J. Agric. Food Chem. 1998, 46, 1577−1582
1577
Synthesis and Cytokinin Activity of New Zeatin Derivatives M’Barek Haidoune,†,‡ Isabelle Raynaud,† Norval O’Connor,† Pascal Richomme,§ Rene´ Mornet,*,† and Michel Laloue| Faculte´ des Sciences, Laboratoire d’Inge´nierie Mole´culaire et Mate´riaux Organiques, 2 Boulevard Lavoisier, 49045 Angers Cedex, France, SCRMN, 16 Boulevard Daviers, 49045 Angers Cedex, France, and Laboratoire de Biologie Cellulaire, INRA, Route de Saint Cyr, 78026 Versailles Cedex, France
The analogue of zeatin bearing a vinylic fluorine atom and its geometrical isomer were synthesized. The fluorine atom exerts a favorable influence on cytokinin activity in the fluoro analogue of ciszeatin, but not in the fluoro analogue of zeatin itself. Another series of zeatin derivatives in which the methyl group was replaced by alkyl (ethyl, propyl, and isopropyl), phenyl, and benzyl groups were also obtained. The ethyl analogue was found to be more active than zeatin, while the others were inactive or slightly active. Keywords: Cytokinins; fluoro compounds; synthesis; biological activity INTRODUCTION
Zeatin (1) and
N6-isopentenyladenine
(2) are the most
widespread natural plant hormones of the cytokinin family. Except for a series of N,N′-disubstituted ureas, the most active cytokinins are N6-substituted adenines. In this latter class of compounds, studies of structureactivity relationships have demonstrated that optimal cytokinin activity was observed when the N6-chain contains five or six carbon atoms and bears a double bond in the 2,3-position (Matsubara, 1990). Analogues of zeatin (1) have been studied, corresponding to various chain isomerisms (Schmitz et al., 1972), hydroxylation or dihydroxylation (Leonard et al., 1968; Letham, 1973; Van Staden and Drewes, 1982) and hydrogenation (Fujii and Ogawa, 1972) of the double bond, and methylation at the C-1 of the chain (Fujii et al., 1989). However, in zeatin (1), neither the influence on the activity of the replacement of the methyl group by other substituents nor that of the replacement of the vinylic proton by halogens has been yet investigated. We recently synthesized two new derivatives (3 and 4) of N6-isopentenyladenine (2), the activities of which were found to be significantly higher than the activity of 2 (Clemenceau et al., 1996). In particular, a near 10fold increase in activity was observed when the vinylic * Author to whom correspondence should be addressed. E-mail:
[email protected]. † Laboratoire d’Inge ´ nierie Mole´culaire et Mate´riaux Organiques. ‡ On leave from the University of Casablanca (Casablanca, Morocco) from 1993 to 1996. § SCRMN. | Laboratoire de Biologie Cellulaire.
hydrogen atom in 2 was substituted for fluorine. Thus, an exceptionnally high cytokinin activity was expected for fluorozeatin (5), since zeatin (1) has been found to be more active than N6-isopentenyladenine (2) in various bioassays (Matsubara, 1990). We describe here the synthesis of this new zeatin analogue and of its geometrical isomer 6 related to cis-zeatin (7) and the results of their cytokinin bioassays. We previously described a method of synthesis of zeatin (1) (Mornet and Gouin, 1977) which could be used for zeatin analogues where the methyl group is substituted for various other groups. To enrich structureactivity relationships in the zeatin series, we have also synthesized compounds 8a-e to evaluate their cytokinin activity.
EXPERIMENTAL PROCEDURES Melting points were measured with a Reichert Thermovar hotstage apparatus. 1H NMR spectra were recorded with TMS as an internal standard, at 270 MHz on a JEOL GSX 270 WB spectrometer or at 60 MHz on a Varian EM 360 apparatus. 13C NMR spectra were obtained from the JEOL spectrometer at 67.9 MHz, and 19F spectra from the same apparatus at 254.2 MHz, using TMS and CFCl3 as internal standards, respectively. EI mass spectra were recorded with a VG Autospec spectrometer. Microanalyses were obtained from the Service d’Analyses du CNRS (Lyon, France). 2-Methyl-1-(trityloxy)-2-propene (9). To a solution of 2-methyl-2-propen-1-ol (11.0 g, 153 mmol), triethylamine (21.6 mL, 153 mmol), and 4-(dimethylamino)pyridine (0.50 g, 4.1 mmol) in dimethylformamide (200 mL) was added trityl chloride (43.4 g, 156 mmol). The reaction mixture was stirred in a nitrogen atmosphere for 24 h. The solution was then diluted with water (350 mL). The white precipitate which was obtained was filtered and extracted with hot cyclohexane (300
S0021-8561(97)00752-8 CCC: $15.00 © 1998 American Chemical Society Published on Web 04/03/1998
1578 J. Agric. Food Chem., Vol. 46, No. 4, 1998 mL). The white crystals obtained on cooling were filtered and dried to give 25.0 g (52%) of compound 9: 1H NMR (270 MHz, CDCl3) δ 1.51 (s, 3H, CH3), 3.30 (s, 2H, CH2O), 4.72 and 5.04 (2 s, 2 × 1H, dCH2), 7.01-7.15 and 7.25-7.30 (2 m, 15H, H-phenyl). 1-Bromo-1-fluoro-2-methyl-2-[(trityloxy)methyl]cyclopropane (10). To a mixture of the ethylenic compound 9 (20.0 g, 0.064 mol), benzyltriethylammonium chloride (0.4 g, 0.88 mmol), sodium hydroxide (25 mL of a 50% aqueous solution), and dichloromethane (250 mL) was added dibromofluoromethane (19.2 g, 0.1 mol) dropwise while the solution was stirred. The mixture was heated to reflux with vigorous stirring for 36 h. After cooling, water (1 L) was added, and the solution was extracted with dichloromethane (3 × 100 mL). The combined organic layers were washed with water and dried over magnesium sulfate. After evaporation of the solvent under low pressure, the residue was chromatographed through a silica gel column with hexane/dichloromethane (8:2) as the eluent. The appropriate fractions were combined and evaporated to leave 19 g of a mixture containing 13.7 g (50%) of 10, as a mixture of diastereomers, and 5.3 g of the starting compound 9 (according to 1H NMR determination). 10 was not further purified: 1H NMR (270 MHz, CDCl3) δ 0.90-1.10 and 1.20-1.40 (2 m, 2 × 1H, CH2-ring), 1.34 (d, 3H, 4JHF ) 2.1 Hz, CH3), 3.06-3.14 (m, 2H, CH2O), 7.10-7.30 and 7.417.47 (2 m, 15H, H-phenyl). N-[2-Fluoro-3-methyl-4-(trityloxy)-2-butenyl]phthalimides (11Z and 11E). Crude cyclopropane 10 (15 g containing 10.79 g of 10, 26 mmol) and potassium phthalimide (9 g, 48.6 mmol) were dissolved in dimethylformamide (100 mL). The mixture was heated to 110 °C for 72 h. After cooling, water (200 mL) was added, and the mixture was extracted with dichloromethane (3 × 100 mL). The combined organic extracts were washed with water (100 mL) and dried over magnesium sulfate. After evaporation of the solvent, the residue was chromatographed through a silica gel column with hexane/ ethyl acetate (8:2) as the eluent. Two fractions were isolated which contained respectively the isomers 11Z and 11E as major products. After evaporation of the solvents from these fractions, the residues were recrystallized from pentane. Pure 11Z was obtained as a white powder (6.0 g, 47%): mp 175 °C; 1H NMR (270 MHz, CDCl ) δ 1.96 (d, 3H, 4J 3 HF ) 2.8 Hz, CH3), 3.73 (d, 2H, 4JHF ) 3.3 Hz, CH2O), 4.44 (d, 2H, 3JHF ) 20.2 Hz, CH2N), 7.10-7.35 and 7.40-7.50 (2 m, 15H, H-triphenyl), 7.72 and 7.83 (2 m, 2 × 2H, H-phthalimide). Elemental analysis found: C, 77.94; H, 5.20; F, 3.74; N, 2.81. Calcd for C32H26FNO3: C, 78.19; H, 5.33; F, 3.86; N, 2.85. 11E was obtained as a white powder containing about 10% of its isomer 11Z (3.0 g, 23.5%): 1H NMR (270 MHz, CDCl3) δ 1.70 (d, 3H, 4J 4 HF ) 3.5 Hz, CH3), 3.79 (d, 2H, JHF ) 1.7 Hz, CH2O), 4.20 (d, 2H, 3JHF ) 19.7 Hz, CH2N), 7.10-7.30 and 7.40 (2 m, 15H, H-triphenyl), 7.63 and 7.75 (2 m, 2 × 2H, H-phthalimide). N-[(Z)-2-Fluoro-4-hydroxy-3-methyl-2-butenyl]phthalimide (12Z). The phthalimide 11Z (3.58 g, 7.3 mmol) was dissolved in methanol (100 mL), and p-toluenesulfonic acid (1.5 g, 7.5 mmol) was added. The solution was stirred for 48 h at room temperature. The solvent was evaporated, and the residue was chromatographed through a silica gel column with dichloromethane/ethanol (95:5) as the eluent. Evaporation of the appropriate fraction left pure 12Z as a white powder (1.20 g, 66%): mp 111 °C; 1H NMR (270 MHz, CDCl3) δ 1.96 (d, 3H, 4J 4 HF ) 2.8 Hz, CH3), 4.22 (d, 2H, JHF ) 3.1 Hz, CH2O), 4.57 (d, 2H, 3JHF ) 20.0 Hz, CH2N), 7.70 and 7.80 (2 m, 2 × 2H, H-phthalimide); 13C NMR (CDCl3) δ 13.3 (d, 3JCF ) 4.2 Hz, CH3), 35.2 (d, 2JCF ) 31.2 Hz, CH2N), 59.7 (d, 3JCF ) 10.4 Hz, CH2O), 116.7 (d, 2JCF ) 11.4 Hz, CdCF), 149.0 (d, 1JCF ) 250.0 Hz, dCF). Elemental analysis found: C, 62.59; H, 4.72; F, 7.54; N, 5.64. Calcd for C13H12FNO3: C, 62.65; H, 4.85; F, 7.62; N, 5.62. N-[(E)-2-Fluoro-4-hydroxy-3-methyl-2-butenyl]phthalimide (12E). This compound was obtained (300 mg, 52%) with the same procedure used for 12Z from the impure phthalimide 11E (1.139 g, 2.32 mmol). It was contaminated by a small amount (
