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-isopropylphosphoramido- thioate 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 micro- somal 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 micro- somal 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 com- pound 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.