Etify A. Bakhite,*, Aly A. Abd-Ella, Mohamed E. A. El-Sayed, and Shaban A. A. Abdel-Raheem INTRODUCTION MATERIALS AND METHODS

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1 pubs.acs.org/jafc Pyridine Derivatives as Insecticides. Part 1: Synthesis and Toxicity of Some Pyridine Derivatives Against Cowpea Aphid, Aphis craccivora Koch (Homoptera: Aphididae) Etify A. Bakhite,*, Aly A. Abd-Ella, Mohamed E. A. El-Sayed, and Shaban A. A. Abdel-Raheem Chemistry Department, Faculty of Science, and Plant Protection Department, Faculty of Agriculture, Assiut University, Assiut, Egypt Soil, Water, and Environment Research Institute, Agriculture Research Center, Cairo, Egypt ABSTRACT: Five pyridine derivatives, namely, N-morpholinium 7,7-dimethyl-3-cyano-4-(4 -nitrophenyl)-5-oxo-1,4,5,6,7,8- hexahydroquinoline-2-thiolate (1), sodium 5-acetyl-3-amino-4-(4 -methoxyphenyl)-6-methylthieno[2,3-b] pyridine-2-carboxylate (2), piperidinium 3,5-dicyano-2-oxo-4-spirocyclopentane-1,2,3,4-tetrahydropyridine-6-thiolate (3), piperidinium 5-acetyl-3- cyano-4-(4 -methoxyphenyl)-6-methylpyridine-2-thiolate (4), and piperidinium 5-acetyl-4-(4 -chlorophenyl)-3-cyano-6-methylpyridine-2-thiolate (5) were prepared in pure state and subjected to the title study. The bioassay results indicated that the insecticidal activity of compound 1 is about 4-fold that of acetamiprid insecticide. The rest of the tested compounds possess moderate to strong aphidicidal activities. KEYWORDS: pyridines, piperidinium thiolates, spiro compounds, thienopyridines, acetamiprid, insecticides INTRODUCTION Many pyridine derivatives are known to possess a wide range of biological and pharmacological activities as well as low toxicity toward mammals. 1 4 Recently, selective insecticides (e.g., neonicotinoids) were introduced into the market instead of traditional insecticides because insect pests became resistant to the most conventional insecticides and are increasingly replacing the organophosphates and carbamates. 5 The favorable selectivity of the neonicotinoids occurs largely at the target level, which is the agonist binding site of the nicotinic acetylcholine receptor. 5,6 Neonicotinoids, exemplified by the major imidacloprid, thiamethoxam, acetamiprid, and dinotefuran, are the most important new class of insecticides of the past 3 decades. They are effective against homopteran pests, such as aphids. 7 On the other hand, neonicotinoid insecticides have many common molecular features. The notable feature is that the compounds contain four sections: aromatic heterocycle, flexible linkage, hydroheterocycle or guanidine/amidine, and electron-withdrawing group. 8 In view of the above observations, the present study was planned to synthesize some heterocyclic compounds containing a pyridine moiety (as aromatic heterocycle), piperidine or morpholine ring (as a hydroheterocycle), and cyano/acetyl group (as an electron-withdrawing group) and to study their toxicity as insecticides against cowpea aphid, Aphis craccivora Koch (Homoptera: Aphididae) hoping to obtain compounds with higher insecticidal activity and being more safe toward aquatic life. MATERIALS AND METHODS Instrumentation and Chemicals. Melting points were determined on an APP Digital ST 15 melting point apparatus and are uncorrected. Elemental analyses (C, H, N, and S) were conducted using a Vario EL C, H, N, S analyzer; their results were found to be in good agreement with the calculated values. The infrared (IR) spectra were obtained on a Pye-Unicam SP3-100 spectrophotometer using a KBr disc technique (v max,incm 1 ). 1 H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 400 MHz spectrometer with chemical shifts given in δ (ppm) and coupling constants (J) given in Hz, using tetramethylsilane (TMS) as an internal reference. The purity of the synthesized compounds was checked by thin-layer chromatography (TLC). Pyridine derivatives 1 5, which were prepared in our laboratory, and neonicotinoid insecticide, (E)-N 1 - [(6-chloro-3-pyridyl)methyl]-N 2 -cyano-n 1 -methylacetamidine (acetamiprid, purity of >98%), were purchased from Sigma-Aldrich Chemicals (France). Compounds 1 5 and acetamiprid were tested against aphid nymphs and adults of cowpea aphids, A. craccivora. Synthetic Procedure for N-Morpholinium 7,7-Dimethyl-3- cyano-4-(4 -nitrophenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-2-thiolate (1). To a mixture of α-thiocarbamoyl-β-(4 - nitrophenyl)acrylonitrile (A) (2.3 g, 10 mmol) and dimedone (1.4 g, 10 mmol) in ethanol (25 ml), 0.9 ml (10 mmol) of morpholine was added. The reaction mixture was heated under reflux for 4 h. The product that formed after cooling was collected by filtration and recrystallized from 1-propanol in the form of yellow plates. Yield: 76%. Melting point (mp): C (literature value of 205 C). 9 IR (ν) (KBr), cm 1 : 3276 (NH), 2168 (CN), 1616 (CO). 1 H NMR (DMSOd 6 ) δ: 8.72 (s, 1H, NH), (d, J = 8 Hz, 2H, Ar-H), (d, J = 8 Hz, 2H, Ar-H), 4.39 (s, 1H, C (4) H), (m, 4H, CH 2 OCH 2 ), (t, 4H, CH 2 NCH 2 ), 2.35 (s, 2H,C (8) H 2 ), (d, 1H, C (6) H), (d, 1H, C (6) H), 0.99 (s, 3H, CH 3 ), 0.86 (s, 3H, CH 3 ). Elemental analysis calculated for C 22 H 26 N 4 O 4 S (%): C, 59.71; H, 5.92; N, 12.66; S, Found (%): C, 59.75; H, 6.21; N, 12.52; S, Synthetic Method of Sodium 5-Acetyl-3-amino-4-(4 -methoxyphenyl)-6-methylthieno[2,3-b]pyridine-2-carboxylate (2). Compound B (0.38 g, 10 mmol) in 7% ethanolic sodium hydroxide solution (25 ml) was heated under reflux for 4 h and left to cool. The precipitated solid was collected and recrystallized from ethanol as pale yellow crystals of compound 2. Yield: 75%. mp: 243 Received: July 5, 2014 Accepted: September 16, 2014 Published: September 16, American Chemical Society 9982

2 244 C. IR (ν) (KBr), cm 1 : 3500, 3350 (NH 2 ), 1690 (CO), 1660 (CO). 1 H NMR (CDCl 3 ) δ: (m, 4H, Ar-H), 5.6 (br s, 2H, NH 2 ), 3.8 (s, 3H, OCH 3 ), 2.0 (s, 3H, CH 3 ), (t, 3H, CH 3 ). Elemental analysis calculated for C 18 H 15 N 2 NaO 4 S (%): C, 57.14; H, 4.00; N, 7.40; S, Found (%): C, 57.39; H, 4.44; N, 7.57; S, Synthetic Procedure for Piperidinium 3,5-Dicyano-2-oxo-4- spirocyclopentane-1,2,3,4-tetrahydro-pyridine-6-thiolate (3). To a mixture of cyclopentanone (0.88 ml, 10 mmol), cyanothioacetamide (1.0 g, 10 mmol), and ethyl cyanoacetate (1.06 ml, 10 mmol) in ethanol (15 ml), 1.0 ml (10 mmol) of piperidine was added. The reaction mixture was stirred at room temperature for 2 h. The resulting precipitate was filtered off and recrystallized from ethanol as white needles of compound 3. Yield: 76%. mp: C. IR (ν) (KBr), cm 1 : 3195 (NH), 2181, 2250 (2CN), 1674 (CO). 1 H NMR (DMSOd 6 ) δ: 9.41 (s, 1H, NH), 8.30 (s, 2H NH 2 ), 4.13 (s, 1H, C (5) H), (t, 4H, N(CH 2 ) 2 ), (m, 14H, 7CH 2 ). Elemental analysis calculated for C 16 H 22 N 4 OS (%): C, 60.35; H, 6.96; N, 17.59; S, Found (%): C, 59.99; H, 6.95; N, 17.50; S, General Synthetic Procedure for Compounds 4 and 5. To a suspension of compound C or D (10 mmol) in ethanol (25 ml), 1.0 ml (10 mmol) of piperidine was added. The reaction mixture was heated under reflux for 5 min and then allowed to cool. The crystalline solid that formed was collected by filtration, washed with ethanol, and air-dried to give yellow needles of compounds 4 or 5. Piperidinium 5-Acetyl-3-cyano-4-(4 -methoxyphenyl)-6- methylpyridine-2-thiolate (4). Yield: 87%. mp: C. IR (ν) (KBr), cm 1 : 2217 (CN), 1704 (CO). 1 H NMR (DMSO-d 6 ) δ: (d, J = 8 Hz, 2H, Ar-H), (d, J = 8 Hz, 2H, Ar-H), 3.81 (s, 3H, OCH 3 ), (t, 3H, CH 2 N + CH 2 ), 2.21 (s, 3H, CH 3 CO), 1.70 (s, 3H, CH 3 ), (m, 10H, 5CH 2 ). Elemental analysis calculated for C 21 H 25 N 3 O 2 S (%): C, 65.77; H, 6.57; N, 10.96; S, Found (%): C, 65.86; H, 6.93; N, 10.77; S, Piperidinium 5-Acetyl-4-(4 -chlorophenyl)-3-cyano-6-methylpyridine-2-thiolate (5). Yield: 84%. mp: C. IR (ν) (KBr), cm 1 : 2217 (CN), 1704 (CO). 1 H NMR (CDCl 3 ) δ: 7.99 (br s, 2H, N + H 2 ), (d, J = 8 Hz, 2H, Ar-H), (d, J =8 Hz, 2H, Ar-H), (t, 4H, CH 2 NCH 2 ), 2.27 (s, 3H, CH 3 CO), (m, 4H, 2CH 2 ), 1.75 (s, 3H, CH 3 ), (m, 2H, CH 2 ). Elemental analysis calculated for C 20 H 22 ClN 3 OS (%): C, 61.92; H, 5.72; N, 10.83; S, Found (%): C, 61.78; H, 5.68; N, 10.69; S, Insect Field Strain. The field strain of cowpea aphids, A. craccivora, was collected from fava bean, Vicia faba, fields of Assiut University Experimental Farm during the 2013/2014 season. Laboratory Bioassay. The activities of insecticidal compounds against nymphs and adults of cowpea aphid were tested by the leaf dip bioassay method. 10 Reported here are the results of laboratory tests to determine the concentration of these chemical compounds that is required to kill 50% (LC 50 ) of nymphs and adults, with a modification in the toxicity tests. Six concentrations of aqueous solution of each compound plus 0.1% Triton X-100 as a surfactant were used. A total of 20 apterous adults and 20 nymphs, approximately of the same size, were dipped for 10 s in each concentration 3 times. The treated aphids were allowed to dry at room temperature for about 0.5 h. Control batches of aphids were similarly dipped in a solution of distilled water plus 0.1% Triton X-100. After the treated batches of aphids had dried, they were individually transferred to Petri dishes (9 cm diameter) and held for 24 h at C, % relative humidity, and photoperiod of 12:12 (light/dark). Aphid mortality was recorded 24 and 48 h after treatment with a binocular microscope. An aphid was considered dead if it was incapable of coordinated forward movement. The toxicity experiment of each compound was repeated twice, and the results were corrected by Abbott s formula. 11 Median lethal concentrations (LC 50 ) and slope values of chemical compounds were determined by the Probit regression analysis program and expressed in parts per million (ppm) RESULTS AND DISCUSSION Synthesis. The synthetic procedures for the title compounds are outlined in Scheme 1. The intermediate compounds A, 13 B, 14 C, and D 15 were prepared following the procedures reported previously. Scheme 1 The structure of the synthesized compounds was elucidated and confirmed on the basis of their spectral and elemental analyses. Toxicity Test for the Cowpea Aphid Nymphs. Insecticidal activities against the nymphs of cowpea aphid are shown in Table 1 and Figure 1. One of the tested compounds showed excellent insecticidal activities against nymphs of cowpea aphid, and others exhibit moderate to strong activities compared to acetamiprid. The LC 50 values of compounds 1, 2, 3, 4, and 5 were 0.004, 0.013, 0.180, 0.570, and ppm, respectively, while that of acetamiprid was ppm after 24 h of treatment. It was found that all compounds showed higher toxicity after 48 h treatment, with LC 50 values of , , , , and , respectively, whereas that of acetamiprid was ppm. The above results revealed that the insecticidal activity of compound 1 against nymphs of cowpea aphid was 2.5- and 4.0-fold that of acetamiprid after 24 and 48 h of treatment. Toxicity Test for the Cowpea Aphid Adults. Insecticidal activities against the adults of cowpea aphid are shown in Table 2 and Figure 2. All of the tested compounds showed low to excellent insecticidal activities against cowpea aphid adults after 24 h of treatment. Compound 1, which has a LC 50 value of ppm, was more active than acetamiprid itself, which

3 Table 1. Insecticidal Activity of Acetamiprid and Compounds 1, 2, 3, 4, and 5 against the Nymphs of Cowpea Aphid, A. craccivora, after 24 and 48 h of Treatment 24 h 48 h compound slope ± SE LC 50 (ppm) toxic ratio a slope ± SE LC 50 (ppm) toxic ratio a acetamiprid 0.24 ± ± ± ± ± ± ± ± ± ± ± ± a The toxic ratio is defined as the ratio of the LC 50 value of acetamiprid for baseline toxicity and the LC 50 value of the compound. Figure 1. Insecticidal activities of acetamiprid and compounds 1, 2, 3, 4, and 5 against the nymphs of cowpea aphid, A. craccivora, after 24 and 48 h of treatment. exhibits a LC 50 value of ppm. The compounds 2, 3, 4, and 5 showed low activity, with LC 50 values of 3.330, 2.270, , and ppm, respectively. Compounds 1, 2, 3, 4, and 5 and acetamiprid showed higher toxicity after 48 h of treatment, with LC 50 values of , , , , , and , respectively. The Insecticidal activity of compound 1 against cowpea aphid adults was about 4.0- and 6.0-fold that of acetamiprid after 48 h of treatment. Finally, It is interesting to note that (i) the highest activity associated with compound 1 may be due to the presence of the nitro group in its molecular structure because a number of neonicotinoids contain this group, (ii) the presence of the cyano group has no effect on the insecticidal activity of the tested compounds because compound 2, which lack this cyano group, showed higher activity than each of compounds 3 (which possess two cyano groups), 4, and/or 5, (iii) the thienopyridine moiety in compound 2 may anticipate the insecticidal activity because this compound ranks second in the order of toxicity after compound 1, and (iv) compound 4, which contains the methoxyl group, showed relatively higher activity than its chloro analogue 5, and this is an unexpected result, where the presence of the chlorine atom in the molecule often promoted the activity. 9984

4 Table 2. Insecticidal Activity of Acetamiprid and Compounds 1, 2, 3, 4, and 5 against the Adults of Cowpea Aphid, A. craccivora, after 24 and 48 h of Treatment 24 h 48 h compound slope ± SE LC 50 (ppm) toxic ratio a slope ± SE LC 50 (ppm) toxic ratio a acetamiprid ± ± ± ± ± ± ± ± ± ± ± ± a The toxic ratio is defined as the ratio of the LC 50 value of acetamiprid for baseline toxicity and the LC 50 value of the compound. Figure 2. Insecticidal activities of acetamiprid and compounds 1, 2, 3, 4, and 5 against the adults of cowpea aphid, A. craccivora, after 24 and 48 h of treatment. AUTHOR INFORMATION Corresponding Author * ebakhite@yahoo.com. Notes The authors declare no competing financial interest. REFERENCES (1) Liu, M. C.; Lin, T. S.; Cory, J. G.; Cory, A. H.; Sartorelli, A. C. Synthesis and biological activity of 3- and 5-amino derivatives of pyridine-2-carboxaldehyde thiosemicarbazone. J. Med. Chem. 1996, 39, (2) Finkelstein, B. L.; Martz, M. A.; Strock, C. Synthesis and insecticidal activity of novel pyridine methanesulfonates. J. Pestic. Sci. 1997, 50, (3) Li, G.; Qian, X.; Cui, J.; Huang, Q.; Zhang, R.; Guan, H. Synthesis and herbicidal activity of novel 3-aminocarbonyl-2- oxazolidinethione derivatives containing a substituted pyridine ring. J. Agric. Food Chem. 2006, 54, (4) Jo, Y. W.; Im, W. B.; Rhee, J. K.; Shim, M. J.; Kim, W. B.; Choi, E. C. Synthesis and antibacterial activity of oxazolidinones containing pyridine substituted with heteroaromatic ring. Bioorg. Med. Chem. 2004, 12, (5) Tomizawa, M.; Maltby, D.; Medzihradszky, K. F.; Zhang, N.; Durkin, K. A.; Presly, J.; Talley, T. T.; Taylor, P.; Burlingame, A. L.; 9985

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