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 of the , 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 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 ), 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. cabbage and chinese cabbage were germinated and grown in soil contained in flats in a green house. Isolation of unknown metabolites. Isolation and The plants were used at about the 3rd to 5th leaf stage identification of the metabolites were conducted with of growth. To follow the movement of the radiolabel the shoots of bean plants. The upper shoots were after foliar application, 1.5 ml of aqueous solution of dipped in the nutrient solution containing 500 ppm isoxathion (1.6 ƒÊCi) in 0.03% Tween 20 was applied to of unlabeled 3-hydroxy-5-phenylisoxazole. After 7 the surface of leaves of bean, cabbage and chinese cab days the shoots were collected and homogenized in bage plants. After exposure, treated plants were dry ice acetone with glass homogenizer, then filtered Metabolism of Isoxathion in Plants 2139

through a glass filter. The filtrate was concentrated at 4045°C in vacuo. The aqueous concentrate was extracted with chloroform to remove soluble materials. The aqueous layer was lyophilized and the residue was column chromatographed (3•~40cm) with silica gel using benzene, benzene-methanol (8:2, 7:3, 1:1, v/v) and methanol as the solvent systems. A small portion from each fraction was chromatographed on thin-layer chromatography with standards for M1 and M2, and spots were visualized under UV light. For M3 and M4, radiochemically isolated ones were used for standards,

and spots visualized under UV light were compared

with those of radioactivity detected by autoradiography. An earliar eluate (Ml and M2) was evaporated to

remove solvent. Silica gel column chromatography

(2 x 30 cm) eluted with methylethylketone as the solvent was successively performed for separation of M1 and

M2. A latter eluate (M3 and M4) was evaporated

to remove solvent and the residue was again column chromatographed with silica gel using ethyl formate, and finally purified by thin-layer chromatography with

solvent systems (C) and (G).

Acetylations of M1, M2 and M3. Twenty mg of Ml (or M2, M3) was acetylated in 2 ml of acetic an

hydride-pyridine (1: 3, v/v) for 24 hr at room tempera ture. The reaction mixture was transferred to a sepa ratory funnel and 20 ml each of water and ethyl acetate

was added. After shaking for 5 min, the ethyl acetate layer was separated and dried up. FIG. 1. Persistence and Absorption of Foliarly applied Isoxathion in Bean, Cabbage and Chinese RESULTS AND DISCUSSION Cabbage plants.

Absorption and translocation

As indicated in Fig. 1, time course experi

ments showed that isoxathion applied on leaves

was absorbed by all examined species. Radi als had entered the bean roots gradually but

oactivity deposited on the surface gradually translocation into stems and leaves during 7

decreased with time, while radioactivity in days was only 3.6%. The absorption from

chloroform soluble, water soluble fractions surface of leaves was more rapidly than that

and unextractable residue material increased. from the roots.

These results showed that isoxathion deposited

on leaf surface was absorbed gradually to plant Metabolism

tissues, then metabolized to water soluble Radioactive materials of chloroform soluble

compounds, and finally in part to bound and water soluble fractions in bean plants

materials. treated with isoxathion were chromatographed

The uptake of isoxathion via the roots of the on thin layer plates of silica gel with solvent

intact bean seedlings appeared to be very slow systems (A), (B), (C), (D), (E), (F) and (G).

as indicated by the autoradiograms (Fig. 2). Almost all radioactivity in chloroform soluble

Radioactivity absorbed from the roots did not fraction was characterized as unchanged iso

localize to the apical meristem region (top xathion. Three major metabolites together

leaves) in the plant, but distributed uniformly. with at least three minor ones were detected

Also as indicated in Fig. 3, radioactive materi- in water soluble fraction, as a representative 2140 M. ANDO, Y. IWASAKI and M. NAKAGAWA

FIG. 3. Distribution of Radioactivity in Bean Plants after Root Absorption of Isoxathion.

FIG. 4. Chromatographic Behavior of Water-soluble Metabolites in Leaves of Isoxathion.

Solvent: benzene-ethanol-water (6: 5: 1, v/v/v). A: isoxathion treatment O-G: 3-(ƒÀ-D-glucopyranosyloxy)- FIG. 2. Autoradiograms of Bean Plants 3 days (A) 5-phenylisoxazole, N-G: 2-(ƒÀ-D-glucopyranosyl)-5- phenyl-4-isoxazolin-3-one, BAA: benzoylaspartic acid, and 9 days (B) after Treatment of 14C-Isoxathion. ƒÀ -BG: 1-(O-benzoyl)-ƒÀ-D-glucopyranose , PI: 3-hy droxy-5-phenylisoxazole. chromatogram is reproduced in Fig. 4. The major metabolites of isoxathion in bean plants of bean plants are shown in Table I. Water were the same as those in cabbage and chinese soluble extracts of bean plants treated with cabbage plants. Relative contents of the meta 3-hydroxy-5-phenylisoxazole in nutrient solu bolites of isoxathion in water soluble fraction tion also yielded the same metabolites, al- Metabolism of Isoxathion in Plants 2141

TABLE I. PERCENTAGE OF FOLIARLY-ABSORBEDISOXATHION AND ITS METABOLITES IN BEAN SEEDLING

though the amounts varied. Therefore, the hydrochloric acid or 1 N sodium hydroxide. It isolation of the metabolites was conducted with remained unaffected with ƒÀ-glucosidase. Spot stem treatment of 3-hydroxy-5-phenylisoxa test with p-nitrobenzenediazonium fluoroborate zole. The major metabolites (Ml, M2 and of M3 was positive, but ninhydrin reaction M3) were isolated and identified, whereas at- was negative. IR spectrum of M3 was almost tempts to determine structures for others were identical with that of an authentic 2-(ƒÀ-D- unsuccessful mainly because they were obtained glucopyranosyl)-5-phenyl-4-isoxazolin-3-one in small amounts. (Fig. 5), indicating that the former is a

Ml produced 3-hydroxy-5-phenylisoxazole readily upon hydrolysis in 1 N hydrochloric acid or 1 N sodium hydroxide or ƒÀ-glucosidase, and ninhydrin reaction on this metabolite was negative. Co-chromatographic identity of

Ml with an authentic sample supported that the structure of M 1 is 3-(ƒÀ-D-glucopyranosyl oxy)-5-phenylisoxazole. IR spectrum of M 1

and NMR and IR spectra of acetylated M1 were all identical with those of synthesized

(III) and (I).

M2 produced only a trace of 3-hydroxy-5- FIG. 5. Infrared Spectra of N-G and M3.

phenylisoxazole upon hydrolysis in 1 N hydro- N-G:2-(ƒÀ-D-glucopyranosyl)-5-phenyl-4-isoxazolin-3- chloric acid or 1 N sodium hydroxide. When one. the metabolite was treated with ƒÀ-glucosidase,

it remained almost unaltered. Co-chromato derivative of the latter. In NMR spectrum

graphic identity of M2 with an authentic (60 MHz) of M3 (Fig. 6), an A2B2 signal at sample supported that the structure of M2 7.48 ppm (2H, J=9 Hz) and 6.88 ppm (2H, is 2-(ƒÀ-D-glucopyranosyl)-5-phenyl-4-isoxa J=9 Hz) was clear enough to show the presence

zolin-3-one. NMR and IR spectra of the of p-substituted phenyl group."' Also the

purified M2 and the acetylated M2 were NMR spectrum showed a single at 6.03 ppm identical with those of synthesized (IV) and (II). (1H, C=CH (4)) and a doublet at 5.26 ppm

M3 produced a trace of an unknown com- (1H, J=8.5 Hz, anomeric), indicating the

pound (presumably 3-hydroxy-5-(p-hydroxy presence of the isoxazole ring and a sugar con phenyl) isoxazole) upon hydrolysis in 1 N jugate with ƒÀ-configuration. NMR spectrum 2142 M. ANDO, Y. IWASAKI and M. NAKAGAWA

chloric acid or 1 N sodium hydroxide or fl. glucosidase. M4 may be therefore a glycoside of 3-hydroxy-5-phenylisoxazole. Desethyl iso xathion, desethyl-oxon and oxon were not detected. 3-Hydroxy-5-phenylisoxazole, the hydrolytic compound of isoxathion, were barely detectable, presumably because of its rapid glucosidation. Previous studies on the metabolism in rats," and in soils" have shown that 3-hydroxy-5- phenylisoxazole partially undergoes a reduc FIG. 6. Nuclear Magnetic Resonance Spectrum of tive cleavage of the isoxazole ring to benzoic M3 (solvent CD3OD, 60 MHz). acid via the formation of benzoylacetamide. In the present study, when the water soluble

(100 MHz) of acetylated M3 revealed a single materials of cabbage and chinese cabbage acetyl group at 2.35 ppm, in addition to four plants were hydrolyzed with 6 N hydrochloric equivalent ones at 2.00 ppm due to substitu acid followed by extraction with ether, small tion on a sugar moiety. In view of the posi amounts of radioactive benzoic acid was tive result of M3 on the spot test for phenolic detected in the ether extract along with 3- derivatives, the former acetyl group was ap hydroxy-5-phenylisoxazole and an unidentified parently a substituent on the phenyl group. compound (presumably 3-hydroxy-5-(p-hydro From these results, M3 was assigned as 2-(ƒÀ-D- xyphenyl) isoxazole). This suggests the glucopyranosyl)-5-p-hydroxyphenyl-4-isoxa presence of the same metabolic pathway of 3- zolin-3-one. hydroxy-5-phenylisoxazole in higher plants as M4 was readily hydrolyzed to afford 3- in rats and soil microbes. However, the hydroxy-5-phenylisoxazole with 1 N hydro expected conjugates of benzoic acid such as

FIG. 7. Proposed Metabolic Pathways of Isoxathion in Plants. Metabolism of Isoxathion in Plants 2143

1-O-benzoyl-ƒÀ-D-glucopyranosel4) and benzoyl REFERENCES aspartic acid15) could not be identified owing to the low level of radioactivity. 1) For biological activity, see Shinnoyaku (Sankyo The metabolism of isoxathion was chara Co.), 27, No. 1 (1973) and 28, No. 1 (1974). cterized by rapid cleavage of the phosphoric 2) Y. Shimada and H. Tamaki, unpublished data. 3) M. Ando, T. Nakamura and M. Nakagawa, Agr. acid linkage to afford 3-hydroxy-5-phenyliso Biol. Chem., 38, 2451 (1974). xazole as an initial product. The isoxazole 4) S. Kamimura, M. Nishikawa, H. Saeki and Y. then underwent the rapid glucosidation to Takahi, Phytopath., 64, 1273 (1974). form 3-(ƒÀ-D-glucopyranosyloxy)-5-phenyliso 5) T. Nakanishi, Y. Takahi and K. Tomita, Ann. xazole and 2-(ƒÀ-D-glucopyranosyl)-5-phenyl-4- Phytopath. Soc. Japan, 40, 383 (1974). 6) M. Nakagawa, T. Nakamura and K. Tomita, isoxazolin-3-one. Apparently, the latter is Agr. Biol. Chem., 38, 2205 (1974). readily oxidized to the principal metabolite, 7) M. Ando, M. Nakagawa, T. Nakamura and K. 2-(ƒÀ-D-glucopyranosyl)-5-p-hydroxyphenyl-4- Tomita, ibid., 39, 803 (1975). isoxazolin-3-one. The hydroxylation of the 8) M. Nakagawa, M. Ando and Y. Obata, ibid.,

former glucoside may also occur among uni 39,1763 (1975). 9) R.U. Lemieux, "Methods in Carbohydrate dentified minor products. Anothei metabolic Chemistry," Vol. II, ed. by R. L. Whistler and pathway of 3-hydroxy-5-phenylisoxazole via M. L. Wolform, Academic Press, N. Y., 1963, the formation of benzoic acid to its conjugates p. 221. was negligible, and the isoxazole skeleton was 10) H. G. Fletcher, ibid., Vol. 11, 1963, p. 231.

quite stable in these plants. 11) E. Fischer, Berichte der Deutchen Chemischen Gesellschaft, 32, 2451 (1899). Summarizing the above results, the metabolic 12) A. Kobayashi, K. Watanabe and K. Yamashita, pathways of isoxathion are proposed in Fig. 7. Agr. Biol. Chem., 36, 2151 (1972).

Acknowledgement. We are grateful to Miss H. 13) R. E. Richards, Y. P. Schaeffer, Trans. Farady Tamari and Mr. Y. Sudo for technical assistance, to Soc., 54, 1280 (1958). 14) W. A. Andereae and Norman E. Good, Plant Mr. H. Oka for providing us with authentic samples of the related compounds of isoxathion, and to Dr. K. Physiology, 32, 566 (1957). Tomita of Sankyo Co. for constant encouragement. 15) H. D. Klambt, Nature, 192, 491 (1962).