25
50
0
89 11*
105 18*
55 14
8
46
9
59
5
25
50
0
87 22*
90 15*
45 12
44 17
58 21
37 7*
35 14*
61 16
54 16
75
53
9
8
12
10
4
53
5
42
41
5
5
25
50
0
66 12
41 10
47 10
103 17
100 14
114 21
111 30
100 19
110 17
123 17*
100 16
126 21*
131 20*
100 21
142 33*
140 21*
82 11*
57 10
53
8
56
57
8
8
38
49
5
5
44 4
25
50
0
71 17
89 17*
52 11
57 11
68 8*
93 11*
44 14
64 13
54 12
43
52
47
63
7
7
9
9
35
38
37
50
37
46
40
8
3
4
8
4
4
4
25
50
0
40
45
55
8
7
9
45
6
5
63 17
25
50
0
103 11*
105 15*
59 12
52 24
56 13
47 10
75 17
43 12
64 14
48 10
69 11
75 14*
11
49 9
25
50
109 22*
124 21*
54 11
63 13
a) The values represent the average length of the roots as a percent of the untreated control. b) IAA, indole-3-acetic acid. c) 2,4-D, 2,4-dichlorophenoxyacetic acid. d) NAA,
1-naphthalene acetic acid. e) PCIB, p-chlorophenoxyisobutyric acid. f) AOPP, L-2-aminooxy-3-phenylpropionic acid. *p<0.05 for a pair comparison of each antagonist to the
untreated group by Tukey–Kramer’s test.
1, four compounds, 2, 6, 7, and 8 were picked up as candidates concentrations were used for the assay. As shown in Table 2,
for cis-CA selective suppressor because they could restore compound 2 exhibited suppressive activity on cis-CA in the
the root growth after inhibition by cis-CA treatment without range of 1–30µM, and the optimal concentration of 2 seemed
any effect on the IAA-induced effects or elongating activity to be 25µM. Compounds 6 and 7 also showed obvious activity
of themselves. Those four compounds were then subjected to but failed to suppress 30µM of cis-CA. Compound 8 was only
further experimentations.
effective on the inhibition induced by 3µM of cis-CA. To con-
To confirm the activity against cis-CA, various cis-CA firm the selectivity against cis-CA, various IAA concentra-

June 2014
603
Table 2. Effects of cis-Cinnamic Acid (cis-CA) Derivatives on Various Concentrations of cis-CA in Lettucea) (Lactuca sativa cv. Great Lakes 366)
cis-CA
Suppressor candidates
µM
Control
1µM
3µM
10µM
30µM
2
0
25
50
0
100 20
117 26
94 21
74 16
108 22*
88 17
55
8
41
6
27
6
112 11*
90 7*
77 19*
79 10*
50 9*
47 15
6
7
8
100 22
98 15
67 14
53 14
37
8
22 6
25
50
0
105 13*
95 14*
87 10*
95 16*
51 11
59 14*
79 10*
28 11
34 11
105
7
100 13
86 10
96 14
100 20
100 13
85 21
58
7
35
6
22
27
6
8
25
50
0
83 12*
94 8*
71 17
88 12
91 19
71 14*
86 13*
66 13*
68 15*
36 10
27
33 10
29
52
7
43
9
8
25
50
74 16*
77 16*
41 12
52 16
8
a) The values represent the average length of the roots as a percent of the untreated control. *p<0.05 for a pair comparison of each antagonist to the untreated group by
Tukey–Kramer’s test.
Table 3. Effects of cis-Cinnamic Acid (cis-CA) Derivatives on Various Concentrations of Indole-3-acetic Acid (IAA) in Lettucea) (Lactuca sativa cv.
Great Lakes 366)
IAA
Suppressor candidates
µM
Control
0.1µM
0.3µM
1µM
3µM
2
0
25
50
0
100 20
122 27
106 19
100 12
122 16
117 30
100 14
107 12
110 14
100 27
117 16
93 22
70
8
57 12
53 11
52 17
50 22
40 12
35 19
44 10
43 19
67 16
52 12
68 17
90 22*
78 28
66 24
99 16
62 16
62 12
36 8*
92 20
76 19
77 13
69 12
77 24
68 12
64 16
69 10
83 19
70 19
54 19*
6
7
8
62
9
53 8
25
50
0
56 12
61 21
40 13
40 10
56 17
30 9*
53 21
61 15
44 20
31 7*
50
7
25
50
0
42 11
49 15
70 14
53 16
37 16*
25
50
a) The values represent the average length of the roots as a percent of the untreated control. *p<0.05 for a pair comparison of each antagonist to the untreated group by
Tukey–Kramer’s test.
Table 4. Effects of cis-Cinnamic Acid (cis-CA) Derivatives on cis-CA-Like Growth Inhibitors in Lettucea) (Lactuca sativa cv. Great Lakes 366)
Growth inhibitors
Suppressor candidates
µM
Control
cis-CA 5µM
46
14 3µM
15 3µM
2
0
25
50
0
100 18
102 28
84 20
9
50 13
83 19*
91 15*
48 11
56 15
91 14*
96 22*
59 15
74 10
85 9*
51 13
82 13*
82 18*
66 17
77 13
71 17
92 16*
83 22*
6
7
8
100 18
97 17
49 6
25
50
0
89 16*
88 19*
62 9
113 17
100 23
103 16
103 11
100 28
106 19
98 19
86 15*
50 18
72 9*
79 13*
52
8
25
50
0
85 11*
85 13*
43
9
46 9
25
50
71 21*
69 21*
63 12
60 15
a) The values represent the average length of the roots as a percent of the untreated control. *p<0.05 for a pair comparison of each antagonist to the untreated group by
Tukey–Kramer’s test.

604
Vol. 62, No. 6
tions were used for the assay. All of these compounds failed to cis-CA as a possible novel plant hormone. cis-CA selective
mitigate the root growth inhibition induced by IAA (Table 3). suppressors will play an important role in elucidating the
Interestingly, only the combination of 3µM of IAA and 50µM mechanisms of plant growth regulation.
of 6 showed a preventative activity. Compound 8 magnified
the inhibitory activity of IAA at higher concentrations. These
results suggested that these four compounds could be cis-CA
selective suppressors.
Experimental
Chemicals Each synthetic method for the newly prepared
compounds and their spectral data are described below. The
Two cis-CA derivatives, 14 and 15, having strong inhibitory other compounds were synthesized as previously reported.
activity15) (Fig. 2) were recruited for further confirmation as IAA, 2,4-D, and PCIB were purchased from Tokyo Chemi-
candidates that effected cis-CA-like activities (Table 4). Com- cal Industry Co., Ltd. (Tokyo, Japan). NAA, and AOPP were
pounds 2 and 7 significantly ameliorated the root shortening purchased from Wako Pure Chemical Industries, Ltd. (Osaka,
by each tested cis-CA derivatives. Compounds 6 and 8 failed Japan). trans-CA was purchased from Nacalai Tesque, Inc.
to restore growth after inhibition by 14 and 15, except at a (Kyoto, Japan). Other chemicals used were commercially
high concentration of 6.
Compounds 2, 6, 7, and 8 clearly exhibited cis-CA selective
available.
Analysis of Synthesized Chemicals The melting points
suppressive activity (Tables 2, 3). The order of their strength were measured on a Yazawa micromelting point BY-1. The
of suppressive activity appears to be 2>7>6>8. Compound 1H- and 13C-NMR spectra were recorded using a JNM
8 showed the weakest suppressive activity among these and EX-270 (270 and 67.5MHz, JEOL) and a JNM AL-400 (400,
promoted the inhibition of IAA. Furthermore, compound 8 100MHz, JEOL). Chemical shifts were reported in ppms
has been reported as an auxin-like growth regulator, similar to downfield from the peak of the internal standard Me4Si (tetra-
cis-CA,2) in the pea split-stem curvature test,16) so it may work methylsilane (TMS)). Splitting patterns are designed as “s,
through different pathways from the other compounds.
d, t, q, and m,” indicating “singlet, doublet, triplet, quartet,
and multiplet,” respectively. The IR spectra were recorded
on a Shimadzu FT/IR-8300 spectrometer using a KBr disk
Conclusion
According to these results, at least three compounds 2, 6, or a NaCl cell. Mass and high-resolution mass spectra were
and 7 can be called cis-CA selective suppressors. Previous obtained on a JMS-700 or a JMS-T100CS (JEOL). Column
reports suggesting that cis-CA acts via both ethylene- and chromatography was performed on silica gel (Kanto Chemical
auxin-independent signaling pathways4,5) are supported by our Co.). Thin-layer chromatography was performed on pre-coated
highly selective suppressors.
plates (0.25mm, silica gel Merck 60 F254). Reaction mixtures
This is the first study on cis-CA suppressors even though were stirred magnetically.
the bioactivities of CA, IAA and anti-auxin17) have been
Syntheses of cis-CA Analogs The cis-CA analogs 1,
reported previously. We have found some cis-CA selective 2, 3, 6, 8, 9, 10, 11, 14, and 15 were synthesized as previ-
suppressors against growth inhibition that have never been ously reported.14,15,18) Syntheses of the analogs 4, 5, 7, and
reported among inactive cis-CA derivatives. There are numer- 12 were also performed mainly by the Z-selective olefination
ous questions surrounding plant growth regulation, especially of the corresponding aldehydes with the Ando–Emmons re-
(a) Ethyl 2-[bis(2-isopropylphenoxy)phosphoryl]acetate, Triton B, tetrahydrofuran (THF), −78°C, 82–99%, (b) 10% NaOH aq., EtOH, rt, 77–96%, (c) n-butyllithium
(BuLi), chlorotrimethylsilane, diethyl ether (Et2O)/hexane, rt, 89%, (d) n-BuLi, Et2O/hexane, −78°C to rt, then dimethyl formamide, Et2O, 56%, (e) K2CO3, 1-iodopentane,
acetone, reflux, 75%, (f) NaH, ethyl 2-(diethoxyphosphoryl)acetate, Et2O, rt, 76%.
Chart 1. Syntheses of cis-CA Acid Analogs 4, 5, 7, 12, and 13

June 2014
605
agents,19,20) followed by the hydrolysis (Chart 1). The commer-
To
a
solution of (3-bromophenyl)trimethylsilane (19)
cially unavailable starting aldehydes 21 and 23 were prepared (0.860g, 3.77mmol) in Et2O (5.7mL), n-BuLi (2.30M in hex-
according to the literature.21,22) The trans-analog 13 was syn- ane, 1.64mL, 3.77mmol) was added dropwise at −78°C under
thesized via the usual Honor–Emmons reaction.22)
an argon atmosphere, and then the reaction was stirred for
(2Z,2′Z)-3,3′-(1,3-Phenylene)diacrylic Acid (4) To a solu- 20min. The reaction was warmed to room temperature and
tion of ethyl 2-[bis(2-isopropylphenoxy)phosphoryl]acetate19,20) stirred for 1.5h. A solution of N,N-dimethylformamide (DMF)
(0.921g, 2.28mmol) in tetrahydrofuran (THF) (10mL), Triton (0.320mL, 6.03mmol) in Et2O (2.8mL) was added drop-
B (40% in methanol, 1.15mL, 2.91mmol) at −78°C under an wise to the solution. After 12h of stirring, the mixture was
argon atmosphere was added dropwise. After 15min of stir- quenched with H2O and extracted with CHCl3. The organic
ring, a solution of isophthalaldehyde (16) (0.139g, 1.04mmol) layer was washed with saturated aqueous NaHCO3 and brine,
in THF (3.5mL) was added dropwise to the solution. After dried over MgSO4, filtered, and concentrated in vacuo. The
10h of stirring, the mixture was quenched with saturated crude product was purified by silica gel flash column chroma-
aqueous NH4Cl and extracted with ethyl acetate (EtOAc). tography (EtOAc–hexane, 10:90) to provide 3-(trimethylsilyl)-
The organic layer was washed with H2O, saturated aqueous benzaldehyde (20) (0.376g, 2.11mmol, 56%) as a colorless oil:
NaHCO3 and brine, dried over MgSO4, filtered and concen- 1H-NMR (CDCl3, 400MHz) δ: 0.31 (9H, s, –CH3), 7.52 (1H, t,
trated in vacuo. The crude product was purified by silica gel J=7.6Hz, Ar-H), 7.78, 7.85 (each 1H, d, J=7.6Hz, Ar-H), 8.02
flash column chromatography (EtOAc–hexane, 5:95) to afford (1H, brs, Ar-H), 10.0 (1H, s, –CHO). The spectral data were in
(2Z,2′Z)-diethyl-3,3′-(1,3-phenylene)diacrylate (17) (0.274g, agreement with those in the literature.23)
1
0.998mmol, 96%, Z:E=96:4, determined by H-NMR spec-
The Z-selective olefination of 20 was performed using
1
trum) as a colorless oil: H-NMR (CDCl3, 400MHz) δ: 1.23 the procedure described above to provide (Z)-ethyl-3-(3-
(6H, t, J=7.4Hz, –CH3), 4.16 (4H, q, J=7.4Hz, –CH2–CH3), (trimethylsilyl)phenyl)acrylate (82%, Z:E=95:5, determined
5.96 (2H, d, J=12.8Hz, =CH–CO2–), 6.93 (2H, d, J=12.8Hz, by 1H-NMR spectrum) (silica gel column chromatography,
1
Ar-CH=), 7.34 (1H, t, J=7.8Hz, Ar-H), 7.59 (2H, d, J=7.8Hz, EtOAc–hexane, 5:95) as a colorless oil: H-NMR (CDCl3,
Ar-H), 7.73 (1H, brs, Ar-H). 13C-NMR (CDCl3, 100MHz) δ: 400MHz) δ: 0.27 (9H, s, –CH3), 1.23 (3H, t, J=6.8Hz,
14.0 (q, –CH3), 60.2 (t, –CH2–), 120.2 (d, =CH–CO2–), 127.6 –CH2–), 4.17 (2H, q, J=6.8Hz, –CH2–CH3), 5.95 (1H, d,
(d, Ar), 130.1 (d, Ar), 131.1 (d, Ar), 134.6 (s, Ar), 142.4 (d, J=12.6Hz, =CH–CO2–), 6.95 (1H, d, J=12.6Hz, Ar-CH=),
Ar-CH=), 166.0 (s, C=O).
7.34 (1H, t, J=7.6Hz, Ar-H), 7.47, 7.62 (each 1H, d, J=7.6Hz,
To a solution of 17 (0.249g, 0.908mmol) in EtOH (1.7mL), Ar-H), 7.63 (1H, brs, Ar-H).
10% aqueous NaOH (3.6mL) was added at room temperature.
The hydrolysis of (Z)-ethyl 3-(3-(trimethylsilyl)phenyl)-
After stirring for 12h, the mixture was adjusted to pH 1.0 acrylate was performed using the procedure described
with 1M aqueous HCl, and then extracted with EtOAc. The above to afford 5 (90%) as a single isomer: colorless solid
organic layer was washed with brine, dried over MgSO4, 1H-NMR (CDCl3, 400MHz) δ: 0.27 (9H, s, –CH3), 5.98 (1H,
filtered and concentrated in vacuo. The residue was purified d, J=12.8Hz, =CH–CO2–), 7.08 (1H, d, J=12.8Hz, Ar-CH=),
by recrystallization to afford 4 (0.190g, 0.872mmol, 96%) as 7.35 (1H, t, J=7.6Hz, Ar-H), 7.50, 7.62 (each 1H, d, J=7.6Hz,
a single isomer: colorless needles, mp 156–157°C (toluene); Ar-H), 7.74 (1H, brs, Ar-H). 13C-NMR (CDCl3, 100MHz) δ:
1H-NMR (CDCl3, 400MHz) δ: 5.92 (2H, d, J=12.6Hz,=CH– −1.25 (q, –CH3), 118.4 (d, =CH–CO2–), 127.4 (d, Ar), 130.3
CO2–), 6.95 (2H, d, J=12.6Hz, Ar-CH=), 7.31 (1H, t, (d, Ar), 133.5 (s, Ar), 134.3 (d, Ar), 135.1 (d, Ar), 140.3 (s,
J=7.8Hz, Ar-H), 7.581, 7.577 (each 1H, d, J=7.8Hz, Ar-H), Ar), 146.2 (d, Ar-CH=), 171.2 (s, C=O). IR (KBr) cm−1: 1624,
7.74 (1H, brs, Ar-H). 13C-NMR (CDCl3, 100MHz) δ: 121.5 1697. Electron ionization (EI)-MS m/z: 220 (M+), 205 (M+−
(d, =CH–CO2–), 128.7 (d, Ar), 131.0 (d, Ar), 132.2 (d, Ar), Me). High resolution (HR)-EI-MS m/z: 220.0916 (M+, Calcd
136.3 (d, Ar), 142.8 (d, Ar-CH=), 169.7 (s, C=O). IR (KBr) for C12H16O2Si: 220.0920).
cm−1: 1699. Electrospray ionization (ESI)-MS m/z: 217
(M+−H); Anal. Calcd for C12H10O4: C, 66.05; H, 4.62. Found: tion of K2CO3 (3.40g, 24.6mmol) in acetone (10mL), 3-hy-
C, 65.86; H, 4.59. droxybenzaldehyde (21) (2.00g, 16.4mmol) was added at 0°C
(Z)-3-[3-(Pentyloxy)phenyl]acrylic Acid (7) To a solu-
(Z)-3-[3-(Trimethylsilyl)phenyl]acrylic Acid (5) To a under an argon atmosphere and then the mixture was stirred
solution of 1,3-dibromobenzene (18) (1.00g, 4.24mmol) in for 15min. After 1-iodopentane (6.50g, 32.8mmol) was added
diethyl ether (Et2O) (8.5mL), n-butyllithium (BuLi) (2.30M in to the solution, the resulting mixture was refluxed for 3.5h.
hexane, 1.84mL, 4.24mmol) was added dropwise at −78°C The reaction was filtered and the filtrate was evaporated to
under an argon atmosphere. After 15min of stirring, chlorotri- eliminate acetone. The resulting residue was diluted with H2O
methylsilane (0.570mL, 4.45mmol) was added dropwise to the and extracted with EtOAc. The organic layers were washed
solution. After the mixture was warmed to room temperature, with brine, dried over MgSO4, filtered, and concentrated in
the reaction was stirred for 1.5h. The mixture was quenched vacuo. The residue was purified by silica gel column chro-
with H2O and extracted with Et2O. The organic layer was matography (EtOAc–hexane, 30:70) to afford 3-(pentyloxy)-
washed with brine, dried over MgSO4, filtered and concentrat- benzaldehyde (22) (2.36g, 12.3mmol, 75%) as a colorless oil:
ed in vacuo. The crude product was purified by silica gel flash 1H-NMR (CDCl3, 270MHz) δ: 0.94 (3H, t, J=7.0Hz, –CH3),
column chromatography (hexane) to give (3-bromophenyl)- 1.33−1.52 (4H, m, –CH2–), 1.75−1.87 (2H, m, –CH2–), 4.02
trimethylsilane (19) (0.863g, 3.78mmol, 89%) as a colorless (2H, t, J=6.6Hz, –OCH2–), 7.17 (1H, m, Ar-H), 7.38 (1H,
1
oil: H-NMR (CDCl3, 400MHz) δ: 0.27 (9H, s, –CH3), 7.22 m, Ar-H), 7.41−7.48 (2H, m, Ar-H), 9.97 (1H, s, –CHO). The
(1H, t, J=8.1Hz, Ar-H), 7.42, 7.47 (each 1H, d, J=8.1Hz, spectral data were in agreement with those in the literature.21)
Ar-H), 7.61 (1H, brs, Ar-H); The spectral data were in agree-
The Z-selective olefination of 22 was performed using
the procedure described above to provide (Z)-ethyl
ment with those in the literature.23)

606
Vol. 62, No. 6
3-[3-(pentyloxy)phenyl]acrylate (99%, Z:E=95:5, deter- to afford (2E,4E)-ethyl-4-methyl-5-phenylpenta-2,4-dienoate
mined by 1H-NMR spectrum) (silica gel column chroma- (1.15g, 5.32mmol, 76%, E only, determined by 1H-NMR
1
1
tography, EtOAc–hexane, 5:95) as a colorless oil: H-NMR spectrum) as a colorless oil; H-NMR (CDCl3, 400MHz) δ:
(CDCl3, 270MHz) δ: 0.93 (3H, t, J=6.9Hz, –CH3), 1.25 1.33 (3H, t, J=7.2Hz, –CH3), 2.05 (3H, brs, =CH(CH3)–),
(3H, t, J=7.2Hz, –CO2–CH2–CH3), 1.26–1.56 (4H, m, 4.25 (2H, q, J=7.2Hz, –CH2–), 5.99 (1H, d, J=16.0Hz, =CH–
–CH2–), 1.67–1.90 (2H, m, –CH2–), 3.96 (2H, t, J=6.5Hz, CO2–), 6.86 (1H, s, Ar-CH=), 7.23–7.48 (5H, m, Ar-H), 7.50
–OCH2–), 4.18 (2H, q, J=7.2Hz, –CO2–CH2–), 5.93 (1H, d, (1H, d, J=16.0Hz, =C(CH3)–CH=).
J=12.5Hz,=CH–CO2–), 6.86 (1H, m, Ar-H), 6.90 (1H, d,
The hydrolysis of (2E,4E)-ethyl-4-methyl-5-phenylpenta-2,4-
J=12.5Hz, Ar-CH=), 7.11 (1H, m, Ar-H), 7.17−7.30 (2H, m, dienoate was performed using the procedure described above
Ar-H).
to afford 13 (89%) as a single isomer: colorless needles:
The hydrolysis of (Z)-ethyl-3-[3-(pentyloxy)phenyl]acrylate mp 113–114°C (CH2Cl2–hexane, 5:95); 1H-NMR (CDCl3,
was performed using the procedure described above to af- 400MHz) δ: 2.08 (3H, s, –CH3), 5.99 (1H, d, J=15.6 Hz,=CH–
ford 7 (77%) as a single isomer: colorless needles: mp CO2–), 6.90 (1H, s, Ar-CH=), 7.19–7.46 (5H, m, Ar-H),
35–36°C (toluene); H-NMR (CDCl3, 400MHz) δ: 0.93 (3H, 7.59 (1H, d, J=15.6Hz, =C(CH3)–CH=). 13C-NMR (CDCl3,
1
t, J=7.2Hz, –CH3), 1.29–1.52 (4H, m, –CH2–), 1.70–1.89 (2H, 100 MHz) δ: 13.7 (q, –CH3), 116.6 (d, =CH–CO2–), 127.9 (d,
m, –CH2–), 3.96 (2H, t, J=6.6Hz, –OCH2–), 5.96 (1H, d, Ar), 128.4 (d, Ar), 129.5 (d, Ar), 134.0 (s, =C(CH3)–), 136.5
J=12.8Hz, =CH–CO2–), 6.89 (1H, dd, J=2.4, 8.1Hz, Ar-H), (s, Ar), 140.1 (d, Ar-CH=), 152.2 (d,=C(CH3)–CH=), 173.0 (s,
7.02 (1H, d, J=12.8Hz, Ar-CH=), 7.13 (1H, d, J=8.1Hz, C=O). IR (KBr) cm−1: 1724. FAB-MS m/z: 188 (M+). Anal.
Ar-H), 7.20–7.34 (2H, m, Ar-H). 13C-NMR (CDCl3, 100MHz) Calcd for C12H12O2: C, 76.57; H, 6.43. Found: C, 76.44; H,
δ: 14.0 (q, –CH3), 22.4 (t, –CH2–), 28.2 (t, –CH2–), 28.9 6.41.
(t, –CH2–), 68.0 (t, –OCH2–), 115.5 (d, Ar), 116.2 (d, =CH–
The Root Growth Inhibition Assay The assay was
CO2–), 118.7 (d, Ar), 122.4 (d, Ar), 129.0 (d, Ar), 135.5 (d, performed according to the Hiradate’s method with minor
Ar), 145.7 (d, Ar-CH=), 158.8 (s, Ar), 171.4 (s, C=O). IR modification.11) An ϕ27 filter paper was placed in a glass petri
(KBr) cm−1: 1699. ESI-MS m/z: 233 (M+−H). Anal. Calcd for dish and methanol solution containing the test compounds and
C14H18O3: C, 71.77; H, 7.74. Found: C, 71.83; H, 7.71.
growth inhibitors were added into the dish simultaneously
(2Z,4Z)-4-Bromo-5-phenylpenta-2,4-dienoic Acid (12) (70µL each, concentrated for desired final concentration).
The Z-selective olefination of (Z)-2-bromo-3-phenylacrylalde- The solvent was removed from the filter paper under reduced
hyde (23) was performed using the procedure described above pressure. After the addition of 700µL distilled water, six pre-
to provide (2Z,4Z)-ethyl-4-bromo-5-phenylpenta-2,4-dienoate germinated lettuce seedlings (Lactuca sativa cv. Great Lakes
1
(98%, Z:E=88:12, determined by H-NMR spectrum) (sil- 366) were placed on the filter paper. The pre-germination
ica gel column chromatography, EtOAc–hexane, 5:95) as state was induced using distilled water at 25°C, 60% relative
1
a colorless oil: H-NMR (CDCl3, 270MHz) δ: 1.26 (3H, t, humidity for 24h in the dark. Two dishes were prepared for
J=7.0Hz, –CH3), 4.19 (2H, q, J=7.0Hz, –CH2–), 5.81 (1H, d, each concentration (n=12). The dishes were incubated at 25°C,
J=11.9 Hz,=CH–CO2–), 6.61 (1H, dd, J=1.1, 11.9Hz, =CBr– 60% relative humidity for 48h in a dark. The root growth was
CH=), 7.24 (1H, brs, Ar-CH=), 7.26–7.50 (3H, m, Ar-H), evaluated by measuring the length of each root and comparing
7.60–7.76 (2H, m, Ar-H).
it with the roots of vehicle control.
The hydrolysis of (2Z,4Z)-ethyl-4-bromo-5-phenylpenta-2,4-
Statistical Analysis Results were expressed as the mean
dienoate was performed using the procedure described above S.D. The statistical analysis was performed using a one-way
to afford 12 (84%) as a single isomer: colorless needles: ANOVA followed by Tukey–Kramer’s test to assess the sig-
mp 64 to 66°C (CH2Cl2–hexane, 5:95); 1H-NMR (CDCl3, nificance of differences in mean values between the drug
400MHz) δ: 5.82 (1H, d, J=12.0Hz, =CH–CO2–), 6.73 (1H, treatment groups and suitable referential group. Significance
d, J=12.0Hz, =CBr–CH–), 7.28 (1H, brs, Ar-CH=), 7.31–7.45 was accepted when the p value was less than 0.05.
(3H, m, Ar-H), 7.60–7.70 (2H, m, Ar-H). 13C-NMR (CDCl3,
100 MHz) δ: 114.8 (s, =CBr–), 119.8 (d, =CH–CO2–), 128.2
Acknowledgments This work is supported by the Pro-
(d, Ar), 128.7 (d, Ar), 129.3 (d, Ar), 134.2 (d, Ar-CH=), 134.9 gram for Promotion of Basic and Applied Research for In-
(s, Ar), 144.1 (d, =CBr–CH=), 170.8 (s, C=O). IR (KBr) cm−1: novations in the Bio-oriented Industry (BRAIN), and Science
1699. EI-MS m/z: 252 (M+−H). HR EI-MS m/z: 251.9788 (M+, and Technology Research Promotion Program for Agriculture,
Calcd for C11H9BrO2: 251.9786).
Forestry, Fisheries and Food Industry.
(2E,4E)-4-Methyl-5-phenylpenta-2,4-dienoic Acid (13)
To a solution of NaH (60% oil solution, 0.504g, 12.6mmol)
in Et2O (5.0mL), a solution of ethyl 2-(diethoxyphosphoryl)-
acetate (1.67g, 7.40mmol)21) in Et2O (5.0mL) was added at
0°C under an argon atmosphere, and then the solution was
stirred for 15min. A solution of (E)-2-methyl-3-phenylacryl-
aldehyde (24) (1.02g, 7.00mmol) in Et2O (4.0mL) was added
to the solution at the same temperature. After the mixture
was warmed to room temperature, the reaction was stirred for
5min. The mixture was quenched with H2O, extracted with
Et2O, washed with H2O and brine, dried over MgSO4, filtered
and concentrated in vacuo. The crude product was purified
by silica gel column chromatography (EtOAc–hexane, 3:97)
References
1) Haagen-Smit A. J., Went F. W., Proc. Kon. Akad. Wet. Amst., 38,
852–857 (1935).
2) Koepfli J. B., Thimann K. V., Went F. W., J. Biol. Chem., 122,
763–780 (1938).
3) Ugochukwu E. N., Wain R. L., Ann. Appl. Biol., 61, 121–130 (1968).
4) Yang X. X., Choi H. W., Yang S. F., Li N., Aust. J. Plant Physiol.,
26, 325–335 (1999).
5) Wong W. S., Guo D., Wang X. L., Yin Z. Q., Xia B., Li N., Plant
Physiol. Biochem., 43, 929–937 (2005).
6) Zimmerman P. W., Hitchcock A. E., Contrib. Boyce Thompson Inst.
Plant Res., 10, 197–200 (1939).
7) van Overbeek J., Blondeau R., Horne V., Am. J. Bot., 38, 589–595

June 2014
(1951).
607
Phytochemistry, 96, 132–147 (2013).
8) Masuda Y., Tanimoto E., Plant Cell Physiol., 8, 459–465 (1967).
9) Dixon R. A., Nature (London), 411, 843–847 (2001).
10) Yin Z., Wong W., Ye W., Li N., Chin. Sci. Bull., 48, 555–558 (2003).
16) Veldstra H., Booij H. L., Biochim. Biophys. Acta, 3, 278–312 (1949).
17) Ricci A., Maggiali C. A., Torelli A., Amorosi S., Ronchini F.,
Branca C., Plant Sci., 117, 151–158 (1996).
11) Hiradate S., Morita S., Sugie H., Fujii Y., Harada J., Phytochemistry, 18) Nishikawa K., Fukuda H., Abe M., Nakanishi K., Tazawa Y.,
65, 731–739 (2004).
12) Guo D., Wong W. S., Xu W. Z., Sun F. F., Qing D. J., Li N., Plant
Mol. Biol., 75, 481–495 (2011).
13) Wasano N., Sugano M., Nishikawa K., Okuda K., Shindo M., Abe
H., Park S. Y., Hiradate S., Kamo T., Fujii Y., Plant Biotechnol., 30,
465–471 (2013).
14) Abe M., Nishikawa K., Fukuda H., Nakanishi K., Tazawa Y., Tani-
guchi T., Park S. Y., Hiradate S., Fujii Y., Okuda K., Shindo M.,
Phytochemistry, 84, 56–67 (2012).
Yamaguchi C., Hiradate S., Fujii Y., Okuda K., Shindo M.,
Phytochemistry, 96, 223–234 (2013).
19) Ando K., Oishi T., Hirama M., Ohno H., Ibuka T., J. Org. Chem.,
65, 4745–4749 (2000).
20) Ando K., J. Org. Chem., 62, 1934–1939 (1997).
21) Runyon S. P., Mosier P. D., Roth B. L., Glennon R. A., Westkaem-
per R. B., J. Med. Chem., 51, 6808–6828 (2008).
22) Wadsworth W. S., Emmons W. D., J. Am. Chem. Soc., 83, 1733–
1738 (1961).
15) Nishikawa K., Fukuda H., Abe M., Nakanishi K., Taniguchi T., No-
mura T., Yamaguchi C., Hiradate S., Fujii Y., Okuda K., Shindo M.,
23) Ye B. H., Naruta Y., Tetrahedron, 59, 3593–3601 (2003).