J. Agric. Food Chem. 2003, 51, 3049−3055
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Phytoene Desaturase Inhibition by O-(2-Phenoxy)ethyl-N-aralkylcarbamates SHINPEI OHKI,† ROSWITHA MILLER-SULGER,† KO WAKABAYASHI,‡ WOLFGANG PFLEIDERER,§ AND PETER BO¨ GER*,† Lehrstuhl fu¨r Physiologie und Biochemie der Pflanzen, Universita¨t Konstanz, D-78457 Konstanz, Germany; Graduate School of Agriculture, Tamagawa University, Machida-shi, Tokyo, 194-8610 Japan; and Fachbereich Chemie, Universia¨t Konstanz, P.O. Box 5560, D-78457 Konstanz, Germany
O-[1-Ethyl-2-(3-trifluoromethylphenoxy)]ethyl-N-benzylcarbamate exhibits a marked inhibition of carotenoid biosynthesis. Forty-one analogues were synthesized and assayed for plant-type phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS) inhibition in a cell-free system using recombinant enzymes obtained from Escherichia coli transformants. The target enzyme of all carbamates synthesized in this study is PDS and not ZDS; no inhibition of ZDS was observed using a 10-4 M inhibitor concentration. Four compounds, O-[1-ethyl-2-(3-trifluoromethylphenoxy)]ethyl-N-(2-phenylethyl)carbamate (23), O-[1-ethyl-2-(3-trifluoromethylphenoxy)]ethyl-N-(2-chlorobenzyl)carbamate (25), O-[1-ethyl-2-(3-trifluoromethylphenoxy)]ethyl-N-(2-chlorobenzyl)carbamate (26), and O-[1-methyl-2(3-trifluoromethylphenoxy)]ethyl-N-benzylcarbamate (30), were the most potent PDS inhibitors. Their pI50 values, the negative logarithms of the molar concentration that produces a 50% inhibition, were 7.5, representing the same inhibitory activity as norflurazon. With respect to a structure-activity relationship the oxygen atom of the phenoxy group and a carbamate structure in O-(1-ethyl-2phenoxy)ethyl-N-aralkylcarbamates studied were found to be essential for strong PDS inhibitors. Also, introduction of an ethyl group at the R-position of the ethylene bridge between the phenoxy group and the carbamate was important for a strong PDS inhibitor. Substituents at the 2- and/or 3-position of the phenoxybenzene ring were found to be favorable to a strong PDS inhibition of the analogues. KEYWORDS: Phytoene desaturase inhibitors; O-(2-phenoxy)ethyl-N-aralkylcarbamates
INTRODUCTION
The carotenoid biosynthesis pathway is an important site of herbicide actions. Heterocyclic compounds, such as norflurazon, flurochloridone, or LS80707, prevent the formation of carotenoids, and some of them have been already commercialized as herbicides (see Figure 1) (1). Most of such herbicides belonging to different chemical classes, namely, diphenylpyridones, diphenylpyridines, phenoxynicotinamides, phenylpyridazinones, and others (2-4), were reported as phytoene desaturase (PDS) inhibitors. Recently, 1,1′-biphenyl derivatives (5), diphenylpyrimidines (6), and diphenylpyrrolidinones (7) have been characterized as new PDS inhibitors. Mitchell (8) proposed a model of the herbicidal binding site of PDS using superimposition with five structurally different inhibitors. Although structure-activity relationship (SAR) studies have been carried out (4, 9), it is still not known which structural elements of the inhibitors are essential for potent PDS inhibition. * Author to whom correspondence should be addressed [fax (+49)-7531883042; e-mail
[email protected]]. † Lehrstuhl fu ¨ r Physiologie und Biochemie der Pflanzen, Universita¨t Konstanz. ‡ Tamagawa University. § Fachbereich Chemie, Universia ¨ t Konstanz.
In the long run, such an SAR study should be performed using PDS inhibitors belonging to different chemical classes and the PDS inhibitory data of the compounds obtained by a cell-free assay. O-(2-Phenoxy)ethyl-N-benzylcarbamates were developed by Imperial Chemical Industry Ltd. (10) and Ciba-Geigy AG (11). Their compounds showed good herbicidal activity in a preemergence application against AVena fatua, Echinochloa crusgalli, Setaria Viridis, Ipomoea purpurea, Abutilon theophrasti, and Brassica kaber. In our preliminary mode of action studies, O-[1-ethyl-2-(3-trifluoromethylphenoxy)]ethyl-N-benzylcarbamate exhibited a marked inhibition of carotenoid biosynthesis. Accordingly, we synthesized a number of analogues and assayed their inhibition of PDS and ζ-carotene desaturase (ZDS) using recently developed cell-free assays (12, 13) to clarify their mode of action and to obtain more information for the molecular design of new carotenoid biosynthesis inhibitors. MATERIALS AND METHODS Syntheses. The carbamate and benzoate derivatives synthesized in this study are listed in Tables 1-3. 2-Phenoxyethanols (10, 11), 2-(3chlorophenylthio)-1-ethylethanol (14), 2-ethyl-2-(3-trifluoromethylphenoxy)ethanol (15), and 2-(N-acylanilino)-1-ethylethanols (16) were
10.1021/jf0262413 CCC: $25.00 © 2003 American Chemical Society Published on Web 04/10/2003
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Ohki et al.
Figure 1. Structures of inhibitors that affect desaturation of phytoene and ζ-carotene and prevent the formation of colored carotenoids. prepared according to standard methods. Corresponding chloroformates were obtained by reaction of the appropriate alcohols with excess phosgene in an inert solvent, such as dichloromethane, in the presence of triethylamine. The product carbamates were synthesized by reaction of the chloroformate intermediates with the appropriate amines in an inert solvent using a base such as a tertiary amine or pyridine (10, 11). [2-(3-Chlorophenoxy)-1-ethyl]ethyl benzoate was prepared by the reaction of [2-(3-chlorophenoxy)-1-ethyl]ethanol with benzoyl chloride in the presence of triethylamine. All reaction products were purified through recrystallization and/or flash column chromatography, and their structures were confirmed by NMR, mass spectroscopy, and elemental analysis. Melting points are uncorrected. 1H NMR and 13C NMR spectra were obtained using a Bruker AC250 FT-NMR at 250 and 62.5 MHz, respectively. Mass spectra data were measured by a Varian MAT 312 for EI techniques. Elemental analyses (C, H, N) were performed by an elementar Vario eL, and the results obtained were within (0.3 of the calculated percentage. [2-(3-Chlorophenoxy)-1-ethyl]ethanol. 3-Chlorophenol (12.0 g, 90 mmol), 1,2-butylene oxide (7.2 g, 100 mmol), and lithium hydroxide monohydrate (0.4 g, 10 mmol) were heated in a sealed tube (pressure reactor) for 16 h to 160 °C. After the reactor had cooled, the reaction mixture was poured into water (50 mL) and extracted with ethyl acetate (3 × 50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated. The residue was flash-chromatographed on silica gel with ethyl acetate/petroleum ether (9:1, v/v): yield, 13.7 g (76%); liquid; 1H NMR (250 MHz, DMSO) δ 0.90 (3H, t, JH ) 7.4 Hz), 1.40 and 1.51(2H, m), 3.67 (1H, m), 3.84 (2H, m), 4.85 (1H, d, JH ) 5.1 Hz), 6.94 (3H, m), 7.28 (1H, t, JH ) 8.1 Hz). [2-(3-Chlorophenylthio)-1-ethyl]ethanol. To 1,2-butylene oxide (2.9 g, 40 mmol) was added 3-chlorothiophenol (5.0 g, 35 mmol) and lithium hydroxide monohydrate (0.2 g, 5 mmol) at 0 °C, and the mixture was stirred for 2 h at room temperature. The reaction mixture was poured onto ice-water (50 mL) and extracted with ethyl acetate (3 × 50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated. The desired compound was separated using silica gel flash chromatography with ethyl acetate/petroleum ether (10:1, v/v): yield,
6.0 g (85%); liquid; 1H NMR (250 MHz, DMSO) δ 0.86 (3H, t, JH ) 7.4 Hz), 1.29-1.64 (2H, m), 3.00 (2H, dd, JH ) 2.0 and 6.0 Hz), 3.53 (1H, m), 4.94 (1H, d, JH ) 5.3 Hz), 7.14-7.37 (4H, m). [2-(N-Acetylanilino)-1-ethyl]ethanol. [2-(3-Chlorophenylamino)-1ethyl]ethanol (yield, 56%; mp, 32-35 °C) was prepared according to the same synthetic method of [2-(3-chlorophenoxy)-1-ethyl]ethanol. To a solution of [2-(3-chloroanilino)-1-ethyl]ethanol (2.0 g, 10 mmol) in tetrahydrofuran (10 mL) was added acetic anhydride (2.0 g, 20 mmol) at 5 °C. The reaction mixture was stirred for 4 h at room temperature and concentrated by evaporation. Ethyl acetate (50 mL) was added to the residue and washed with sodium hydrogencarbonate solution (3 × 50 mL) and saturated NaCl solution (3 × 50 mL). The organic phase was dried over anhydrous sodium sulfate and evaporated. Flash chromatography of the residue on silica gel with ethyl acetate/petroleum ether (1:3, v/v) provided the desired compound in a pure state: yield, 1.3 g (54%); liquid; 1H NMR (250 MHz, DMSO) δ 0.81 (3H, t, JH ) 7.3 Hz), 1.15-1.40 (2H, m), 1.75 (3H, s), 3.45 (2H, m), 3.62 (1H, m), 4.63 (1H, s), 7.32-7.53 (4H, m). [1-Ethyl-1-(3-trifluoromethylphenoxy)]ethanol. Ethyl 2-(3-trifluoromethylphenoxy)butanoate was synthesized by the reaction of 3-trifluoromethylphenol with ethyl 2-bromobutanoate according to the method of Wimmer et al. (15). 2-(3-Trifluoromethylphenoxy)butanoic acid was prepared by a hydrolysis of ethyl 2-(3-trifluoromethylphenoxy)butanoate using sodium hydroxide in methanolic solution. 2-(3Trifluoromethylphenoxy)butanoic acid (6.9 g, 30 mmol) in diethyl ether (30 mL) was added to a suspension of LiAlH4 (1.2 g, 30 mmol) in dry diethyl ether (30 mL) at 0 °C. The reaction mixture was stirred for 3 h at room temperature and refluxed for 3 h. Excess LiAlH4 was decomposed by dropwise addition of water, and the resulting white suspension was filtered. After concentration of the filtrate, pure [1-ethyl1-(3-trifluoromethylphenoxy)]ethanol was obtained: yield, 5.5 g (78.3%); liquid; 1H NMR (250 MHz, DMSO) δ 0.89 (3H, t, JH ) 7.4 Hz), 1.63 (2H, m), 3.54 (2H, t, JH ) 5.3 Hz), 4.33 (1H, m), 4.86 (1H, t, JH ) 5.7 Hz), 7.15-7.32 (3H, m), 7.51 (1H, t, JH ) 12.5 Hz). O-[2-(3-Chlorophenoxy)-1-ethyl]ethyl-N-benzylcarbamate (4). A solution of [2-(3-chlorophenoxy)-1-ethyl]ethanol (6.0 g, 30 mmol) and triethylamine (3.0 g, 31 mmol) in tetrahydrofuran (15 mL) was added
PDS Inhibition by O-(2-Phenoxy)ethyl-N-aralkylcarbamates
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Table 1. Physical Data of O-(1-Ethyl-2-phenoxy)ethyl-N-benzylcarbamates and Their Phytoene Desaturase and ζ-Carotene Desaturase Inhibitory
Activities
no.
R1
mp (°C)
pI50 (PDS)
pI50 (ZDS)
1
3-CF3
60−61
6.9
