Metabolism of Isoxathion, O,O-Diethyl O-(5-Phenyl-3-Isoxazolyl)
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Agr. Biol. Chem., 39 (11), 21372143, 1975 Metabolism of Isoxathion, O,O-Diethyl O-(5-Phenyl-3-isoxazolyl)- phosphorothionate in Plants Mitsuru ANDO,Yukiko IWASAKIand Masayuki NAKAGAWA SankyoCo., Ltd., AgriculturalChemicals Research Laboratories 2-58, l-chome, Hiromachi, Shinagawa-ku, Tokyo, Japan Received May 15, 1975 The absorption and metabolism of the insecticide, Isoxathion, on bean, cabbage and chinese cabbage plants were examined using carbon-14 labeled compound. Isoxathion penetrated into plant tissues was hydrolyzed to produce 3-hydroxy-5-phenylisoxazole, which was then rapidly converted to water soluble compounds. Among them, 3-(ƒÀ-3-D-glucopyranosyl oxy)-5-phenylisoxazole, 2-(ƒÀ-D-glucopyranosyl)-5-phenyl-4-isoxazolin-3-one and 2-(ƒÀ-D-gluco pyranosyl)-5-p-hydroxy-phenyl-4-isoxazolin-3-one were unequivocally identified as the major metabolites. Another metabolic pathway of 3-hydroxy-5-phenylisoxazole via a reductive cleavage of the isoxazole ring to form benzoic acid was negligible. Isoxathion, Karphos_??_, O, O-diethyl O-(5- phenylisoxazole (I), 2-(2•L,3•L,4•L,6•L-tetra-O-acetyl-ƒÀ-D- glucopyranosyl)-5-phenyl-4-isoxazolin-3-one (II), 3- phenyl-3-isoxazolyl)-phosphorothionate, is a (ƒÀ-D-glucopyranosyloxy)-5-phenylisoxazole (III) and 2- registered insecticide of a broad spectrum.'' ƒÀ-D-glucopyranosyl)-5-phenyl-4-isoxazolin-3-one (IV) The toxicity of the compound is as follows: were synthesized as follows: a mixture of 60g (ca. acute (oral) LD,0 to rats is 112 mg/kg" and 0.15M) of 2,3,4,6-tetra-O-acetyl-ƒ¿-D-glucopyranosyl non-effect level to rats in 90 days consecutive bromide," 24.2g (0.15M) of 3-hydroxy-5-phenyliso xazole, 16g (ca. 0.15M) of sodium carbonate, 60g feeding test is approximately 1 mg/kg/day. of Drierite and 500 ml of anhyd. acetonitrile was re Regarding the agricultural chemicals having fluxed with stirring for 8 hr with exclusion of moisture. isoxazole nucleus, fate of hymexazol (3- The mixture was cooled and filtered to remove inorganic hydroxy-5-methylisoxazole) in rats ,3) higher materials. Solvent was evaporated, and remaining plants,4) soils,5) photolysis,6) and of isoxathion sirup was dissolved in 500 ml of benzene. The solu in rats," soils"' has been reported. Hymexazol tion was washed with aq. saturated sodium bicarbonate and then with water, and dried over anhyd. sodium treated in plants was rapidly absorbed and sulfate. The solvent was removed in vacuo, and a transformed to 2-(ƒÀ-9-D-glucopyranosyl)-5-me sirupy residue was chromatographed on a column of thyl-4-isoxazolin-3-one and 3-(ƒÀ-D-glucopyra silica gel with ethyl acetate-n-hexane (2:3 and 4:1, nosyloxy)-5-methyli soxazole.4) v/v). Eluates were monitored by thin layer chromato In the present paper, isoxathion labeled with graphy of silica gel, and spots visualized under UV light. Evaporation of an earlier eluate gave 15.0 g of (I). carbon-14 was treated in plants to determine It was recrystallized from acetone to afford colorless the absorption and metabolic fate of the needles melting at 158°C. [ƒ¿]20D-10.5•‹ (c=1.00, chloro compound. form). Anal. Found: C, 56.36; H, 5.18; N, 2.57. Calcd. for C23H25NO11: C, 56.21; H, 5.13; N, 2.85%. IR ƒËKBrmax cm-1: 3150 (C=CH), 1750 (OCOCH3), 1625 MATERIALS AND METHODS (C=C), 1510 (C=N). The latter eluate gave 3.1 g of (II). Chemicals. Radioactive O,O-diethyl O-(5-phenyl- It was recrystallized from n-propanol to afford colorless 3-isoxazolyl)-phosphorothionate-5-14C and 3-hydroxy- needles melting at 75°C. [a]1 -48.l° (c=1.00, chloro 5-phenylisoxazole-5-14C were synthesized and purified form). Anal. Found: C, 56.50; H, 5.24; N, 2.76 %. as described elsewhere.7) The specific activity of each IR ƒËKBrmax cm-1: 3120 (C=CH), 1750 (OCOCH3), 1700 compound was 1.00 mCi/mM, and no radioactive im (C=O), 1625 (C=C). To a solution of 7.5 g of (I) in purity was detected by thin layer chromatography. 60 ml of methanol was added 60 mg of sodium metho 3-(2•L,3•L,4•L,6•L-Tetra-O-acetyl-ƒÀ-D-glucopyranosyloxy)-5- xide. The mixture was stirred at room temperature 2138 M. ANDO, Y. IWASAKI and M. NAKAGAWA for 6 hr, and then treated with Dowex 50W-X8 (H+ washed thoroughly in running acetone to remove radio- form) to neutralize the solution. After removal of the activity not absorbed. Each tissue was, homogenized resin by filtration, methanol was evaporated to dryness, with glass homogenizer in dry-ice acetone, then filtered giving 4.6 g of crude (111). Recrystallization from through a glass filter. The filtrate was concentrated ethanol afforded 4.0 g of (III) as colorless needles melt- at 40-45°C in vacuo. The aqueous concentrate was ing at l85•`186•Ž. [a]n -62.0•‹(c=0.20, H2O). Anal. transferred to a separatory funnel and chloroform was Found: C, 55.64; H, 5.71; N, 4.09. Calcd. for added. After shaking for 10 min, the layers were C15H17NO7: C, 55.72; H, 5.30; N, 4.33%. IR ƒËKBr separated and made up to volume for radioassay. max cm-1: 3400 (OH), 3140 (C=CH), 1625 (C=C), 1520 Sediments on the glass filter were dried, then a portion (C=N). NMR 8 (60 MHz, d6-DMSO): 7.95-7.45 from each sample was burned in an Automatic Sample (5H, m, aromatic), 6.85 (1H, s, C-CH(4)), 5.16 (1H, d, Oxidizer (Packard) and radioassayed. On water J=7.0 Hz, anomeric). To a solution of 1.5 g of (11) culture experiments, bean plants were transferred to in 15 ml of methanol was added 12 mg of sodium meth jars containing Knop's solution. One day was allowed oxide. The mixture was treated with the same pro for adaptation to the new conditions, then treatment cedures as described above to give 0.9 g of crude (IV). was started by transfer of each plant to 5 ml of aqueous Recrystallization from ethanol afforded 0.8 g of (IV) solution containing 1.6 ƒÊCi of isoxathion in 0.03 as fine prisms, melting at 239°C. [ƒ¿]20D-27.9•‹(c=0.70, Tween 20. When the solution had been almost ab H2O). Anal. Found: C, 55.62, H, 5.30; N, 4.20%. sorbed, the nutrient solution was added. The individual IR ƒËKBrmax cm-1: 3450 (OH), 3130 (C=CH), 1650 (C-O), plant was sampled 1, 3, 5 and 7 days after the initiation (1620) (C=C). NMR 3(60 MHz, d6-DMSO): 7.95•` 7.52 of treatment. Radioactivity in shoots and roots was (5H, m, aromatic), 6.44 (1H, s, C=CH(4)), 5.13 (1H, d, separately assayed after homogenization and extraction J=9.0 Hz, anomeric). 1-O-Benzoyl- ƒÀ-D-glucopyra by the same manner as described above. For auto- nose10) melting at 196°C (lit.10) mp 191-192°C (cor.)) radiography of whole bean plants, the plants were and benzoylaspartic acidly melting at 180°C were pre sampled periodically, separately dried and then sub pared by the reported procedures. Attempts to jected to autoradiography with Sakura Type N X-ray prepare the corresponding a-type ester by modifying film for constant 7 days. the reported method12) for 1-0-(2•L,4•L-dichlorophenoxy acetyl)-ƒ¿-D-glueopyranose was unsuccessful, because Separation of isoxathion and its metabolites. Thin in basic conditions a spontaneous migration of the layer chromatography with silica gel GF254 (Merck, thickness 0.25 mm) was conducted to separate the radio- benzoyl group from C, to C2 occured upon benzoyla tion of 4,6-O-benzylidene glucose. O,O-Diethyl O- active metabolites. Solvent systems used were (A) benzene-acetic acid (8: 1, v/v) (B) n-hexane-ethyl (5-phenyl-3-isoxazolyl)phosphate (abbreviated as oxon), the sodium salt of O-ethyl O-hydrogen O-(5-phenyl-3- acetate-formic acid (70: 30: 1, v/v/v) (C) benzene ethanol-water (6: 5: 1, v/v/v) (D) n-propanol-conc. isoxazolyl)phosphorothionate (abbreviated as des ethyl-isoxathion) and the potassium salt of O-ethyl ammonium hydroxide-acetone (60: 20: 3, v/v/v) (E) O-hydrogen O-(5-phenyl-3-isoxazolyl)phosphate (ab chloroform-methanol-conc. ammonium hydroxide breviated as desethyl-oxon) were provided by H. Oka (4: 4: 1, v/v/v) (F) n-butanol-acetic acid-water (8:1:1, v/v/v) (G) ethyl acetate-methylethylketone-formic acid- of Sankyo Co. water (5: 3: 1: 1, v/v/v/v). Locations of the radio- Determination of radioactivity. Radioactivity was active metabolites were detected by autoradiography measured by liquid scintillation counting with a Beck- with Sakura Type N X-ray film. Radioactivity in man Liquid Scintillation Spectrometer, Model LS-250. each spot was determined by scraping the correspond- The scintillation solution used was composed of 8.0 g ing silica gel area from the plates. of PPO and 0.2 g of dimethyl-POPOP in a mixture of Hydrolyses of three metabolites (Ml, M2 and M3). 500 ml each of ethanol and toluene. Counting effi Radiochemically isolated Ml (or M2, M3) was hydroly ciency for each sample was determined by using the zed for 1 hr on a steam bath with 1 N hydrochloric acid external standard technique. or I N sodium hydroxide. Enzymatic hydrolyses with ƒÀ-glucosidase (Sigma Chemical Co .), in 0.1 M acetate Plant materials and treatments. Seeds of bean, buffer (pH 5.0), were also carried out at 37°C for 4 hr.