Bioactivation of N-Alkyl Substituted Phosphor- Amidothioate Insecticides

J. Pesticide Sci. 9, 675-680 (1984)

Original Article

Bioactivation of N-Alkyl Substituted Phosphor- amidothioate Insecticides

Masako UEJI and Chojiro ToMIzAWA

National Institute of Agro-Environmental Sciences, Yatabe, Tsukuba-gun, Ibaraki 305, Japan

(Received May 15, 1984)

The insecticidal activity of O-ethyl O-2-isopropoxycarbonylphenyl N-alkylphoshor- amidothioates was examined with reference to their activation in biologicalsystems. Toxicity to the adzuki bean weevil varied with different N-alkyl groups. N-isopropylphosphoramidothioate was the most toxic of the compounds tested, and N-unsubstituted, N-methyl- and N-ethylphosphoramidothioates were more toxic than fenitrothion. However, N-propyl and N-butyl homologs were less active than fenitrothion with the exception of the N-isopropyl homolog. LD50 values of the phosphoramidothioates for the insect were not correlated with in vitro anti-AChE activity of the phosphoramidates. 150 of N-isopropylphosphoramidate for acetylcholinesterases from adzuki bean weevil and bovine serum was higher than 10-3 M. When the phosphoramidothioates and phosphoramidate were incubated with rat liver microsomal system, AChE activity of bovine serum was strongly inhibited in the presence of NADPH. Inhibition of AChE activity was reduced by addition of SKF 525-A to the microsomal system. The compounds became also potent inhibitors for AChE by treatment with m-chloroperbenzoic acid. From these results, it was concluded that phosphoramidothioates and their oxons were activated oxidatively to inhibit AChE by the microsomal system as well as chemical treatment with peracid.

of amide groups of phosphoramidothioate or INTRODUCTION phosphoramidate insecticides except schradan Bioactivity of phosphoramidothioates is vari- (octamethylpyrophosphoramidate). Moreover, able with structural changes. Isofenphos (0- these compounds are chiral at the phosphoru ethyl O-2-isopropoxycarbonylphenyl N-isopro- atom, and it is expected that differences in pylphosphoramidothioate) and amidothioate chirality may affect their bioactivity. This (O-2-chloro-4-methylthiophenyl 0-methyl N- report is concerned with synthesis, examina- ethylphosphoramidothioate) have been used tion of insecticidal activity and bioactivation as insecticides, while butamif os (O-ethyl O-6- of N-alkyl substituted phosphoramidothioates nitro -2 -methylphenyl N-sec-butylphosphor- and their oxons with emphasis on isofenphos. amidothioate) and amiprophos-methyl (0- methyl O-2-nitro-4-methylphenyl N-isopropyl- MATERIALSAND METHODS phosphoramidothioate) have been used as 1. Synthesis of O-ethyl O-2-isoj5yopoxycarbonyl- herbicides. Efficacy of the phosphoramido- phenyl N-alkylphosphoramidothioates (Fig. 1) thioates to target pests depends significantly A mixture of 0.08 mol each of isopropyl on the types of substituted phenyl group. salicylate and sodium hydroxide in 22 ml of Isof enphos acts as a contact and stomach distilled water were added slowly to 0.1 mol poison and is applied mainly to control insects 0-ethyl phosphorodichloridothioate in toluene. infesting soils,1) and has exceptional residual The reaction mixture was stirred for 3 hr activity against the corn rootworm. 2) There at 40C and then washed with cold water. has been little information on the bioactivation Organic layer was concentrated under reduced 676 日本農薬学会誌第9巻第4号昭和59年11月

[M-C3H7]+, 303 [M-C4H8]+;N-tert-butyl com-

R = H, CH3, CZH5, pound 359 [M]+, 344 [M-CH3]+, 316 [M-C3H7]+, 303 [M-C4H6]+. C3H7 or, C4H9 PMR signals SMsdhasDgi3:NH2 compound 1.34

Figs 1 Chemical structures of compounds. [3 H, t, OCH2CH3], 1.37 [6 H, d, OCH(CH3)2], 3.66-3. 82 [2 H, br. m, NHCH], 4. 22 [2 H, dq, OCH2CH3], 5.20 [1 H, hep, OCH(CH3)2], 7. 18- pressure to obtain O-ethyl 0-2-isopropoxy- 7.92 [4 H, m, aromatic]; N-isopropyl com- carbonylphenyl phosphorochloridothioate and pound 1. 10 [6 H, dd, NHCH(CH3)2], 1.30 [3 H, purified by preparative thin layer chromato- t, OCH2CH3], 1.32 [6 H, d, OCH(CH3)2],3. 40- plates (TLC) with n-hexane-acetone (10/1) as 3.85 [2 H, br. m, NHCH], 4. 60 [2 H, dq, OCH2- developing solvent. CH3], 5. 18 [1 H, hep, OCH(CH3)2], 7.20-7. 84 NH2 compound: Excess ammonia evolved [4 H, m, aromatic]. from ammonium chloride and sodium hydrox- Synthesis of O-ethyl O-2-isopropoxycarbonyl- ide in acetone-water (9: 1) was added to 0- phenyl N-alkylphosphoramidates (oxon analogs): ethyl O-2-isopropoxycarbonylphenyl phos- Phosphoramidates were prepared according to phorochloridothioate (0.1 mol) in toluene at the method of Herriott.3) m-Chloroperbenzoic 0-5C and stirred for 3 hr at 40C. After acid (MCPBA, 1.0 mol) in dichloromethane washing with cold water, organic layer was was added slowly to a stirred solution of each concentrated under reduced pressure and 0-ethyl O-2-isopropoxycarbonylphenyl N-al- purified by preparative TLC with n-hexane- kylphosphoramidothioate (1.0 mol) in dichloro- acetone (10/1). methane at 0-5C. The reaction mixture was N-methyl compound: Methylamine evolved concentrated under reduced pressure and an from methylamine chloride was used in place appropriate volume of n-hexane was added to of ammonia and the following method was the remove m-chlorobenzoic acid. The filtrate was same as for synthesis of the NH2 compound. purified by preparative TLC with n-hexane- N-ethyl, -n-propyl, -isopropyl, -n-butyl, -iso- ether (1/4). butyl, -sec-butyl and -tert-butyl compounds: El-MS (m/z); All the oxon analogs synthe- These amines (0.22 mol) in toluene were added sized have common peaks, namely, 271 [C2H5- to 0.1 mol of O-ethyl O-2-isopropoxycarbonyl- OP(0)OC6H4000C3H7]+, 229 [C2H5OP(O)OC6- phenyl phosphorochloridothioate in toluene. H4000H]+, 213 [C2H5OPOC6H4COOH]+,201 Reaction and purification methods were the [HOP(O)OC6H4COOH]+,183 [OPOC6H4COO]+, same as for synthesis of the NH2 compound. 138 [HOC6H40OOH], 121 [HOC6H4CO]+. El-MS (m/z): All the phosphoramidothio- Peaks in addition to the preceding ones were ates synthesized have common peaks, namely, as follows: NH2-oxon compound 287 [M]+; N- 287 [C2H5OP(S)OC6H40OOC3H7]+,255 [C2H5- methyl-oxon compound 301[M]; N-ethyl-oxon OPOC6H4000C3H7]+, 245 [C2H5OP(S)OC6H4- compound 315 [M]+, 300 [M-CH3]+; N-n-pro- 000H]+, 217 [HOP(S) OC6H40OOH]+, 213 pyl-oxon compound 329 [M]+, 314 [M-CH3]+, [C2H5OPOC6H4COOH]+, 185 [HOPOC6H4CO- 300 [M-C2H5]+; N-isopropyl-oxon compound OH]+, 138 [HOC6H40OOH], 121 [HOC6H4CO]+, 329 [M]+, 314 [M-CH3]+;N-n-butyl-oxon com- 96 [HOP(S)O]+. Peaks in addition to the pound 343 [M]+, 314 [M-C2H5]+, 300 [M-C3- preceding ones were as follows: NH2 com- H7]+; N-isobutyl-oxon compound 343 [M]+, 328 pound 303 [M]+; N-methyl compound 317 [M-CH3]+, 300 [M-CaH7]+; N-sec-butyl-oxon [M]+; N-ethyl compound 331 [M]+; N-n-propyl compound 343 [M]+, 328 [M-CH3]+, 314 [M- compound 345 [M]+, 316 [M-C2H5]+, 303 [M- C2H5]+, 300 [M-C3H7]+;N-tert-butyl-oxon com- C3H6]+; N-isopropyl compound 345 [M]+, 330 pound 343 [M]+, 328 [M-CH3]+, 300 [M-C3H7]t. [M-CH3], 303 [M-C3H6]+; N-n-butyl com- PMR signals QMDgi3: N-ethyl-oxon compound 359 [M]+, 344 [M-CH3], 316 [M-C3H7]+, pound 1. 12 [3 H, t, NHCH2CH3], 1.41 [3 H, t, 303 [M-C4H6]+; N-isobutyl compound 359 OCH2CH3], 1.43 [6 H, d, OCH(CH3)2], 2. 82- [M]+, 344 [M-CH3]+, 303 [M-C4H6]+; N-sec- 3.25 [3 H, br. m, NHCH2CH3], 4.22 [2 H, dq, butyl compound 359 [M]+, 330 [M-C2H5]+,316 OCH2CH3], 5. 28 [1 H, hep, OCH (CH3)2],7.08- Journal of Pesticide Science 9 (4), November 1984 67?

7.88 [4 H, m, aromatic]; N- isopropyl-oxon the reaction was stopped by addition of compound 1.10 [6 H, dd, NHCH(CH3)2], 1.30 Amberlite CG-120 resin suspended in dioxane. [3 H, t, OCH2CH3], 1.32 [6 H, d, OCH2(CH3)2], Aliquots of dioxane were taken into vials for 3. 16-3. 50 [2 H, br. m, NHCH], 4. 22 [2 H, dq, radioassay. After adding scintillation solution, OCH2CH3], 5. 21 [1 H, hep, OCH(CH3)2], 7.02- radio activity was determined by an Aloka 7. 77 [4 H, m, aromatic]. LSC-673 liquid scintillation spectrometer.

2. Toxicity Test 4. Microsomal Oxidation Insecticidal activity was determined with the Liver microsomal fraction was prepared adzuki bean weevil (Callosobruchus chinensis. from male rat (Wister strain, 200g). A 20% L). The test tube method designed by rat liver homogenate in 0.25 M sucrose, 0.01 M Suwanai4' was utilized with a slight modifica- phosphate buffer, pH 7.4, and 1x10 M tion as described below. A series of half- EDTA was centrifuged at 8000xg for 30 min. concentrations of phosphoramidothioates and The supernatant fraction was further centri- their oxon analogs were prepared by dissolving fuged at 105,000xg for 60 min at 5C. The each test compound in acetone. One hundred microsomal pellet was suspended in 0.1 M al of acetone solution was poured into a test phosphate buffer containing 1.15% KCl and tube (17.6 x 0.7 cm) and the acetone evaporated centrifuged again at 105,000xg for 30 min. spontaneously. After drying, twenty 2-day- A mixture containing 0.2ml of 0-ethyl old insects were put into each test tube. Test 0-2- isopropoxycarbonylphenyl N - alkylphos- tubes were plugged with cotton and held at phoramidothioates (N-isopropyl and -tert- 25C. Mortality was determined 48 hr after butyl: 1x10-3 M; NH2, N-n-propyl, -n-butyl, treatment. LD50 values were estimated by -isobutyl and -sec-butyl: 1x10-4 M) or oxon the computer system designed for probit anal- homolog (1x103 M), 1.6 ml of microsomal ysis from the results of three or four replica- enzyme preparation (equivalent to 106 mg of tions. protein) in 0.1 M phosphate buffer (pH 7.4) and 0.2ml of 5x102 M NADPH was incubated at 3. In vitro Acetylcholinesterase (ACNE) Inhibi- 37C for 60min. In the examination on the tion effect of SKF 525-A as inhibitor of the micro- The molar concentration for 50% inhibition somal mixed function oxidases, final concentra- (I50)was determined for acetylcholinesterases of tion of 10-4 M SKF 525-A was added to some adzuki bean weevil and bovine serum. Adult incubation mixtures 10 min before the addition adzuki bean weevils, 48 hr after emergence, of phosphoramidothioates or oxon homolog. were homogenized with ten times their volume After incubation, 0.15 ml of each mixture was of 0.1 M phosphate buffer (pH 7.4) and filtered assayed for the activity of bovine serum through three fold of gauze. The filtrate was ACNE as mentioned above. used as enzyme source. Bovine blood was centrifuged at 8000xg to remove erythrocytes, 5. Chemical Oxidation and the supernatant dissolved in 0.1 M phos- A dichloromethane solution of m-chloro- phate buffer (pH 7.4) solution (1: 1) was used perbenzoic acid (MCPBA) or m-chlorobenzoic as enzyme source. ACNE activity was deter- acid (MCBA) was added slowly to equi- or mined by the radiometric method of Siakotos hemi-molar O-ethyl O-2-isopropoxycarbonyl- et al., 5) using [1-14C] acetylcholine chloride phenyl N-isopropylphosphoramidothioate or (55.3 mCi/mmol) which was purchased from its oxon at 0C. After mixing at room tempera- Amersham, England. The reaction mixture ture for 10 min, the solvent was evaporated by consisted of 0.3 ml of 0.1 M phosphate buffer, nitrogen stream, and 0.9ml of distilled water pH 7.4, 0.3 ml of the enzyme solution, 0.3 ml and 0.3ml of the enzyme solution derived of inhibitor solution dissolved in acetone-distill- from the adzuki bean weevil were added to the ed water (1: 10), 0.3 ml of 2.5 x 10-2 M [1-14C] reaction mixture. The mixtures were pre- acetylcholine chloride and 0.3 ml of distilled incubated at 37C for 10 min, and after adding water. After incubation at 37C for 30 min, 0.3 ml of 2.5x10-2 M [1-14C] acetylcholine 678 日本農薬学会誌第9巻第4号昭和59年11月

chloride, were incubated at 37C for 30 min Table 1 Toxicity of O-ethyl O-2-isopropoxy- to determine AChE activity. MCPBA and carbonylphenyl N-alkylphosphoramidothioates MCBA did not inhibit ACNE activity at the and their phosphoramidates (oxon analog) to concentrations used. adzuki bean weevil.

6. Chromatography TLC was carried out on silica gel 60F254 chromatoplates (E. Merck, Germany). Chro- matoplates were visualized by exposure to UV light at 254 nm, and 0.5% palladium chloride in N HCI was used as chromogenic reagent for sulfur-containing compounds. Determination of mass spectra was carried out on Shimadzu LKB 9000 B GC-mass spectrometer. Operat- ing conditions were: column, 1mx2.5 mm (i.d. ) glass column packed with 1% silicone OV-1 on Chromosorb W (60-80 mesh); tempera- ture, column 165C, injector port 220C, ionization source 290C; flow rate, He 30ml/min; accerating, 3.5 kV; ionization voltage, 70 eV. Table 2 Inhibition by O-ethyl O-2-isopropoxy- 7. Other Method carbonylphenyl N-alkylphosphoramidates of acetylcholinesterase of adzuki bean weevil and PMR spectra were recorded on a JEOL FX- bovine serum. 90Q spectrometer at 90 MHz in deuteriochloro- form solution containing tetramethylsilane as internal standard. RESULTS AND DISCUSSION 1. Insecticidal Activity of Phosphoramidothio- ates and Their Oxons Results of the toxicological evaluations of 0-ethyl O-2- isopropoxycarbonylphenyl N- alkylphosphoramidothioates and their phosphoramidates (oxon analogs) to the adzuki bean weevil are shown in Table 1. LD5o values changed by the difference of N-alkyl groups, with phosphoramidothioates being more toxic than the oxon analogs, except n-propyl and n- butyl compounds. The toxicity of the com- 2. ACNE Inhibition by Phosphoramidates pounds having the same number of carbons in As shown in Table 2, phosphoramidates are the N-alkyl group varied with different struc- generally poor inhibitors of the adzuki bean tures. Of the phosphoramidothioates, the N- weevil and bovine serum AChE. Selectivity isopropyl compound was approximately 13-fold between two sources of enzyme was not found. more toxic than the N-n-propyl compound, 150 values of phosphoramidates with NH2, N- and of the N-butyl group, the sec-butyl com- methyl and N-ethyl groups were in the range pound was the most toxic, the order of toxic- of 10-5 to 10-6 M, and ACNE inhibition by ity being: sec->n->iso->tert-butyl. N-Iso- these phosphoramidates was lower than those propylphosphoramidothioate, isofenphos, was of dialkyl phenyl phosphates which show the most toxic among those tested, being ap- comparatively high toxicity.6) 150 values of proximately 4-times more toxic than fenitro- N-propyl- and N-butyl-oxons were above thion which was used for comparison. 10 M. In spite of strong biological activity Journal of Pesticide Science 9 (4), November 1984 679 of N-isopropylphosphoramidothioate, I50 value Table 3 Effect of NADPH and SKF 525-A on of AChE of its oxon was the lowest, >10-3 M. oxidative activation by rat liver microsomal Compared to the fenitrothion, phosphor- system of O-ethyl O-2-isopropoxycarbonylphenyl amidates were relatively poor inhibitors of N-isopropylphosphoramidothioate and its phos- AChE, and it was evident that anti-AChE ac- phoramidate. tivity of the oxons was too low to explain their insecticidal activity. Moreover, it was found that phosphoramidothioates were transformed easily to the corresponding phosphoramidates in biological systems such as mammals, insects and plants.7) Therefore, it was suggested that phosphoramidothioates require metabolic activation to a more potent AChE inhibitor.

3. Oxidative Activation of Phosphoramido-

thioates and Their Oxons by Microsomal- Table 4 Oxidative activation by rat liver micro- NADPH System and MCPBA somal-NADPH system of O-ethyl O-2-isopro- Table 3 shows the inhibition of bovine serum poxycarbonylphenyl N-alkylphosphoramidothio- AChE by N-isopropylphosphoramidothioate ates. and its oxon incubated with rat liver microsomal system. Both compounds became powerful inhibitors of AChE in the presence of NADPH, and it seemed that inhibition of AChE activity by phosphoramidothioate was slightly higher than that by its oxon. Addition of SKF 525-A reduced the inhibition of bovine serum AChE. This result suggested that bioactivation of the compounds was catalyzed by the mixed-function oxidase system. As shown in Table 4, the degree of inhibition of AChE by a) N-isopropyl- and N-test-butylphosphoramido- N-alkylphosphoramidothioates incubated with thioate 1x10-a M and other compounds 1x rat liver microsomal system varied with differ- 10-4 M. ent N-alkyl groups. High insecticidal activity of the N-isopropyl compound and low insecti- Table 5 Oxidative activation by chemical cidal activity of the N-tent-butyl compound agents of O-ethyl O-2-isopropoxycarbonylphenyl N-isopropylphosphoramidothioate and its phos- seems to be correlated with the degree of activation by the microsomal-NADPH system. phoramidate. However, N-sec-butyl compound having comparatively high insecticidal activity was less activated. The results shown in Table 5 indicate that N-isopropylphosphoramidothioate and its oxon were activated by treatment with MCPBA. The degree of activation of the oxon was lower than that of the corresponding phosphoramidothioate, suggesting that the active metabolite of the oxon in a reaction system was less stable than that of the phosphoramidothioate. Ac- a) amount of MCPBA was equi-molar (+) and tivation by MCPBA was further increased by twice-molar (-H-), MCBA was equi-molar. addition of twice molar equivalent of MCPBA b) phosphoramidothioate and phosphoramidate , and no appreciable activation was observed 3.3x10-4M. 680 日本農薬学会誌第9巻第4号昭和59年11月

by treatment with MCBA. It was evident that ACKNOWLEDGEMENTS the compounds tested were also activated The authors wish to express their sincere thanks chemically by peracid. to Dr. T. Shishido for his valuable suggestion and High insecticidal activity of N-alkylphos- Miss M. Yoshida for the determination of PMR phoramidothioates on the adzuki bean weevil spectra both of National Institute of Agro-Environ- could not be explained by the relatively poor mental Sciences. anti-AChE activity of their oxons. The results REFERENCES from this study show that the phosphoramido- 1) E. Cantu & D. A. Wolfenbarger: Tex., Agric. thioates are oxidatively activated by the micro- Exp. Stn., Progr. Rep. 2844, 57 (1972) somal-NADPH system or by treatment with 2) B. Homeyer: Meded. Fac. Landbouwwet., MCPBA. In addition, it was found that Rijksuniv. Gent 39, 789 (1974) schradan, a phosphoramidate which lacks anti- 3) A. W. Herriott: J. Am. Chem. Soc. 93, 3304 (1971) AChE activity, is oxidatively converted to a 4) M. Suwanai: Bull. Int. Inst. Agric. Sci., Ser. higher effective anti-AChE agent through C7, 113 (1957) enzymatic or chemical oxidation process. 8)9) 5) A. N. Siakotos, M. Filbert & R. Hester: Bio- Although an oxidized compound of schradan chem. Med. 3, 1 (1969) 6) T. R. Fukuto & R. L. Metcalf : J. Agric. Food has not been isolated, it was reported that Chem. 4, 930 (1956) schradan is oxidized to the amido oxide and 7) M. Ueji & C. Tomizawa: unpublished data is converted into a N-methyloyl group which 8) J. E. Casida & M. A. Stahmann: J. Agric. then undergoes elimination as formaldehyde. Food Chem. 1, 883 (1953) 9) J. E. Casida, T. C. Allen & M. A. Stahmann : Moreover, it was postulated that the presence J. Biol. Chem. 210, 607 (1954) of oxygen in the dimethylamido moiety was 10) H. Tsuyuki, M. A. Stahmann & J. E. Casida: responsible for the increased anti-AChE ac- J. Agric. Food Chem. 3, 922 (1955) tivity of the metabolite. 10-12) By analogy, it is 11) E. Y. Spencer, R. D. O'Brien & R. W. White: J. Agric. Food Chem. 5, 123 (1957) suggested that phosphoramidothioates are ac- 12) R. D. O'Brien: Can. J. Biochem. 34, 1131 (1956) tivated by oxidation at a site close to the 13) R. Kato, A. Takanaka & Y. Omori : Jpn. J. nitrogen atom to a potent inhibitor of AChE. Pharmacol. 17, 509 (1967) In the microsomal-NADPH system, the inhibi- 14) M. Eto, S. Okabe, Y. Ozoe & K. Maekawa : tion of AChE was reduced by addition of SKF Pestic. Biochem. Physiol. 7, 364 (1977) 525-A. It was found that the toxicity of schradan is increased by treatment with 要約 phenobarbital and decreased by SKF 525-A A-Alkyl phosphoramidothioate系殺虫剤の生 or other inhibitors of microsomal enzymes. 13) 体内活性化 As shown in Table 5, chemical oxidation of N- 上路雅子, 富澤長次郎 isopropylphosphoramidothioate and its oxon O-ethyl O-2-isopropoxycarbonylphenyl N-alkylphos- by treatment with MCPBA increased the phoramidothioatesの殺虫活性について検討した. アズ inhibition of AChE as well as the activation by キゾウムシに対する殺虫力は, N-アルキル置換同族体間 the microsomal-NADPH system. Anti-AChE activity by the conversion product of meth- で大きな差異があり, N-イソプロピル同族体が最も強い amidophos (O,S-dimethyl phosphoramidothio- 殺虫力を示した. N-アルキルオキソンのin vitroでの late) obtained by treatment with MCPBA in- アセチルコリンエステラーゼ阻害作用は概して弱く, と creased at least seven fold, but the in vitro くにN-イソフ.ロピル, tert-ブチルのI50値は10-3 M以 activation by the microsomal-NADPH system 上であった. N-アルキル同族体およびN-イソプロピル was less marked that by MCPBA. 14) The chemical structures of the active me- オキソンはミクロゾームの酸化酵素系によって, より強 tabolites of schradan and methamidophos 力なアセチルコリンエステラーゼ阻害剤となり, 酸化剤 remain to be elucidated, because they would be m-クロル過安息香酸によっても同様の結果を得た. こ too unstable to be isolated. Similarly, the れらの結果から, phosphoramidothioateおよびオキソ active metabolites of the present N-alkyl- ンは酸化的に活性化され, 強力なアセチルコリンエステ phosphoramidothioates produced by biological or chemical treatment may be unstable inter- ラーゼ阻害効果を発現し, 高い殺虫活性を示すことが示 mediates. 唆された.