J. Pesticide Sci. 23, 250-254 (1998)

Original Article

Synthesis and Insecticidal Activity of Alkylated N-Benzoyl-N'- Phenylureas and Their Toxicity to Aquatic Invertebrate

Tohru KOYANAGI, Masayuki MORITA and Yasuhiro FUJII

Central Research Institute, Ishihara Sangyo Kaisha, Ltd., Nishi-Shibukawa, Kusatsu 525-0025, Japan

(Received September 19, 1997; Accepted March 26, 1998)

Alkylation of the nitrogen atoms in the urea moiety of N-benzoyl-N'-phenylureas was extensively carried out, and the alkylated molecules showed the remarkably decreased toxicity against the aqueous invertebrate. From the 1H-NMR studies, it was suggested that the intramolecular association complex was formed in the solution of the non-alkylated N-benzoyl-N'-phenylurea, while such an association was not observed in the alkylated compounds. And it was assumed that the lack of these interactions might be related to the decrease in the toxicity to the aqueous invertebrate. Among the alkylated derivatives, N-benzoyl-N'-methyl-N'-phenylurea showed the highest Insecticidal activity. As to the phenyl portion, phenoxyphenyl or biphenyl group substituted with lipophilic groups was favorable.

phenylureas with alkyl halides in the presence of sodium INTRODUCTION hydride in dimethyl sulfoxide (Eq. 1). Ureas alkylated N-Benzoyl-N'-phenylureas are insect growth regula- at the nitrogen atom adjacent to the benzoyl group were tors, which can interfere with deposition of chitin in synthesized by the reaction of N-alkyl-benzamides with insects.1) In Table 1, structures of commercialized N- the corresponding phenyl isocyanates in the presence of benzoyl-N'-phenylureas are shown with the toxicities to n-butyl lithium in ether (Eq. 2). Ureas alkylated at the insect (diamondback moth, Plutella xylostella) and nitrogen atom adjacent to the phenyl group were aquatic invertebrate (Daphnia magma Straus). obtained by the reaction of the alkylated anilines with the Generally these compounds show high pesticidal activ- corresponding benzoyl isocyanates (Eq. 3). ities against lepidpterous insects, however, toxicities to Typical examples of the synthetic procedure are as aquatic invertebrates are also remarkably high. We follows. have carried out alkylation of the nitrogen atoms in the 2.1 N-[4-(3-Chloro-5-trifluoromethylpyridin-2-yloxy)- urea moiety of N-benzoyl-N'-phenylureas extensively, 3,5-dichlorophenyl]-N'-(2,6-difluorobenzoyl)-N, and studied the influence of alkylation on the Insecticidal N'-dimethylurea (4) activity and the toxicity to aquatic invertebrate. To a stirred suspension of 0.72g of 60% sodium hydride in oil (18mmol) in DMSO (25ml) at room MATERIALS AND METHODS temperature, was added 2.16g (4mmol) of N-[4-(3- 1. Apparatus chloro-5-trifluoromethylpyridin-2-yloxy)-3, 5- All melting points were determined on a Mettler FP62 dichlorophenyl]-N'-(2,6-difluorobenzoyl)urea micro melting point apparatus and are uncorrected. (chlorfluazuron, 1) in 10ml DMSO. After 30min, NMR spectra were recorded on a Jeol JNM-GSX400 or methyl iodide (5.6g, 39mmol) was added to the above a JNM-PMX60 spectrometer with tetramethylsilane as an solution, keeping the temperature below 40C. The internal standard. High performance liquid chroma- stirring was continued for 2h, and the reaction was tography (HPLC) was performed on a Jasco TRI quenched with ice water. The mixture was extracted ROTAR V, using a Finepak SIL C18 column. Solvent with EtOAc, and the extract was washed successively system for chromatography was McOH-H20 (4:1, v/v). with water and brine, and dried over anhydrous Na2SO4. Concentration followed by column chromatography on 2. Synthesis of the Compounds silica gel with n-hexane-EtOAc (7:3, v/v) gave 1.4g Synthetic schemes of alkylated N-benzoyl-N'- (62% yield) of the title compound (4) as colorless crystals: phenylureas are shown in Fig. 1. N,N'-Dialkylureas mp 153-154C; iH NMR (400MHz, CDC13) S ppm: 3.13 were prepared by the reaction of N-benzoyl-N'- (3H, s, CH3), 3.34 (3H, s, CH3), 6.91-6.95 (3H, m, Journal of Pesticide Science 23 (3) August 1998 251

Table 1 Insecticidal activity and toxicity to aquatic invertebrate of commercialized N-benzoyl-N'-phenylureas.

a)Insecticidal activity against Plutella xylostella. b)Immobilization con- centration against Daphnia magma Straus.

(Eq. 1)

(Eq. 2)

(Eq. 3)

Fig. 1 Synthetic routes of alkylated N-benzoyl-N'-phenylureas. phenyl), 7.34-7.42 (2H, m, phenyl), 8.01(1H, s, pyridine- m, phenyl), 7.70-7.71 (2H, m, phenyl), 8.10 (1H, s, C4-H), 8.16 (1H, s, pyridine-Cs-H). pyridine-C4-H), 8.22 (1H, s, pyridine-Cs-H), 11.42 (1 H, 2.2 N [4-(3-Chloro-5-trifluoromethylpyridin-2 yloxy)- NH). 3, 5-dichlorophenyl]-N'-(2, 6-difluorobenzoyl)-N'- 2.3 N-[4-(3-Chloro-5-trifluoromethylpyridin-2-yloxy)- methylurea (2) 3,5-dichlorophenyl]-N'-(2, 6-difluorobenzoyl)-N- To a stirred solution of N-methyl-2, 6-difluoro- methylurea (3) benzamide (0.43g, 2.5mmol) in 10ml of Et20 was To a cooled solution (0C) of 4-(3-chloro-5-tri- added 1.88ml (1.6M in hexane) of n-BuLi at -78C. fluoromethylpyridin-2-yloxy)-3, 5-dichloroaniline (3.75g, The resulting slurry was stirred for 30min before 4-(3- 10mmol) in pyridine (20ml) was added acetyl chloride chloro-5-trifluoromethylpyridin-2-yloxy)-3, 5-dichloro- (0.94g, 12mmol) dropwise, and the mixture was stirred phenyl isocyanate (0.96g, 2.5mmol) in 10ml of Et20 for 30 min. The reaction mixture was poured onto a was added. The stirring was continued for 1 hr at room mixture of EtOAc and water. The organic layer was temperature followed by quenching with ice water. The separated, washed successively with dilute hydrochloric mixture was extracted with EtOAc and the extract was acid, water and brine, and dried over anhydrous Na2SO4. washed with brine and dried over anhydrous Na2SO4. Concentration under reduced pressure gave 3.88 g (97% Concentration followed by column chromatography on yield) of N-[4-(3-chloro-5-trifluoromethyl- silica gel with n-hexane-EtOAc (3:2, v/v) gave 0.22g pyridin-2-yloxy)-3, 5-dichlorophenyl] acetamide as color- (16% yield) of the title compound (2) as colorless crystals: less crystals. mp 85-92C; 1H NMR (400 MHz, CDC13) S ppm: 3.26 To a stirred suspension of 0.11g of 60% sodium (3H, s, CH3), 7.02-7.06 (2H, m, phenyl), 7.44-7.51 (1H, hydride in oil (4.5mmol) in DMSO (10ml) at room 252 日本 農 薬 学 会 誌 第23巻 第3号 平 成10年8月 temperature, was added 1.2g (3mmol) of the above leaves were dipped into the respective dispersions for 10 acetamide in 10ml of DMF. After 30min, methyl sec, and then dried in air. A sheet of moistened filter iodide (0.85g, 6mmol) was added at room tempera- paper was placed in a petri dish having a diameter of 9 ture. The stirring was continued for 2h, and the reac- cm, and the dried cabbage leaves were put on the filter tion was quenched with ice water. The mixture was paper. Larvae of diamondback moth (Plutella xylostel- extracted with EtOAc, and the extract was washed succes- la) in second or third instar were released on the leaves, sively with water and brine, and dried over anhydrous and the petri dishes were covered and kept in a thermo- Na2SO4. Concentration under reduced pressure gave stated chamber (26C). On the seventh day after release, 1.06g (85% yield) of N-[4-(3-chloro-5-trifluoromethyl- dead insects were counted. pyridin-2-yloxy)-3, 5-dichlorophenyl] -N-methyl- 3.2 Acute immobilization test against Daphnia magma acetamide as colorless crystals. Straus To a solution of 1.0g (2.4mmol) of the above N- Test solutions were prepared by dispersing an acetone methylacetamide in McOH (20ml), was added concen- solution of each compound in water. Two hundred and trated hydrochloric acid (10 ml). The solution was fifty milliliters of each test solution was introduced into a refluxed for 18h, and the reaction mixture was cooled 500ml beaker. Into each beaker, 10 larvae (first instar) and poured into 100ml of a 10% potassium hydroxide were released and the solution was kept at 23+1C. aqueous solution. The mixture was extracted with The immobilization up to 48hr after the release of the CH2C12, and the extract was washed successively with larvae was investigated in accordance with OECD (Orga- water and brine, and dried over anhydrous Na2SO4. nization for Economic Cooperation and Development) Concentration followed by column chromatography on test guideline.2) The test was repeated 2-3 times in two silica gel with n-hexane-EtOAc (4:1, v/v) gave 0.78g series for each concentration. The 50% immobilization (87% yield) of N-methyl-[4-(3-chloro-5-trifluoromethyl- concentration (EC50, ppb) was obtained by Probit pyridin-2-yloxy)-3, 5-dichloro] aniline. method. To a solution of the above N-methylaniline (0.50g, 1.3 mmol) in dioxane (10ml), was added 0.87g (4.4mmol) RESULTS AND DISCUSSION of 2,6-difluorobenzoyl isocyanate at room temperature. At first, in order to investigate the effects of the posi- The stirring was continued for 4 hr, and the solvent was tion of the alkyl group on the toxicities to insect and evaporated under reduced pressure. The residue was aquatic invertebrate, methyl group was introduced on the purified by column chromatography on silica gel with nitrogen atom of the urea moiety in chlorfluazuron (1). n-hexane-EtOAc (2:1, v/v) to give 0.72g (96% yield) of As shown in Table 2, an insecticidal activity to diamond- the title compound as colorless crystals: mp 165-16TC; back moth decreased extensively by alkylation of the 1H NMR (400 MHz, CDC13) S ppm: 3.26 (3H, s, CH3), nitrogen adjacent to the benzoyl group. On the other 6.92-6.98 (2H, m, phenyl), 7.35-7.43 (3H, m, phenyl), hand, compound (3) alkylated at the nitrogen atom 7.86 (1H, s, NH), 8.04 (1H, s, pyridine-C4-H), 8.22 (1H, adjacent to the phenyl group, or compound (4) alkylated s, pyridine-C6-H). at the both nitrogens did not show so much decrease in an insecticidal activity as in the case of 2. Furthermore, 3. Biological Tests these alkylated compounds showed a remarkable 3.1 Insecticidal activities decrease (about a one-thousandth) in toxicity to an Each formulation containing an active ingredient was aquatic invertebrate, compared to 1. dispersed in water to obtain a dispersion of an active In the next step, in order to investigate the factors ingredient having a various concentration. Cabbage responsible for these behaviors, hydrophobic parameters

Table 2 Effects of methylation of chlorfluazuron on physical properties, insecticidal activity and toxicity to aquatic invertebrate.

a)Insecticidal activity against Plutella xylostella. b)Immobilization con - centration against Daphnia magma Straus. Journal of Pesticide Science 23 (3) August 1998 253

Table 3 Concentration dependence of the 'H- NMR chemical shifts of N-benzoyl-N'- phenylureas.

Fig. 2 Schematic drawing of a possible model for the molecular association of N-benzoyl-N'-phenylurea.

cules are suggested to be more subject to hydrolysis than 1. a) The NH proton adjacent to the benzoyl group. Furthermore, as shown in Table 2, the N-alkylated b) The NH proton adjacent to the phenyl group. molecules differ somewhat from 1 in certain physico- chemical properties (mp, log P). Variations in these (log P) of these compounds were obtained by using controlling factors can also be responsible for the HPLC method.3) As shown in Table 2, alkylated deriv- decrease in the toxicity to the aqueous invertebrate. atives (3, 4) showed decrease in log P compared with 1, In the next step, since the NHS-methylated compound while 2 gave larger value. (3) showed a higher insecticidal activity than dimethylat- Presence of the specific intermolecular association ed (4) or NHa-methylated one(2), effects of the structures complex between N-acyl urea derivatives was suggested of the alkyl groups were examined for the derivatives of by Endo et al. by the spectroscopic studies (NMR, IR).4) 3. As shown in Table 4, methyl- and ethyl-substituted According to their studies, when the intermolecular compounds (3, 5) exhibited the higher activity than other hydrogen bonding is formed between molecules, their alkyl- or haloalkyl-substituted derivatives. Though the NMR chemical shifts generally vary with the concentra- varieties of substituents are not enough to conclude the tion of the solution. On the other hand, a large varia- substituent effects, bulkiness and the susceptibility to the tion in the chemical shifts is not observed in the presence metabolism seem to control the insecticidal activity. of the intramolecular association. From the data in Tables 2 and 4, the N-benzoyl-N'- Accordingly, the influence of the concentration on methyl-N'-phenylurea is shown to be the most favorable, proton NMR shifts was examined for these N-benzoyl- taking into consideration of the insecticidal activity and N'-phenylurea derivatives in CDC13. As shown in the toxicity to the aqueous invertebrate. Table 3, by diluting the solution of 1 from 0.02M to In the final step, substituent effects on the phenyl 0.005M, the NH proton adjacent to the benzoyl group moiety were examined, and these data are shown in (NHa) was largely shifted upfield (about 0.6ppm), while Table 5. On the toxicities to the aqueous invertebrate, the NH proton adjacent to the phenyl group (NHS) all these compounds showed the weaker activities (about underwent small shift (about 0.04ppm). On the other a two-hundredths to a one-thousandth) than 1. As to hand, in the methylated molecules (2, 3) only the small the insecticidal activities, phenoxyphenyl or biphenyl changes were observed in the chemical shifts of NHa or

NHS by diluting the solution. Consequently, in N- Table 4 Physical properties and insecticidal activity benzoyl-N'-phenylurea both NHa and NHS protons are of N-alkyl-N'-benzoyl-N-phenylureas. indispensable to form the intermolecular association complex. These results suggest that 1 is conformational- ly fixed by the formation of the intramolecular hydrogen bond, and then the intermolecular association takes place, as shown in Fig. 2. These associations are effective to stabilize the molecule in the solution. From the effects of the concentration on NMR shifts, it is estimated that the intermolecular association cannot be present in the N-alkylated molecules. Since the absence of the intermolecular association results in destabiliza- tion of the molecule in the solution, N-alkylated mole- a) Insecticidal activity against Plutella xylostella. 254 日本 農 薬 学 会 誌 第23巻 第3号 平 成10年8月

Table 5 Physical properties, insecticidal activity and toxicity to aquatic invertebrate of N-benzoyl-N'-methyl-N'-phenylureas.

a) Insecticidal activity against Plutella xylostella. b) Immobilization con- centration against Daphnia magma Straus.

derivatives with lipophilic substituents (9, 10, 11) were higher than pyridyloxyphenyl or phenyl analogues (3, 要 約 12, 13). And these highly active compounds possessed the equally high insecticidal activities as the conventional ア ル キル 化N-ベ ン ゾ イル-N'-フ エニ ル ウ レア 誘 導 N-benzoyl-N'-phenylureas in Table 1. 体 の合 成 と殺 虫 活性 及 び水 棲 動 物 に対 す る毒性 Consequently, methylation of the nitrogen adjacent to 小柳 徹, 森 田雅之, 藤井康 弘 the phenyl group of the N-benzoyl-N'-phenylurea mole- N-ベ ンゾ イル-N'-フ ェニ ル ウレアの窒素 原 子の ア ル キ cule is shown to be quite effective for lowering the tox- ル化 を広範 囲の誘導体 に対 して検討 した ところ, ア ルキル icity to aquatic invertebrate, while maintaining the in- 化 した化合物 の水 棲動物 に対 す る毒性 が, 著 し く減少す る secticidal activity. こ とが認め られた.1H-NMRス ペ ク トルの研究 か ら, 溶液 中で非ア ルキル化N-ベ ンゾイル-N'-フ ェニ ル ウレア化合 REFERENCES 物 間に, 会合錯体 が生成す るのに対 して, アルキル化 した 1) L. C. Post & W. R. Vincent: Naturwissen. 9, 431 (1973) 化合物 では, その ような錯体 は生成 しない こ とが示 唆 され 2) "DECD Guidelines for Testing of Chemicals," Vol. 1, た.さ らに, この ような会合錯体 生成 の有 無が水棲 動物 に Japanese Ed., Dai-ichi Hoki Publishing, Tokyo, pp. 843- 対す る毒 性 に関係 して いるこ とが推定 され た.ア ル キル化 856, 1981 した化合 物 の中 では、N-ベ ンゾ イル-N'-メ チル-N'-フ ェ 3) A. Hulshoff & J. H. Perrin: J. Chromatogr.129, 263 (1976) ニ ルウ レア誘導体 が最 も高 い殺 虫活性 を示 した.フ ェニ ル 4) T. Endo, Y. Takeda, T. Orii, Y. Kaneko & M. Kondo: Chem. Lett. 1979, 1455 (1979) 部分 の構 造一活性相関 を検討 した とこ ろ, 脂 溶性 の基 が置換 したフェ ノキシフェニル あるいは ビフェニル基が好 ましい 結果 を与 えた.