2-Fluoroabscisic acid analogues: Their synthesis and biological activities

Article abstract of DOI:10.1021/jf980265l

Fluorine was introduced into the 2-position of the side chain of abscisic acid (ABA) analogues by Wittig reaction of α-ionone derivatives with ethyl triethylphosphono-2-fluoroacetate. The effects of the fluorinated analogues were evaluated on inhibition o

Full text of DOI:10.1021/jf980265l

J. Agric. Food Chem. 1999, 47, 313−317
313
2-F lu or oa bscisic Acid An a logu es: Th eir Syn th esis a n d Biologica l
Activities
Bum Tae Kim,† Yong Ki Min,† Tadao Asami,*,‡ No Kyun Park,† Oh Young Kwon,§
Kwang Yun Cho,† and Shigeo Yoshida‡
Korea Research Institute of Chemical Technology, P.O. Box 107, Yusong, Taejon 305-606, Korea, The
Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako 351-01, J apan, and Department
of Chemistry, Soong-Sil University, 1-1 Sang Do 5 Dong, Dong J ak Gu, Seoul 156-743, Korea
Fluorine was introduced into the 2-position of the side chain of abscisic acid (ABA) analogues by
Wittig reaction of R-ionone derivatives with ethyl triethylphosphono-2-fluoroacetate. The effects of
the fluorinated analogues were evaluated on inhibition of cress seed germination and inhibition of
gibberellin-inducible R-amylase induction in embryoless barley half-seeds. (2E,4E)-2-Fluoro-5-(1′-
hydroxy-2′,6′,6′-trimethyl-2′-cyclohexen-1′-yl)-3-methyl-2,4-pentadienoic acid (5b) showed potent
inhibitory activity at the same level as ABA in the cress seed germination test, and 5b also inhibited
gibberellin-inducible R-amylase induction at 4 × 10-6, 3 times the concentration of ABA (1 × 10-6
)
for 50% inhibition of R-amylase production. 5b also showed dehydrin induction activity. These results
indicate that fluorinated ABA analogues mimic ABA action and can be a lead for a plant growth
regulator which regulates plant growth or protects plants from environmental stresses.
Keyw or d s: Inhibition of cress seed germination; inhibition of amylase induction; dehydrin induction;
aleurone; 2-fluoroabscisic acid; abscisic acid
INTRODUCTION
Abscisic acid (ABA, 1; Figure 1) is a plant hormone
that regulates physiological processes (Ohkuma et al.,
1963; Zeevaart et al., 1988) such as the acceleration of
abscission, induction of dormancy, inhibition of rooting,
and stimulation of stomatal closure. In addition to these
hormonal activities, ABA has attracted considerable
attention due to the role it plays in the response to
environmental stress such as drought (Davies et al.,
1993) and cold (Prasad et al., 1994; Anderson et al.,
1994). Therefore, if ABA analogues mimic ABA action
and have a commercial advantage over ABA to field
application, they could be developed as plant growth
regulators. Since the first synthesis of ABA (Cornforth
et al., 1965), varieties of ABA analogues have been
reported, but none of them has been developed as a
plant growth regulator. A fluorine atom has been
increasingly used as a label for obtaining structural
information and mechanistic details of biologically ac-
tive molecules (Welch, 1987; Kim et al., 1995; Todoroki
et al., 1995). Substitution of hydrogen with fluorine
changes not only the molecule’s size and shape slightly
but also the electronic nature of the molecule greatly
due to the strong electronegativity of fluorine. For
example, a pyrethric acid analogue in which fluorine is
introduced into the double bond shows strong insecti-
cidal activity (Martel et al., 1984). Kiyota et al. (1996)
introduced fluorine at C-2 of epoxy-â-ionylideneacetic
acid (2), which was as active as the parent compound
F igu r e 1. Structures of ABA and its active derivative.
of epoxy-â-ionylideneacetic acid. Considering these re-
sults, it is likely worthwhile to introduce a fluorine atom
into ABA analogues and evaluate their activities. As in
some assays ABA analogues, which can be prepared
through less expensive methods than ABA, show ABA-
like activities (Oritani and Yamashita, 1983; Van der
Meulen et al., 1993), we tried to introduce a fluorine
atom onto such ABA analogues. In this paper, we
describe the synthetic method for obtaining the target
molecules 3, 5, and the corresponding isomers 4 and 6
and the inhibitory activities of these compounds on
germination and R-amylase induction or the dehydrin
induction activity of 5b, which is the most active
compound in the former assays.
RESULTS AND DISCUSSION
Syn th esis. Modification of the cyclohexenone ring
system was done by oxidation reaction. Compound 7b
(1′-hydroxy group) was synthesized by the hydrolysis
of the 1′,2′-epoxy compound (8), which was prepared by
epoxidation of â-ionone with m-chloroperoxybenzoic acid
in high yields (Eschenmoser et al., 1981; Kakeya et al.,
1991). Compound 7c (4′-oxo group) was synthesized by
J ones oxidation of R-ionone (7a ) (Widmer et al., 1982).
2-Fluoroabscisic acid analogues 3a -c and 4a -c were
prepared by the Wittig-Horner reaction from various
ionone derivatives 7a -c with ethyl triethylphosphono-
* Author to whom correspondence should be addressed
(telephone +81-48-467-9527; fax +81-48-462-4674; e-mail
tasami@postman.riken.go.jp).
† Korea Research Institute of Chemical Technology.
‡ The Institute of Physical and Chemical Research (RIKEN).
§ Soong-Sil University.
10.1021/jf980265l CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/29/1998

314 J. Agric. Food Chem., Vol. 47, No. 1, 1999
Kim et al.
F igu r e 3. Effects of 2-fluoro-ABA analogues on gibberellin-
inducible amylase induction.
Oritani and Yamashita (1974) reported that the parent
compound of 5b, 4′-deoxo-ABA, was far less active than
ABA in inhibition of lettuce seed germination and azuki
bean seedlings. Actually, in the cress test reported here,
the activity of 4′-deoxo-ABA was about one-sixth that
of ABA. The lesser effectiveness of 4′-deoxo-ABA com-
pared with that of 5b was also demonstrated in the
R-amylase induction test (see results below and Figure
3). Taking these results into consideration, the reason
for the potent activity of 5b could be due to the
introduction of the fluorine atom onto the 2-position of
4′-deoxo-ABA. Because a replacement of hydrogen by
fluorine changes not only the electronic properties in
its environment but also the lipophilicity and geometry,
at present, the main reason for the effect of fluorine on
this activity enhancement is not clear. No enhancement
of the activity was found by the introduction of fluorine
atom onto the 2-position of the side chain of other
derivatives (3a -c, 5a ,c).
Inhibition of gibberellin-inducible R-amylase produc-
tion is also a typical activity of ABA and has been used
to determine whether chemicals have ABA-like activity.
2-Fluoroabscisic acid analogues (3a -c, 5a -c) showed
inhibition of the gibberellin-inducible R-amylase produc-
tion in embryoless barley (Hordeum vulgare L. cv.
Senbonhadaka) half-seeds (Figure 3). Ester derivatives
were less active than acid derivatives, and 5b also
showed the highest inhibitory activity in the compounds
tested here. The concentration of 5b required for 50%
inhibition (4 × 10-6) of R-amylase induction in aleurone
layers was ∼4 times higher than that of ABA (1 × 10-6).
The fact that the ethyl esters 3a and 3b are inactive
and the 1′-deoxy acid ethyl ester 3c shows some activity,
at least at high concentrations, may point to the
importance of lipophilicity for the activity of esters.
Here, 5b was far more active than 4′-deoxo-ABA, as was
shown in the cress seed germination assay.
F igu r e 2. Synthesis of 2-fluoro-ABA analogues.
Ta ble 1. p I50 Va lu e of 2-F lu or o-ABA An a logu esa for
In h ibition of Cr ess Seed Ger m in a tion
compd
X
Y
R
pI50
3a
3b
3c
4a
5a
5b
5c
6a
H2
H2
O
H2
H2
H2
O
H
OH
H
H
H
OH
H
H
Et
Et
Et
Et
H
H
H
H
4.1
4.5
5.1
<3.0
5.2
6.0
4.6
<3.0
H2
a
pI50 of ABA ) 6.1; pI50 of 4′-deoxo-ABA ) 5.3.
2-fluoroacetate (Pawson et al., 1979), which was pre-
pared by the Arbusov reaction of ethyl bromofluoro-
acetate with triethyl phosphite. The Wittig-Horner
reaction gave a 1:1 isomeric mixture of (2E,4E)-3a -c
and (2Z,4E)-4a -c. These isomers were separated by
silica gel column chromatography and hydrolyzed to
give the acids (2E,4E)-5a -c and (2Z,4E)-6a -c (Figure
2).
Biologica l Activities. The biological activity of the
synthesized compounds was examined by cress seed
(Lepidium sativum) germination (Table 1), which is one
of the typical bioassay systems for ABA activity. The
geometry (2Z,4E) of the 2,4-pentadienoic acid moiety of
ABA is essential for its hormonal activities, and when
the side chain is isomerized to the 2E,4E isomer, ABA
becomes inactive (Millborrow, 1970). The same relation-
ship between the side-chain geometry of 2-F-ABA and
the activity was observed in the cress seed assay; that
is, the 2E,4E isomers (3, 5) showed potent activity,
whereas the 2Z,4E isomers (4a , 6a ) were inactive. This
result suggests that the geometrical isomers (4a -c, 6a -
c) will be inactive in other assays. Therefore, the
biological activities of these isomers were not examined
in other assays. Ethyl ester derivatives (3) inhibited
germination, but they were less active than acid deriva-
tives (5). Among the acid derivatives, 5b showed the
highest inhibitory activity, and it was as active as ABA.
ABA is known to mediate rapid physiological re-
sponses to water stress such as stomatal closure (Ze-
evaart and Creelman, 1988) and to be involved in slower
responses such as dehydrin synthesis (Chandler and
Robertson, 1994). To determine whether 5b has an
ABA-like activity in plants, a “positive” biological re-
sponse induced by ABA should be included. Alterna-
tively, 5b may inhibit steps in signal transduction
between GA perception and the expression of R-amylase
gene by a mechanism different from ABA. For example,
okadaic acid, a protein phosphatase inhibitor, blocks
GA3-induced R-amylase production and also greatly

2-Fluoroabscisic Acid Analogues
J. Agric. Food Chem., Vol. 47, No. 1, 1999 315
for a new plant growth regulator that regulates plant
growth or protects plants from environmental stresses.
MATERIALS AND METHODS
Cr ess Seed Ger m in a tion Assa y. Cress (L. sativum) seeds
were purchased locally. The germination test following the
method of Taylor and Burden (1973) was carried out on
triplicates of 25 seeds for each concentration of the samples.
The test seeds were placed on Toyo No. 2 filter papers (5 cm)
in plastic Petri dishes (5 cm i.d., Eizai Co., J apan) and kept in
the dark at 25 °C. The germination rate was counted 48 h after
sowing.
r-Am yla se In d u ction Assa y. Ten embryoless barley (H.
vulgare L. cv. Senbonhadaka) half-seeds were incubated in 4
mL of buffer (2 mM acetate and 2 mM CaCl2, pH 5.4), with
GA3 (100 nM) plus ABA analogues (1, 10, or 100 µM) for 48 h
at 25 °C. The activity of R-amylase secreted under these
conditions was measured according to the procedure of J ones
and Varner (1967).
Isola tion of Ba r ley Aleu r on e P r otop la sts a n d Tr a n -
sien t Assa y. Barley seeds, H. vulgare cv. Himalaya, harvested
in 1994 in Canberra, Australia, were used. The procedure used
for the isolation of barley aleurone protoplasts was based on
the previous procedure described by Lin et al. (1996) with
minor modifications. Here, we incubated 10 aleurone layers
in a flask with 1.5 mL of enzyme solution. After protoplasts
had been isolated, pelleted protoplasts were resuspended in 1
mL of IM (0.5 M mannitol, 0.11 M glucose, 0.055 M sucrose,
14 mM L-arginine, 10 mM MES, 0.32% B5-Gamborg salt, pH
5.5) and mixed gently with 100 µg of salmon sperm DNA and
100 µg of Hv41(-935)-IGN plasmid DNA (Robertson et al.,
1995). The suspension was left undisturbed for 1 min. Proto-
plast transfection medium (PTM) consisting of 17.3% (w/v)
polyethylene glycol 6000 (PEG), 10 mM Tris-HCl, pH 9.0, 0.67
M mannitol, and 0.133 M Ca(NO3)2‚4H2O was filter sterilized.
Three volumes of PTM was added to the protoplast suspension,
mixed, and left at room temperature for 20 min with occasional
swirling. Forty milliliters of IM was then added in 10 mL
aliquots with 2 min between additions. The protoplasts were
collected by centrifugation for 2 min at 50g and washed twice
in 30 mL of IM. Pelleted protoplasts were resuspended in an
appropriate volume (generally 15 mL from 150 grains) of IM
containing 50 units/mL nystatin, 150 µg/mL cefotaxime, 20
mM CaCl2, 1.5 µg/mL aprotinin, and 1.5 µg/mL leupeptin. One
milliliter of the protoplast preparation was aliquoted into
flasks. These samples were incubated in the dark at 25 °C for
24 h in the presence of 0 or 10 µM ABA or 100 µM 5b, in
triplicate for each sample. Fluorometric assays of GUS activity
were performed in triplicate for each sample, as described by
J efferson (1988).
F igu r e 4. Activation of the dehydrin promoter by (+)-ABA
and analogues. Promoter activity was measured by GUS
reporter gene activity in barley aleurone protoplast transient
assays. Protoplasts were treated with 10 µM (+)-ABA and 5b
and 100 µM 5b.
reduces the accumulation of R-amylase mRNA but does
not lead to the accumulation of ABA-inducible mRNA
(Kuo et al., 1996). Therefore, we measured the expres-
sion of dehydrin genes (Close et al., 1989; Robertson et
al., 1995) because it is up-regulated by ABA. The effect
of 5b on dehydrin gene expression was tested in aleu-
rone protoplasts by measuring the dehydrin promoter
activity in transient assays (Figure 4). ABA increased
the GUS activity by ∼30-fold over the control when
protoplasts were treated with 10 µM ABA. 5b also
increased the GUS activity significantly but was less
active than ABA even at the concentration 10 times
higher than ABA. 4′-Deoxo-ABA was almost inactive at
the concentration of 100 µM, suggesting that the
introduction of fluorine was also effective in enhancing
the activity of 4′-deoxo-ABA.
In this study, it was found that 2-F-ABA analogues
mimicked ABA action in three assay systems, and one
of the analogues, 5b, showed the most potent activities.
A fluorine atom at C-2 enhanced activity as shown in
the cress seed germination assay, R-amylase induction
assay, and dehydrin induction assay, when compared
with its nonfluorinated compound, 4′-deoxo-ABA. Con-
sidering the more potent activity of 5b relative to that
of 4′-deoxo-ABA in these assay systems, the introduction
of fluorine onto the side chain seems to be effective for
activity exhibition. The differences in activities between
ABA and 5b vary in the assay systems: in the germina-
tion test 5b is as active as ABA; in the R-amylase
induction test the activity of 5b is about one-fourth that
of ABA; and in the dehydrin induction test the activity
of 5b is far less than one-tenth that of ABA. It can be
thought that ABA receptors in different tissues could
have different structural requirements for their ligand;
therefore, it is worthwhile to test ABA analogues
reported here in other assays for ABA activities. This
would be helpful for finding a potency of these analogues
Ch em ica ls. 1H NMR spectra were recorded with a J EOL
PMX 60 SI (60 MHz) or a Varian Gemini 200 (200 MHz), with
tetramethylsilane as an internal standard. Mass spectra were
recorded with a Shimadzu GC-MS QP-1000 spectrometer and
HR-MS with a J EOL AX-505 mass spectrometer.
4-(1′,2′-Epoxy-2′,6′,6′-trimethyl-2′-cyclohex-1′-yl)-3-buten-2-
one (8). To a solution of 9.62 g (0.05 mol) of â-ionone in 100
mL of methylene chloride being cooled in an ice bath was
added 17.26 g (0.05 mol, 50% purity) of m-chloroperoxybenzoic
acid. The reaction mixture was stirred at room temperature
for 20 h and then filtered and washed with aqueous NaHCO3
and then 10% sodium bisulfite. The organic phase was dried
with magnesium sulfate and evaporated under reduced pres-
sure. The residue was purified on column chromatography
(silica gel, ethyl acetate/n-hexane ) 1:4) to give 8.84 g of 8
(yield ) 85%): 1H NMR (200 MHz, CDCl3) δ 0.93 (s, 3H), 1.14
(s, 6H), 1.38-1.95 (m, 6H), 2.28 (s, 3H), 6.28 (d, 1H, J ) 15.7
Hz), 7.02 (d, 1H, J ) 15.7 Hz); MS, m/z 208 (M+, 5), 193 (18),
165 (13), 135 (100), 123 (100), 107 (51), 95 (52), 79 (25), 69
(30), 43 (100). Anal. Calcd (%) for C13H20O2: C, 74.96; H, 9.68;
O, 15.36. Found: C, 74.90; H, 9.70; O, 15.40.
4-(1′-Hydroxy-2′,6′,6′-trimethyl-2′-cyclohexen-1′-yl)-3-buten-
2-one (7b). Under N2 gas, 25 mL (0.0625 mol of 2.5 M solution)

316 J. Agric. Food Chem., Vol. 47, No. 1, 1999
Kim et al.
of n-butyllithium was added to a solution of 4.39 g (0.06 mol)
of diethylamine in 100 mL of dry diethyl ether at -78 °C. To
the reaction mixture was added 4.16 g (0.02 mol) of 8, and the
mixture was stirred for 2 h. The reaction mixture was poured
into ice and extracted with 150 mL of diethyl ether, and then
the organic phase was dried with magnesium sulfate and
evaporated under reduced pressure. The purification of the
residue on column chromatography (silica gel, ethyl acetate/
n-hexane ) 1:2) gave 0.975 g of 7b (yield ) 23%): 1H NMR
(200 MHz, CDCl3) δ 0.91 (s, 3H), 1.01 (s, 3H), 1.45-1.68 (m,
2H), 1.59 (s, 3H), 2.00-2.15 (m, 3H), 2.29 (s, 3H), 5.56 (bs,
1H), 6.35 (d, 1H), 6.81 (d, 1H); MS, m/z 208 (M+, 4), 193 (9),
181 (5), 165 (21), 152 (11), 123 (20), 109 (100), 81 (22), 43 (39).
Anal. Calcd (%) for C13H20O2: C, 74.96; H, 9.68; O, 15.36.
Found: C, 74.93; H, 9.69; O, 15.38.
4-(4′-Oxo-2′,6′,6′-trimethyl-2′-cyclohexen-1′-yl)-3-buten-2-
one (7c). To a solution of 1.5 g (7.8 mmol) of R-ionone in 40
mL of acetone was added dropwise 265 mL (0.75 mol solution)
of J ones reagent (chromic acid in diluted sulfuric acid). The
reaction mixture was stirred at room temperature for 10 h and
then filtered, and the solvent was evaporated under reduced
pressure. The purification of the residue on column chroma-
tography (silica gel, ethyl acetate/n-hexane ) 1:2) gave 0.45 g
of 7c (yield ) 28%): 1H NMR (200 MHz, CDCl3) δ 1.01 (s, 3H),
1.08 (s, 3H), 1.90 (d, 3H, J ) 1.0 Hz), 2.29 (s, 3H), 2.15-2.41
(m, 2H), 2.72 (d, 1H, J ) 9.5 Hz), 5.98 (s, 1H), 6.19 (d, 1H, J
) 15.8 Hz), 6.68 (dd, 1H, J ) 15.8 Hz, 9.5 Hz); MS, m/z 206
(M+, 2), 191 (2), 164 (2), 150 (44), 135 (6), 108 (100), 43 (100).
Anal. Calcd (%) for C13H18O2: C, 75.69; H, 8.79; O, 15.51.
Found: C, 75.73; H, 8.80; O, 15.46.
Gen er a l P r oced u r e for Wittig-Hor n er Rea ction a n d
Sep a r a tion of Isom er s. To a solution of 1.33 g (5.5 mmol) of
ethyl triethylphosphono-2-fluoroacetate in 50 mL of anhydrous
tetrahydrofuran was added 165 mg (5.5 mmol of 80% disper-
sion in mineral oil) of sodium hydride under a flow of nitrogen.
After the mixture had been stirred at room temperature for 1
h, 5 mmol of 7a -c was added and then refluxed for 12 h. The
reaction mixture was poured into crushed ice and extracted
with ether, dried over anhydrous magnesium sulfate, and
concentrated under reduced pressure. Column chromatogra-
phy (silica gel, ethyl acetate/n-hexane ) 1:4) of the residue
gave 3a -c (2E,4E) and 4a -c (2Z,4E).
(2E,4E)-Ethyl 2-fluoro-3-methyl-5-(2′,6′,6′-trimethyl-2′-cyclo-
hexen-1′-yl)-2,4-pentadienoate (3a ): yield ) 37%; 1H NMR (200
MHz, CDCl3) δ 0.82 (s, 3H), 0.92 (s, 3H), 1.15-1.45 (m, 2H),
1.35 (t, 3H), 1.58 (d, 3H), 1.90-2.10 (m, 2H), 1.98 (d, 3H), 2.29
(d, 1H), 4.31 (q, 2H), 5.45 (s, 1H), 5.89 (dd, 1H), 7.29 (d, 1H);
MS, m/z (relative intensity) 280 (M+, 5), 224 (15), 175 (5), 157
(100), 144 (40), 131 (30), 107 (5); HR-MS, m/z 280.1903, calcd
for C17H25FO2, 280.1838.
Gen er a l P r oced u r e of Hyd r olysis. A mixture of 0.75
mmol of ethyl ester 3a -c or 4a -c and 0.41 g of potassium
hydroxide in 5 mL of ethanol and 5 mL of water was refluxed
for 1 h. Ethanol was then evaporated under reduced pressure.
The residue was acidified with 2 N HCl solution and then
extracted with ethyl acetate three times. The organic phase
was washed with water, dried over anhydrous magnesium
sulfate, and concentrated under reduced pressure. Column
chromatography (silica gel, ethyl acetate/n-hexane ) 1:4) of
the residue gave 5a -c (2E,4E) and 6a -c (2Z,4E).
(2E,4E)-2-Fluoro-3-methyl-5-(2′,6′,6′-trimethyl-2′-cyclohexen-
1
1′-yl)-2,4-pentadienoic acid (5a ): yield ) 78%; H NMR (200
MHz, CDCl3) δ 0.82 (s, 3H), 0.91 (s, 3H), 1.15-1.50 (m, 2H),
1.57 (d, 3H, J ) 1.5 Hz), 1.90-2.10 (m, 2H), 2.00 (d, 3H, J )
3 Hz), 2.29 (d, 1H, J ) 9.5 Hz), 5.44 (s, 1H), 5.93 (dd, 1H, J )
15.6, 9.5 Hz), 7.27 (d, 1H), 9.2 (bs, 1H); MS, m/z (relative
intensity) 252 (M+, 4), 237 (3), 196 (50), 176 (34), 163 (18),
151 (48), 129 (100), 122 (29), 117 (10), 107 (30); HR-MS, m/z
252.1557, calcd for C15H21FO2, 252.1525.
(2E,4E)-2-Fluoro-5-(1′-hydroxy-2′,6′,6′-trimethyl-2′-cyclohexen-
1′-yl)-3-methyl-2,4-pentadienoic acid (5b): yield ) 79%; 1H
NMR (200 MHz, CDCl3) δ 0.93 (s, 3H), 1.01 (s, 3H), 1.41-
1.72 (m, 2H), 1.68 (s, 3H), 1.99 (d, 3H), 1.80-2.10 (m, 3H),
5.61 (bs, 1H), 6.05 (d, 1H), 7.55 (d, 1H), 9.8 (bs, 1H); MS, m/z
(relative intensity) 268 (M+, 5), 251 (12), 194 (5), 169 (3), 149
(5), 135 (9), 122 (100), 107 (60), 45 (20); HR-MS, m/z 268.1522,
calcd for C15H21FO3, 268.1475.
(2E,4E)-2-Fluoro-3-methyl-5-(4′-oxo-2′,6′,6′-trimethyl-2′-cy-
clohexen-1′-yl)-2,4-pentadienoic acid (5c): yield ) 81%; 1H NMR
(200 MHz, CDCl3) δ 0.99 (s, 3H), 1.07 (s, 3H), 1.93 (d, 3H, J )
1.2 Hz), 2.02 (d, 3H, J ) 4.4 Hz), 2.37-2.45 (m, 2H), 2.75 (d,
1H, J ) 9.5 Hz), 5.87-6.05 (m, 2H), 7.48 (d, 1H, J ) 15.7 Hz),
9.9 (bs, 1H); MS, m/z (relative intensity) 266 (M+, 28), 210 (17),
190 (100), 164 (19), 138 (42), 124 (21); HR-MS, m/z 266.1357,
calcd for C15H19FO3, 266.1318.
(2Z,4E)-2-Fluoro-3-methyl-5-(2′,6′,6′-trimethyl-2′-cyclohexen-
1
1′-yl)-2,4-pentadienoic acid (6a ): yield ) 75%; H NMR (200
MHz, CDCl3) δ 0.83 (s, 3H), 0.93 (s, 3H), 1.20-1.51 (m, 2H),
1.58 (d, 3H, J ) 1.5 Hz), 1.87-2.10 (m, 2H), 2.23 (d, 3H, J )
3 Hz), 2.31 (d, 1H, J ) 9.6 Hz), 5.47 (s, 1H), 5.99 (dd, 1H, J )
15.7 Hz, 9.7 Hz), 6.63 (d, 1H, J ) 15.6 Hz), 9.80 (bs, 1H); MS,
m/z (relative intensity) 252 (M+, 13), 237 (8), 196 (72), 176 (58),
151 (100), 131 (55), 129 (41), 109 (30), 91 (58), 77 (44); HR-
MS, m/z 252.1491, calcd for C15H21FO2, 252.1525.
ABBREVIATIONS USED
ABA, abscisic acid; GA, gibberellin; GUS, â-glucu-
ronidase.
(2E,4E)-Ethyl 2-fluoro-5-(1′-Hydroxy-2′,6′,6′-trimethyl-2′-cy-
clohexen-1′-yl)-3-methyl-2,4-pentadienoate (3b): yield ) 38%;
1H NMR (200 MHz, CDCl3) δ 0.90 (s, 3H), 0.99 (s, 3H), 1.35 (t,
3H), 1.61 (s, 3H), 1.41-1.71 (m, 2H), 1.98 (d, 3H, J ) 4.4 Hz),
1.80-2.08 (m, 3H), 4.29 (q, 2H), 5.55 (bs, 1H), 6.07 (d, 1H, J
) 16 Hz), 7.45 (d, 1H, J ) 16 Hz); MS, m/z (relative intensity)
296 (M+, 15), 279 (100), 233 (14), 181 (11), 165 (25), 147 (85),
139 (23), 123 (13), 109 (17), 91 (12), 55 (13), 43 (25); HR-MS,
m/z 296.1763, calcd for C17H25FO3, 296.1788.
(2E,4E)-Ethyl 2-fluoro-3-methyl-5-(4′-oxo-2′,6′,6′-trimethyl-
2′-cyclohexen-1′-yl)-2,4-pentadienoate (3c): yield ) 36%; 1H
NMR (200 MHz, CDCl3) δ 0.98 (s, 3H), 1.02 (s, 3H), 1.33 (t,
3H), 1.95 (s, 3H), 1.97 (s, 3H), 2.05-2.40 (m, 2H), 2.67 (d, 1H),
4.32 (q, 2H), 5.94-6.12 (m, 2H), 7.50 (d, 1H); MS, m/z (relative
intensity) 294 (M+, 13) 296 (M+, 15), 277 (100), 165 (25), 147
(85), 139 (23), 123 (17), 109 (21), 91 (15), 55 (17), 43 (35); HR-
MS, m/z 294.1675, calcd for C17H23FO3, 294.1631.
(2Z,4E)-Ethyl 2-fluoro-3-methyl-5-(2′,6′,6′-trimethyl-2′-cyclo-
hexen-1′-yl)-2,4-pentadienoate (4a ): yield ) 39%; 1H NMR (200
MHz, CDCl3) δ 0.82 (s, 3H), 0.92 (s, 3H), 1.15-1.45 (m, 2H),
1.35 (t, 3H), 1.58 (d, 3H), 1.90-2.10 (m, 2H), 2.23 (d, 3H, J )
3.2 Hz), 2.30 (d, 1H), 4.31 (q, 2H), 5.45 (s, 1H), 5.89 (dd, 1H,
J ) 15.5, 9.5 Hz), 6.59 (d, 1H, J ) 15.7 Hz); MS, m/z (relative
intensity) 280 (M+, 12), 224 (37), 175 (15), 157 (100); HR-MS,
m/z 296.1773, calcd for C17H25FO2, 280.1838.
ACKNOWLEDGMENT
We deeply thank Mr. Shin Yamamoto for his skillful
bioassays.
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Received for review March 16, 1998. Revised manuscript
received November 9, 1998. Accepted November 11, 1998. This
work was supported by a grant from the Korean Ministry of
Science and Technology and a Grant-in-Aid for Scientific
Research (C) to T.A. (No.09660121) from the Ministry of
Education, Science, Sports and Culture of J apan.
J F980265L