Molecular Features of Neonicotinoid Pharmacophore Variants

Jan 29, 2009 - ... crop protection, accounting for approximately one-fifth of the current global ...... Sgard , F., Fraser , S. P., Katkowska , M. J.,...
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476

Chem. Res. Toxicol. 2009, 22, 476–482

Molecular Features of Neonicotinoid Pharmacophore Variants Interacting with the Insect Nicotinic Receptor Ikuya Ohno,† Motohiro Tomizawa,‡ Kathleen A. Durkin,§ Yuji Naruse,| John E. Casida,‡ and Shinzo Kagabu*,† Departments of Chemistry, Faculties of Education and Engineering, Gifu UniVersity, Gifu 501-1193, Japan, and EnVironmental Chemistry and Toxicology Laboratory, Department of EnVironmental Science, Policy, and Management, and Molecular Graphics and Computational Facility, College of Chemistry, UniVersity of California, Berkeley, California 94720 ReceiVed NoVember 12, 2008

Molecular interactions of neonicotinoid insecticides with the nicotinic acetylcholine receptor have been mapped by chemical and structural neurobiology approaches, thereby encouraging the biorational design of novel nicotinic ligands. This investigation designs, prepares, and evaluates the target site potency of neonicotinoid analogues with various types of electronegative pharmacophores and subsequently predicts their molecular recognition in the ligand-binding pocket. The N-nitroimino (dNNO2) neonicotinoid pharmacophore is systematically replaced by N-nitrosoimino (dNNO), N-formylimino [dNC(O)H], N-alkyl- and N-arylcarbonylimino [dNC(O)R], and N-alkoxy- and N-aryloxycarbonylimino [dNC(O)OR] variants. The dNNO analogues essentially retain the binding affinity of the dNNO2 compounds, while the isosteric dNC(O)H congeners have diminished potency. The dNC(O)R and dNC(O)OR analogues, where R is methyl, trifluoromethyl, phenyl, or pyridin-3-yl, have moderate to high affinities. Orientation of the tip oxygen plays a critical role for binding of the dNNO and dNC(O)H pharmacophores, and the extended dNC(O)R and dNC(O)OR moieties are embraced by unique binding domains. Introduction Neonicotinoids such as imidacloprid (IMI)1 and thiacloprid (THIA) (Figure 1) along with five analogues, acting at the insect nicotinic acetylcholine receptor (nAChR), are extensively used for crop protection, accounting for approximately one-fifth of the current global insecticide market (1-3). Their unique molecular feature is an electronegative N-nitro or N-cyano substituent, which is coplanar with the guanidine or amidine plane, yielding electronic conjugation to facilitate partial negative charge (δ-) flow toward the tip, enabling hydrogen bonding with the receptor subsite (4-7). The binding site interactions of IMI and THIA have been defined with mollusk acetylcholine binding protein (AChBP), which is a suitable structural surrogate of the extracellular ligand binding domain of the nAChR, by chemical and structural neurobiology approaches with adequate resolution to understand the recognition properties of the ligand binding pocket (8-11). These studies prompted receptor structure-guided drug design to explore novel binding cavities or different interaction mechanisms that are not accessible or observed by the present N-nitroimino and N-cyanoimino neonicotinoids. The goal of this investigation is to design, prepare, and evaluate the target site potency of neonicotinoids bearing N-nitrosoimine (dNNO), N-formylimine [dNC(O)H], N-alkyl* To whom correspondence should be addressed. Tel: +81-58-293-2253. Fax: +81-58-293-2207. E-mail: [email protected]. † Faculty of Education, Gifu University. ‡ Department of Environmental Science, Policy, and Management, University of California. § College of Chemistry, University of California. | Faculty of Engineering, Gifu University. 1 Abbreviations: AChBP, acetylcholine binding protein; CPM, 6-chloropyridin-3-ylmethyl; CTM, 2-chloro-1,3-thiazol-5-ylmethyl; IMI or 3 [ H]IMI, imidacloprid or its tritiated radioligand; Ki, inhibition constant; nAChR, nicotinic acetylcholine receptor; THIA, thiacloprid; VDW, van der Waals.

Figure 1. Structures of neonicotinoid insecticides IMI and THIA and systematic variants of the electronegative pharmacophore.

or N-arylcarbonylimine [dNC(O)R], and N-alkoxy- or Naryloxycarbonylimine [dNC(O)OR] electronegative pharmacophore variants (Figure 1) and ultimately to predict their molecular recognition at the insect nAChR structural model focusing on the pharmacophore-subsite interactions.

Materials and Methods General. All melting points (mp) are uncorrected. Elemental analyses performed on a Yanaco CHN CORDER MT-6 Elemental Analyzer (Kyoto, Japan) gave C, H, and N results within 0.4% of calculated values (data not shown). IR spectra were measured with a Perkin-Elmer FTIR 1600 spectrometer (Waltham, MA) using KBr tablets. 1H and 13C NMR spectra were recorded at 500 and 125

10.1021/tx800430e CCC: $40.75  2009 American Chemical Society Published on Web 01/29/2009

Neonicotinoid Pharmacophore Variants Scheme 1. Synthesis of N-Formylimino, N-Alkyl-, or N-Arylcarbonylimino and N-Alkoxy- or N-Aryloxycarbonylimino Neonicotinoid Variantsa

a Five N-alkoxycarbonylimino compounds were obtained by a conventional manner using chloroformates (17-20) except for compound 21 with carbonic acid di-3-pyridyl ester.

MHz, respectively, for solutions in CDCl3 unless stated otherwise using a JEOL ECA-500 spectrometer (Tokyo, Japan). Mass spectra were determined at 70 eV with the JEOL JMS-700 instrument. Starting materials 1-(6-chloropyridin-3-ylmethyl)-2-iminoimidazolidine and the analogues with thiazolidine, thiazoline, and pyrrolidine rings were prepared according to described procedures (12) (Scheme 1). 3-(2-Chloro-1,3-thiazol-5-ylmethyl)-2-imino-1,3-thiazolidine and the pyrrolidine analogue were prepared according to the patent descriptions (13), and the following spectral data support their structures. 3-(2-Chloro-1,3-thiazol-5-ylmethyl)-2-imino-1,3thiazolidine: liquid. IR νmax: 3271, 1605, 1415, 1277, 1055 cm-1. 1 H NMR δ (CDCl3): 3.18 (m, 2H), 3.53 (m, 2H), 4.66 (s, 2H), 7.42 (s, 1H). 13C NMR δ (CDCl3): 26.9, 41.9, 50.8, 135.5, 140.1, 152.8, 164.0. EI-LRMS m/z (%): 233 (M+, 3), 198 (M+-Cl, 51), 170 (46), 132 (87), 59 (100). 3-(2-Chloro-1,3-thiazol-5-ylmethyl)2-iminopyrrolidine: liquid. IR νmax: 3258, 1627, 1275, 1125 cm-1. 1 H NMR δ (CDCl3): 1.95 (m, 2H), 2.49 (m, 2H), 3.30 (m, 2H), 4.59 (s, 2H), 7.41 (s, 1H). 13C NMR δ (CDCl3): 19.5, 32.4, 40.1, 48.8, 136.6, 139.6, 152.1, 168.6. EI-LRMS m/z (%): 215 (M+, 13), 180 (M+-Cl, 100), 152 (77), 132 (35). Preparations of New Products (Scheme 1). 1-(6-Chloropyridin3-ylmethyl)-2-formyliminoimidazolidine (1). A mixture of 1-(6chloropyridin-3-ylmethyl)-2-iminoimidazolidine (210 mg, 1.0 mmol) and 10 mL of ethyl formate was heated at reflux temperature for 5 h. The residual liquid after evaporation was subjected to column chromatography on SiO2 with ethyl acetate as the eluent. The product was rinsed with ether. Yield, 5.4%; liquid. IR νmax: 3332, 1730, 1609, 1567, 1462, 1388, 1371, 1286 cm-1. 1H NMR δ (CDCl3): 3.39 (2H, t, J ) 8.6 Hz), 3.69 (2H, t, J ) 8.6 Hz), 4.58 (2H, s), 7.33 (1H, d, J ) 8.2 Hz), 7.67 (1H, d, J ) 8.2 Hz), 8.32 (1H, s), 8.58 (1H, s), 8.61 (1H, bs). 13C NMR δ (CDCl3): 40.9, 44.3, 44.9, 124.7, 130.9, 139.0, 149.3, 151.3, 163.1, 174.5. LRMS m/z (%): 238 (M+, 70), 209 (M+ - CHO, 95), 175 (M+ - Cl, CHO, 28), 126 (100). HRMS for C10H11ClN4O: calcd, 238.0621; found, 238.0631. The following formamides were also prepared with the above methodology. 3-(6-Chloropyridin-3-ylmethyl)-2-formylimino-1,3-thiazolidine (2). Yield, 13%; mp, 99-100 °C. IR νmax: 1617, 1536, 1401, 1380 cm-1. 1H NMR δ (CDCl3): 3.25 (2H, dd, J ) 8.1 Hz, J ) 7.7 Hz), 3.64 (2H, dd, J ) 8.1 Hz, J ) 7.7 Hz), 4.85 (2H, s), 7.34 (1H, d, J ) 8.1 Hz), 7.69 (1H, dd, J ) 8.1 Hz, J ) 2.6 Hz), 8.33

Chem. Res. Toxicol., Vol. 22, No. 3, 2009 477 (1H, d, J ) 2.6 Hz), 8.97 (1H, s). 13C NMR δ (CDCl3): 27.0, 47.5, 49.2, 124.7, 129.9, 139.0, 149.3, 151.5, 172.9, 173.0. LRMS m/z (%): 255 (M+, 57), 226 (M+ - CHO, 75), 126 (100). HRMS for C10H10ClN3OS: calcd, 255.0233; found, 255.0261. 1-(6-Chloropyridin-3-ylmethyl)-2-formyliminopyrrolidine (3). Yield, 85%; liquid. IR νmax: 1687, 1630, 1593, 1459, 1386 cm-1. 1 H NMR δ (CDCl3): 2.17 (2H, m), 3.06 (2H, t, J ) 8.1 Hz), 3.60 (2H, t, J ) 7.3 Hz), 4.82 (2H, s), 7.37 (1H, d, J ) 7.3 Hz), 7.72 (1H, dd, J ) 7.3 Hz, J ) 2.6 Hz), 8.30 (1H, s), 8.35 (1H, d, J ) 2.6 Hz). 13C NMR δ (CDCl3): 18.9, 31.9, 46.6, 53.1, 125.2, 128.3, 139.5, 149.5, 152.1, 166.4, 169.1. LRMS m/z (%): 237 (M+, 1), 208 (M+ - CHO, 100), 174 (M+ - HCl, CHO, 29), 126 (23). HRMS for C11H12ClN3O: calcd, 237.0669; found, 237.0672. 3-(2-Chloro-1,3-thiazol-5-ylmethyl)-2-formylimino-1,3-thiazolidine (4). Yield, 12%; mp, 76 °C. IR νmax: 1620, 1520, 1411, 1383 cm-1. 1H NMR δ (CDCl3): 3.25 (2H, t, J ) 8.1 Hz, J ) 7.7 Hz), 3.69 (2H, dd, J ) 8.1 Hz, J ) 7.7 Hz), 4.89 (2H, s), 7.48 (1H, s), 8.97 (1H, s). 13C NMR δ (CDCl3): 27.1, 43.1, 49.0, 133.8, 140.8, 153.7, 172.4, 172.6. LRMS m/z (%): 261 (M+, 16), 226 (M+ - Cl, 100), 198 (M+ - Cl, CO, 94), 132 (75). HRMS for C8H8ClN3OS2: calcd, 260.9797; found, 260.9768. 1-(2-Chloro-1,3-thiazol-5-ylmethyl)-2-formyliminopyrrolidine (5). Yield, 92%, liquid. IR νmax: 1684, 1600, 1416, 1352 cm-1. 1H NMR δ (CDCl3): 2.18 (2H, m), 3.06 (2H, m, J ) 8.0 Hz), 3.67 (2H, t, J ) 7.1 Hz), 5.07 (2H, s), 7.63 (1H, s), 8.36 (1H, s). 13C NMR δ (acetone-d6): 18.8, 31.7, 41.2, 53.0, 133.4, 142.3, 151.9, 156.2, 169.1. LRMS m/z (%): 243 (M+, 1), 215 (M+ - CO, 6), 180 (M+ - HCl, CO, 100), 132 (18). HRMS for C9H10ClN3OS: calcd, 243.0233; found, 243.0196. 3-(6-Chloropyridin-3-ylmethyl)-2-acetylimino-1,3-thiazolidine (10). To a mixture of 3-(6-chloropyridin-3-ylmethyl)-2-imino-1,3-thiazolidine (227 mg, 1.0 mmol) and triethylamine (110 mg, 1.1 mmol) in 10 mL of acetonitrile was added dropwise a solution of acetyl chloride (78.5 mg, 1.0 mmol) in 5 mL of acetonitrile with ice water cooling. The mixture was stirred overnight at ambient temperature. The solvent was evaporated, and the residual semisolid was dissolved in ethyl acetate (∼20 mL), which was successively washed with 2 N HCl, water, saturated NaHCO3 solution, and finally with water and dried. After the solvent was evaporated, the residue was subjected to column chromatography on SiO2 with ethyl acetate and hexane (1:1 to 1:2 in volume) as the eluent. The product was rinsed with hexane. Yield, 58%; mp, 108-110 °C. IR νmax: 1626, 1525, 1396, 1270 cm-1. 1H NMR δ (CDCl3): 2.23 (s, 3H), 3.11 (m, 2H), 3.53 (t, 2H, J ) 7.8 Hz), 4.81 (s, 2H), 7.33 (d, 1H, J ) 8.2 Hz), 7.65 (dd, 1H, J ) 8.2 Hz, J ) 2.7 Hz), 8.34 (d, 1H, J ) 2.7 Hz). 13C NMR δ (CDCl3): 26.9, 27.5, 47.6, 48.8, 124.7, 130.6, 139.0, 149.5, 151.4, 171.2, 182.7. EI-LRMS m/z (%): 269 (M+, 99), 254 (M+ - CH3, 25), 226 (M+ - COCH3, 40), 199 (29), 126 (ClPyCH2, 100). EI-HRMS for C11H12ClN3OS: calcd, 269.0390; found, 269.0370. Similarly, the benzoylimino compound was synthesized with benzoyl chloride. 3-(6-Chloropyridin-3-ylmethyl)-2-benzoylimino-1,3-thiazolidine (14). Yield, 65%; mp, 133 °C. IR νmax: 1615, 1522, 1455, 1401 cm-1. 1H NMR δ (CDCl3): 3.19 (2H, dd, J ) 8.3 Hz, J ) 7.5 Hz), 3.62 (2H, dd, J ) 8.3 Hz, J ) 7.5 Hz), 4.98 (2H, s), 7.32 (1H, d, J ) 8.2 Hz), 7.43 (2H, m), 7.50 (1H, m), 7.72 (1H, dd, J ) 8.2 Hz, J ) 2.6 Hz), 8.26 (2H, m), 8.40 (1H, d, J ) 2.6 Hz). 13C NMR δ (CDCl3): 26.9, 47.8, 48.9, 124.7, 128.2, 129.1, 130.6, 132.1, 136.3, 138.8, 149.3, 151.3, 172.5, 175.8. LRMS m/z (%): 331 (M+, 82), 226 (M+ - COC6H5, 66), 190 (M+ - COC6H5, -Cl, 28), 105(100). HRMS for C16H14ClN3OS: calcd, 331.0546; found, 331.0560. 3-(6-Chloropyridin-3-ylmethyl)-2-acetylimino-1,3-thiazoline (11). To a mixture of 3-(6-chloropyridin-3-ylmethyl)-2-imino-1,3-thiazoline (225 mg, 1.0 mmol) and triethylamine (122 mg, 1.2 mmol) in 3 mL of pyridine was added dropwise acetyl chloride (75 mg, 1.0 mmol) with ice water cooling. The mixture was stirred overnight at ambient temperature. The reaction mixture was poured into water and extracted with chloroform (3 × 15 mL). The combined organic layers were successively washed with 1% HCl, saturated NaHCO3 solution, and finally with water and dried. After the solvent was evaporated, the residue was subjected to TLC on SiO2 with ethyl

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Ohno et al.

Table 1. Potency of Neonicotinoids with Various Electronegative Pharmacophores as Displacers of [3H]IMI Binding to the Drosophila nAChR

compound no.

receptor potency

B

1 2 3 4 5 6 7 8 9

CPM CPM CPM CTM CTM CPM CPM CPM CPM

1 2 3 2 3 1 2 4 5

N-formyl- and N-nitrosoimino NC(O)H NC(O)H NC(O)H NC(O)H NC(O)H NNO NNO NNO NNO

1040 ( 320 92 ( 8 280 ( 30 180 ( 52 150 ( 3 30 ( 3a 2.8 ( 0.8a 41 ( 4a 5.1 ( 0.1a

10 11 12 13 14 15 16

CPM CPM CPM CPM CPM CPM CPM

2 5 4 5 2 5 5

N-alkyl- or N-arylcarbonylimino NC(O)CH3 NC(O)CH3 NC(O)CF3 NC(O)CF3 NC(O)C6H5 NC(O)C6H5 NC(O)-pyridin-3-yl

160 ( 10 83 ( 11 4.5 ( 0.4a 1.8 ( 0.6a 120 ( 4 71 ( 4b 2.8 ( 0.1b

17 18 19 20 21

CPM CPM CPM CPM CPM

N-alkoxy- or N-aryloxycarbonylimino 2 NC(O)OCH3 5 NC(O)OCH3 2 NC(O)OC6H5 5 NC(O)OC6H5 5 NC(O)O-pyridin-3-yl

58 ( 5 36 ( 2 27 ( 1 7.9 ( 0.4 17 ( 1

a

X

Ki ( SD, nM (n ) 3)

A

Data from ref 6. b Data from ref 21.

acetate as the eluent. The crude product was rinsed with ether. Yield, 38%; mp, 125-126 °C. IR νmax: 1599, 1562, 1487, 1388, 1368 cm-1. 1H NMR δ (CDCl3): 2.30 (s, 3H), 5.33 (s, 2H), 6.63 (d, 1H, J ) 4.6 Hz), 6.95 (d, 1H, J ) 4.6 Hz), 7.33 (d, 1H, J ) 8.6 Hz), 7.66 (dd, 1H, J ) 8.6 Hz, J ) 2.3 Hz), 8.42 (d, 1H, J ) 2.3 Hz). 13 C NMR δ (CDCl3): 27.0, 48.8, 110.0, 124.6, 124.7, 130.4, 138.9, 149.4, 151.7, 167.3, 180.5. EI-LRMS m/z (%): 267 (M+, 58), 252 (M+ - CH3, 16), 224 (M+ - COCH3, 20), 126 (ClPyCH2, 100). EI-HRMS for C11H10ClN3OS: calcd, 267.0233; found, 267.0247. The phenoxycarbonylimino compound was prepared by the reaction of the thiazolidine with phenyl chloroformate. 3-(6-Chloropyridin-3-ylmethyl)-2-(phenoxycarbonylimino)-1,3thiazolidine (19). Yield, 67%; mp, 169 °C. IR νmax: 1673, 1570, 1460, 1445, 1423, 1265, 1236, 1198 cm-1. 1H NMR δ (CDCl3): 3.19 (2H, dd, J ) 8.2 Hz, J ) 7.5 Hz), 3.62 (2H, dd, J ) 8.2 Hz, J ) 7.5 Hz), 4.84 (2H, s), 7.19-7.25 (3H, m), 7.34-7.40 (3H, m), 7.64 (1H, dd, J ) 8.2 Hz, J ) 2.0 Hz), 8.33 (1H, d, J ) 2.0 Hz). 13 C NMR δ (CDCl3): 26.6, 47.6, 49.7, 121.8, 124.7, 125.3, 129.2, 130.0, 139.0, 149.3, 151.5, 151.7, 162.0, 175.3. LRMS m/z (%): 347 (M+, 0.1), 254 (M+ - OC6H5, 50), 226 (M+ - CO2C6H5, 38), 174 (63), 146 (67), 126 (100). HRMS for C16H14ClN3O2S: calcd, 347.0495; found, 347.0508. 3-(6-Chloropyridin-3-ylmethyl)-2-(methoxycarbonylimino)-1,3thiazolidine (17). A mixture of 3-(6-chloropyridin-3-ylmethyl)-2-imino-1,3-thiazolidine (227 mg, 1.0 mmol) and methyl chloroformate (114 mg, 1.0 mmol) in 15 mL of acetonitrile was treated with potassium carbonate (165 mg, 1.2 mmol) portionwise, and the mixture was stirred at ambient temperature overnight. The solid was filtered off and washed with ethyl acetate on the filter paper. The filtrate was condensed in vacuum, and the residual solid was recrystallized from ethyl acetate. Yield, 42%; mp, 128-130 °C. IR νmax: 1662, 1555, 1459, 1440, 1264 cm-1. 1H NMR δ (CDCl3): 3.18 (2H, dd, J ) 8.0 Hz, J ) 7.4 Hz), 3.60 (2H, dd, J

) 8.0 Hz, J ) 7.4 Hz), 3.79 (3H, s), 4.81 (2H, s), 7.32 (1H, d, J ) 8.0 Hz), 7.67 (1H, dd, J ) 8.0 Hz, J ) 2.3 Hz), 8.31 (1H, d, J ) 2.3 Hz). 13C NMR δ (CDCl3): 26.5, 47.3, 49.4, 52.9, 124.5, 130.1, 138.8, 149.1, 151.2, 163.5, 173.8. EI-LRMS m/z (%): 285 (M+, 100), 254 (M+ - OCH3, 20), 126 (ClPyCH2, 66). HRMS for C11H12ClN3O2S: calcd, 285.0339; found, 285.0354. The following two compounds were prepared as above by the reaction of 3-(6chloropyridin-3-ylmethyl)-2-imino-1,3-thiazoline with phenyl chloroformate and methyl chloroformate, respectively. 3-(6-Chloropyridin-3-ylmethyl)-2-(methoxycarbonylimino)-1,3thiazoline (18). Yield, 40%; mp, 178-180 °C. IR νmax: 1646, 1515, 1439, 1287 cm-1. 1H NMR δ (CDCl3): 3.83 (s, 3H), 5.28 (s, 2H), 6.63 (d, 1H, J ) 5.0 Hz), 6.86 (d, 1H, J ) 5.0 Hz), 7.32 (d, 1H, J ) 8.2 Hz), 7.64 (dd, 1H, J ) 8.2 Hz, J ) 2.3 Hz), 8.36 (d, 1H, J ) 2.3 Hz). 13C NMR: δ (CDCl3): 48.1, 53.1, 109.0, 124.8, 125.2, 130.2, 138.8, 149.1, 151.8, 163.5, 170.6. EI-LRMS m/z (%): 283 (M+, 57), 252 (M+ - OCH3, 10), 224 (M+ - CO2CH3, 52), 126 (ClPyCH2, 100). EI-HRMS for C11H10ClN3O2S: calcd, 283.0182; found, 283.0195. 3-(6-Chloropyridin-3-ylmethyl)-2-(phenoxycarbonylimino)-1,3thiazoline (20). Yield, 17%; mp, 110-112 °C. IR νmax: 1660, 1519, 1204 cm-1. 1H NMR δ (CDCl3): 5.30 (s, 2H), 6.68 (d, 1H, J ) 5.2 Hz), 6.91 (d, 1H, J ) 5.2 Hz), 7.21-7.23 (m, 3H), 7.33 (d, 1H, J ) 8.0 Hz), 7.39 (m, 2H), 7.62 (dd, 1H, J ) 8.0 Hz, J ) 2.3 Hz), 8.37 (d, 1H, J ) 2.3 Hz). 13C NMR δ (CDCl3): 48.4, 109.9, 121.9, 124.8, 125.4, 125.5, 129.3, 130.0, 139.0, 149.3, 151.9, 152.0, 161.8, 171.4. EI-LRMS m/z (%): 345 (M+, 0.7), 252 (M+ - OC6H5, 92), 224 (M+ - CO2C6H5, 4) 126 (ClPyCH2, 100). EI-HRMS for C16H12ClN3O2S: calcd, 345.0339; found, 345.0337. 3-(6-Chloropyridin-3-ylmethyl)-2-(3-pyridyloxycarbonylimino)1,3-thiazoline (21). To a solution of 3-(6-chloropyridin-3-ylmethyl)2-imino-1,3-thiazoline (225 mg, 1.0 mmol) in 20 mL of acetonitrile was added carbonic acid di-3-pyridyl ester (216 mg, 1.0 mmol). The mixture was stirred for 12 h at ambient temperature. To complete the reaction, 140 mg (1.0 mmol) of powdered potassium carbonate was added, and the mixture was stirred for an additional 1 h. The reaction mixture was filtered, and the filtrate was evaporated. The residue was subjected to column chromatography on Al2O3 with ethyl acetate/hexane (1:1). The crude product was recrystallized from ether. Yield, 49%; mp, 129-130 °C. IR νmax: 1670, 1516, 1276, 1209 cm-1. 1H NMR δ (CDCl3): 5.32 (s, 2H), 6.74 (d, 1H, J ) 5.2 Hz), 6.96 (d, 1H, J ) 5.2 Hz), 7.33-7.36 (m, 2H), 7.60 (d, 1H, J ) 8.9 Hz), 7.63 (dd, 1H, J ) 8.3 Hz, J ) 2.8 Hz), 8.38 (s, 1H), 8.47 (d, 1H, J ) 4.1 Hz), 8.56 (d, 1H, J ) 2.8 Hz). 13C NMR δ (CDCl3): 48.5, 110.0, 123.8, 124.9, 125.7, 129.4, 129.7, 138.8, 143.9, 146.4, 148.7, 149.2, 152.0, 161.0, 171.5. EILRMS m/z (%): 346 (M+, 0.2), 252 (M+ - OPy, 100), 126 (ClPyCH2, 99). EI-HRMS for C15H11ClN4O2S: calcd, 346.0291; found, 346.0262. Radioligand Binding. The potency of test compounds as inhibitors of [3H]IMI binding to the native Drosophila brain nAChR was determined according to Tomizawa et al. (14). IC50 values (molar concentrations of test compounds necessary for 50% inhibition of specific [3H]IMI binding) were determined by iterative nonlinear least-squares regression using Sigmaplot software (SPSS Inc., Chicago, IL). IC50 values were finally converted to inhibition constants (Ki) using the equation of Cheng and Prusoff (15), that is, Ki ) IC50/(1 + [L]/Kd) where the concentration of radioligand [L] was 4 nM and the dissociation constant (Kd) of [3H]IMI was 5.7 nM (16). Calculations. Quantum mechanics calculations for several model structures were performed at B3LYP/6-311+G(2d,2p) using Gaussian 03 (17). The structural homology model used for the interfacial agonist-binding domain of the insect nAChR from peach-potato aphid Myzus persicae (R2β1) (18-20) was based on the crystal structure of Aplysia californica AChBP, which is sensitive to neonicotinoids (9, 21). This model is representative of insect nAChRs since the important amino acids forming the binding pocket are conserved in all of the known insect nAChR subunits (11). Docking calculations were carried out using AutoDock 4 (22, 23). The receptor was treated as rigid while flexible ligands were docked

Neonicotinoid Pharmacophore Variants

Chem. Res. Toxicol., Vol. 22, No. 3, 2009 479

Table 2. Relative Electronic Energies of Possible Conformers for Four Sets (a-d) of 2-Nitrosoimino and 2-Formylimino Model Structures

a Relative energies [calculated at B3LYP/6-311+G(2d,2p)] can be compared only within each conformer set. Conformations of 1,3-thiazolidines shown as c3, c4, d3, and d4 are not applicable in the corresponding actual compound with a CPM or CTM substituent.

Results and Discussion

Figure 2. Preferred geometries [optimized at B3LYP/6-311+G(2d,2p)] of 1,3-thiazoline model structures with 2-acetylimino (e), 2-trifluoroacetylimino (f), 2-benzoylimino (g), 2-pyridin-3-oylimino (h), 2-(methoxy)-carbonylimino (i), 2-(phenoxy)-carbonylimino (j), and 2-(pyridin3-oxy)-carbonylimino (k) substituents. For h or k, the alternative conformation (the pyridine nitrogen flipped 180°) is less favorable by ∼0.5 kcal/mol.

in a 15 Å cubic grid centered on the active site. In each case, a multistep Lamarkian Genetic Algorithm search was performed. Good quality hits were those with binding energies below -7 kcal/ mol. Visualizations were performed with Gaussview4 and Maestro 8.5 (Schro¨dinger, L.L.C.).

Synthesis. Transformation of 1-benzyl-2-cyanoimino-1,3thiazolidine to the 2-formylimino derivative using diisobutylaluminium hydride (24) was considered as a possible route to the desired products. However, this reduction failed in the 6-chloropyridin-3-ylmethyl (CPM) and 2-chloro-1,3-thiazol-5ylmethyl (CTM) analogues, resulting in almost quantitative recovery of starting substances. Instead, we found that treating known 1-hetarylmethyl-2-imino-1,3-thiazolidines with ethyl formate is simple and successful for not only 1,3-thiazolidinyl formamides (2 and 4) but also substituted-pyrrolidinyl (3 and 5) and -imidazolidinyl formamides (1) (Scheme 1). 1,3Thiazolidinyl and 1,3-thiazolinyl acylimines were accessible by adding the corresponding acyl chloride to the imines in the presence of a base. Five carbamates were obtained in a conventional way using chloroformates (17-20) or a carbonic acid di-3-pyridyl ester (21). The spectral data supported the structures of these products. Binding Affinity (Table 1). Neonicotinoid analogues with the N-formylimino [dNC(O)H] moiety (1–5) were examined because the isosteric N-nitroso (dNNO) compounds (6–9) retain the high affinity of the corresponding N-nitroimino (dNNO2) derivatives (6). The dNC(O)H analogues with CPM and imidazolidine moieties (1) had greatly diminished potency, whereas compounds with either CPM or CTM and 1,3thiazolidine or pyrrolidine counterparts (2–5) unexpectedly showed moderate levels of affinity (92-280 nM). The stability half-lives of these dNC(O)H compounds in a phosphatebuffered saline (pH 7.4) was far over 24 h (data not shown).

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Figure 3. Structural models for binding site interactions of N-nitrosoimino (7), N-pyridin-3-oylimino (16), N-methoxycarbonylimino (18), and N-phenoxycarbonylimino (20) nicotinic ligands with the R-β subunit interfacial agonist-binding domain of the insect nAChR. Relevant amino acids in yellow are from the R-subunit (loop B W174; loop C Y224, C226, and C227) and in aquamarine are from the β-subunit (loop D W79 and R81; loop E N131, L141, and I143). C226 and C227 are omitted, as they would obscure other important substituents. R81 is relevant only for ligand 16.

Next, the dNC(O)H moiety was replaced by N-alkyl- or N-arylcarbonylimino [dNC(O)R] with a small methyl (10, 11) or trifluoromethyl (12 and 13) group or bulky phenyl (14, 15) or 3-pyridinyl (16) aromatic ring in combination with 1,3thiazolidine or 1,3-thiazoline as the CPM counterpart. The acetylimino analogues (10 and 11) showed medium potency (83-160 nM) similar to the dNC(O)H compounds. In marked contrast, the trifluoroacetylimino compounds (12 and 13) had greatly enhanced affinity (1.8-4.5 nM) (6). The benzoylimino compounds (14 and 15) were moderately potent (71-120 nM), while the 3-pyridinoylimino compound (16) was highly active (2.8 nM), suggesting that the pyridine nitrogen atom serves a key role such as an H-acceptor at the target site. Finally, the N-nitroimino (dNNO2) pharmacophore was transformed to N-alkoxy- or N-aryloxycarbonylimino [dNC(O)OR], where R is methyl (17, 18), phenyl (19, 20), or pyridin-3-yl (21). These modifications gave high affinity compounds (7.9-58 nM). In contrast to the relationship between benzoylimino and pyridinoylimino analogues (15 and 16), the pyridinoxy (21) substitution does not enhance the affinity of the phenoxy analogue (20), suggesting a different interaction mechanism for dNC(O)OR 20 and 21 as compared to that for dNC(O)R 15 and 16. Pharmacophore Orientations. The oxygen tip of the dNC(O)H or dNNO pharmacophore has two possible orientations in compounds 1-9. Quantum mechanics calculations using model structures were therefore performed to determine energetically favorable orientations (Table 2). In the model structures with a 1,3-thiazolidine ring, there are four possible conformations, although two of them [dNC(O)H and dNNO near the thiazolidine NH (c3, c4, d3, and d4)] are not applicable in the

actual compounds since the NH hydrogen is substituted by CPM or CTM. In the N-nitrosoimino structures, the a1 or c1 orientation is energetically preferable over the a2 or c2 orientation, and this result matches the crystal structures of N-nitrosoimino neonicotinoids 6 and 7 (6). In contrast, the oxygen tip of N-formylimino favors facing the heterocyclic ring (b2 and d2). In particular, the b2 orientation is dominated by an intramolecular H-bond between the imidazolidine NH hydrogen and the NC(O)H oxygen. While b1 is quite high in energy relative to b2, other reasonably low energy forms such as d1 and d2 are presumably attainable under biological conditions. Therefore, these calculation results and the present structure-activity relationship, along with the defined molecular interactions of N-nitroimino or N-cyanoimino neonicotinoids (8, 10), clearly suggest that the tip oxygen in the descending direction (as a1, b1, c1, or d1) is the active conformation enabling H-bonding with the receptor subsite(s). Moreover, conformational flexibility of the tip oxygen orientations in the binding pocket may determine the final binding constant as an average. Preferred geometries were further considered for 1,3-thiazoline model structures with dNC(O)R and dNC(O)OR substituents (Figure 2). For 2-acetylimino- and 2-trifluoroacetylimino1,3-thiazolines e and f, the alternate orientation with the amide bond flipped such that the CH3 or CF3 is near the thiazoline sulfur is greatly higher in energy (10.76 and 10.86 kcal/mol, respectively) than when the amide bond is as shown in Figure 2. This favorable conformation (e and f) is identical to that in the crystal structure of the dNC(O)CF3 neonicotinoid (13) (6). In this orientation, the descending CH3 tip is prevented from

Neonicotinoid Pharmacophore Variants

H-bonding with the receptor subsite(s). In contrast, the CF3 head in this direction provides both van der Waals (VDW) contacts and H-bonding abilities and creates a novel binding mechanism conferring high affinity (21). Furthermore, the benzoylimino and pyridine-3-carbonylimino structures (g and h) are only stable in the conformation shown (Figure 2). Calculations show that the conformation with the amide bond flipped is very high in energy. In 2-(methoxy)-, 2-(phenoxy)-, or 2-(pyridin-3-oxy)carbonylimino-1,3-thiazoline (i, j, or k), the conformation shown with carbonyl oxygen in the vicinity of the sulfur is preferred by 4.86, 4.88, or 4.94 kcal/mol, respectively, over the alternate amide orientation (the CdO points away from the sulfur). With this lower energy gap, the biologically active geometry for these O-hinged (rotatable) methyl, phenyl, or pyridin-3-yl moieties could ultimately be determined by the regional binding domain involving H-bonds and other stabilizing interactions. Prediction of Binding Site Interactions (Figure 3). Docking simulations for several representative compounds were performed to predict the binding site interactions. For all compounds simulated here, as with IMI or THIA and their analogues (8-10, 21), the CPM chlorine atom can have favorable VDW interactions with the backbone of loop E N131 and L141 and nearby residues; the pyridine N H-bonds with the backbone of loop E I143 possibly via water bridge(s); the electronically conjugated amidine plane π-stacks with the loop C Y224 aromatic side chain and also interacts via stacking or hydrophobic interactions with other aromatic residues like the loop B W174 indole moiety. Relative to the N-nitrosoimino compound (7), the tip oxygen atom can H-bond with the loop C C226 backbone NH and the loop D W79 side chain NH. This N-nitrosoimino analogue (7) is perfectly superimposable onto the isosteric N-formylimino compound (2) as the predicted active conformation (not shown). The pyridin-3-oyl nitrogen atom of compound 16 can H-bond with the guanidine NH2 of the conformationally flexible side chain of loop D R81; the NC(O) oxygen H-bonds to the W79 indole NH. Thus, the pyridinoylimino pharmacophore is embraced by the loop D niche (extending toward R81) on the β subunit, which is inaccessible to the N-nitroimino, N-cyanoimino, N-nitrosoimino, or N-formylimino group. The OCH3 oxygen of compound 18 H-bonds to the C226 backbone NH and the W79 indole NH; the CdO oxygen undergoes H-bonding with the W79 indole NH [exactly the same as for O from the NO2 of IMI (9) (not shown)]. In the receptor model liganded with phenoxycarbonylimino compound 20, the CdO and phenoxy oxygen atoms H-bond with the W79 aromatic NH and/or the C226 backbone NH (not shown); the phenyl ring of 20 and the W79 indole form a T-shaped aromatic interaction, which can offer about as much stabilization as the more classical π-stacking (both are on the order of 2 kcal/mol) (25). Concluding Remarks. Exploration of nicotinic compounds with novel pharmacophores and looking for different interaction mechanisms from the present N-nitroimino or N-cyanoimino neonicotinoids may help expand the insecticidal spectrum and circumvent possible resistance issues. In the present study, systematic modification of the neonicotinoid pharmacophore (from N-nitroimino or N-nitrosoimino to N-formylimino, Nalkyl- or N-arylcarbonylimino, or N-alkoxy- or N-aryloxycarbonylimino) identified several compounds with high affinity to the insect nAChR. In molecular recognition, the oxygen tip orientation plays an important role in the binding of the dNNO or dNC(O)H pharmacophore. The extended dNC(O)R and dNC(O)OR substituents orient in a different direction and/or interact with unique receptor subsites. These structural models

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are predictive of the observed structure-activity relationships. Accordingly, this investigation demonstrates a reasonable strategy in ligand design combining the chemorational approach and calculation data with the binding site structure, assisting lead generation and discovery of novel insecticides with diverse interaction mechanisms. Acknowledgment. The research of M.T. and J.E.C. was supported by NIEHS Grant R01 ES08424 and the William Muriece Hoskins Chair in Chemical and Molecular Entomology, and the research of K.A.D. was supported by NSF Grant CHE0233882.

References (1) Kagabu, S. Molecular design of neonicotinoids: Past, present, and future. In Chemistry of Crop Protection; Voss, A., Ramos, G., Eds.; Wiley-VCH: Weinheim, 2003; pp 193-212. (2) Tomizawa, M., and Casida, J. E. (2003) Selective toxicity of neonicotinoids attributable to specificity of insect and mammalian nicotinic receptors. Annu. ReV. Entomol. 48, 339–364. (3) Tomizawa, M., and Casida, J. E. (2005) Neonicotinoid insecticide toxicology: Mechanisms of selective action. Annu. ReV. Pharmacol. Toxicol. 45, 247–268. (4) Kagabu, S., and Matsuno, H. (1997) Chloronicotinyl insecticides. 8. Crystal and molecular structures of imidacloprid and analogous compounds. J. Agric. Food Chem. 45, 276–281. (5) Tomizawa, M., Lee, D. L., and Casida, J. E. (2000) Neonicotinoid insecticides: Molecular features conferring selectivity for insect versus mammalian nicotinic receptors. J. Agric. Food Chem. 48, 6016–6024. (6) Tomizawa, M., Zhang, N., Durkin, K. A., Olmstead, M. M., and Casida, J. E. (2003) The neonicotinoid electronegative pharmacophore plays the crucial role in the high affinity and selectivity for the Drosophila nicotinic receptor: An anomaly for the nicotinoid cation-π interaction model. Biochemistry 42, 7819–7827. (7) Kagabu, S., Ishihara, I., Hieda, Y., Nishimura, K., and Naruse, Y. (2007) Insecticidal and neuroblocking potencies of variants of the imizolidine moiety of imidacloprid-related neonicotinoids and relationship to partition coefficient and charge density on the pharmacophore. J. Agric. Food. Chem. 55, 812–818. (8) Tomizawa, M., Talley, T. T., Maltby, D., Durkin, K. A., Medzihradszky, K. F., Burlingame, A. L., Taylor, P., and Casida, J. E. (2007) Mapping the elusive neonicotinoid binding site. Proc. Natl. Acad. Sci. U.S.A. 104, 9075–9080. (9) Tomizawa, M., Maltby, D., Talley, T. T., Durkin, K. A., Medzihradszky, K. F., Burlingame, A. L., Taylor, P., and Casida, J. E. (2008) Atypical nicotinic agonist bound conformations conferring subtype selectivity. Proc. Natl. Acad. Sci. U.S.A. 105, 1728–1732. (10) Talley, T. T., Harel, M., Hibbs, R. H., Radic´, Z., Tomizawa, M., Casida, J. E., and Taylor, P. (2008) Atomic interactions of neonicotinoid agonists with AChBP: Molecular recognition of the distinctive electronegative pharmacophore. Proc. Natl. Acad. Sci. U.S.A. 105, 7606–7611. (11) Tomizawa, M.; Casida, J. E. Molecular recognition of neonicotinoid insecticides: the determinants of life or death. Acc. Chem. Res. 2009, 42, in press (DOI: 10.1021/ar800131p). (12) Latli, B., D’Amour, K., and Casida, J. E. (1999) Novel and potent 6-chloro-3-pyridinyl ligands for the R4β2 neuronal nicotinic acetylcholine receptor. J. Med. Chem. 42, 2227–2234. (13) Imoto, M.; Iwanami, T.; Akabane, M.; Tani, Y. Heterocyclic compounds having effect of activating nicotinic acetylcholine R4β2 receptor. WO 2000053582 A1 20000914 (Chem. Abstr. 2000, 133, 222740). (14) Tomizawa, M., Latli, B., and Casida, J. E. (1996) Novel neonicotinoidagarose affinity column for Drosophila and Musca nicotinic acetylcholine receptors. J. Neurochem. 67, 1669–1676. (15) Cheng, Y.-C., and Prusoff, W. H. (1973) Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50% inhibition (I50) of an enzyme reaction. Biochem. Pharmacol. 22, 3099–3108. (16) Zhang, N., Tomizawa, M., and Casida, J. E. (2004) Drosophila nicotinic receptors: evidence for imidacloprid insecticide and R-bungarotoxin binding to distinct sites. Neurosci. Lett. 371, 56–59. (17) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene,

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M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A.; Gaussian 03, Revision D.01; Gaussian, Inc.: Wallingford, CT, 2004. (18) Sgard, F., Fraser, S. P., Katkowska, M. J., Djamgoz, M. B. A., Dunbar, S. J., and Windass, J. D. (1998) Cloning and functional characterization of two novel nicotinic acetylcholine receptor R subunits from the insect pest Myzus persicae. J. Neurochem. 71, 903–912. (19) Huang, Y., Williamson, M. S., Devonshire, A. L., Windass, J. D., Lansdell, S. J., and Millar, N. S. (1999) Molecular characterization and imidacloprid selectivity of nicotinic acetylcholine receptor subunits from the peach-potato aphid Myzus persicae. J. Neurochem. 73, 380– 389. (20) Huang, Y., Williamson, M. S., Devonshire, A. L., Windass, J. D., Lansdell, S. J., and Millar, N. S. (2000) Cloning, heterologous

Ohno et al.

(21)

(22)

(23) (24)

(25)

expression and co-assembly of Mpβ1, a nicotinic acetylcholine receptor subunit from the aphid Myzus persicae. Neurosci. Lett. 284, 116– 120. Tomizawa, M., Kagabu, S., Ohno, I., Durkin, K. A., and Casida, J. E. (2008) Potency and selectivity of trifluoroacetylimino and pyrazinoylimino nicotinic insecticides and their fit at a unique binding site niche. J. Med. Chem. 51, 4213–4218. Morris, G. M., Goodsell, D. S., Halliday, R. S., Huey, R., Hart, W. E., Belew, R. K., and Olson, A. J. (1998) Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J. Comput. Chem. 19, 1639–1662. Huey, R., Morris, G. M., Olson, A. J., and Goodsell, D. S. (2007) A semiempirical free energy force field with charge-based desolvation. J. Comput. Chem. 28, 1145–1152. Iwata, C., Fujimoto, M., Watanabe, M., Kawakami, T., Maeda, N., Imanishi, T., and Tanaka, T. (1991) Regioslective hydride reduction of 2-(N-cyanoimino)thiazolidine derivatives. Heterocycles 32, 2471– 2478. McGaughey, G. B., Gagne´, M., and Rappe´, A. K. (1998) π-Staking interactions. Alive and well in proteins. J. Biol. Chem. 273, 15458–15463.

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