cis-Cinnamic Acid Selective Suppressors Distinct from Auxin Inhibitors

Abstract

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 inhibited; 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 suppressors 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.

cis-Cinnamic acid (cis-CA; Fig. 1) is known to inhibit root growth of an Arabidopsis, Avena, wheat and flax, and to cause epinastic curvature in pea and tomato seedlings as do the plant hormones auxin and ethylene.^1–5) Naturally occurring CA has been found in both trans- and cis-forms. trans-CA is generally considered to be a weak antagonist of auxin in higher plants.^2,6–8) The biosynthesis of trans-CA from l-phenylalanine is catalyzed by phenylalanine ammonia-lyase (PAL; EC 4.3.1.5). It is then converted to 4-hydroxycinnamic acid by cinnamic acid 4-hydroxylase (EC 1.14.13.11), which serves as the precursor for the biosynthesis of many phenolic compounds such as flavonoids, phytoalexin, salicylic acid and lignin.⁹⁾ cis-CA can be an ultraviolet light-mediated isomerization product of trans-CA.^4,5) The presence of natural cis-CA in plants has been reported in Brassica parachinensis¹⁰⁾ and its glycosides, 1-O-cis-cinnamoyl-β-d-glucopyranose and 6-O-(4′-hydroxy-2′-methylene-butyroyl)-1-O-cis-cinnamoyl-β-d-glucopyranose, as allelochemicals in Spiraea thunbergii.¹¹⁾ While cis-CA has an inhibitory activity on root growth, it showed a growth-promoting activity on aerial moieties such as pea stem and avena coleoptile like auxin.¹⁾ The most representative natural auxin is indole-3-acetic acid (IAA, Fig. 1). Because of the similar activities for plants, cis-CA has long been believed to be an analog of auxin.

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.

Further studies using an ethylene perception-deficient or an ethylene biosynthesis-deficient tomato plant,⁴⁾ and two auxin-insensitive Arabidopsis mutants⁵⁾ showed that cis-CA acts on plant cells through both ethylene- and auxin-independent signaling pathways. Recently, cis-CA-upregulating Arabidopsis genes, MLPL1 (AT2G01520) and MLPL2 (AT2G01530), were identified and the up-regulation occurred in auxin perception-deficient mutants.¹²⁾ Furthermore, the treatment of cis-CA induced some early auxin responsive genes only in roots without affecting the SMALL AUXIN-UP RNA (SAUR) families.¹³⁾ Although these studies suggested that cis-CA was not a member of the auxins, little is known about its physiological functions and mechanism of action for cis-CA.

We have been taking particular note of cis-CA bioactivity and reported some structure–activity relationships studies.^14,15) During these investigations, we synthesized more than 250 cis-CA derivatives and evaluated their inhibitory effects on root growth in lettuce. In this study, the compounds that showed low- or no-activity on root growth inhibition were recruited as candidates for a suppressor against cis-CA and/or auxin (Fig. 2). Suppressors of cis-CA would aid in elucidating the inhibitory mechanism as well as the unique compounds having the inhibitory activity against cis-CA. It would also become evident that cis-CA and auxin regulate plant growth through distinct signaling pathways.

Fig. 2. Structures of Cinnamic Acid (CA) Derivatives

The concentrations in the brackets represent IC₅₀ values of root growth inhibition.

Results and Discussion

The assay for suppressive activity was conducted by adding a test compound and a root growth inhibitor simultaneously. cis-CA and IAA were nominated as representative inhibitors, and 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthaleneacetic acid (NAA) were also used as authentic auxins. We initially screened for effects on the inhibitory activities of cis-CA, IAA, 2,4-D, and NAA (Table 1). The concentration of the inhibitors was set according to preliminary experiments to get the maximum response from a suppressor.

Table 1. Effects of cis-Cinnamic Acid (cis-CA) Derivatives on cis-CA and Auxins in Lettuce^a) (Lactuca sativa cv. Great Lakes 366)

Suppressor candidates	µm	Control	Growth inhibitors
			cis-CA 5 µm	IAAb) 0.5 µm	2,4-Dc) 0.5 µm	NAAd) 0.5 µm
trans-CA	0	100±12	59±17	61±10	43±4	46±7
	25	109±23	63±9	64±13	46±4	52±6
	50	102±16	53±10	57±11	47±4	50±6
1	0	100±15	47±7	50±5	41±6	44±6
	25	93±27	55±6	52±12	44±4	54±5
	50	99±16	55±9	54±8	43±5	55±8
3	0	100±16	51±6	48±10	41±6	50±6
	25	111±37	61±20	41±8	40±17	73±13
	50	118±53	79±23	40±8	65±7	76±16
13	0	100±19	57±9	61±11	46±9	55±11
	25	84±28	61±10	64±15	41±6	52±7
	50	105±44	77±19	64±15	54±11	58±8
9	0	100±19	64±7	63±9	54±9	72±8
	25	109±31	77±14	54±9	41±6	59±7
	50	113±32	69±13	60±9	40±6	65±6
PCIBe)	0	100±22	56±9	55±11	38±6	47±6
	25	138±28*	89±15*	81±11*	49±9	63±5
	50	142±26*	99±19*	84±14*	71±12*	71±10
AOPPf)	0	100±19	56±10	56±7	45±4	55±5
	25	135±28*	93±13*	85±14*	51±6	71±11
	50	133±31*	105±17*	86±15*	59±8	74±15
2	0	100±21	59±14	61±14	49±6	54±10
	25	114±17	105±16*	51±16	54±8	80±15*
	50	106±19	93±17*	41±10	59±13	72±16
6	0	100±18	56±16	50±6	40±6	43±6
	25	97±18	85±16*	37±8	38±4	35±6
	50	102±16	93±8*	45±12	41±8	50±13
7	0	100±20	59±9	62±13	50±6	54±10
	25	107±16	89±11*	51±18	57±9	60±12
	50	104±19	105±18*	45±15	58±9	71±15
8	0	100±19	55±14	46±9	48±5	59±5
	25	110±24	87±22*	45±12	49±9	61±16
	50	108±18	90±15*	44±17	54±16	75±9
12	0	100±26	53±5	58±21	42±5	53±8
	25	97±14	66±12	37±7*	41±5	41±10
	50	103±17	82±11*	35±14*	57±10	47±10
10	0	100±14	53±8	56±8	38±5	44±4
	25	114±21	71±17	57±8	49±5	68±8*
	50	111±30	89±17*	64±13	54±12	93±11*
4	0	100±19	52±11	43±7	35±8	44±14
	25	110±17	57±11	52±7	38±3	40±8
	50	123±17*	45±6	47±9	37±4	45±7
5	0	100±16	63±17	63±9	50±8	55±9
	25	126±21*	103±11*	59±12	37±4	43±12
	50	131±20*	105±15*	52±24	46±4	64±14
11	0	100±21	49±9	56±13	40±4	48±10
	25	142±33*	109±22*	47±10	54±11	69±11
	50	140±21*	124±21*	75±17	63±13	75±14*

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.

In the absence of growth inhibitors, there was no tested compounds that made roots shorter because the selected compounds previously showed low or no inhibitory activity. p-Chlorophenoxyisobutyric acid (PCIB) and l-2-aminooxy-3-phenylpropionic acid (AOPP), well known as anti-auxins, promoted lettuce root growth as much as 140% compared with the vehicle controls because the intrinsic auxin function was eliminated by the anti-auxins. AOPP might decrease the amount of intrinsic auxin because of its inhibitory activity of auxin biosynthesis. Compounds 4, 5, and 11 also showed promoting activity; however, the activity of 4 appeared weaker than that of the others because only a high-concentration (50 µm) was effective.

In the presence of cis-CA, root growth was inhibited about 50% and was restored by the addition of anti-auxins and cis-CA derivatives 2, 5, 6, 7, 8, 10, 11, and 12. Using compounds 2, 5, 7, 11, PCIB and AOPP, the recovery was complete and control-level growth was achieved. Compounds 10 and 12 exhibited weak suppressive activity, with 90% growth restored at high concentrations. These root length recovery by compounds 5, 11, PCIB and AOPP, which were also found to possess elongating activity, could not be differentiated whether this recovery was the result of elongating activity or 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 activity (data not shown).

When IAA was used as an inhibitor, it strongly inhibited the root growth even at one-tenth of the concentration used for the screening assay. As shown in Table 1, only two anti-auxins PCIB and AOPP were able to reduce the effect of IAA significantly and the restoration levels were at most 84% and 86%, respectively. Compound 12 seemed to promote inhibitory activity of IAA. In the case of the authentic auxins, 2,4-D and NAA were used as inhibitors, a few compounds affected their inhibitory activities. Among all of the tested compounds, only PCIB, not AOPP, ameliorated the effect of 2,4-D. Compounds 2, 10 and 11 showed recovery against NAA induced inhibition, but neither PCIB nor AOPP did.

trans-CA and compounds 1, 3, 9, and 13 did not have any activity on the lettuce root regardless of the presence or absence of the inhibitor. According to the results shown in Table 1, four compounds, 2, 6, 7, and 8 were picked up as candidates for cis-CA selective suppressor because they could restore the root growth after inhibition by cis-CA treatment without any effect on the IAA-induced effects or elongating activity of themselves. Those four compounds were then subjected to further experimentations.

To confirm the activity against cis-CA, various cis-CA concentrations were used for the assay. As shown in Table 2, compound 2 exhibited suppressive activity on cis-CA in the range of 1–30 µm, and the optimal concentration of 2 seemed to be 25 µm. Compounds 6 and 7 also showed obvious activity but failed to suppress 30 µm of cis-CA. Compound 8 was only effective on the inhibition induced by 3 µm of cis-CA. To confirm the selectivity against cis-CA, various IAA concentrations were used for the assay. All of these compounds failed to mitigate the root growth inhibition induced by IAA (Table 3). Interestingly, only the combination of 3 µm of IAA and 50 µm 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.

Table 2. Effects of cis-Cinnamic Acid (cis-CA) Derivatives on Various Concentrations of cis-CA in Lettucea) (Lactuca sativa cv. Great Lakes 366)

Suppressor candidates	µm	Control	cis-CA
			1 µm	3 µm	10 µm	30 µm
2	0	100±20	74±16	55±8	41±6	27±6
	25	117±26	108±22*	112±11*	77±19*	50±9*
	50	94±21	88±17	90±7*	79±10*	47±15
6	0	100±22	67±14	53±14	37±8	22±6
	25	98±15	105±13*	87±10*	59±14*	28±11
	50	105±7	95±14*	95±16*	79±10*	34±11
7	0	100±13	58±7	51±11	35±6	22±6
	25	86±10	83±12*	71±14*	66±13*	27±8
	50	96±14	94±8*	86±13*	68±15*	36±10
8	0	100±20	71±17	52±7	43±9	27±8
	25	100±13	88±12	74±16*	41±12	33±10
	50	85±21	91±19	77±16*	52±16	29±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 Lettuce^a) (Lactuca sativa cv. Great Lakes 366)

Suppressor candidates	µm	Control	IAA
			0.1 µm	0.3 µm	1 µm	3 µm
2	0	100±20	70±8	57±12	50±22	44±10
	25	122±27	92±20	53±11	40±12	43±19
	50	106±19	76±19	52±17	35±19	67±16
6	0	100±12	77±13	62±9	53±8	52±12
	25	122±16	69±12	56±12	40±13	68±17
	50	117±30	77±24	61±21	40±10	90±22*
7	0	100±14	68±12	50±7	56±17	78±28
	25	107±12	64±16	42±11	30±9*	66±24
	50	110±14	69±10	49±15	53±21	99±16
8	0	100±27	83±19	70±14	61±15	62±16
	25	117±16	70±19	53±16	44±20	62±12
	50	93±22	54±19*	37±16*	31±7*	36±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.

Two cis-CA derivatives, 14 and 15, having strong inhibitory activity¹⁵⁾ (Fig. 2) were recruited for further confirmation as candidates that effected cis-CA-like activities (Table 4). Compounds 2 and 7 significantly ameliorated the root shortening by each tested cis-CA derivatives. Compounds 6 and 8 failed to restore growth after inhibition by 14 and 15, except at a high concentration of 6.

Table 4. Effects of cis-Cinnamic Acid (cis-CA) Derivatives on cis-CA-Like Growth Inhibitors in Lettuce^a) (Lactuca sativa cv. Great Lakes 366)

Suppressor candidates	µm	Control	Growth inhibitors
			cis-CA 5 µm	14 3 µm	15 3 µm
2	0	100±18	46±9	50±13	56±15
	25	102±28	92±16*	83±19*	91±14*
	50	84±20	83±22*	91±15*	96±22*
6	0	100±18	49±6	48±11	59±15
	25	97±17	89±16*	62±9	74±10
	50	113±17	88±19*	86±15*	85±9*
7	0	100±23	52±8	50±18	51±13
	25	103±16	85±11*	72±9*	82±13*
	50	103±11	85±13*	79±13*	82±18*
8	0	100±28	43±9	46±9	66±17
	25	106±19	71±21*	63±12	77±13
	50	98±19	69±21*	60±15	71±17

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.

Compounds 2, 6, 7, and 8 clearly exhibited cis-CA selective suppressive activity (Tables 2, 3). The order of their strength of suppressive activity appears to be 2>7>6>8. Compound 8 showed the weakest suppressive activity among these and promoted the inhibition of IAA. Furthermore, compound 8 has been reported as an auxin-like growth regulator, similar to cis-CA,²⁾ in the pea split-stem curvature test,¹⁶⁾ so it may work through different pathways from the other compounds.

Conclusion

According to these results, at least three compounds 2, 6, and 7 can be called cis-CA selective suppressors. Previous reports suggesting that cis-CA acts via both ethylene- and auxin-independent signaling pathways^4,5) are supported by our highly selective suppressors.

This is the first study on cis-CA suppressors even though the bioactivities of CA, IAA and anti-auxin¹⁷⁾ have been reported previously. We have found some cis-CA selective suppressors against growth inhibition that have never been reported among inactive cis-CA derivatives. There are numerous questions surrounding plant growth regulation, especially cis-CA as a possible novel plant hormone. cis-CA selective suppressors will play an important role in elucidating the mechanisms of plant growth regulation.

Experimental

Chemicals

Each synthetic method for the newly prepared compounds and their spectral data are described below. The other compounds were synthesized as previously reported. IAA, 2,4-D, and PCIB were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). NAA, and AOPP were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). trans-CA was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Other chemicals used were commercially available.

Analysis of Synthesized Chemicals

The melting points were measured on a Yazawa micromelting point BY-1. The ¹H- and ¹³C-NMR spectra were recorded using a JNM EX-270 (270 and 67.5 MHz, JEOL) and a JNM AL-400 (400, 100 MHz, JEOL). Chemical shifts were reported in ppms downfield from the peak of the internal standard Me₄Si (tetramethylsilane (TMS)). Splitting patterns are designed as “s, 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 or a NaCl cell. Mass and high-resolution mass spectra were obtained on a JMS-700 or a JMS-T100CS (JEOL). Column chromatography was performed on silica gel (Kanto Chemical Co.). Thin-layer chromatography was performed on pre-coated plates (0.25 mm, silica gel Merck 60 F254). Reaction mixtures were stirred magnetically.

Syntheses of cis-CA Analogs

The cis-CA analogs 1, 2, 3, 6, 8, 9, 10, 11, 14, and 15 were synthesized as previously reported.^14,15,18) Syntheses of the analogs 4, 5, 7, and 12 were also performed mainly by the Z-selective olefination of the corresponding aldehydes with the Ando–Emmons reagents,^19,20) followed by the hydrolysis (Chart 1). The commercially unavailable starting aldehydes 21 and 23 were prepared according to the literature.^21,22) The trans-analog 13 was synthesized via the usual Honor–Emmons reaction.²²⁾

Chart 1. Syntheses of cis-CA Acid Analogs 4, 5, 7, 12, and 13

(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%.

(2Z,2′Z)-3,3′-(1,3-Phenylene)diacrylic Acid (4)

To a solution of ethyl 2-[bis(2-isopropylphenoxy)phosphoryl]acetate^19,20) (0.921 g, 2.28 mmol) in tetrahydrofuran (THF) (10 mL), Triton B (40% in methanol, 1.15 mL, 2.91 mmol) at −78°C under an argon atmosphere was added dropwise. After 15 min of stirring, a solution of isophthalaldehyde (16) (0.139 g, 1.04 mmol) in THF (3.5 mL) was added dropwise to the solution. After 10 h of stirring, the mixture was quenched with saturated aqueous NH₄Cl and extracted with ethyl acetate (EtOAc). The organic layer was washed with H₂O, saturated aqueous NaHCO₃ and brine, dried over MgSO₄, filtered and concentrated in vacuo. The crude product was purified by silica gel flash column chromatography (EtOAc–hexane, 5 : 95) to afford (2Z,2′Z)-diethyl-3,3′-(1,3-phenylene)diacrylate (17) (0.274 g, 0.998 mmol, 96%, Z : E=96 : 4, determined by ¹H-NMR spectrum) as a colorless oil: ¹H-NMR (CDCl₃, 400 MHz) δ: 1.23 (6H, t, J=7.4 Hz, –CH₃), 4.16 (4H, q, J=7.4 Hz, –CH₂–CH₃), 5.96 (2H, d, J=12.8 Hz, =CH–CO₂–), 6.93 (2H, d, J=12.8 Hz, Ar-CH=), 7.34 (1H, t, J=7.8 Hz, Ar-H), 7.59 (2H, d, J=7.8 Hz, Ar-H), 7.73 (1H, br s, Ar-H). ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.0 (q, –CH₃), 60.2 (t, –CH₂–), 120.2 (d, =CH–CO₂–), 127.6 (d, Ar), 130.1 (d, Ar), 131.1 (d, Ar), 134.6 (s, Ar), 142.4 (d, Ar-CH=), 166.0 (s, C=O).

To a solution of 17 (0.249 g, 0.908 mmol) in EtOH (1.7 mL), 10% aqueous NaOH (3.6 mL) was added at room temperature. After stirring for 12 h, the mixture was adjusted to pH 1.0 with 1m aqueous HCl, and then extracted with EtOAc. The organic layer was washed with brine, dried over MgSO₄, filtered and concentrated in vacuo. The residue was purified by recrystallization to afford 4 (0.190 g, 0.872 mmol, 96%) as a single isomer: colorless needles, mp 156–157°C (toluene); ¹H-NMR (CDCl₃, 400 MHz) δ: 5.92 (2H, d, J=12.6 Hz,=CH–CO₂–), 6.95 (2H, d, J=12.6 Hz, Ar-CH=), 7.31 (1H, t, J=7.8 Hz, Ar-H), 7.581, 7.577 (each 1H, d, J=7.8 Hz, Ar-H), 7.74 (1H, br s, Ar-H). ¹³C-NMR (CDCl₃, 100 MHz) δ: 121.5 (d, =CH–CO₂–), 128.7 (d, Ar), 131.0 (d, Ar), 132.2 (d, Ar), 136.3 (d, Ar), 142.8 (d, Ar-CH=), 169.7 (s, C=O). IR (KBr) cm⁻¹: 1699. Electrospray ionization (ESI)-MS m/z: 217 (M⁺−H); Anal. Calcd for C₁₂H₁₀O₄: C, 66.05; H, 4.62. Found: C, 65.86; H, 4.59.

(Z)-3-[3-(Trimethylsilyl)phenyl]acrylic Acid (5)

To a solution of 1,3-dibromobenzene (18) (1.00 g, 4.24 mmol) in diethyl ether (Et₂O) (8.5 mL), n-butyllithium (BuLi) (2.30 m in hexane, 1.84 mL, 4.24 mmol) was added dropwise at −78°C under an argon atmosphere. After 15 min of stirring, chlorotrimethylsilane (0.570 mL, 4.45 mmol) was added dropwise to the solution. After the mixture was warmed to room temperature, the reaction was stirred for 1.5 h. The mixture was quenched with H₂O and extracted with Et₂O. The organic layer was washed with brine, dried over MgSO₄, filtered and concentrated in vacuo. The crude product was purified by silica gel flash column chromatography (hexane) to give (3-bromophenyl)trimethylsilane (19) (0.863 g, 3.78 mmol, 89%) as a colorless oil: ¹H-NMR (CDCl₃, 400 MHz) δ: 0.27 (9H, s, –CH₃), 7.22 (1H, t, J=8.1 Hz, Ar-H), 7.42, 7.47 (each 1H, d, J=8.1 Hz, Ar-H), 7.61 (1H, br s, Ar-H); The spectral data were in agreement with those in the literature.²³⁾

To a solution of (3-bromophenyl)trimethylsilane (19) (0.860 g, 3.77 mmol) in Et₂O (5.7 mL), n-BuLi (2.30 m in hexane, 1.64 mL, 3.77 mmol) was added dropwise at −78°C under an argon atmosphere, and then the reaction was stirred for 20 min. The reaction was warmed to room temperature and stirred for 1.5 h. A solution of N,N-dimethylformamide (DMF) (0.320 mL, 6.03 mmol) in Et₂O (2.8 mL) was added dropwise to the solution. After 12 h of stirring, the mixture was quenched with H₂O and extracted with CHCl₃. The organic layer was washed with saturated aqueous NaHCO₃ and brine, dried over MgSO₄, filtered, and concentrated in vacuo. The crude product was purified by silica gel flash column chromatography (EtOAc–hexane, 10 : 90) to provide 3-(trimethylsilyl)benzaldehyde (20) (0.376 g, 2.11 mmol, 56%) as a colorless oil: ¹H-NMR (CDCl₃, 400 MHz) δ: 0.31 (9H, s, –CH₃), 7.52 (1H, t, J=7.6 Hz, Ar-H), 7.78, 7.85 (each 1H, d, J=7.6 Hz, Ar-H), 8.02 (1H, br s, Ar-H), 10.0 (1H, s, –CHO). The spectral data were in agreement with those in the literature.²³⁾

The Z-selective olefination of 20 was performed using the procedure described above to provide (Z)-ethyl-3-(3-(trimethylsilyl)phenyl)acrylate (82%, Z : E=95 : 5, determined by ¹H-NMR spectrum) (silica gel column chromatography, EtOAc–hexane, 5 : 95) as a colorless oil: ¹H-NMR (CDCl₃, 400 MHz) δ: 0.27 (9H, s, –CH₃), 1.23 (3H, t, J=6.8 Hz, –CH₂–), 4.17 (2H, q, J=6.8 Hz, –CH₂–CH₃), 5.95 (1H, d, J=12.6 Hz, =CH–CO₂–), 6.95 (1H, d, J=12.6 Hz, Ar-CH=), 7.34 (1H, t, J=7.6 Hz, Ar-H), 7.47, 7.62 (each 1H, d, J=7.6 Hz, Ar-H), 7.63 (1H, br s, Ar-H).

The hydrolysis of (Z)-ethyl 3-(3-(trimethylsilyl)phenyl)acrylate was performed using the procedure described above to afford 5 (90%) as a single isomer: colorless solid ¹H-NMR (CDCl₃, 400 MHz) δ: 0.27 (9H, s, –CH₃), 5.98 (1H, d, J=12.8 Hz, =CH–CO₂–), 7.08 (1H, d, J=12.8 Hz, Ar-CH=), 7.35 (1H, t, J=7.6 Hz, Ar-H), 7.50, 7.62 (each 1H, d, J=7.6 Hz, Ar-H), 7.74 (1H, br s, Ar-H). ¹³C-NMR (CDCl₃, 100 MHz) δ: −1.25 (q, –CH₃), 118.4 (d, =CH–CO₂–), 127.4 (d, Ar), 130.3 (d, Ar), 133.5 (s, Ar), 134.3 (d, Ar), 135.1 (d, Ar), 140.3 (s, Ar), 146.2 (d, Ar-CH=), 171.2 (s, C=O). IR (KBr) cm⁻¹: 1624, 1697. Electron ionization (EI)-MS m/z: 220 (M⁺), 205 (M⁺−Me). High resolution (HR)-EI-MS m/z: 220.0916 (M⁺, Calcd for C₁₂H₁₆O₂Si: 220.0920).

(Z)-3-[3-(Pentyloxy)phenyl]acrylic Acid (7)

To a solution of K₂CO₃ (3.40 g, 24.6 mmol) in acetone (10 mL), 3-hydroxybenzaldehyde (21) (2.00 g, 16.4 mmol) was added at 0°C under an argon atmosphere and then the mixture was stirred for 15 min. After 1-iodopentane (6.50 g, 32.8 mmol) was added to the solution, the resulting mixture was refluxed for 3.5 h. The reaction was filtered and the filtrate was evaporated to eliminate acetone. The resulting residue was diluted with H₂O and extracted with EtOAc. The organic layers were washed with brine, dried over MgSO₄, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (EtOAc–hexane, 30 : 70) to afford 3-(pentyloxy)benzaldehyde (22) (2.36 g, 12.3 mmol, 75%) as a colorless oil: ¹H-NMR (CDCl₃, 270 MHz) δ: 0.94 (3H, t, J=7.0 Hz, –CH₃), 1.33−1.52 (4H, m, –CH₂–), 1.75−1.87 (2H, m, –CH₂–), 4.02 (2H, t, J=6.6 Hz, –OCH₂–), 7.17 (1H, m, Ar-H), 7.38 (1H, m, Ar-H), 7.41−7.48 (2H, m, Ar-H), 9.97 (1H, s, –CHO). The spectral data were in agreement with those in the literature.²¹⁾

The Z-selective olefination of 22 was performed using the procedure described above to provide (Z)-ethyl 3-[3-(pentyloxy)phenyl]acrylate (99%, Z : E=95 : 5, determined by ¹H-NMR spectrum) (silica gel column chromatography, EtOAc–hexane, 5 : 95) as a colorless oil: ¹H-NMR (CDCl₃, 270 MHz) δ: 0.93 (3H, t, J=6.9 Hz, –CH₃), 1.25 (3H, t, J=7.2 Hz, –CO₂–CH₂–CH₃), 1.26–1.56 (4H, m, –CH₂–), 1.67–1.90 (2H, m, –CH₂–), 3.96 (2H, t, J=6.5 Hz, –OCH₂–), 4.18 (2H, q, J=7.2 Hz, –CO₂–CH₂–), 5.93 (1H, d, J=12.5 Hz,=CH–CO₂–), 6.86 (1H, m, Ar-H), 6.90 (1H, d, J=12.5 Hz, Ar-CH=), 7.11 (1H, m, Ar-H), 7.17−7.30 (2H, m, Ar-H).

The hydrolysis of (Z)-ethyl-3-[3-(pentyloxy)phenyl]acrylate was performed using the procedure described above to afford 7 (77%) as a single isomer: colorless needles: mp 35–36°C (toluene); ¹H-NMR (CDCl₃, 400 MHz) δ: 0.93 (3H, t, J=7.2 Hz, –CH₃), 1.29–1.52 (4H, m, –CH₂–), 1.70–1.89 (2H, m, –CH₂–), 3.96 (2H, t, J=6.6 Hz, –OCH₂–), 5.96 (1H, d, J=12.8 Hz, =CH–CO₂–), 6.89 (1H, dd, J=2.4, 8.1 Hz, Ar-H), 7.02 (1H, d, J=12.8 Hz, Ar-CH=), 7.13 (1H, d, J=8.1 Hz, Ar-H), 7.20–7.34 (2H, m, Ar-H). ¹³C-NMR (CDCl₃, 100 MHz) δ: 14.0 (q, –CH₃), 22.4 (t, –CH₂–), 28.2 (t, –CH₂–), 28.9 (t, –CH₂–), 68.0 (t, –OCH₂–), 115.5 (d, Ar), 116.2 (d, =CH–CO₂–), 118.7 (d, Ar), 122.4 (d, Ar), 129.0 (d, Ar), 135.5 (d, Ar), 145.7 (d, Ar-CH=), 158.8 (s, Ar), 171.4 (s, C=O). IR (KBr) cm⁻¹: 1699. ESI-MS m/z: 233 (M⁺−H). Anal. Calcd for C₁₄H₁₈O₃: C, 71.77; H, 7.74. Found: C, 71.83; H, 7.71.

(2Z,4Z)-4-Bromo-5-phenylpenta-2,4-dienoic Acid (12)

The Z-selective olefination of (Z)-2-bromo-3-phenylacrylaldehyde (23) was performed using the procedure described above to provide (2Z,4Z)-ethyl-4-bromo-5-phenylpenta-2,4-dienoate (98%, Z : E=88 : 12, determined by ¹H-NMR spectrum) (silica gel column chromatography, EtOAc–hexane, 5 : 95) as a colorless oil: ¹H-NMR (CDCl₃, 270 MHz) δ: 1.26 (3H, t, J=7.0 Hz, –CH₃), 4.19 (2H, q, J=7.0 Hz, –CH₂–), 5.81 (1H, d, J=11.9 Hz,=CH–CO₂–), 6.61 (1H, dd, J=1.1, 11.9 Hz, =CBr–CH=), 7.24 (1H, br s, Ar-CH=), 7.26–7.50 (3H, m, Ar-H), 7.60–7.76 (2H, m, Ar-H).

The hydrolysis of (2Z,4Z)-ethyl-4-bromo-5-phenylpenta-2,4-dienoate was performed using the procedure described above to afford 12 (84%) as a single isomer: colorless needles: mp 64 to 66°C (CH₂Cl₂–hexane, 5 : 95); ¹H-NMR (CDCl₃, 400 MHz) δ: 5.82 (1H, d, J=12.0 Hz, =CH–CO₂–), 6.73 (1H, d, J=12.0 Hz, =CBr–CH–), 7.28 (1H, br s, Ar-CH=), 7.31–7.45 (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 (d, Ar), 128.7 (d, Ar), 129.3 (d, Ar), 134.2 (d, Ar-CH=), 134.9 (s, Ar), 144.1 (d, =CBr–CH=), 170.8 (s, C=O). IR (KBr) cm⁻¹: 1699. EI-MS m/z: 252 (M⁺−H). HR EI-MS m/z: 251.9788 (M⁺, Calcd for C₁₁H₉BrO₂: 251.9786).

(2E,4E)-4-Methyl-5-phenylpenta-2,4-dienoic Acid (13)

To a solution of NaH (60% oil solution, 0.504 g, 12.6 mmol) in Et₂O (5.0 mL), a solution of ethyl 2-(diethoxyphosphoryl)acetate (1.67 g, 7.40 mmol)²¹⁾ in Et₂O (5.0 mL) was added at 0°C under an argon atmosphere, and then the solution was stirred for 15 min. A solution of (E)-2-methyl-3-phenylacrylaldehyde (24) (1.02 g, 7.00 mmol) in Et₂O (4.0 mL) was added to the solution at the same temperature. After the mixture was warmed to room temperature, the reaction was stirred for 5 min. 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) to afford (2E,4E)-ethyl-4-methyl-5-phenylpenta-2,4-dienoate (1.15 g, 5.32 mmol, 76%, E only, determined by ¹H-NMR spectrum) as a colorless oil; ¹H-NMR (CDCl₃, 400 MHz) δ: 1.33 (3H, t, J=7.2 Hz, –CH₃), 2.05 (3H, br s, =CH(CH₃)–), 4.25 (2H, q, J=7.2 Hz, –CH₂–), 5.99 (1H, d, J=16.0 Hz, =CH–CO₂–), 6.86 (1H, s, Ar-CH=), 7.23–7.48 (5H, m, Ar-H), 7.50 (1H, d, J=16.0 Hz, =C(CH₃)–CH=).

The hydrolysis of (2E,4E)-ethyl-4-methyl-5-phenylpenta-2,4-dienoate was performed using the procedure described above to afford 13 (89%) as a single isomer: colorless needles: mp 113–114°C (CH₂Cl₂–hexane, 5 : 95); ¹H-NMR (CDCl₃, 400 MHz) δ: 2.08 (3H, s, –CH₃), 5.99 (1H, d, J=15.6 Hz,=CH–CO₂–), 6.90 (1H, s, Ar-CH=), 7.19–7.46 (5H, m, Ar-H), 7.59 (1H, d, J=15.6 Hz, =C(CH₃)–CH=). ¹³C-NMR (CDCl₃, 100 MHz) δ: 13.7 (q, –CH₃), 116.6 (d, =CH–CO₂–), 127.9 (d, Ar), 128.4 (d, Ar), 129.5 (d, Ar), 134.0 (s, =C(CH₃)–), 136.5 (s, Ar), 140.1 (d, Ar-CH=), 152.2 (d,=C(CH₃)–CH=), 173.0 (s, C=O). IR (KBr) cm⁻¹: 1724. FAB-MS m/z: 188 (M⁺). Anal. Calcd for C₁₂H₁₂O₂: C, 76.57; H, 6.43. Found: C, 76.44; H, 6.41.

The Root Growth Inhibition Assay

The assay was performed according to the Hiradate’s method with minor modification.¹¹⁾ An ϕ27 filter paper was placed in a glass petri dish and methanol solution containing the test compounds and growth inhibitors were added into the dish simultaneously (70 µL each, concentrated for desired final concentration). The solvent was removed from the filter paper under reduced pressure. After the addition of 700 µL distilled water, six pre-germinated lettuce seedlings (Lactuca sativa cv. Great Lakes 366) were placed on the filter paper. The pre-germination state was induced using distilled water at 25°C, 60% relative humidity for 24 h in the dark. Two dishes were prepared for each concentration (n=12). The dishes were incubated at 25°C, 60% relative humidity for 48 h in a dark. The root growth was evaluated by measuring the length of each root and comparing it with the roots of vehicle control.

Statistical Analysis

Results were expressed as the mean±S.D. The statistical analysis was performed using a one-way ANOVA followed by Tukey–Kramer’s test to assess the significance of differences in mean values between the drug treatment groups and suitable referential group. Significance was accepted when the p value was less than 0.05.

Acknowledgment

This work is supported by the Program for Promotion of Basic and Applied Research for Innovations in the Bio-oriented Industry (BRAIN), and Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry.

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