J. Pesticide Sci. 15, 175-187 (1990) Original Article Aryl N, N-Dirnethylcarbamates, Synergists for Organo- phosphorus Insecticides against Organophosphorus- resistant Rice Stem Borers* Yasuhiko KoNNO and Takashi SHISHIDO Division of Pesticides, National Institute of Agro-Environmental Sciences, Kannondai, Tsukuba 305, Japan (Received September 1, 1989) One hundred noninsecticidal carbamates were synthesized and evaluated as synergists for fenitrothion and pirimiphos-methyl against the OP-resistant rice stem borer. Substituted aryl (including phenyl and heterocyclic groups) N,N-dimethylcarbamates had a synergistic activity. In substituted phenyl esters, the order of positional effectiveness of the substituent group on the benzene ring was para meta>ortho. 3-Methyl-4-nitrophenyl (SK-2) and 3-methyl-4- methylthiophenyl (SK-9) esters were excellent synergists. In substituted heterocyclic esters, 5-phenyl-3-isoxazolyl (SK-40) and 2-dimethylamino-6-methyl-4-pyrimidinyl (SK-102) esters were extremely effective, and the latter reduced the resistance level against pirimiphos-methyl from 1202 to 1.1-fold. When the aryl esters of N,N-dimethylcarbamate were changed to corresponding N, N-diethyl-, N, N-dimethylthio-, and N, N-dimethylthiolcarbamates, their synergistic activities decreased from 1/18 to 1/2 that of N, N-dimethylcarbamates. Aryl esters of N-methyl-, N-ethyl- and N-phenylcarbamates, and aliphatic esters of N, N-dimethylcarba- mates were inactive. SK-2, -9, -40 and -102 had no synergistic activity against the susceptible strain. The synergistic mechanism of SK-102 for fenitrothion was strong inhibition of fenitroxon detoxication by binding protein and hydrolysis. a level equal to that of the susceptible strain. INTRODUCTION On the other hand, we have revealed that Several synergism studies against the or- the OP-resistant rice stem borer shows high ganophosphorus (OP)-resistant rice stem borer, levels of resistance to 0,0-dimethyl 0-aryl Chilo suppressalis WALKER,have been done phosphorothioates and phosphates such as since it appeared in Okayama prefecture in fenitrothion, pirimiphos-methyl and their 1978, and such chemicals as IBP,1,2) edifen- oxons.4,6) Our in vivo and in vitro studies phos1) and pirimicarb3) were found as syner- have shown that increased detoxication of gists. We have tested various noninsecticidal fenitroxon by cleavage of the P(O)-O-aryl OP compounds as synergists for f enitrothion, bond is the main mechanism of f enitrothion and found that K-2 and DEF have a synergistic resistance,4,7) and that both protein-binding acti.vity.4,5) Although K-2 and DEF were and hydrolysis of fenitroxon in a soluble frac- more active than IBP, edifenphos and pirimi- tion from the OP-resistant rice stem borer carb, neither of them was capable of increasing play an important role in the fenitroxon detoxi- the susceptibility of the OP-resistant strain to cation. 8) These facts suggest that oxon de- * This work was supported in part by a Grant-in- toxication sites of the binding protein and Aid (Bio Media Program) from the Ministry of hydrolysis in the OP-resistant rice stem borer Agriculture, Forestry and Fisheries (BMP 90-IV- had a high affinity for the structure of 0, 0- 2-1) dimethyl 0-aryl phosphate. 176 日本 農 薬 学 会 誌 第15巻 第2号 平 成2年5月 In synergism theory, analog synergists are shimacho, Saitama prefecture in 1971 by the defined as synergists whose structure closely Institute of Physical and Chemical Research. resemble that of an insecticide they synergize. The rearing methods of the two strains were An analog synergist competes with the in- described previously.5) Fifth instar larvae secticide for the same detoxication site and (body weight: 60 to 70 mg) of the two strains exhibits synergism by occupying and blocking were used in this study. the detoxication site.9) One extensive study on the evaluation of noninsecticidal carbamates 2. Chemicals as synergists for Isolan and carbaryl has done A considerable number of N,N-dialkyl- by using houseflies.10) carbamates or -thionocarbamates were pre- We attempted to obtain analog syner- pared by reacting respective N,N-dialkyl- gists from these points of view. The N,N- carbamoyl chlorides or N,N-dimethylthio- dimethylcarbamoyloxy group was selected carbamoyl chlorides with appropriate phenols, as a fundamental skelton, because the alcohols and isoxazols in an excess of dry (CH3)2NC (O) O-moiety of carbamate ester re- pyridine (Method A).11) Heterocyclic esters sembles the (CH3O)2P(O)O-moiety of OP ester of N,N-dialkylcarbamates were prepared by in structure and function, and an N,N-di- treating N, N-dialkylcarbamoyl chlorides either methylcarbamate insecticide, pirimicarb (2-di- with heterocyclic enol in the presence of an methylamino-5, 6-dimethyl - 4 -pyrimidinyl N, acid-consuming agent, anhydrous potassium N-dimethylcarbamate), is already known to be carbonate in dry acetone solution (Method active as a synergist against the OP-resistant B)12) or directly with sodium enolate in dry rice stem borer.3) Furthermore, in order to toluene solution (Method C-1)13,14)and in dry enhance an affinity for the detoxication site, methyl ethyl keeone solution (Method C-2). the substituted phenyl group of OP esters, to Alkyl or thiophenyl esters of N, N-dimethyl- which the OP-resistant rice stem borer showed carbamates were prepared from N, N-dimethyl- the high level of resistance, was chosen as the carbamoyl chloride and sodium salts of phenyl moiety of N,N-dimethylcarbamate, aliphatic alcohols and thiophenols (Method that is, the 3-methyl-4-nitrophenyl group of C-1).13) N-Alkyl- or N-phenylcarbamates fenitrothion and the 3-methyl-4-methylthio- were prepared by reacting the appropriate phenyl group of f enthion. We found that phenol with alkyl isocyanate or phenyl iso- SK-2 (3-methyl-4-nitrophenyl N,N-dimethyl- cyanate in dry isopropyl ether with triethyl- carbamate) and SK-9 (3-methyl-4-methylthio- amine catalyst or in dry toluene (Method D).1" phenyl N,N-dimethylcarbamate) had a re- The synthesized compounds were purified ap- markable synergistic activity as novel syner- propriately by recrystalization or preparative gists against the OP-resistant rice stem borer. TLC. Yields were from 40 to 90%. Structures This paper deals with the relation of chemical of the compounds were confirmed by GC-MS structures of various noninsecticidal car- (a Shimadzu LKB 9000B GC-MS spectro- bamates to synergistic activity and with their meter) analysis. Their melting points and action mechanism. refractive indices are listed in Tables 2, 3 and 4. MATERIALS AND METHODS The following heterocyclic enols were 1. Insects synthesized by known methods: 2, 6-dimethyl- The following Hata-f and S strains of rice 4-hydroxypyrimidine, 16) 4-hydroxy-6-methyl- stem borers were used. An OP-resistant Hata-f 2-phenylpyrimidine,17) 4-hydroxy-6-methyl-2- strain was obtained from an OP-resistant methylthiopyrimidine,18) 4-hydroxy-6-methyl- Hata strain by selection with fenitrothion for 2-methoxypyrimidine,19) 4-hydroxy-6-methyl- three successive generations. The original 2-methylaminopyrimidine,20) 2-anilino-4-hy- Hata strain was collected at Hata in Soj a, droxy-6-methylpyrimidine,21) 2-N,N-dimethyl- Okayama prefecture in 1983, and maintained amino-4-hydroxy-6-methylpyrimidine,22) and 6- for four years without exposure to insecticides. hydroxy-2-phenyl-3-pyridazinon.23) Other he- A susceptible S strain was collected at Kawa- terocyclic enols, phenols or alcohols used in Journal of Pesticide Science 15 (2), May 1990 177 Table 1 Toxicity of OP insecticides to the Hata- methyl-4-nitrophenyl phosphate, 1.6 mCi/ f and S strains of rice stem borers. mmol, >99% purity) dissolved in 10 ul of ethanol was added, and the mixture was in- cubated at 27C for 30 mm. The detoxication activity of fenitroxon by binding protein and hydrolysis was determined by the method of Konno & Shishido.8) RESULTS 1. Synergistic Activity of Substituted Phenyl N, N-Dimethylcarbamates for Fenitrothion a ) Figures in parentheses indicate 95% confidence against the Hata f Strain of Rice Stem limits. Borers Results are summarized in Table 2. Syner- gism was found in almost all test compounds. this experiments were obtained from com- An unsubstituted phenyl ester of N,N-di- mercial sources. methylcarbamate (1) gave a synergistic ratio of 5.8. The position, type, size and number 3. Toxicity and Synergism Tests of substituents attached to the aromatic Toxicity and synergism tests were conducted nucleus of phenyl N,N-dimethylcarbamate according to the methods described previous- greatly affected synergistic effect for f eni- ly.5,6) Since all of the test carbamates used as trothion. synergists are nontoxic to rice stem borers at In the monosubstituted group, the order of the test dose, synergistic effect is simply ex- the positional effectiveness of synergism for pressed as the following ratio. methyl (2-4), nitro (8-10) and chloro (11-13) Synergistic ratio (SR) substituents was para = meta>ortho. Syner- LD50 of OP insecticide alone gistic activity was weaker in the presence of ortho substituents (2, 8 and 11: SR= 4.3-5.1) LD50 of OP insecticide in mixture than unsubstituted ester (1). Especially, the OP insecticides used in this study were feni- presence of more bulky substituents such as trothion, pirimiphos-methyl and f enitroxon, sec-butyl (6) and isopropoxycarbonyl (19) and Table 1 shows their LD 5o values for the two groups in the ortho-position of ring significantly strains. decreased synergism (SR=1.8 and 0.9, re- spectively). Esters containing such strongly 4. Enzyme Preparation electron attractive substituents as nitro (9, 10), Whole bodies of 5th instar larvae were chloro (12, 13) and cyano (14) groups in the homogenized (20% w/v) with 1 mM EDTA- meta- or para-position showed a marked 5 mM 2-mercaptoethanol-0.1 M phosphate synergistic activity (SR = 8.2-14) compared buffer, pH 7.4 in an ice bath.