Design and synthesis of conformationally constrained analogues of cis-cinnamic acid and evaluation of their plant growth inhibitory activity

Article abstract of DOI:10.1016/j.phytochem.2013.10.001

1-O-cis-Cinnamoyl-β-d-glucopyranose is known to be one of the most potent allelochemical candidates and was isolated from Spiraea thunbergii Sieb by Hiradate et al. (2004), who suggested that it derived its strong inhibitory activity from cis-cinnamic acid, which is crucial for phytotoxicity. In this study, key structural features and substituent effects of cis-cinnamic acid (cis-CA) on lettuce root growth inhibition was investigated. These structure-activity relationship studies indicated the importance of the spatial relationship of the aromatic ring and carboxylic acid moieties. In this context, conformationally constrained cis-CA analogues, in which the aromatic ring and cis-olefin were connected by a carbon bridge, were designed, synthesized, and evaluated as plant growth inhibitors. The results of the present study demonstrated that the inhibitory activities of the five-membered and six-membered bridged compounds were enhanced, up to 0.27 μM, and were ten times higher than cis-CA, while the potency of the other compounds was reduced.

Full text of DOI:10.1016/j.phytochem.2013.10.001

Phytochemistry 96 (2013) 223–234
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Design and synthesis of conformationally constrained analogues
of cis-cinnamic acid and evaluation of their plant growth inhibitory
activity
Keisuke Nishikawa a, Hiroshi Fukuda b, Masato Abe a, Kazunari Nakanishi b, Yuta Tazawa b,
Chihiro Yamaguchi d, Syuntaro Hiradate c, Yoshiharu Fujii d, Katsuhiro Okuda a, Mitsuru Shindo a,
⇑
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 Biodiversity Division, National Institute for Agro-Environmental Sciences, 3-1-3 Kan-nondai, Tsukuba, Ibaraki 305-8604, Japan
d International Environmental and Agricultural Sciences, Tokyo University of Agriculture and Technology, 3-5-8, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 March 2013
Received in revised form 19 September 2013
Available online 28 October 2013
1-O-cis-Cinnamoyl-b-
D
-glucopyranose is known to be one of the most potent allelochemical candidates and
was isolated from Spiraea thunbergii Sieb by Hiradate et al. (2004), who suggested that it derived its strong
inhibitory activity from cis-cinnamic acid, which is crucial for phytotoxicity. In this study, key structural
features and substituent effects of cis-cinnamic acid (cis-CA) on lettuce root growth inhibition was investi-
gated. These structure–activity relationship studies indicated the importance of the spatial relationship of
the aromatic ring and carboxylic acid moieties. In this context, conformationally constrained cis-CA ana-
logues, in which the aromatic ring and cis-olefin were connected by a carbon bridge, were designed, synthe-
sized, and evaluated as plant growth inhibitors. The results of the present study demonstrated that the
inhibitory activities of the five-membered and six-membered bridged compounds were enhanced, up to
Keywords:
Lettuce
Lactuca sativa
Asteraceae
cis-Cinnamic acid
Plant growth inhibitors
SAR
0.27
l
M, and were ten times higher than cis-CA, while the potency of the other compounds was reduced.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
be an auxin agonist. Although mechanistic studies based on molec-
ular biology have been reported (Chen et al., 2005; Guo et al.,
1-O-cis-Cinnamoyl-b-
D
-glucopyranose (1) (Figure 1), isolated
2011), the molecular mechanisms of these activities have not yet
been elucidated. In this study, herein, the glycosyl ester 1 (Matsuo
et al., 2011) was synthesized, and the structure–activity relation-
ship of cis-2 subsequently investigated. It was found that the struc-
tural features essential for bioactivity were a cis-configuration of
the alkene or cyclopropane, a carboxylic acid or its esters, and a
planar ring including a phenyl group (Abe et al., 2012). Further-
more, the substituent effect on the aromatic ring of cis-2 was also
established, in which meta-substituents, especially sterically
unhindered hydrophobic ones, were not critical for potency,
whereas several cis-2 analogues 3–5, such as m-iodo, m-methoxy,
or m-trifluoromethyl substituted ones, were more potent than that
of cis-CA (Figure 2) (Nishikawa et al., 2013).
During the course of examining the structure–activity relation-
ship (SAR) of cis-2, the planar ring and the carboxylic acid moiety
were found to be essential for potency. However, these moieties
could not be in the same plane, due to steric interference between
the ortho-substituent and the carboxylate. Therefore, although the
three-dimensional arrangement between the aromatic ring and
carboxylate was expected to be crucial for bioactivity, the most
effective three-dimensional arrangement could not be determined
readily (Figure 3) because the C–C bond connecting the aromatic
from Spiraea thunbergii Sieb (Hiradate et al., 2004) as a potent allel-
ochemical candidate, has been identified in 56 species of woody
plants grown in Japan by growth-inhibitory activity tests on root
elongation in germinated seedlings of lettuce (Lactuca sativa L.)
(Morita et al., 2001). The part of the cis-cinnamic acid (cis-CA)
structure (cis-2) considered essential for bioactivity is the aglycone
of 1, because cis-CA inhibited lettuce root growth as effectively as
1, whereas inhibition of growth by trans-cinnamic acid (trans-CA)
(trans-2) was markedly less than that by the cis-isomer (Hiradate
et al., 2005). The trans-2 isomer has generally been considered to
be physiologically inactive and is also a weak antagonist of auxin,
a plant hormone that regulates growth in the roots and stems
(Koepfli et al., 1938; van Overbeek et al., 1951; Ferro et al.,
2010). On the other hand, the cis-2 isomer was shown to inhibit
the root growth of Avena sativa, Triticum aestivum, and Arabidopsis
thaliana, and also induced epinastic curvature in Solanum lycopersi-
cum seedlings (Koepfli et al., 1938; van Overbeek et al., 1951; Yang
et al., 1999; Wong et al., 2005). Therefore, it is widely considered to
⇑
Corresponding author. Tel.: +81 92 583 7802; fax: +81 92 583 7875.
E-mail address: shindo@cm.kyushu-u.ac.jp (M. Shindo).
0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.10.001

224
K. Nishikawa et al. / Phytochemistry 96 (2013) 223–234
Fig. 1. Structures of 1-O-cis-cinnamoyl-b-
(cis- and trans-2).
D-glucopyranose (1) and cis- and trans-CA
Fig. 3. Design of conformationally constrained analogues cis-6 to cis-13.
2003). After separation of these products (37 and 38) with column
chromatography, each iodotetralone was olefinated via Peterson
olefination (Novák et al., 1992) to afford a mixture of cis- and
trans-olefins along with an endo-olefin. After these products (39
and 40) were separated by column chromatography, the pure es-
ters were individually hydrolyzed to provide the corresponding
carboxylic acids 12 and 13, respectively.
The endo-alkenyl bridged analogue 16 was also synthesized via
a Reformatsky reaction of 29 with the bromoacetic acid ethyl ester
(Johnson and Glenn, 1949; Miyashi et al., 1986; Chavan et al.,
1992), followed by the dehydration and hydrolysis of ethyl ester
16 (Scheme 7).
ring and olefin could rotate freely. Therefore, conformationally
constrained cis-2 analogues may provide useful information on
the conformation critical for potency. Herein is described the syn-
thesis and evaluation of various kinds of conformationally con-
strained bridged cis-2 analogues 6–13.
2. Results and discussion
2.1. Design and synthesis of cis-cinnamic acid analogues
Bridged cis/trans-CA analogues 6–9 were designed to under-
stand the spatial relevance between the aromatic ring and carbox-
ylic acid, because these were expected to be conformationally
constrained by bridging of the aromatic ring and the olefin. Ana-
logues cis-6ꢀcis-9 were thus synthesized via olefination with the
Horner–Wadswoth–Emmons reaction of the corresponding ke-
tones 14–17, followed by separation and hydrolysis of the cis/
trans-isomers 18–21 (Scheme 1). Benzocyclobutenone (14) was
prepared as shown in Scheme 2 (Aidhen and Ahuja, 1992).
Whereas the endo-isomer 7 was also obtained as a by-product of
olefination of the corresponding ketone 15 and its subsequent
hydrolysis.
The non-bridged analogue cis-24, which had the same number
of carbons as that in 8, was also synthesized from 2-methylbenzal-
dehyde (25) by the following transformations (Scheme 3): (1) addi-
tion of EtMgI to the aldehyde group of 25, (2) Jones oxidation of
secondary alcohol 26, (3) Peterson olefination of ketone 27 (Novák
et al., 1992), (4) separation of cis- and trans-28, and (5) hydrolysis.
From results of the previous study, the cis-alkenyl group could
be replaced by a cis-cyclopropyl group without significant loss in
activity (Abe et al., 2012). The cyclopropyl analogues cis- and
trans-10 of the bridged CA were also prepared by Rh-catalyzed
cyclopropanation (Cordi et al., 2001) of 1-methylene-1,2,3,4-tetra-
hydronaphthalene (30), followed by hydrolysis (Scheme 4). The
stereochemistries of cis- and trans-10 were then determined by
NOE experiments.
2.2. Bioassay and discussion
The growth inhibitory activity of the cis-CA analogues against
the root-growth of lettuce (Lactuca sativa cv.) was measured as
described previously (Hiradate et al., 2005). EC50 values, which
indicate the effective concentration required to induce a half-
maximum effect, are shown in Tables 1–3.
Bridged CA analogues 6–13 were conformationally constrained
compounds that were expected to provide useful information on
the conformation critical for the inhibitory activity of cis-CA (Ta-
ble 1). The dihedral angle (°) between the aromatic plane and
alkenyl plane in particular may be fixed by bridging (Fig. 4). Con-
formation searches and dihedral angles of bridged analogues
were calculated with MMFF using the CONFLEX module to locate
the global minimum (Goto and Osawa, 1993). As shown in Ta-
ble 1, cis-7 and cis-8 were found to be markedly stronger inhibi-
tors than cis-2 itself, with cis-7 in particular showing bioactivity
that was ten times higher than that of cis-2. The four-membered
ring analogue cis-6, which had a 0 dihedral angle, also exhibited
potent activity, while that of the seven-membered ring analogue
cis-9, the dihedral angle of which was 66°, was markedly reduced.
These calculated dihedral angles were 66°, 34°, 18°, and 0° as the
bridged ring size decreased from 7 to 4, which indicated that the
optimal angle may be located between 0° and 18°. The potencies
of trans-cinnamic acid analogues 6–9, bridged by four, six, and se-
ven-membered rings, were also reduced. Since non-bridged ana-
logue cis-24, which had the same carbon number, did not
exhibit inhibitory activity, the high potency of the six-membered
bridged analogue may only be slightly related to the hydrophobic
factor.
It was previously shown in a SAR study on cis-CA (Abe et al.,
2012) that the cis-alkene could be replaced with the cis-cyclopro-
pane skeleton, indicating the importance of the spacial relationship
between the aromatic ring and carboxylate. Therefore, the alkene
in the bridged analogues could be replaced by cyclopropane in a
similar manner to that described above. Based on this hypothesis,
two kinds of bridged cis- and trans-cyclopropane analogues 10 and
11, as shown in Table 2, were designed and their growth inhibitory
activities were examined. However, these compounds, including
either the cis- or trans-form, were not potent, which may have been
due to an unsuitable spatial relationship.
Also designed and synthesized were the ‘‘doubly bridged’’ com-
pounds cis- and trans-11, as shown in Scheme 5 (Cordi et al., 2001).
These compounds were bridged by a dotted bond indicated by an
arrow. The stereochemistries of cis- and trans-11 were also deter-
mined by NOE experiments.
The meta-iodinated bridged CA analogues 12 and 13 were pre-
pared as follows (Scheme 6): phenylbutyric acid 34 underwent
iodination (Plati et al., 1943), and the resulting mixture of isomers
35 and 36 was subjected to a Friedel–Crafts reaction to afford a
mixture of 7-iodo and 5-iodotetralones (37 and 38) (Cui et al.,
Based on the aforementioned substituent effects, both meta-
iodo-substituted and six-membered bridged analogues improved
Fig. 2. More potent cis-CA analogues 3–5.

K. Nishikawa et al. / Phytochemistry 96 (2013) 223–234
225
Scheme 1. Synthesis of bridged CA analogues cis- and trans-6–9 and endo-7: (a) NaH, ethyl 2-(diethoxyphosphoryl)acetate, THF, (b) 10% NaOH aq., EtOH, rt. cis-18: 36%, trans-
18: 33%, cis-6: 67%, trans-6: 68%, cis-19: 16%, trans-19: 11%, endo-19: 22%, cis-7: 92%, trans-7, 87%, endo-7: 91%, cis-20: 17%, trans-20: 33%, cis-8: 83%, trans-8: 84%, cis-21: 75%,
trans-21: 13%, cis-9: 61%, trans-9: 76%.
Scheme 2. Preparation of benzocyclobutenone (14): (a) N,O-dimethylhydroxyl-
amine hydrochloride, DCC, DMAP, Et3N, CH2Cl2, reflux, 87%, (b) tBuLi, THF, ꢀ40 °C,
40%.
inhibitory potency. Therefore, the combination of these modifica-
tions was expected to synergistically improve inhibitory activity.
Scheme 3. Synthesis of non-bridged cis-CA analogues 24: (a) EtMgI, Et2O, 0 °C, 98%,
However, the cis-meta-iodo six-membered bridged cinnamic acid
analogue 12 did not exhibit strong inhibitory activity, and the cor-
responding trans-analogue 12 was completely inactive (Table 3).
(b) Jones reagent, acetone, 0 °C, 93%, (c) diisopropylamine, nBuLi, ethyl(trimethyl-
silyl)acetate, THF, ꢀ78 to ꢀ20 °C, cis-28: 18%, trans-28: 41%, (d) 10% NaOH aq.,
EtOH, rt, 83% (from cis-28).

226
K. Nishikawa et al. / Phytochemistry 96 (2013) 223–234
multiplicities were indicated with the following abbreviations: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad.
IR spectra were recorded on a Shimadzu FT/IR-8300 spectrometer
using a KBr disk or NaCl cell. Mass spectra were obtained on a JEOL
JMS-700 or JEOL JMS-T100CS spectrophotometer. High-resolution
mass spectra were obtained on a JEOL JMS-700 or JEOL JMS-
T100CS spectrophotometer. Column chromatography (CC) was
performed on silica gel (Kanto Chemical Co.). Thin-layer chroma-
tography (TLC) was performed on pre-coated plates (0.25 mm, sil-
ica gel Merck 60 F254). Reaction mixtures were stirred
magnetically. Stereochemistry was determined by NOE experi-
ments, unless otherwise noted.
Scheme 4. Synthesis of cyclopropyl analogues cis-10 and trans-10: (a) methyltri-
phenylphosphonium bromide, nBuLi, THF, rt, 80%, (b) Rh2(OAc)4, ethyl diazoacetate,
CH2Cl2, rt, cis-31: 31%, trans-31: 33%, (c) 10% NaOH aq., EtOH, rt, cis-10: 96% (from
cis-31), trans-10: 93% (from trans-31).
4.2. Synthesis
4.2.1. (Z)-2-(Bicyclo[4.2.0]octa-1,3,5-trien-7-ylidene)acetic acid (cis-
6) and (E)-2-(bicyclo[4.2.0]octa-1,3,5-trien-7-ylidene)acetic acid
(trans-6) (Aidhen and Ahuja, 1992)
N,O-Dimethylhydroxylamine
hydrochloride
(0.447 g,
4.58 mmol), DCC (0.947 g, 4.58 mmol), DMAP (46.7 mg,
0.382 mmol), and Et3N (1.92 mL, 13.8 mmol) were added to a
solution of 2-(2-iodophenyl)acetic acid (22) (1.00 g, 3.82 mmol)
in CH2Cl2 (38 mL) at room temperature under Ar. After the mixture
was heated until reflux began, this being maintained for 4 h, the
solution was cooled to room temperature, with H2O (10 mL) and
CH2Cl2 (10 mL) next added. The resulting mixture was extracted
with CH2Cl2, washed (brine), dried (Na2SO4), filtered, and concen-
trated in vacuo. The crude product was purified by silica gel CC
(EtOAc/n-hexane, 20:80) to give 2-(2-iodophenyl)-N-methoxy-N-
methylacetamide (23) (1.01 g, 3.31 mmol, 87%) as a colorless oil.;
1H NMR (CDCl3, 400 MHz) d: 3.23 (s, 3H, –CH3), 3.73 (s, 3H,
–OCH3), 3.93 (s, 2H, –CH2–), 6.95 (dt, J = 2.4, 7.6 Hz, 1H, Ar-H),
7.29–7.33 (m, 2H, Ar-H), 7.84 (d, J = 8.4 Hz, 1H, Ar-H).
Scheme 5. Synthesis of cyclopropyl analogues cis- and trans-11: (a) Rh2(OAc)4,
ethyl diazoacetate, CH2Cl2, cis-33: 45%, trans-33: 22%, (b) 10% NaOH aq., EtOH, rt,
cis-11: 89% (from cis-33), trans-11: 98% (from trans-33).
On the other hand, endo-alkenyl bridged analogues 12, 8, and 7 had
higher potencies. From the viewpoint of structural similarities,
these partially saturated naphthalene-type compounds may be re-
garded as analogues of naphthaleneacetic acid (41) and indole-3-
acetic acid, which are known as synthetic and natural auxins,
respectively.
tBuLi (1.69 M in pentane, 2.30 mL, 3.84 mmol) was added drop-
wise to a solution of 23 (0.558 g, 1.83 mmol) in THF (18 mL) at
ꢀ78 °C under Ar. After the mixture was warmed to ꢀ40 °C, the
reaction was stirred for 2 h. The mixture was quenched with satu-
rated aqueous NH4Cl (10 mL), extracted with EtOAc (3 ꢁ 10 mL),
washed with brine, dried (Na2SO4), filtered, and concentrated in
vacuo. The crude product was purified by silica gel CC (EtOAc/hex-
ane, 20:80) to provide benzocyclobutanone (14) (97.1 mg,
0.732 mmol, 40%) as a yellow oil; 1H NMR (CDCl3, 400 MHz) d:
3.99 (s, 2H, c-butene), 7.34–7.41, 7.52–7.54 (m, each 2H, Ar-H).
Ethyl 2-(diethoxyphosphoryl)acetate (0.180 g, 0.805 mmol) was
added to a solution of NaH (60% oil, 40.4 mg, 1.01 mmol) in THF
(0.80 mL) at ꢀ78 °C under Ar. The mixture was stirred for
15 min, and a solution of 14 (79.3 mg, 0.671 mmol) in THF
(0.40 mL) was added. After the mixture was stirred for 30 min, sat-
urated aqueous NH4Cl was added to the reaction. The mixture was
then extracted with CH2Cl2 (3 ꢁ 10 mL), washed (brine), dried
(Na2SO4), filtered, and concentrated in vacuo. The crude product
was purified by silica gel CC (EtOAc/hexane, 10:90) to give (Z)-
ethyl 2-(bicyclo[4.2.0]octa-1(6),2,4-trien-7-ylidene)acetate (cis-
18) (major, 47.3 mg, 0.251 mmol, 36%) and (E)-ethyl 2-(bicy-
clo[4.2.0]octa-1(6),2,4-trien-7-ylidene)acetate (trans-18) (minor,
42.2 mg, 0.224 mmol, 33%) as a colorless oil; cis-18: 1H NMR
(CDCl3, 600 MHz) d: 1.37 (t, J = 7.2 Hz, 3H, –CH3), 3.73 (s, 2H, c-bu-
tene), 4.29 (q, J = 7.2 Hz, 2H, –CH2–), 5.69 (s, 1H, @CH–CO2–), 7.24
(d, J = 7.2 Hz, 1H, Ar-H), 7.31–7.38 (m, 2H, Ar-H), 7.81 (d, J = 7.2 Hz,
1H, Ar-H); trans-18: 1H NMR (CDCl3, 600 MHz) d: 1.32 (t, J = 7.2 Hz,
3H, –CH3), 4.02 (s, 2H, c-butene), 4.23 (q, J = 7.2 Hz, 2H, –CH2–),
6.02 (d, J = 1.8 Hz, 1H, @CH–CO2–), 7.23–7.34 (m, 4H, Ar-H).
An aqueous solution of 0.52 M NaOH (0.63 mL) was added to a
solution of cis-18 (47.3 mg, 0.251 mmol) in EtOH (0.62 mL) at room
temperature. The mixture was stirred for 12 h, diluted with H2O,
and the acidity of the solution was adjusted with 1 M HCl to pH
3. Conclusions
New plant growth inhibitors derived from cis-CA was designed
and synthesized. Conformational fixation by bridging suggested an
important three-dimensional relationship between the aromatic
ring and carboxylic acid moiety. The inhibitory activities of the
five-membered and six-membered bridged compounds were en-
hanced, up to 0.27 lM, and were ten times higher than that of
the original cis-CA. These bridged analogues may work in a similar
manner to naphthaleneacetic acid, because of their structural
similarities.
This study on cis-CA may be very useful for designing and syn-
thesizing molecular probes for mechanistic investigations and also
for new potent inhibitors targeting new types of agrochemicals.
4. Experimental section
4.1. Materials
Optical rotation values were obtained with Horiba SEPA-300.
Melting points were measured at Yazawa micromelting point BY-
1. 1H and 13C NMR spectra were recorded using JNM EX-270
(270 and 67.5 MHz), JEOL JNM AL-400 (400 and 100 MHz), and
JNM ECA-600 (600 and 150 MHz) spectrometers, respectively.
Chemical shifts were reported in ppm downfield from the peak
of Me4Si (TMS), which was used as internal standard. Peak

K. Nishikawa et al. / Phytochemistry 96 (2013) 223–234
227
Scheme 6. Synthesis of iodinated bridged CA analogues cis-, trans-, and endo-12 and 13: (a) H5IO6, I2, CH3CO2H, 2 M H2SO4, 79% (a mixture of 35 and 36), (b) polyphosphoric
acid (PPA), 90 °C, 37: 56%, 38: 17%, (c) diisopropylamine, nBuLi, ethyl(trimethylsilyl)acetate, THF, ꢀ20 °C to rt, cis-39: 20%, trans-39: 19%, endo-39: 35% (from 37), cis-40: 65%,
trans-40: 14% (from 38) (d) 10% NaOH aq., EtOH, rt, cis-12: 87% (from cis-39), trans-12: 88% (from trans-39), endo-12: 65% (from endo-39), cis-13: 87% (from cis-40), trans-13:
79% (from trans-40).
Table 1
Growth inhibitory activities and dihedral angles of cis- and trans-bridged CA
analogues 6–9 and non-bridged analogue cis-24.
Scheme 7. Synthesis of the bridged CA analogue endo-8: (a) Zn powder, bromo-
acetic acid ethyl ester, THF, 60 °C, then 1 M HCl, 0 °C, 63%, (b) 10% NaOH aq., EtOH,
rt, 88%.
1.0. The mixture was extracted with EtOAc (3 ꢁ 10 mL), washed
(brine), dried (MgSO4), and filtered. After the solvent was removed
in vacuo, the crude product was purified by recrystallization to give
Entry
(No)
n
Dihedral angles
EC50 (lM)
cis-6 (26.7 mg, 0.167 mmol, 67%) as colorless needles: mp
139–140 °C (CH2Cl2/hexane, 5:95); 1H NMR (CDCl3, 400 MHz) d:
3.78 (s, 2H, c-butene), 5.73 (s, 1H, @CH–CO2–), 7.26 (d, J = 6.4 Hz,
1H, Ar-H), 7.42–7.33 (m, 2H, Ar-H), 7.81 (d, J = 6.8 Hz,
1H, Ar-H); 13C NMR (CDCl3, 100 MHz) d: 38.9 (t, c-butene),
108.7 (d, @CH–CO2–), 122.7 (d, Ar), 125.2 (d, Ar), 128.2
(d, Ar), 132.2 (d, Ar), 142.8 (s, Ar), 147.1 (s, Ar), 156.4
(s, c-butene) 171.3 (s, C@O); IR (KBr) 1703 cmꢀ1; EI-MS m/z
160 (M+); Anal. calcd for C10H8O2: C, 74.99; H, 5.03. Found: C,
75.01; H, 5.09.
1
2
3
4
5
6
7
8
9
cis-2
cis-6
cis-7
cis-8
–
1
2
3
4
1
2
3
4
–
120°
0°
18°
34°
66°
9°
2.2
8.9
0.27
0.64
>500
99
cis-9
trans-6
trans-7
trans-8
trans-9
cis-24
6°
36
18°
62°
92°
240
>500
>500
10

228
K. Nishikawa et al. / Phytochemistry 96 (2013) 223–234
(t, c-butene), 107.5 (d, @CH–CO2–), 120.2 (d, Ar), 123.2 (d, Ar),
127.9 (d, Ar), 132.1 (d, Ar), 142.8 (s, Ar), 147.6 (s, Ar), 158.1 (s, c-bu-
tene) 171.4 (s, C@O); IR (KBr) 1701 cmꢀ1; EI-MS m/z 160 (M+);
HRMS (EI) calcd for C10H8O2 160.0524. Found 160.0522.
Fig. 4. Dihedral angles h of cis- and trans-bridged CA analogues 6–9.
Table 2
Doubly bridged cis-CA analogues 10 and 11.
4.2.2. (Z)-2-(2,3-Dihydro-1H-inden-1-ylidene)acetic acid (cis-7), (E)-
2-(2,3-dihydro-1H-inden-1-ylidene)acetic acid (trans-7) and 2-(1H-
inden-3-yl)acetic acid (endo-7)
Horner-Wadsworth-Emmons (HWE) olefination of 2,3-dihydro-
1H-inden-1-one (15) was performed using the procedure described
above to afford (Z)-ethyl 2-(2,3-dihydro-1H-inden-1-ylidene)ace-
tate (cis-19) (16%) and a mixture of (E)-ethyl 2-(2,3-dihydro-1H-
inden-1-ylidene)acetate (trans-19) and ethyl 2-(1H-inden-3-
yl)acetate (endo-19) (35%, 1:2) (silica gel CC, EtOAc/hexane, 5/95)
as colorless oils. The mixture was purified by preparative HPLC
(C18-column, EtOAc/n-hexane, 5:95) to afford trans-19 (11%) and
endo-19 (22%) as colorless oils; cis-19: 1H NMR (CDCl3, 400 MHz)
d: 1.32 (t, J = 7.2 Hz, 3H, –CH3), 2.90–2.93, 2.98–2.93 (m, each 2H,
c-pentene-H), 4.22 (q, J = 7.2 Hz, 2H, –CH2–), 5.97 (t, J = 2.0 Hz,
1H, @CH–CO2–), 7.27–7.34 (m, 3H, Ar-H), 8.82 (d, J = 8.0 Hz, 1H,
Ar-H); trans-19: 1H NMR (CDCl3, 400 MHz) d: 1.33 (t, J = 7.2 Hz,
3H, –CH3), 3.08 (t, J = 6.4 Hz, 2H, c-pentene-H), 3.28–3.32 (m, 2H, c-
pentene-H), 4.23 (q, J = 7.2 Hz, 2H, –CH2–), 6.31 (t, J = 2.4 Hz, 1H,
@CH–CO2–), 7.24–7.27 (m, 1H, Ar-H), 7.35 (d, J = 4.0 Hz, 2H, Ar-
H), 7.60 (d, J = 7.6 Hz, 1H, Ar-H); endo-19: 1H NMR (CDCl3,
400 MHz) d: 1.26 (t, J = 7.2 Hz, 3H, –CH3), 3.38 (s, 2H, –CH2–CO2–
), 3.59 (d, J = 1.6 Hz, 2H, c-pentadiene-H), 4.18 (q, J = 7.2 Hz, 2H,
–CH2–), 6.44 (s, 1H, c-pentadiene-H), 7.21, 7.30 (t, J = 7.2 Hz, each
1H, Ar-H), 7.38, 7.46 (d, J = 7.2 Hz, each 1H, Ar-H).
Entry
(No)
EC50 (lM)
1
2
3
4
cis-10
trans-10
cis-11
>500
230
222
trans-11
>500
Hydrolysis of trans-18 was performed using the procedure de-
scribed above to afford trans-6 (68%) as colorless needles: mp
184–185 °C (CH2Cl2/hexane, 5:95); 1H NMR (CDCl3, 400 MHz) d:
4.05 (s, 2H, c-butene), 6.04 (s, 1H, @CH–CO2–), 7.26–7.30,
7.35–7.39 (m, each 2H, Ar-H); 13C NMR (CDCl3, 100 MHz) d: 40.7
Table 3
Combination and endo-olefin type cis-CA analogues 7, 8, 12, and 13.
Entry
(No)
EC50 (lM)
1
2
3
4
5
6
7
8
cis-12
cis-13
470
450
>500
>500
4.2
0.74
10
0.38
trans-12
trans-13
endo-12
endo-8
endo-7
41

K. Nishikawa et al. / Phytochemistry 96 (2013) 223–234
229
Hydrolysis of cis-19 was performed using the procedure de-
scribed above to afford cis-7 (92%) as colorless needles: mp 88–
89 °C (CH2Cl2/hexane, 5:95); 1H NMR (CDCl3, 600 MHz) d: 2.97,
3.01 (d, J = 7.2 Hz, each 2H, c-pentene), 6.02 (s, 1H, @CH–CO2–),
7.30 (t, J = 7.8 Hz, 1H, Ar-H), 7.33 (d, J = 7.8 Hz, 1H, Ar-H), 7.37 (t,
J = 7.8 Hz, 1H, Ar-H), 8.80 (d, J = 7.8 Hz, 1H, Ar-H); 13C NMR (CDCl3,
150 MHz) d: 29.6 (t, c-pentene), 36.1 (t, c-pentene), 109.6 (d, @CH–
CO2–), 125.1 (d, Ar), 126.7 (d, Ar), 129.1 (d, Ar), 131.2 (d, Ar), 137.3
(s, Ar), 151.5 (s, Ar), 163.5 (s, c-pentene), 171.1 (s, C@O); IR (KBr)
1680 cmꢀ1; ESI-MS m/z 173 (M+ꢀH); Anal. calcd for C11H10O2: C,
75.84; H, 5.79. Found: C, 75.98; H, 5.69.
Hydrolysis of trans-19 was performed using the procedure de-
scribed above to afford trans-7 (87%) as colorless needles: mp
195–197 °C (CH2Cl2/n-hexane, 5:95); 1H NMR (CDCl3, 400 MHz)
d: 3.10 (t, J = 6.4 Hz, 2H, c-pentene-H), 3.33 (dt, J = 2.8, 6.4 Hz, 2H,
c-pentene-H), 6.35 (t, J = 2.8 Hz, 1H, @CH–CO2–), 7.28 (m, 1H, Ar-
H), 7.38–7.39 (m, 2H, Ar-H), 7.64 (d, J = 8.0 Hz, 1H, Ar-H); 13C
NMR (CDCl3, 100 MHz) d: 30.6 (t, c-pentene), 31.5 (t, c-pentene),
107.0 (d, @CH–CO2–), 122.0 (d, Ar), 125.7 (d, Ar), 126.9 (d, Ar),
131.3 (d, Ar), 140.0 (s, Ar), 150.0 (s, Ar), 165.9 (s, c-pentene),
172.7 (s, C@O); IR (KBr) 1684 cmꢀ1; ESI-MS m/z 173 (M+ꢀH); Anal.
calcd for C11H10O2: C, 75.84; H, 5.79. Found: C, 75.56; H, 5.82.
Hydrolysis of endo-19 was performed using the procedure de-
scribed above to afford endo-7 (91%) as colorless needles: mp
88–89 °C (CH2Cl2/hexane, 5:95); 1H NMR (CDCl3, 400 MHz) d:
3.40 (s, 2H, –CH2–CO2–), 3.64, 6.48 (brs, each 1H, c-pentadiene),
7.22, 7.31 (t, J = 7.2 Hz, each 1H, Ar-H), 7.38, 7.47 (d, J = 7.2 Hz, each
1H, Ar-H); 13C NMR (CDCl3, 100 MHz) d: 33.7 (t, –CH2–CO2–), 38.0
(t, c-pentadiene), 119.1 (d, c-pentadiene), 123.8 (d, Ar), 125.0 (d,
Ar), 126.2 (d, Ar), 132.4 (d, Ar), 135.9 (s, Ar), 143.9 (s, Ar), 144.1
(s, c-pentene), 177.4 (s, C@O); IR (KBr) 1697 cmꢀ1; ESI-MS m/z
173 (M+ꢀH); Anal. calcd for C11H10O2: C, 75.84; H, 5.79. Found:
C, 75.96; H, 5.59.
2H, c-Hexene-H), 5.81 (s, 1H, @CH–CO2–), 7.10–7.14 (m, 2H, Ar-
H), 7.28 (m, 1H, Ar-H), 7.64 (d, J = 7.6 Hz, 1H, Ar-H); 13C NMR
(CDCl3, 100 MHz) d: 22.9 (t, c-Hexene), 29.0 (t, c-Hexene), 35.3 (t,
c-Hexene), 113.5 (d, @CH–CO2–), 124.9 (d, Ar), 128.4 (d, Ar),
129.8 (d, Ar), 132.9 (s, Ar), 139.2 (s, Ar), 156.5 (s, c-Hexene),
172.2 (s, C@O); IR (KBr) 1691 cmꢀ1; ESI-MS m/z 187 (M+ꢀH); Anal.
calcd for C12H12O2: C, 76.57; H, 6.43. Found: C, 76.49; H, 6.40.
Hydrolysis of trans-20 was performed using the procedure de-
scribed above to afford trans-8 (84%) as colorless needles: mp
168–169 °C (CH2Cl2/n-hexane, 5:95); 1H NMR (CDCl3, 400 MHz)
d: 1.87 (quintet, J = 6.4 Hz, 2H, c-Hexene-H), 2.81, 3.20 (t,
J = 6.4 Hz, each 2H, c-Hexene-H), 6.38 (s, 1H, @CH–CO2–), 7.17 (d,
J = 7.6 Hz, 1H, Ar-H), 7.29–7.33 (m, 2H, Ar-H), 7.63 (d, J = 8.4 Hz,
1H, Ar-H); 13C NMR (CDCl3, 100 MHz) d: 22.6 (t, c-hexene), 28.3
(t, c-hexene), 30.1 (t, c-hexene), 111.8 (d, 1H, @CH–CO2–), 125.1
(d, Ar), 126.4 (d, Ar), 129.2 (d, Ar), 134.0 (s, Ar), 140.6 (s, Ar),
157.5 (s, c-hexene), 172.5 (s, C@O); IR (KBr) 1684 cmꢀ1; ESI-MS
m/z 187 (M+ꢀH); Anal. calcd for C12H12O2: C, 76.57; H, 6.43. Found:
C, 76.47; H, 6.43.
4.2.4. (Z)-2-(6,7,8,9-Tetrahydro-5H-benzo[7]annulen-5-ylidene)acetic
acid (cis-9) and (E)-2-(6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-
ylidene)acetic acid (trans-9) (Lee et al., 1989)
HWE olefination of 6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-
one (17) was performed using the procedure described above to af-
ford (Z)-ethyl 2-(6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-yli-
dene)acetate (cis-21) (major, 75%) and (E)-ethyl 2-(6,7,8,9-
tetrahydro-5H-benzo[7]annulen-5-ylidene)acetate
(trans-21)
(minor, 13%) as colorless oils: cis-21: 1H NMR (CDCl3, 400 MHz)
d: 1.06 (t, J = 7.2 Hz, 3H, –CH3), 1.73, 1.92, 2.39 (brs, each 2H, c-hep-
tene-H), 2.73–2.74 (m, 2H, Ar-H), 3.98 (q, J = 7.2 Hz, 2H, –CH2–),
5.95 (s, 1H, @CH–CO2–), 7.05 (d, J = 7.8 Hz, 1H, Ar-H), 7.12–7.22
(m, 3H, Ar-H); spectroscopic data were in agreement with those
in the literature (Lee et al., 1989); trans-21: 1H NMR (CDCl3,
400 MHz) d: 1.32 (t, J = 7.2 Hz, 3H, –CH3), 1.68–1.81 (m, 4H, c-hep-
tene-H), 2.75 (brs, 2H, c-heptene-H), 2.98 (brs, 4H, c-heptene-H),
4.21 (q, J = 7.2 Hz, 2H, –CH2–), 5.87 (s, 1H, @CH–CO2–), 7.11 (d,
J = 7.2 Hz, 1H, Ar-H), 7.18–7.23 (m, 3H, Ar-H); spectroscopic data
were in agreement with those in the literature (Lee et al., 1989).
Hydrolysis of cis-21 was performed using the procedure de-
scribed above to afford cis-9 (61%) as colorless needles: mp 149–
150 °C (CH2Cl2/hexane, 5:95); 1H NMR (CDCl3, 400 MHz) d: 1.72–
1.74 (m, 2H, c-heptene-H), 1.89–1.95 (m, 2H, c-heptene-H), 2.41,
2.74 (t, J = 5.8 Hz, each 2H, c-heptene-H), 5.96 (s, 1H, @CH–CO2–),
7.06 (d, J = 8.4 Hz, 1H, Ar-H), 7.14–7.19 (m, 3H, Ar-H); 13C NMR
(CDCl3, 100 MHz) d: 27.5 (t, c-heptene), 32.4 (t, c-heptene), 35.8
(t, c-heptene), 39.0 (t, c-heptene), 116.6 (d, @CH–CO2–), 125.7 (d,
Ar), 127.7 (d, Ar), 127.9 (d, Ar), 128.9 (d, Ar), 138.8 (s, Ar), 140.6
4.2.3. (Z)-2-[3,4-Dihydronaphthalen-1(2H)-ylidene]acetic acid (cis-8)
and (E)-2-[3,4-dihydronaphthalen-1(2H)-ylidene]acetic acid (trans-8)
HWE olefination of
a-tetralone (16) was performed using the
procedure described above to afford (Z)-ethyl 2-[3,4-
dihydronaphthalen-1(2H)-ylidene]acetate (cis-20) (minor, 17%)
and (E)-ethyl 2-[3,4-dihydronaphthalen-1(2H)-ylidene]acetate
(trans-20) (major, 33%) as colorless oils: cis-20: 1H NMR (CDCl3,
400 MHz) d: 1.25 (t, J = 7.2 Hz, 3H, –CH3), 1.97 (quintet, J = 6.8 Hz,
2H, c-pentene-H), 2.51 (t, J = 6.8 Hz, 2H, c-pentene-H), 2.86 (t,
J = 6.8 Hz, 2H, c-pentene-H), 4.16 (q, J = 7.2 Hz, 2H, –CH2–), 5.79
(s, 1H, @CH–CO2–), 7.12–7.18 (m, 3H, Ar-H), 7.59 (d, J = 7.2 Hz,
1H, Ar-H); trans-20: 1H NMR (CDCl3, 400 MHz) d: 1.32 (t,
J = 7.2 Hz, 3H, –CH3), 1.86 (quintet, J = 6.4 Hz, 2H, c-pentene-H),
2.80 (t, J = 6.4 Hz, 2H, c-pentene-H), 3.20 (dt, J = 2.2, 6.4 Hz 2H, c-
pentene-H), 4.21 (q, J = 7.2 Hz, 2H, –CH2–), 6.33 (s, J = 2.2 Hz, 1H,
@CH–CO2–), 7.14–7.21 (m, 2H, Ar-H), 7.26–7.30 (m, 1H, Ar-H),
7.66 (d, J = 8.4 Hz, 1H, Ar-H).
(s, Ar), 164.9 (s, c-hexene), 170.3 (s, C@O); IR (KBr) 1697 cmꢀ1
;
ESI-MS m/z 201 (M+ꢀH); Anal. calcd for C13H14O2: C, 77.20; H,
6.98. Found: C, 77.22; H, 7.00.
Hydrolysis of trans-21 was performed using the procedure de-
scribed above to afford trans-9 (76%) as colorless needles: mp
104–106 °C (CH2Cl2/hexane, 5:95); 1H NMR (CDCl3, 400 MHz) d:
1.79–1.80 (m, 4H, c-heptene-H), 2.76 (t, J = 5.6 Hz, 2H, c-heptene-
H), 3.02 (brs, 2H, c-heptene-H), 5.92 (s, 1H, @CH–CO2–), 7.13
Hydrolysis of cis-20 was performed using the procedure de-
scribed above to afford cis-8 (83%) as colorless needles: mp 72–
74 °C (CH2Cl2/n-hexane, 5:95); 1H NMR (CDCl3, 400 MHz) d: 1.98
(quintet, J = 6.8 Hz, 2H, c-Hexene-H), 2.53, 2.86 (t, J = 6.8 Hz, each

230
K. Nishikawa et al. / Phytochemistry 96 (2013) 223–234
(d, J = 7.2 Hz, 1H, Ar-H), 7.25–7.21 (m, 3H, Ar-H); 13C NMR (CDCl3,
100 MHz) d: 26.5 (t, c-heptene), 26.7 (t, c-heptene), 31.3 (t, c-hep-
tene), 33.9 (t, c-heptene), 116.9 (d, @CH–CO2–), 126.4 (d, Ar), 127.1
(d, Ar), 128.6 (d, Ar), 129.0 (d, Ar), 139.4 (s, Ar), 142.8 (s, Ar), 167.2
(s, c-hexene), 171.4 (s, C@O); IR (KBr) 1684 cmꢀ1; ESI-MS m/z 201
(M+ꢀH); Anal. calcd for C13H14O2: C, 77.20; H, 6.98. Found: C,
77.00; H, 6.98.
Ar-CH3), 2.95 (q, J = 7.6 Hz, 2H, –CH2–), 4.21 (q, J = 7.2 Hz, 2H,
–CO2–CH2–), 5.68 (s, 1H, @CH–CO2–), 7.04 (d, J = 7.2 Hz, 1H, Ar-
H), 7.18–7.21 (m, 3H, Ar-H).
Hydrolysis of cis-28 was performed using the procedure
described above to afford cis-24 (83%) as colorless needles: mp
85–88 °C (toluene); 1H NMR (CDCl3, 400 MHz) d: 1.06 (t,
J = 7.4 Hz, 3H, –CH2–CH3), 2.15 (s, 3H, Ar-CH3), 2.37 (q, J = 7.4 Hz,
2H, –CH2–), 5.89 (t, J = 1.6 Hz, 1H, @CH–CO2–), 6.89 (m, 1H, Ar-
H), 7.11–7.19 (m, 3H, Ar-H); 13C NMR (CDCl3, 100 MHz) d: 11.6
(q, –CH2–CH3), 19.2 (q, Ar-CH3), 33.6 (t, –CH2–), 116.3 (d,
@CH–CO2–), 125.3 (d, Ar), 126.2 (d, Ar), 127.2 (d, Ar), 129.7 (d,
Ar), 133.8 (s, Ar), 139.9 (s, Ar), 163.8 (s, ArC(Et)@), 170.4 (s,
C@O); IR (KBr) 1697 cmꢀ1; EI-MS m/z 190 (M+); Anal. calcd for
C12H14O2: C, 75.76; H, 7.42. Found: C, 75.58; H, 7.46.
4.2.5. (Z)-3-(o-Tolyl)pent-2-enoic acid (cis-24) (Novák et al., 1992)
25 (24.0 mL, 20.8 mmol) was added to a solution of EtMgI
(17.4 g, 62.4 mmol) in Et2O (100 mL) at 0 °C under Ar. After 1 h
of stirring, the reaction was quenched with saturated aqueous NH4-
Cl and extracted with CH2Cl2. The organic layers were washed with
H2O and brine, dried (MgSO4), filtered, and concentrated in vacuo.
The residue was purified by silica gel CC (EtOAc/n-hexane, 10:90)
to afford 1-(o-tolyl)propan-1-ol (26) (3.06 g, 20.4 mmol, 98%) as a
yellow oil; 1H NMR (CDCl3, 400 MHz) d: 0.98 (t, J = 7.2 Hz, 3H,
–CH2–CH3), 1.70–1.81 (m, 2H, –CH2–), 2.34 (s, 3H, Ar-CH3), 4.85
(t, J = 6.2 Hz, 1H, Ar-CH(OH)-), 7.12–7.18 (m, 2H, Ar-H), 7.23 (t,
J = 7.2 Hz, 1H, Ar-H), 7.46 (d, J = 7.6 Hz, 1H, Ar-H); spectroscopic
data were in agreement with those in the literature (Ueno et al.,
2011).
Jones reagent (15.5 mL, 30.0 mmol) was added to a solution of
26 (3.00 g, 20.0 mmol) acetone (200 mL) at 0 °C under Ar. After
1 h stirring, 2-propanol (30 mL) was added to the mixture. After
1.5 h stirring, the resulting mixture was concentrated in vacuo, di-
luted with H2O at 0 °C, extracted with CH2Cl2 (3 ꢁ 30 mL), washed
(H2O) and brine, dried (MgSO4), filtered, and concentrated in vacuo.
The crude product was purified by silica gel CC (EtOAc/hexane,
5:95) to give 1-(o-tolyl)propan-1-one (27) (2.76 g, 18.6 mmol,
93%) as yellow needles; 1H NMR (CDCl3, 400 MHz) d: 1.20 (t,
J = 7.2 Hz, 3H, –CH2–CH3), 2.49 (s, 3H, Ar-CH3), 2.92 (q, J = 7.2 Hz,
2H, –CH2–), 7.23–7.27 (m, 2H, Ar-H), 7.36 (m, 1H, Ar-H), 7.62 (d,
J = 7.6 Hz, 1H, Ar-H); spectroscopic data were in agreement with
those in the literature (Cahiez et al., 2004).
4.2.6. (1RS, 2SR)-30,40-Dihydro-20H-spiro(cyclopropane-1,10-
naphthalene)-2-carboxylic acid (cis-10) and (1RS, 2RS)-30,40-dihydro-
20H-spiro(cyclopropane-1,10-naphthalene)-2-carboxylic acid (trans-
10) (Cordi et al., 2001)
n-BuLi (1.67 M in hexane, 8.26 mL, 13.8 mmol) was added drop-
wise to
a solution of methyltriphenylphosphonium bromide
(5.17 g, 14.5 mmol) in THF (56 mL) at 0 °C under Ar. After 15 min
stirring, a solution of 29 (1.06 g, 7.24 mmol) in THF (15 mL) was
added. After 10 h stirring at room temperature, the mixture was
quenched with saturated aqueous NH4Cl and extracted with EtOAc
(3 ꢁ 30 mL). The organic layer was washed with brine, dried (Na2-
SO4), filtered, and concentrated in vacuo. The crude product was
purified by silica gel CC (EtOAc/n-hexane, 3:97) to give 1-methy-
lene-1,2,3,4-tetrahydronaphthalene (30) (0.831 g, 5.76 mmol,
80%) as a colorless oil: 1H NMR (CDCl3, 400 MHz) d: 1.88 (quintet,
J = 6.4 Hz, 2H, c-hexene-H), 2.55 (dt, J = 1.6, 6.4 Hz, 2H, c-hexene-
H), 2.85 (t, J = 6.4 Hz, 2H, c-hexene-H), 4.95 (d, J = 1.6 Hz, 1H,
@CH2), 5.47 (s, 1H, @CH2), 7.10 (m, 1H, Ar-H), 7.14–7.17 (m, 2H,
Ar-H), 7.65 (m, 1H, Ar-H).
A solution of ethyl diazoacetate (1.51 mL, 14.4 mmol) in THF
(1.7 mL) was slowly added to a solution of 30 (0.830 g, 5.76 mmol)
and Rh2(OAc)4 (25.4 mg, 57.6 lmol) in CH2Cl2 (3.5 mL) at room
nBuLi (1.64 M in hexane, 20.7 mL, 4.60 mmol) was added to a
solution of diisopropylamine (4.80 mL, 34.0 mmol) in THF
(70 mL) at ꢀ78 °C under Ar. After 20 min stirring, ethyl(trimethyl-
silyl)acetate (5.86 mL, 32.0 mmol) was added. After 10 min
stirring, the resulting mixture was added to 27 (2.37 g, 16.0 mmol)
in THF (6.0 mL). After 2 h stirring, the reaction temperature was
warmed to ꢀ20 °C and stirred for an additional 2 h. The reaction
was quenched with H2O at 0 °C, extracted with EtOAc
(3 ꢁ 30 mL), then washed with H2O, 1 M HCl, successively
saturated aqueous NaHCO3, and brine, respectively, dried (MgSO4),
filtered, and concentrated in vacuo. The crude product was purified
by silica gel CC (EtOAc/n-hexane, 10:90) to give (Z)-ethyl 3-(o-to-
lyl)pent-2-enoate (cis-28) (minor, 0.624 mg, 2.86 mmol, 18%) and
(E)-ethyl 3-(o-tolyl)pent-2-enoate (trans-28) (major, 1.42 g,
6.51 mmol, 41%) as yellow oils: cis-28: 1H NMR (CDCl3, 400 MHz)
d: 1.02, 1.10 (t, J = 7.2 Hz, each 3H, –CH3), 2.18 (s, 3H, Ar-CH3),
2.39 (q, J = 7.2 Hz, 2H, –CH2–), 3.95 (q, J = 7.2 Hz, 2H, –CO2–CH2–
), 5.93 (s, 1H, @CH–CO2–), 6.93 (d, J = 6.4 Hz, 1H, Ar-H), 7.15–7.17
(m, 3H,
temperature under an argon atmosphere. After 15 min, the reac-
tion was concentrated in vacuo. The residue was purified by silica
gel column chromatography (EtOAc/n-hexane, 5:95) to afford
(1RS,2SR)-ethyl 30,40-dihydro-20H-spiro(cyclopropane-1,10-naph-
thalene)-2-carboxylate (cis-31) (minor, 0.411 g, 1.79 mmol, 31%)
and (1RS,2RS)-ethyl 30,40-dihydro-20H-spiro(cyclopropane-1,10-
naphthalene)-2-carboxylate (trans-31) (major, 0.437 g, 1.90 mmol,
33%) as colorless oils: cis-31: 1H NMR (CDCl3, 400 MHz) d: 0.95 (t,
J = 6.6 Hz, 3H, –CH3), 1.19–1.30 (m, 2H, c-Hexene-H, c-Pr-H), 1.83
(dd, J = 6.4, 8.0 Hz, 1H, c-Pr-H), 1.90 (m, 1H, c-hexene-H), 2.05–
2.16 (m, 3H, c-hexene-H, c-Pr-H), 2.82–2.94 (m, 2H, c-hexene-H),
3.84 (q, J = 6.6 Hz, 2H, –CH2–), 7.05–7.15 (m, 4H, Ar-H); trans-31:
1H NMR (CDCl3, 400 MHz) hb: 1.25 (t, J = 7.2 Hz, 3H, –CH3), 1.53–
1.61 (m, 2H, c-Pr-H), 1.72, 1.88 (m, each 1H, c-hexene-H), 1.93–
2.04 (m, 3H, c-hexene-H, c-Pr-H), 2.87 (t, J = 6.4 Hz, 2H, c-hexene-
H), 4.11–4.20 (m, 2H, –CH2–), 6.72 (m, 1H, Ar-H), 7.09–7.11 (m,
3H, Ar-H).
Ar-H); trans-28: 1H NMR (CDCl3, 400 MHz) d: 0.99 (t, J = 7.6 Hz, 3H,
–CH3), 1.31 (t, J = 7.2 Hz, 3H, –CO2–CH2–CH3), 2.28 (s, 3H,
Hydrolysis of cis-31 was performed using the procedure de-
scribed above to afford cis-10 (96%) as colorless needles: mp

K. Nishikawa et al. / Phytochemistry 96 (2013) 223–234
231
179–181 °C (CH2Cl2/n-hexane, 50:50); 1H NMR (CDCl3, 400 MHz)
d: 1.18 (td, J = 4.0, 12.8 Hz, 1H, c-hexene-H), 1.26 (dd, J = 5.6,
8.0 Hz, 1H, c-Pr-H), 1.76 (dd, J = 6.0, 8.0 Hz, 1H, c-Pr-H), 1.89 (m,
1H, c-hexene-H), 1.99–2.07 (m, 2H, c-hexene-H, c-Pr-H), 2.10 (dt,
J = 4.6, 12.0 Hz, 1H, c-hexene-H), 2.81–2.94 (m, 2H, c-hexene-H),
7.01–7.11 (m, 4H, Ar-H); 13C NMR (CDCl3, 100 MHz) d: 18.4 (t, c-
hexene), 21.8 (t, c-hexene), 29.7 (t, c-hexene), 31.9 (d, c-Pr), 33.3
(s, c-Pr), 35.7 (t, c-Pr), 124.5 (d, Ar), 126.2 (d, Ar), 126.5 (d, Ar),
128.3 (d, Ar), 133.9 (s, Ar), 138.8 (s, Ar), 175.8 (s, C@O); IR (KBr)
1697 cmꢀ1; ESI-MS m/z 201 (M+ꢀH); Anal. calcd for C13H14O2: C,
77.20; H, 6.98. Found: C, 76.92; H, 6.92.
Hydrolysis of trans-31 was performed using the procedure de-
scribed above to afford trans-10 (93%) as colorless needles: mp
175–176 °C (CH2Cl2/n-hexane, 50:50); 1H NMR (CDCl3, 400 MHz)
d: 1.58 (dd, J = 5.2, 6.4 Hz, 1H, c-Pr-H), 1.67 (dd, J = 5.2, 8.8 Hz,
1H, c-Pr-H), 1.78 (m, 1H, c-hexene-H), 1.88 (m, 1H, c-hexene-H),
1.97–2.05 (m, 3H, c-hexene-H, c-Pr-H), 2.88 (t, J = 6.4 Hz, 2H, c-
hexene-H), 6.73 (m, 1H, Ar-H), 7.08–7.13 (m, 3H, Ar-H); 13C NMR
(CDCl3, 100 MHz) d: 22.1 (t, c-hexene), 23.1 (t, c-Pr), 27.6 (t, c-hex-
ene), 30.4 (t, c-hexene), 30.4 (s, c-Pr), 31.8 (d, c-Pr), 121.8 (d, Ar),
126.0 (d, Ar), 126.3 (d, Ar), 129.1 (d, Ar), 138.0 (s, Ar), 139.0 (s,
Ar), 177.6 (s, C@O); IR (KBr) 1683 cmꢀ1; ESI-MS m/z 201 (M+ꢀH);
Anal. calcd for C13H14O2: C, 77.20; H, 6.98. Found: C, 77.38; H, 7.13.
Hydrolysis of cis-33 was performed using the procedure de-
scribed above to afford cis-11 (89%) as colorless needles: mp
162–163 °C (CH2Cl2/n-hexane, 20:80); 1H NMR (CDCl3, 400 MHz)
d: 1.18 (ddt, J = 2.8, 6.0, 14.0 Hz, 1H, c-Hexene-H), 2.08 (t,
J = 3.6 Hz, 1H, c-Pr-H), 2.18–2.25 (m, 2H, c-Hexene-H, c-Pr-H),
2.46 (td, J = 14.0, 6.4 Hz, 1H, c-Hexene-H), 2.61–2.68 (m, 2H, c-Hex-
ene-H, c-Pr-H), 7.02 (d, J = 6.4 Hz, 1H, Ar-H), 7.10–7.17 (m, 2H, Ar-
H), 7.28 (m, 1H, Ar-H); 13C NMR (CDCl3, 100 MHz) d: 18.6 (t, c-hex-
ene), 23.0 (d, c-Pr), 25.1 (d, c-Pr), 25.2 (t, c-hexene), 27.3 (d, c-Pr),
126.2 (d, Ar), 126.3 (d, Ar), 128.6 (d, Ar), 128.9 (d, Ar), 133.7 (s,
Ar), 134.5 (s, Ar), 179.7 (s, C@O); IR (KBr) 1678 cmꢀ1; ESI-MS m/z
187 (M+ꢀH); Anal. calcd for C13H14O2: C, 77.20; H, 6.98. Found:
C, 76.92; H, 6.92.
The hydrolysis of trans-33 was performed using the procedure
described above to afford trans-11 (98%) as colorless needles: mp
159–161 °C (toluene/n-hexane, 50:50); 1H NMR (CDCl3, 400 MHz)
d: 1.95–1.98 (m, 2H, c-Pr-H), 2.09–2.13 (m, 2H, c-hexene-H), 2.45
(t, J = 8.8 Hz, 1H, c-Pr-H), 2.54 (td, J = 5.6, 15.6 Hz, 1H, c-hexene-
H), 2.75 (td, J = 8.0, 15.6 Hz, 1H, c-hexene-H), 7.04 (d, J = 6.8 Hz,
1H, Ar-H), 7.07–7.13 (m, 2H, Ar-H), 7.20 (m, 1H, Ar-H); 13C NMR
(CDCl3, 100 MHz) d: 17.9 (t, c-hexene), 19.4 (d, c-Pr), 21.5 (d, c-
Pr), 25.5 (d, c-Pr), 27.9 (t, c-hexene), 126.1 (d, Ar), 126.5 (d, Ar),
127.8 (d, Ar), 129.8 (d, Ar), 131.7 (s, Ar), 138.5 (s, Ar), 173.6 (s,
C@O); IR (KBr) 1695 cmꢀ1; ESI-MS m/z 211 [(M+Na)+]; Anal. calcd
for C13H14O2: C, 76.57; H, 6.43. Found: C, 76.38; H, 6.36.
4.2.8. (Z)-2-[7-Iodo-3,4-dihydronaphthalen-1(2H)-ylidene]acetic acid
(cis-12), (E)-2-[7-iodo-3,4-dihydronaphthalen-1(2H)-ylidene]acetic
acid (trans-12), and 2-[7-iodo-3,4-dihydronaphthalen-1-yl]acetic acid
(endo-12) (Plati et al., 1943; Novák et al., 1992; Cui et al., 2003)
AcOH (35 mL) and 2.0 M H2SO4 (5 mL) were added to a mixture
of 4-phenylbutanoic acid (34) (5.00 g, 30.5 mmol), H5OI6 (2.78 g,
12.2 mmol), and I2 (4.60 g, 18.3 mmol) at room temperature. After
the mixture was warmed to 75 °C, the reaction was stirred for 4 h.
The reaction was cooled to room temperature and quenched with
H2O. The resulting precipitates were collected by filtration, then,
dissolved in CH2Cl2, washed with H2O and saturated aqueous
Na2S2O3, dried (Na2SO4), filtered, and concentrated in vacuo. The
residue was purified by silica gel CC (EtOAc/hexane, 5:95) to give
4.2.7. (1SR,1aRS,7bSR)-1a,2,3,7b-tetrahydro-1H-cyclopropa[a]naphthalene-
1-carboxylic acid (cis-11) and (1aRS, 7bSR)-1a,2,3,7b-tetrahydro-1H-
cyclopropa[a]naphthalene-1-carboxylic acid (trans-11) (Cordi et al., 2001)
A solution of ethyl diazoacetate (0.482 mL, 4.60 mmol) in THF
(0.69 mL) was slowly added to a solution of 1,2-dihydronapthalene
(32) (0.299 g, 2.30 mmol) and Rh2(OAc)4 (10.2 mg, 23.0 lmol) in
Et2O (1.4 mL) at room temperature under Ar. After 15 min, the
reaction was concentrated in vacuo. The residue was purified by sil-
ica gel CC (EtOAc/n-hexane, 5:95) to afford (1SR,1aRS,7bSR)-ethyl
1a,2,3,7b-tetrahydro-1H-cyclopropa[a]naphthalene-1-carboxylate
(cis-33) (major, 0.224 g, 1.04 mmol, 45%) and (1aRS,7bSR)-ethyl
1a,2,3,7b-tetrahydro-1H-cyclopropa[a]naphthalene-1-carboxylate
(trans-33) (minor, 0.109 g, 0.506 mmol, 22%) as colorless oils: cis-
33: 1H NMR (CDCl3, 400 MHz) d: 1.27 (t, J = 7.2 Hz, 3H, –CH3),
1.79 (ddt, J = 3.2, 6.2, 16.0 Hz, 1H, c-hexene-H), 2.07 (t, J = 3.8 Hz,
1H, c-Pr-H), 2.17–2.22 (m, 2H, c-hexene-H, c-Pr-H), 2.46 (dt,
J = 6.2, 14.0 Hz, 1H, c-hexene-H), 2.55 (dd, J = 3.8, 8.8 Hz, 1H, c-Pr-H),
2.65 (dd, J = 6.2, 16.0 Hz, 1H, c-hexene-H), 4.15 (q, J = 7.2 Hz, 2H,
–CH2–), 7.02 (d, J = 7.2 Hz, 1H, Ar-H), 7.09–7.16 (m, 2H, Ar-H),
7.28 (m, 1H, Ar-H); spectroscopic data were in agreement with
those in the literature (Ye et al., 2007); trans-33: 1H NMR (CDCl3,
400 MHz) d: 1.04 (t, J = 6.8 Hz, 3H, –CH3), 1.94 (m, 1H, c-Pr-H),
2.00 (t, J = 8.4 Hz, 1H, c-Pr-H), 2.10–2.20 (m, 2H, c-hexene-H),
2.41 (t, J = 8.4 Hz, 1H, c-Pr-H), 2.54 (td, J = 5.6, 16.0 Hz, 1H, c-hex-
ene-H), 2.77 (td, J = 8.0, 16.0 Hz, 1H, c-hexene-H), 3.92 (q,
J = 6.8 Hz, 2H, –CH2–), 7.05–7.14 (m, 3H, Ar-H), 7.22 (m, 1H, Ar-H).
a
mixture of 4-(4-iodophenyl)butanoic acid (35) and 4-(2-
iodophenyl)butanoic acid (36) (6.97 g, 24.0 mmol, 79%) as yellow
crystals.
Polyphosphoric acid (PAA) (30.0 g) was added to a mixture of 35
and 36 (6.97 g, 24.0 mmol), and the reaction was then stirred for
7 min at 90 °C. Additional PAA (26.0 g) was added to the mixture.
After stirring for 10 min, the reaction was quenched with saturated
aqueous NH4Cl and extracted with EtOAc. The organic layers were
washed with H2O and brine, dried (MgSO4), filtered, and concen-
trated in vacuo. The residue was purified by silica gel CC (Et2O/hex-
ane, 10:90) to afford 7-iodo-3,4-dihydronaphthalen-1(2H)-
one (37) (major, 3.66 g, 13.4 mmol, 56%) and 5-iodo-3,4-
dihydronaphthalen-1(2H)-one (38) (minor, 1.11 g, 4.08 mmol,
17%) as yellow needles: 37: 1H NMR (CDCl3, 400 MHz) d: 2.13
(quintet, J = 6.4 Hz, 2H, c-hexane-H), 2.65, 2.91 (t, J = 6.4 Hz, each

232
K. Nishikawa et al. / Phytochemistry 96 (2013) 223–234
2H, c-hexane-H), 7.01 (d, J = 8.0 Hz, 1H, Ar-H), 7.77 (dd, J = 1.8,
8.0 Hz, 1H, Ar-H), 8.35 (d, J = 1.8 Hz, 1H, Ar-H); spectroscopic data
were in agreement with those in the literature (Cui et al., 2003);
38: mp 49–50 °C (toluene); 1H NMR (CDCl3, 400 MHz) d: 2.16
(quintet, J = 6.4 Hz, 2H, c-hexene-H), 2.65 (d, J = 6.4 Hz, 2H, c-hex-
ene-H), 2.96 (t, J = 6.4 Hz, 2H, c-hexene-H), 7.04 (dd, J = 7.6,
8.0 Hz 1H, Ar-H), 8.02 (dd, J = 1.2, 8.0 Hz, 1H, Ar-H), 8.05 (d,
J = 7.6 Hz, 1H, Ar-H); 13C NMR (CDCl3, 100 MHz) d: 22.5 (t, c-hex-
ene-H), 35.6 (t, c-hexene-H), 38.0 (t, c-hexene-H), 101.4 (s, Ar),
127.5 (d, Ar), 128.2 (d, Ar), 134.2 (s, Ar), 144.0 (d, Ar), 146.4 (s,
Ar), 197.5 (s, C@O); IR (KBr) 1676 cmꢀ1; EI-MS m/z 272 (M+); Anal.
calcd for C10H9IO: C, 44.14; H, 3.33. Found: C, 44.13; H, 3.20.
nBuLi (1.64 M in hexane, 2.80 mL, 4.60 mmol) was added to a
solution of diisopropylamine (0.650 mL, 4.60 mmol) in THF
(4.2 mL) at ꢀ78 °C under N2. After 20 min stirring, ethyl(trimethyl-
silyl)acetate (0.870 mL, 4.80 mmol) was added to the mixture.
After 10 min stirring, the resulting mixture was added to 37
(0.490 g, 1.80 mmol) in THF (6.0 mL). After 2 h at ꢀ20 °C and an
additional 2 h at room temperature, the reaction was quenched
with H2O at 0 °C, extracted with EtOAc (3 ꢁ 10 mL), then succes-
sively washed with H2O, 5% H2SO4, satd aq. NaHCO3, and brine,
dried (MgSO4), filtered, and concentrated in vacuo. The crude prod-
uct was purified by silica gel CC (EtOAc/n-hexane, 10:90) to give
(Z)-ethyl 2-[7-iodo-3,4-dihydronaphthalen-1(2H)-ylidene]acetate
(cis-39) (89.0 mg, 0.260 mmol, 20%), and (E)-ethyl 2-[7-iodo-3,4-
dihydronaphthalen-1(2H)-ylidene]acetate (trans-39) (88.0 mg,
0.257 mmol, 19%) as yellow oil and ethyl 2-(7-iodo-3,4-
dihydronaphthalen-1-yl)acetate (endo-39) (0.225 g, 0.658 mmol,
35%) as yellow needles: cis-39: 1H NMR (CDCl3, 400 MHz) d: 1.23
(t, J = 7.2 Hz, 3H), 1.95 (quintet, J = 6.8 Hz, 2H, c-hexene-H), 2.47
(m, 2H, c-hexene-H), 2.78 (t, J = 6.8 Hz, 2H, c-hexene-H), 4.18 (q,
J = 7.2 Hz, 2H, –CH2–), 5.80 (s, 1H, @CH–CO2–), 6.88 (d, J = 8.0 Hz,
1H, Ar-H), 7.55 (dd, J = 2.0, 8.0 Hz, 1H, Ar-H), 7.87 (d, J = 2.0 Hz,
1H, Ar-H); 13C NMR (CDCl3, 100 MHz) d 14.2 (q, –CH3), 22.8 (t, –
CH2–), 28.8 (t, –CH2–), 34.3 (t, –CH2–), 60.4 (t, –CH2–), 89.4 (s,
Ar), 115.8 (d, @CH–CO2–), 130.2 (d, Ar), 135.6 (s, Ar), 137.8 (d,
Ar), 137.9 (d, Ar), 138.5 (s, Ar), 150.2 (s, c-hexene), 166.8 (s,
C@O); IR (neat) 1715 cmꢀ1; EI-MS m/z 342 (M+); HRMS (FAB) calcd
for C14H16IO6 343.1843, found 343.1852; trans-39: 1H NMR (CDCl3,
400 MHz) d: 1.32 (t, J = 7.2 Hz, 3H, –CH3), 1.84 (quintet, J = 6,4 Hz,
2H, c-hexene-H), 2.73 (t, J = 6.4 Hz, 2H, c-hexene-H), 3.15 (dt,
J = 2.0, 6.4 Hz, 2H, c-hexene-H), 4.21 (q, J = 7.2 Hz, 2H, –CH2–),
6.27 (t, J = 2.0 Hz, 1H, @CH–CO2–), 6.89 (d, J = 7.6 Hz, 1H, Ar-H),
7.57 (dd, J = 1.6, 7.6 Hz, 1H, Ar-H), 7.96 (d, J = 1.6 Hz, 1H, Ar-H);
13C NMR (CDCl3, 100 MHz) d: 14.4 (q, –CH3), 22.4 (t, –CH2–), 27.7
(t, –CH2–), 29.8 (t, –CH2–), 59.9 (t, –CH2–), 91.2 (s, Ar), 113.6 (d,
@CH–CO2–), 130.9 (d, Ar), 133.7 (d, Ar), 136.6 (s, Ar), 138.1 (d,
Ar), 139.8 (s, Ar), 153.0 (s, c-hexene), 166.7 (s, C@O); IR (neat)
1712 cmꢀ1; EI-MS m/z 342 (M+), HRMS (FAB) calcd for C14H16IO2
343.1843, found 343.1852; endo-39: 1H NMR (CDCl3, 400 MHz) d:
1.27 (t, J = 7.6 Hz, 3H, –CH3), 2.28–2.33 (m, 2H, c-hexadiene-H),
2.73 (t, J = 7.6 Hz, 2H, c-hexadiene-H), 3.39 (s, 2H, –CH2–CO2–),
4.17 (q, J = 7.6 Hz, 2H, –CH2–), 6.03 (t, J = 4.4 Hz, 1H, vinyl-H),
6.87 (d, J = 8.0 Hz, 1H, Ar-H), 7.46 (dd, J = 1.6, 8.0 Hz, 1H, Ar-H),
7.49 (d, J = 1.6 Hz, 1H, Ar-H); 13C NMR (CDCl3, 100 MHz) d:
14.2(q, –CH3), 23.0 (t, –CH2–), 27.5 (t, –CH2–), 39.0 (t, –CH2–),
60.9 (t, –CH2–), 91.3 (s, Ar), 129.2 (s, c-hexadiene), 129.4
(d, c-hexadiene), 130.4 (d, Ar-H), 131.6 (d, Ar-H), 135.8 (d, Ar-H),
135.8 (s, Ar-H), 136.5 (s, Ar-H), 171.5 (s, C@O); IR (neat) 1722
(dd, J = 1.4, 8.4 Hz, 1H, Ar-H), 7.86 (d, J = 1.4 Hz, 1H, Ar-H); 13C NMR
(DMSO-d6, 100 MHz) d: 22.5 (t, c-hexene), 28.2 (t, c-hexene), 33.4
(t, c-hexene), 89.9 (s, Ar), 116.7 (d, @CH–CO2–), 130.6 (d, Ar),
135.4 (s, Ar), 137.0 (d, Ar), 137.2 (d, Ar), 138.4 (s, Ar), 147.8 (s, c-
hexene), 167.7 (s, C@O); IR (KBr) 1681 cmꢀ1; ESI-MS m/z 313
(M+ꢀH); Anal. calcd for C12H11IO2 C 45.88, H 3.53. Found C 45.67,
H 3.49.
Hydrolysis of trans-39 was performed using the procedure de-
scribed above to afford trans-12 (88%) as colorless needles; de-
comp. 241–243 °C (acetone); 1H NMR (THF-d8, 400 MHz) d: 1.79
(quintet, J = 6.4 Hz, 2H, c-Hexene-H), 2.74, 3.15 (t, J = 6.4 Hz, each
2H, c-Hexene-H), 6.32 (s, 1H, @CH–CO2–), 6.93, 7.58 (d, J = 8.0 Hz,
each 1H, Ar-H), 8.02 (s, 1H, Ar-H); 13C NMR (THF-d8, 100 MHz) d:
23.3 (t, c-hexene), 28.2 (t, c-hexene), 30.4 (t, c-hexene), 91.7 (s,
Ar), 114.6 (d, @CH–CO2–), 131.7 (d, Ar), 134.3 (d, Ar), 137.6 (s,
Ar), 138.7 (d, Ar), 140.4 (s, Ar), 152.9 (s, c-hexene), 167.7 (s,
C@O); IR (KBr) 1693 cmꢀ1; EI-MS m/z 315 (M+H); HRMS (EI) calcd
for C12H12IO2 314.9882. Found 314.9882.
Hydrolysis of endo-39 was performed using the procedure de-
scribed above to afford endo-12 (65%) as colorless needles: mp.
144–145 °C (toluene); 1H NMR (CDCl3, 600 MHz) d: 2.29–2.32 (m,
2H, c-hexadiene-H), 2.72 (t, J = 7.6 Hz, 2H, c-hexadiene-H), 3.43
(s, 2H, –CH2–CO2–), 6.05 (t, J = 4.2 Hz, 1H, vinyl-H), 6.87 (d,
J = 8.4 Hz, 1H, Ar-H), 7.46–7.47 (m, 2H, Ar-H); 13C NMR (CDCl3,
150 MHz) d: 23.0 (t, c-hexadiene), 27.4 (t, c-hexadiene-H), 38.3
(t, –CH2–CO2–), 91.4 (s, Ar), 128.6 (s, c-hexadiene), 129.5 (d, c-
hexadiene), 131.0 (d, Ar), 131.4 (d, Ar), 135.8 (s, Ar), 136.0 (d,
Ar), 136.2 (s, Ar), 176.3 (s, C@O); IR (KBr) 1701 cmꢀ1; ESI-MS m/z
313 (M+ꢀH); HRMS (FAB) calcd for C12H14IO2 313.9804, found
313.9804.
4.2.9. (Z)-2-[5-Iodo-3,4-dihydronaphthalen-1(2H)-ylidene]acetic acid
(cis-13) and (E)-2-[5-iodo-3,4-dihydronaphthalen-1(2H)-
ylidene]acetic acid (trans-13) (Novák et al., 1992)
Peterson olefination of 38 was performed according to the
procedure described above to afford (Z)-ethyl 2-[5-iodo-3,4-
dihydronaphthalen-1(2H)-ylidene]acetate (cis-40) (major, 65%)
and
(E)-ethyl
2-[5-iodo-3,4-dihydronaphthalen-1(2H)-yli-
dene]acetate (trans-40) (minor, 14%) as colorless oils: cis-40: 1H
NMR (CDCl3, 400 MHz) d: 1.24 (t, J = 7.2 Hz, 3H, –CH3), 2.02 (quin-
tet, J = 6.4 Hz, 2H, c-hexene-H), 2.44, 2.81 (t, J = 6.4 Hz, each 2H, c-
hexene-H), 4.15 (q, J = 7.2 Hz, 2H, –CH2–), 5.81 (s, 1H, @CH–CO2–),
6.85 (t, J = 8.0 Hz, 1H, Ar-H), 7.53, 7.82 (d, J = 8.0 Hz, each 1H, Ar-
H); 13C NMR (CDCl3, 100 MHz) d: 14.1 (q, –CH3), 23.9 (t, c-hexene),
34.8 (t, c-hexene), 35.9 (t, c-hexene), 60.2 (t, –CH2–), 101.6 (s, Ar),
114.9 (d, @CH–CO2–), 126.3 (d, Ar), 129.7 (d, Ar), 135.1 (s, Ar),
140.2 (d, Ar), 140.6 (s, Ar), 152.9 (s, c-hexene), 166.8 (s, C@O); IR
(neat) 1715 cmꢀ1; EI-MS m/z 342 (M+); HRMS (EI) calcd for
C14H15IO2 342.0117, found 342.0121; trans-40: 1H NMR (CDCl3,
400 MHz) d: 1.32 (t, J = 7.2 Hz, 3H, –CH3), 1.89 (quintet, J = 6.4 Hz,
2H, c-hexene-H), 2.79 (t, J = 6.4 Hz, 2H, c-hexene-H), 3.14 (dt,
J = 1.6, 6.4 Hz, 2H, c-hexene-H), 4.21 (q, J = 7.2 Hz, 2H, –CH2–),
6.27 (s, 1H, @CH–CO2–), 6.91 (dd, J = 7.6, 8.0 Hz, 1H, Ar-H), 7.60
(d, J = 7.6 Hz, 1H, Ar-H), 7.85 (d, J = 8.0 Hz, 1H, Ar-H); 13C NMR
(CDCl3, 100 MHz) d: 14.3 (q, –CH3), 23.1 (t, c-hexene), 27.2
(C@O), 2941 cmꢀ1
14H15IO2 342.1764, found 342.1770.
Hydrolysis of cis-39 was performed using the procedure de-
;
EI-MS 342 (M+), HRMS (FAB) calcd for
C
scribed above to afford cis-12 (87%) as a colorless solid: mp 168–
170 °C (toluene); 1H NMR (DMSO-d6, 400 MHz) d: 1.84 (quintet,
J = 6.4 Hz, 2H, c-hexene-H), 2.42, 2.75 (t, J = 6.4 Hz, each 2H, c-hex-
ene-H), 5.85 (s, 1H, @CH–CO2–), 6.98 (d, J = 8.4 Hz, 1H, Ar-H), 7.59

K. Nishikawa et al. / Phytochemistry 96 (2013) 223–234
233
(t, c-hexene), 36.2 (t, c-hexene), 59.9 (t, –CH2–), 102.8 (s, Ar), 113.5
(d, @CH–CO2–), 125.3 (d, Ar), 127.7 (d, Ar), 136.8 (s, Ar), 140.4 (d,
Ar), 141.9 (s, Ar), 154.7 (s, c-hexene), 166.7 (s, C@O); IR (neat)
1715 cmꢀ1; EI-MS m/z 342 (M+); HRMS (FAB) calcd for C14H16IO2
343.1843, found 343.1852.
ESI-MS m/z 187 (M+ꢀH); Anal. calcd for C12H12O2: C, 76.57; H,
6.43. Found: C, 76.47; H, 6.40.
4.3. Measurement of phytotoxic activities against lettuce root growth
Hydrolysis of cis-40 was performed using the procedure de-
scribed above to afford cis-13 (87%) as colorless needles: mp
115–117 °C (EtOAc); 1H NMR (CDCl3, 400 MHz) d: 2.02 (quintet,
J = 6.8 Hz, 2H, c-hexene-H), 2.46, 2.81 (t, J = 6.8 Hz, each 2H, c-hex-
ene-H), 5.82 (s, 1H, @CH2–CO2–), 6.83 (t, J = 8.0 Hz, 1H, Ar-H), 7.57,
7.83 (d, J = 8.0 Hz, each 1H, Ar-H); 13C NMR (CDCl3, 100 MHz) d:
23.7 (t, c-Hexene), 35.2 (t, c-hexene), 35.8 (t, c-hexene), 101.6 (s,
c-hexene), 113.7 (d, @CH2–CO2–), 126.5 (d, Ar), 130.0 (d, Ar),
134.6 (s, Ar), 140.6 (d, Ar), 140.8 (s, Ar), 156.7 (s, Ar), 171.9 (s,
C@O); IR (KBr) 1684 cmꢀ1; EI-MS m/z 314 (M+); HRMS (EI) calcd
for C12H11IO2 313.9804, found 313.9803.
Phytotoxic activity was measured according to the method de-
scribed by Hiradate et al. (2004). Filter paper was placed in a glass
petri dish. The test compound was dissolved in H2O at various con-
centrations, and a portion of each test solution was added to the fil-
ter paper in the petri dish for each treatment. Fifteen pre-
germinated (25 °C in the dark) seedlings of lettuce (Lactuca sativa
cv. Great Lakes 366) were used as a replicate for each treatment.
The seedlings were incubated for 48 h at 25 °C in the dark, and
the inhibitory activity of each test solution on root elongation
was determined by measuring the length of each root and compar-
ing it with that of the untreated control (using only distilled
water). The EC50 value, which indicates the effective concentration
required to induce a half-maximum effect, was calculated from the
dose response curve of the phytotoxicity for each compound by
applying a statistical model, the probit model (Bliss, 1934), using
the computer program, SPSS 13.0J.
Hydrolysis of trans-40 was performed using the procedure de-
scribed above to afford trans-13 (79%) as colorless needles: mp
220–221 °C (acetone); 1H NMR (THF-d8, 400 MHz) d: 1.85 (quintet,
J = 6.8 Hz, 2H, c-hexene-H), 2.79, 3.14 (t, J = 6.4 Hz, each 2H, c-hex-
ene-H), 6.32 (s, 1H, @CH2–CO2–), 6.94 (dd, J = 7.6, 8.8 Hz, 1H, Ar-H),
7.69 (d, J = 8.8 Hz, 1H, Ar-H), 7.85 (d, J = 7.6 Hz, 1H, Ar-H); 13C NMR
(CDCl3, 100 MHz) d: 24.0 (t, c-hexene), 27.6 (t, c-hexene), 37.1 (t, c-
hexene), 103.1 (s, c-hexene), 114.5 (d, @CH2–CO2–), 126.1 (d, Ar),
128.5 (d, Ar), 137.8 (s, Ar), 141.0 (d, Ar), 142.3 (s, Ar), 154.6 (s,
Acknowledgements
Ar), 167.7 (s, C@O); IR (KBr) 1684 cmꢀ1
; FAB-MS m/z 313
This work is supported by the Program for Promotion of Basic
and Applied Research for Innovations in the Bio-oriented Industry
(BRAIN) and ‘‘Innovations Inspired by Nature Research Support
Program (Sekisui Chemical).’’
(M+ꢀH); HRMS (FAB) calcd for C12H10IO2 312.9725, found
312.9727.
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