Neonicotinoid Nitroguanidine Insecticide Metabolites: Synthesis and

Substrate Specificity of Rabbit Aldehyde Oxidase for Nitroguanidine and Nitromethylene Neonicotinoid Insecticides. Ryan A. Dick, David B. Kanne, and J...
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Chem. Res. Toxicol. 2005, 18, 1479-1484

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Neonicotinoid Nitroguanidine Insecticide Metabolites: Synthesis and Nicotinic Receptor Potency of Guanidines, Aminoguanidines, and Their Derivatives David B. Kanne, Ryan A. Dick, Motohiro Tomizawa, and John E. Casida* Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, Berkeley, California 94720-3112 Received June 15, 2005

Four neonicotinoid nitroguanidine insecticides (imidacloprid, thiamethoxam, clothianidin, and dinotefuran) acting as nicotinic agonists account for 10-15% of worldwide insecticide sales. General methods are needed for synthesis of their guanidine and aminoguanidine metabolites so they may be used as analytical standards and for evaluation of nicotinic receptor potency. The guanidines are obtained by treating the parent nitroguanidines with Fe powder in aqueous C2H5OH containing NH4Cl and isolated by silica chromatography. The aminoguanidines are prepared as mixtures with the guanidines on reaction of the parent nitroguanidines and Zn powder in glacial acetic acid. The imidacloprid aminoguanidine is isolated as the acetone imine or trifluoroacetamide and the clothianidin and dinotefuran aminoguanidines as the acetone imines using silica chromatography. Deprotection under acidic conditions then leads to the aminoguanidine‚HCl salts. Because of stability considerations, a pH partitioning method is used to separate thiamethoxam aminoguanidine and guanidine. An alternate procedure to the aminoguanidine of imidacloprid (but not thiamethoxam, clothianidin, or dinotefuran) is reaction with hydrazine hydrate and NH4Cl in anhydrous C2H5OH. Ambiguities in further biological reactions are clarified by synthesizing authentic standards of three purported metabolites formed via the imidacloprid aminoguanidine: the 1,2,4-triazol-3-one derivative with ethyl chloroformate or ethyl pyrocarbonate, the acetaldehyde imine with acetaldehyde, and the 3-methyl-1,2,4-triazin-4-one derivative with ethyl pyruvate in refluxing toluene. The purported triazolone metabolite is reassigned as the aminoguanidine acetaldehyde imine probably formed as an artifact from acetaldehyde present in the ethyl acetate used for metabolite extraction. Potency at the Drosophila nicotinic receptor is greatly decreased on converting a nitroguanidine to a guanidine or aminoguanidine. In sharp contrast, potency at the vertebrate R4β2 nicotinic receptor is generally increased on conversion from the nitroguanidine to aminoguanidine and particularly guanidine derivatives.

Introduction Insecticides play an important role in the production of food and fiber for an expanding world population and in the protection of humans and tended animals from pest insect attack. Neonicotinoids are the newest major class of insecticides for crop protection and flea control on pets accounting for 10-15% of worldwide insecticide sales (1-3). They are increasingly replacing the organophosphorus and methylcarbamate acetylcholinesterase inhibitors in control of crop pests. The nitroguanidine imidacloprid (1) (Figure 1) is the most important neonicotinoid insecticide (4, 5). Three major analogues are thiamethoxam (11), clothianidin (15), and dinotefuran (19) (Figure 1) (2). The nitroguanidine moiety of these molecules is important for potency and safety. This electronegative pharmacophore confers high selectivity for binding to insect as compared with vertebrate nicotinic acetylcholine receptors (nAChRs)1 (3, 6, 7). The * To whom correspondence should be addressed. Tel: 510-642-5424. Fax: 510-642-6497. E-mail: [email protected]. 1 Abbreviations: HRMS, high-resolution mass spectrometry; mp, melting point; nAChR, nicotinic acetylcholine receptor.

Figure 1. Structures of nitroguanidine insecticides and their guanidine, aminoguanidine, and acetone imine derivatives.

nitroguanidine moiety is also a common site for metabolism via cleavage to the guanidine and reduction to nitrosoguanidine and aminoguanidine derivatives (8-14). These metabolic modifications often result in enhanced potency for vertebrate nAChRs and toxicity (3, 6, 15). The structural relationships between the nitroguanidines (1, 11, 15, and 19), the guanidines (2, 12, 16, and 20), the aminoguanidines (3, 13, 17, and 21), and the acetone imine derivatives of the aminoguanidines (6, 14, 18, and 22) are shown in Figure 1. Imidacloprid aminoguanidine

10.1021/tx050160u CCC: $30.25 © 2005 American Chemical Society Published on Web 08/31/2005

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Kanne et al.

Table 1. Nicotinic Receptor Potency of Nitroguanidines and Their Derivatives HPLC no.

compound

tR

(min)b

IC50 (nM) Drosophila

imidacloprid and derivatives 18.4 4.6 ( 0.5c 10.8 1530 ( 66 10.6 12600 ( 700

R4β2 2600 ( 85c 8.2 ( 1.5 42 ( 2

1 2 3

nitroguanidine guanidine‚HCla aminoguanidine‚HCla

4 5 6 7 8 9 10

trifluoroacetamide acetaldehyde imine acetone imine ethyl carbamate triazolone ethyl pyruvate imine methyltriazinonea

imidacloprid aminoguanidine derivatives 13.9 730 ( 56 13.1 15760 ( 1570 14.0 11700 ( 1720 12.7 24600 ( 3060 13.7 68600 ( 3960 16.9 480 ( 44 13.5 65300 ( 6200

11 12 13

nitroguanidine guanidine‚HCl aminoguanidine‚HCl

thiamethoxam and derivatives 15.3 6200 ( 840 10.8 8200 ( 280 11.2 26900 ( 2470

>100000 (I% ) 36) 500 ( 32 1620 ( 98

15 16 17

nitroguanidine guanidine‚HCla aminoguanidine‚HCla

clothianidin and derivatives 17.5 6.1 ( 0.8c 9.8 2510 ( 614 9.8 51400 ( 4600

3500 ( 500c 486 ( 55 1672 ( 220

19 20 21

nitroguanidine guanidine‚HCla,d aminoguanidine‚HCla,d

dinotefuran and derivatives 10.2 130 ( 6 d 8090 ( 570 d 101000 ( 11 000

4026 ( 403 160 ( 20 840 ( 32 10000 ( 1300 2640 ( 364 283 ( 40 49460 ( 5864

>100000 (I% ) 29) 36000 ( 5000 30600 ( 5000

a Observed as metabolites [8-14 and unpublished studies of metabolism by rabbit liver aldehyde oxidase with N-methylnicotinamide and in vivo in mice (except for 20 and 21) by R. A. Dick, K. A. Ford, D. B. Kanne, and J. E. Casida]. b Luna C18 5 µm 250 mm × 4.6 mm column (Phenomenex, Torrance, CA) using a linear gradient of 5% CH3CN in water with 0.1% trifluoroacetic acid to 40% CH3CN in water with 0.1% trifluoroacetic acid over 20 min at a flow rate of 1 mL/min with detection at 254 nm. c Nitrosoguanidine IC50 values (nM) are as follows for Drosophila and R4β2, respectively: nitrosoimidacloprid, 51 ( 5 and 850 ( 26 (7); nitrosoclothianidin, 437 ( 59 and 87500 ( 5520 (present investigation). d Poor detection by HPLC but LC/MS with single ion monitoring showed no 20 in 21 and no 21 in 20. TLC on silica using 20% CH3OH in CH2Cl2 with iodine visualization also showed the absence of 20 in 21.

(3) is reported to undergo conjugation to form methyltriazinone (10) (8, 9) and purported triazolone (8) (10) derivatives (structures given in Scheme 1). An essential step in defining the metabolic chemistry and toxicology of nitroguanidine insecticides is the synthesis of the aforementioned metabolites and derivatives for validation of structure and determination of bioactivity. Most of them are new compounds or fully described here for the first time. We report the relevant syntheses, starting from the parent nitroguanidines, and the structure-activity relationships for the guanidine and aminoguanidine derivatives as selective ligands for Drosophila and vertebrate R4β2 nAChRs.

Scheme 1. Conversion of the Nitroguanidine Imidacloprid (1) to Its Guanidine (2) and Aminoguanidine (3) Derivatives and of the Aminoguanidine to Its Trifluoroacetamide (4), Acetaldehyde Imine (5), Acetone Imine (6), Ethyl Carbamate (7), Triazolone (8), Ethyl Pyruvate Imine (9), and Methyltriazinone (10) Derivativesa

Chemistry Synthesis of Nitrosoguanidines. Although not detailed here, the conditions for conversion of 1 (RdNO2) (Figure 1) to nitrosoimidacloprid (RdNO) by hydrogenation over Raney Ni in C2H5OH (16) are also applicable to preparing the nitrosoguanidines of 15 and 19. The nitrosoguanidine derivative of 15 is unstable on silica cleaving to the urea (dN-R of clothianidin replaced by dO in Figure 1). Synthesis of Guanidines 2, 12, 16, and 20. A reported reduction of 1 to aminoguanidine 3 (17) with Fe powder and NH4Cl in aqueous C2H5OH (18) was found under the conditions used here to give primarily guanidine 2 (19). The same method was employed to prepare guanidines 12, 16, and 20. The high efficiency of the conversions (>90% based on HPLC) was somewhat compromised by solubility-associated losses on chromatography to obtain the required purity. Guanidines 2, 16,

a Reagents and conditions: (a) Fe powder, NH Cl, C H OH. (b) 4 2 5 Zn powder, gl. CH3C(O)OH or H2NNH2, NH4Cl, C2H5OH anhydrous. (c) [CF3C(O)2O]. (d) Aqueous HCl. (e) CH3CHO or (CH3)2CO, 4 Å molecular sieves. (f) C2H5OC(O)Cl or [C2H5OC(O)]2O. (g) o-Cl2benzene, reflux. (h) Ethyl pyruvate, C2H5OH anhydrous, 4 Å molecular sieves. (i) Ethyl pyruvate, toluene, reflux.

and 20 are observed as metabolites of the corresponding nitroguanidines (Table 1).

Neonicotinoid Insecticide Metabolites

Synthesis of Aminoguanidines 3, 13, 17, and 21. Two procedures were developed to reduce 1 to 3: one by treatment with Zn powder (20) and the other with hydrazine hydrate (21). Zn and glacial acetic acid (20) converted 1 to a mixture of guanidine 2 and aminoguanidine 3 in approximately a 4:1 ratio. Aminoguanidine 3 was stabilized for purification by protection as trifluoroacetamide 4 and acetone imine 6, which are easily deprotected. Efficient conversion of 1 to 3 was also achieved by treatment with hydrazine and NH4Cl in anhydrous C2H5OH at 80 °C. The crude mixture from the hydrazine reaction was of sufficient purity and stability for use without purification in the preparation of imines 5, 6, and 9, carbamate 7, and methyltriazinone 10. The Zn and glacial acetic acid procedure with nitroguanidines 15 and 19 via acetone imines 18 and 22 gave aminoguanidines 17 and 21, respectively. In the case of 11, the acetone imine approach failed at the acid deprotection step, perhaps due to side reaction of the oxadiazinane moiety. However, because of the reduced basicity of aminoguanidine 13 as compared to guanidine 12, it was possible to partition the latter from ether into the aqueous phase at pH 11.5. The organic phase was then subjected to a final chromatography on silica to produce pure 13‚HCl. When the hydrazine method was applied to 11, 15, and 19, significant quantities of product were observed from diaddition (23) (see the Supporting Information). In the case of 11, methylamine and formaldehyde can be expelled from the tetrahedral intermediate while 15 and 19 can lose methylamine. In the case of 1, the potential intermediate 24 was not seen, presumably since the diaddition product can readily reclose to the fivemembered ring.

Aminoguanidines 3, 17, and 21 are observed as metabolites of the corresponding nitroguanidines (Table 1). Synthesis of Triazolone 8 and Reassignment of Purported Imidacloprid Metabolite as Artifact Aminoguanidine Acetaldehyde Imine 5. An unusual metabolite of 1 in a human microsome-NADPH system was tentatively assigned as 1,2,4-triazol-3-one 8 (10) on the basis of LC/MS m/z 252 for [M + H]+ and a fragmentation pattern that was rationalized to the triazolone structure. In addition, one of the products resulting from attempted synthesis of 8 by treating 3 with ethyl chloroformate in C2H5OH had an LC/MS/MS identical to the purported triazolone (10). However, two points were disturbing; that is, the proposed triazolone 8 was somewhat unstable and partially reverted to 3 on attempted isolation and the source for the introduced carboxyl group was unknown (10). On reexamining the synthesis reaction with diethyl pyrocarbonate in C2H5OH or ethyl acetate, the ethyl carbamate (7) was the major compound but there was also a side product possessing LC/MS/MS identical to the purported imidacloprid metabolite. The high-resolution mass spectrometry (HRMS) data, however, were not suitable for triazolone 8 but were appropriate for acetaldehyde imine 5 of the same nominal mass. In the present study, authentic triazolone 8 was

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synthesized by heating the aminoguanidine ethyl carbamate (7) in o-dichlorobenzene based on a general procedure for triazolone formation (22). The purported metabolite was in fact identical to the synthesized acetaldehyde imine 5 not triazolone 8. Generation of 5 in both the enzymatic and the synthetic reactions is attributed to formation of 3, which then reacted with trace amounts of acetaldehyde in the tissue or more likely in the extraction or reaction solvents used. On this basis, aminoguanidine metabolites are a potential source of analytical artifacts and derivatization reactions of biological relevance. The benzaldehyde imine of 3 may be formed on 1 metabolism when benzaldehyde is used as the electron donor in the aldehyde oxidase system (14). The ease of forming the acetaldehyde imine serves as a caution to avoid the presence of aldehydes and ketones in extraction solvents and analytical methods. Synthesis of Methyltriazinone 10 via Ethyl Pyruvate Imine 9. The first approach for synthesis of the imidacloprid metabolite triazinone 10 involved a ring closure of imine 9 in refluxing o-dichlorobenzene as for 8. Although 9 was prepared and isolated without difficulty, it was not cleanly converted to 10 under these conditions. However, a one-step procedure in which 3‚ HCl and ethyl pyruvate are refluxed in toluene (23) provided after chromatography a good yield of pure 10. Methyltriazinone 10 is a metabolite of 1 in rat liver (8, 9) probably formed by reaction of aminoguanidine 3 with pyruvate or a derivative thereof.

Nicotinic Receptor Potency The nitroguanidine parent compounds and their metabolites and derivatives were assayed for binding potency with the Drosophila and vertebrate R4β2 nAChRs (Table 1). Nitroguanidines 1, 15, and 19 (except 11) are very potent with Drosophila but not R4β2 receptors. With the insect receptor, the nitroguanidines (1, 11, 15, and 19) are more effective than the guanidines (2, 12, 16, and 20) and much more so than the aminoguanidines (3, 13, 17, and 21).2 On the contrary, with the mammalian receptor, the nitroguanidines are much less active than their guanidine derivatives and the aminoguanidines (except for 21) are of intermediate potency. Therefore, the conversion of nitroguanidines to guanidines and aminoguanidines is a detoxification for Drosophila nAChR and an activation for R4β2 nAChR. An earlier report that 3 is less potent than 1 at the mouse brain [3H]nicotine binding site (15) may be attributable to decomposition of 3 prior to assay. Seven derivatives of imidacloprid aminoguanidine are less potent than the parent 3 on the R4β2 receptor and generally on the Drosophila receptor except for the trifluoroacetamide (4) and the ethyl pyruvate imine (9).

Experimental Procedures General. Melting points (mp) are uncorrected. Reagents from Aldrich (Milwaukee, WI) and solvents from Fisher Scientific (Tustin, CA) were used without further purification. NMR spectra were obtained on a 300 MHz spectrometer for solutions in CDCl3 (δ ) 7.27 for 1H and 77.0 for 13C), CD3OD (δ ) 3.31 for 1H and 49.0 for 13C), or CD3SOCD3 (δ ) 2.50 for 1H and 39.5 2 The nitroso derivatives of nitroguanidines 1 and 15 are 10- and 200-fold less potent, respectively, than their nitro precursors at the Drosophila nAChR (7 and present study).

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for 13C); readily exchangeable protons are not shown. The conditions for LC/MS have been described (14). HRMS was performed at the University of Notre Dame Mass Spectrometry Laboratory. The purity of the final products for bioassay was >99% based on HPLC or LC/MS (total ion current) under the conditions given in Table 1. 1-[(6-Chloro-3-pyridinyl)methyl]-2-imidazolidinone Imine (Imidacloprid Guanidine) (2) and Its Analogues 12, 16, and 20. To a magnetically stirred suspension of 1 (0.750 g, 2.93 mmol) in 10 mL of C2H5OH was added NH4Cl (0.78 g, 14.6 mmol) dissolved in 1.8 mL of water followed by powdered Fe (0.490 g, 8.79 mmol). The mixture was refluxed for 2.5 h and then filtered hot to remove Fe. The filter cake was washed with 20 mL of CH3OH, and the combined filtrates were concentrated in vacuo. Conversion to the desired guanidine (19) was estimated by HPLC to be >95%, but the yellow solid also contained some NH4Cl. After chromatography on silica (25% CH3OH in CH2Cl2) and evaporation, the residue was taken up in 10% CH3OH in CH2Cl2 and filtered (0.45 µm Teflon) to yield 22 mg (17%) of 2‚HCl. LC/MS, m/z 211 [M + H]+, 252 [M + H + CH3CN]+. Guanidine derivatives 12, 16, and 20 (all as HCl salts), prepared in an analogous fashion, were obtained in high conversion but low yield due to losses on chromatography and recovery to obtain the required purity. Thiamethoxam guanidine (12‚HCl): 1H NMR (CD3SOCD3): δ 7.76 (s, 1H), 4.98 (s, 2H), 4.93 (s, 2H), 4.83 (s, 2H), 2.93 (s, 3H). LC/MS, m/z: 247 [M + H]+, 288 [MH+ + 41, CH3CN adduct]. FAB-HR: [C8H11ClN4OS + H]+ calcd, 247.0420; found, 247.0396. Clothianidin guanidine (16‚ HCl): 1H NMR (CD3OD): δ 7.59 (s, 1H), 4.64 (s, 2H), 2.88 (s, 3H). 13C NMR (CD3OD): δ 158.06, 153.39, 141.07, 138.56, 38.64, 28.51. FAB-HR: [C6H9ClN4S + H]+ calcd, 205.0315; found, 205.0303. Dinotefuran guanidine (20‚HCl): 1H NMR (CDCl3): δ 3.7-3.9 (m, 3H), 3.47-3.53 (m, 1H), 3.15 (m, 2H), 2.83 (s, 3H), 2.5 (m, 1H), 2.1 (m, 1H), 1.63 (m, 1H). 13C NMR (CH3OH): δ 158.42, 71.79, 68.56, 45.20, 39.83, 30.64, 28.40. FAB-HR: [C7H15N3O + H]+ calcd, 158.1293; found, 158.1277. 1-[(6-Chloro-3-pyridinyl)methyl]-2-imidazolidinone Hydrazone (3) (Imidacloprid Aminoguanidine) Prepared by Two Methods. Via 1-[(6-Chloro-3-pyridinyl)methyl]-2-imidazolidinone Hydrazone Trifluoroacetamide (Imidacloprid Aminoguanidine Trifluoroacetamide) (4). Cold glacial acetic acid (12.5 mL) was added to 1 (0.830 g, 3.25 mmol) and Zn powder (1.08 g, 16.5 mmol), and the mixture was stirred for 5 h with ice cooling as necessary to keep the temperature below 40 °C. Filtration through Celite and washing the cake with CH2Cl2 (8 mL) gave a 4:1 mixture of guanidine and aminoguanidine based on HPLC analysis. Trifluoroacetic anhydride (1.95 g, 9.28 mmol) was added to the cold (5 °C) filtrate, and the mixture was stirred at 25 °C for 16 h. The reaction mixture was then partitioned between CH2Cl2 and water (with enough NaHCO3 to bring the pH to ∼8). The aqueous phase was washed (3 × 50 mL) with CH2Cl2. The combined organic phase was dried, filtered, and evaporated to give a dark oil, which was chromatographed on silica (7% CH3OH in CH2Cl2) to afford 178 mg (17% yield over two steps) of 4 as a white solid (mp 216-217 °C). Compound 4: 1H NMR (CDCl ): δ 8.33 (d, J ) 2.1 Hz, 1H), 7.77 (dd, J ) 2.1, 3 8.2 Hz, 1H), 7.37 (d, J ) 8.2 Hz, 1H), 5.0 (br s, 2H), 3.64 (m, 4H). FAB-HR: [C11H11ClF3N5O + H]+ calcd, 322.0682; found, 322.0700. For hydrolysis, a solution of 4 (25 mg, 0.078 mol) in 2 M HCl (1.5 mL) was heated at 100 °C for 45 min and then lyophilized overnight to give 20 mg (98% yield) of 3‚HCl as a white solid. 3‚HCl: 1H NMR (CD3OD): δ 8.39 (d, J ) 2.6 Hz, 1H), 7.86 (dd, J ) 2.6, 8.2 Hz, 1H), 7.54 (d, J ) 8.2 Hz, 1H), 4.56 (s, 2H), 3.69 (s, 4H). 13C NMR (CD3OD): δ 161.9, 151.0, 149.0, 142.0, 132.3, 126.5, 50.0, 46.5, 42.1. FAB-HR: [C9H12ClN5 + H]+ calcd, 226.0859; found, 226.0879. Via Reaction of 1 with Hydrazine. To a solution of 1 (1.00 g, 3.91 mmol) and NH4Cl (214 mg, 4.00 mmol) in anhydrous C2H5OH (12 mL) was added hydrazine hydrate (0.200 g, 4.00 mmol), and the mixture was heated at reflux for 2.5 h. Careful HPLC monitoring is required since side reaction products

Kanne et al. appear if the hydrazine is added beyond the 95% conversion point. The reaction required two further additions of hydrazine hydrate (2 mmol each) and longer time (3.5 h) at reflux for 95% completion. The reaction mixture, purged with nitrogen and stored at -20 °C, was of sufficient purity (∼95%) and stability (no decomposition after 6 months) to be used as a stock solution of 3‚HCl without workup or purification in the preparation of 5-7, 9, and 10. In these syntheses, only the weights of 3‚HCl are given although the presence of residual NH4Cl is understood. All yields of single step reactions involving this stock solution are calculated over two steps from starting material 1. 1-[(6-Chloro-3-pyridinyl)methyl]-2-imidazolidinone Hydrazone Acetaldehyde Imine (5) and Acetone Imine (6). To 0.230 g (0.877 mmol) of 3‚HCl in 2.3 mL of C2H5OH (from stock solution as above) was added 42 mg (0.95 mmol) of acetaldehyde, and the mixture was stirred at room temperature over 4 Å molecular sieves for 24 h. Additional acetaldehyde (42 mg, 0.95 mmol) was needed (due to evaporative losses) for consumption of starting material. After filtration to remove molecular sieves and evaporation, the mixture was partitioned between CH2Cl2 and cold 0.1 M Na2CO3. The organic phase was dried (Na2SO4) and evaporated in vacuo. The residue was chromatographed on silica using 6.5% CH3OH in CH2Cl2 to yield 86 mg (39% for two steps) of an oil. NMR indicated a 1:3.5 ratio of syn and anti imines [the downfield (24) imine proton when methyl is anti (δ ) 7.47) predominates over that when methyl is syn (δ ) 6.79)]. Compound 5: 1H NMR (CD3CN): δ 8.338.38 (pair of doublets, J ) 2.6 Hz, 1H, 7.7-7.8 (pair of dd, J ) 2.6, 8.2 Hz, 1H), 7.47 (q, J ) 5.1 Hz, 1H), 7.37 (d, J ) 8.2 Hz, 1H), 6.79 (q, J ) 5.1 Hz, 1H), 4.43 (two singlets, 1H), 3.2-3.4 (m, 4H), 1.85-1.91 (pair of doublets, J ) 5.1 Hz, 3H). LC/MS, m/z: 252 [M + H]+. FAB-HR: [C11H14ClN5H]+ calcd, 252.1016; found, 252.1023. The imidacloprid aminoguanidine acetone imine (6) was prepared in 42% yield over two steps in an analogous fashion by reaction of 3 with acetone. 1H NMR (CDCl3): δ 8.36 (d, J ) 2.6 Hz, 1H), 7.72 (dd, J ) 2.6, 8.2 Hz, 1H), 7.29 (d, 8.2 Hz, 1H), 4.45 (s, 2H), 3.44 (t, 2H), 3.27 (t, 2H), 2.02 (s, 3H), 1.96 (s, 3H). 13C NMR (CDCl3): δ 160.57, 156.73, 150.38, 149.37, 139.00, 132.32, 124.09, 46.89, 45.85, 40.67, 24.91, 17.24. LC/MS, m/z: 266 [M + H]+. The conditions for hydrolysis of 6 to 3 were the same as those given later for conversion of 18 to 17. 1-[(6-Chloro-3-pyridinyl)methyl]-2-imidazolidinone Hydrazone Ethyl Carbamate (Imidacloprid Aminoguanidine Ethyl Carbamate) (7). To ethyl chloroformate (25 mL) cooled to 5 °C was added 3‚HCl (0.655 g, 2.50 mmol) in 6.5 mL of C2H5OH (from stock solution as above). The mixture was refluxed for 2 h and cooled overnight, and the white solid product was collected by filtration. This HCl salt was partitioned between CH2Cl2 and 0.2 M NaHCO3. The aqueous phase was washed with CH2Cl2 (5 × 25 mL). The combined organic phase was dried, filtered, and evaporated to give 620 mg of a white solid, which was chromatographed on silica (10% CH3OH in CH2Cl2) to afford 475 mg (64% yield over two steps) of 7 as a white solid (mp 126-127 °C). Compound 7: 1H NMR (CDCl3): δ 8.33 (d, J ) 2.05 Hz, 1H), 7.81(dd, J ) 2.05, 8.21 Hz, 1H), 7.32(d, J ) 8.21 Hz, 1H), 4.43(s, 2H), 4.18(q, 2H), 3.42(m, 2H), 3.30(m, 2H), 1.28(t, 3H). 13C NMR (CDCl3): δ 162.8, 157.1, 150.5, 149.1, 139.2, 131.6, 124.3, 61.3, 47.0, 45.6, 40.5, 14.5. LC/MS, m/z: 298 [M + H]+. 7-[(6-Chloro-3-pyridinyl)methyl]-2,5,6,7-tetrahydroimidazo[2,1-c]-1,2,4-triazol-3-one (Imidacloprid Aminoguanidine Triazolone) (8). Carbamate 7 (0.122 g, 0.411 mmol) was added to 6.5 mL of o-dichlorobenzene, and the mixture was refluxed for 1.5 h, cooled overnight, and refrigerated for 3 h. The solid (82 mg) collected by filtration was washed with ether and dried. Chromatography on silica (10% CH3OH in CH2Cl2) afforded 62 mg (60%) of 8 as a white solid (mp 106-107 °C). Compound 8: 1H NMR (CDCl3): δ 8.35 (d, J ) 2.05 Hz, 1H), 7.69 (dd, J ) 2.05, 8.21 Hz, 1H), 7.35 (d, J ) 8.21 Hz, 1H), 4.31 (s, 2H), 3.88 (m, 2H), 3.70 (m, 2H). 13C NMR (CDCl3): δ 154.7,

Neonicotinoid Insecticide Metabolites 152.6, 151.0, 149.0, 139.0, 130.3, 124.4, 53.2, 46.9, 39.0. FABHR: [C10H10ClN5O + H]+ calcd, 252.0652; found, 252.0662. 1-[(6-Chloro-3-pyridinyl)methyl]-2-imidazolidinone Hydrazone Ethyl Pyruvate Imine (Imidacloprid Aminoguanidine Ethyl Pyruvate Imine) (9). To 61 mg (0.23 mmol) of 3‚ HCl in 0.61 mL of C2H5OH (from stock solution as above) was added 54 mg (0.46 mmol) of ethyl pyruvate and 50 mg of 4 Å molecular sieves. After 8 h of stirring at room temperature, another 54 mg of ethyl pyruvate was added and stirring was continued for another 16 h. Most of the solvent was then removed by evaporation. The residue was chromatographed on silica with 2.5% CH3OH in CH2Cl2 to give 11.7 mg (15% yield over two steps) of 9. Compound 9: 1H NMR (CDCl3): δ 8.36 (d, 1H), 7.72 (dd, 1H), 7.30 (d, 1H), 4.56 (s, 2H), 4.28 (q, 2H), 3.55 (t, 2H), 3.42 (t, 2H), 2.20 (s, 3H), 1.35 (t, 3H). LC/MS, m/z: 324 [M + H]+, 365 [M + H + CH3CN]+. FAB-HR: [C14H18ClN5O2 + H]+ calcd, 324.1227; found, 324.1234. 8-[(6-Chloro-3-pyridinyl)methyl]-3-methyl-7,8-dihydro6H-imidazo[2,1-c]-1,2,4-triazin-4-one (Imidacloprid Aminoguanidine Methyltriazinone) (10). To 0.500 g (0.431 mmol) of ethyl pyruvate in 2.0 g of toluene at 110 °C was added dropwise over 5 min 107 mg (0.408 mmol) of 3‚HCl in 1.1 mL of C2H5OH (from stock solution as above) containing 10 mg of triethylamine. The reaction was stirred overnight at a bath temperature of 95 °C and then cooled and partitioned between 0.1 M HCl and ethyl acetate. The aqueous phase was treated with solid Na2CO3 until pH 11 and extracted twice with CH2Cl2 and twice with ethyl acetate. The combined organic layer was dried (Na2SO4) and evaporated to yield 127 mg of a dark amber oil. Chromatography on silica with 3% CH3OH in CH2Cl2 gave 51 mg (45% yield over three steps) of 10 as an oil. Compound 10: 1H NMR (CDCl3): δ 8.36 (d, J ) 2.6 Hz, 1H), 7.77 (dd, J ) 8.2, 2.6 Hz, 1H), 7.34 (d, J ) 8.2 Hz, 1H), 4.64 (s, 2H), 4.13 (t, 2H), 3.59 (t, 2H), 2.37 (s, 3H). 13C NMR (CD3OD): δ 155.6, 154.9, 151.9. 150.4, 140.8, 132.6, 125.7, 46.3, 45.9, 42.3, 16.4 (either the CdN or CdO carbon is not observed). LC/MS, m/z: 278 [M + H]+, 319 [M + H + CH3CN]+. FAB-HR: [C12H12ClN5O + H]+ calcd, 278.0809; found, 278.0826. The 1H NMR spectrum of 10 from this synthesis was identical to those of both a standard prepared by Bayer and an imidacloprid metabolite isolated from the liver of rats [spectra kindly provided by Otto Klein, Bayer CropScience AG (Monheim, Germany)]. Thiamethoxam Aminoguanidine (13) and Acetone Imine (14). To prepare 13, the first step of the Zn reduction was performed as for 17 below but without trapping as the acetone imine. The crude aminoguanidine, after filtration through Celite to remove the Zn, was precipitated by adding ether/HCl and washed three times with ether. The white precipitate was partitioned between water (pH 6) and ether. The aqueous phase containing 12 and 13 was then adjusted to pH 11.5 with Na2CO3 and extracted three times with ether. The combined ether layer was then back extracted four times with pH 11.5 Na2CO3 solution (there is some loss of 13 into the aqueous phase during this back extraction). HPLC at this point indicated virtually all 12 had been removed from the organic layer. The ether layer was dried (Na2SO4) and evaporated in vacuo. A portion of the residue was subjected to final chromatography on silica (12.5% CH3OH in CH2Cl2). Concentration of 13 as the free base leads to decomposition and/or dimerization (25). Accordingly, one equivalent of HCl/ether was added to ensure the absence of free base during final concentration (blown down with nitrogen followed by evaporation under high vacuum overnight) to yield 3 mg of 13‚HCl. Compound 13: 1H NMR (CD3OD): δ 7.65 (s, 1H), 4.90 (s, 2H), 4.85 (s, 2H), 4.81 (s, 2H), 3.03 (s, 3H). LC/MS, m/z: 262 [M + H]+, 303 [M + H + CH3CN]+. Compound 14 was synthesized from 11 via Zn reduction and trapping with acetone followed by chromatography (20% yield over two steps) as below for the preparation of 18 from 17. Compound 14: LC/ MS: 302 [M + H]+. Clothianidin and Dinotefuran Aminoguanidines (17 and 21) and Acetone Imines (18 and 22). To prepare 17, a mixture of 15 (0.250 g, 1.00 mmol) and Zn powder (0.327 g, 5.00

Chem. Res. Toxicol., Vol. 18, No. 9, 2005 1483 mmol) in glacial acetic acid (2.2 g) was stirred at or below 40 °C for 5 h with ice cooling during the first 30 min to keep the temperature at 15-20 °C. The mixture was filtered through Celite, and the cake was washed with CH2Cl2. Most of the CH2Cl2 was removed in vacuo, and 3.0 g of acetone was added and the mixture was stirred with molecular sieves for 72 h. After the molecular sieves were removed by filtration and excess acetone was stripped off, the solution was added to cold ether/ HCl (1.0 M). The white precipitate was then washed with cold ether, and the residue was partitioned between 0.2 M NaHCO3 and ether. The aqueous phase was then extracted three times with CH2Cl2. The organic extracts were combined, dried (Na2SO4), and evaporated in vacuo. Chromatography on silica with 10% CH3OH in CH2Cl2 yielded 50 mg (19% yield over two steps) of 18 as a light yellow oil. Compound 18: 1H NMR (CDCl3): δ 7.37 (s, 1H), 4.57 (s, 2H), 2.81 (s, 3H), 2.07 (s, 3H), 1.97 (s, 3H). 13C NMR (CDCl ): δ 156.55, 156.13, 152.33, 139.48, 138.76, 3 37.97, 28.18, 25.12, 17.87. FAB-HR: [C9H14ClN5S + H]+ calcd, 260.0737; found, 260.0724. An analogous procedure was used to prepare 22 from 19 with 15% yield over two steps. Compound 22: 1H NMR (CDCl3): δ 3.9 (m, 1H), 3.75 (m, 2H), 3.65 (m, 1H), 2.96 (s, NCH3), 2.6 (m, 1H), 2.1 (m, 1H), 2.08 (s, CH3), 1.98 (s, CH3), 1.7 (m, 1H). 13C NMR (CDCl3): δ 157.12, 155.03, 71.34, 67.62, 44.17, 39.41, 29.85, 28.00, 25.00, 17.24. FAB-HR: [C10H20N4O + H]+ calcd, 213.1715; found, 213.1736. Aminoguanidines 17 and 21 were obtained by hydrolysis of acetone imines 18 and 22, respectively. Thus, 18 (18.8 mg) and 2 mL of 1 M HCl were heated at 100 °C for 45 min giving complete conversion (HPLC) to a single new product. The mixture was lyophilized overnight to yield 15 mg (81%) of 17‚ HCl as a white solid. Compound 17: 1H NMR (CDCl3): δ 7.58 (s, 1H), 4.64 (s, 2H), 2.86 (s, 3H). LC/MS, m/z: 220 [M + H]+, 261 [M + H + CH3CN]+. FAB-HR: [C6H10ClN5S + H]+ calcd, 220.0424; found, 220.0419. An analogous procedure was used to prepare 21 from 22. Compound 21: 1H NMR (CDCl3): δ 3.73.9 (m, 3H), 3.47-3.53 (m, 1H), 3.14 (d, 2H), 2.83 (s, 3H), 2.5 (m, 1H), 2.1 (m, 1H), 1.63 (m, 1H). 13C NMR (CH3OH): δ 158.72, 71.79, 68.53, 44.63, 39.83, 30.55, 27.76. FAB-HR: [C7H16N4O + H]+ calcd, 173.1402; found, 173.1413. Diaminoguanidine Derivatives. Although not detailed here, the hydrazine hydrate reaction of 11, 15, and 19 leads first to the corresponding aminoguanidines but then proceeds to new derivatives with a major one tentatively identified as the diaminoguanidine such as 23 obtained from 11 and 15 and the tetrahydrofuryl analogue of 23 from 19 (see the Supporting Information). Compound 23: LC/MS, m/z: 221 [M + H]+. Radioligand Binding. The potency of test compounds for the nAChR from Drosophila melanogaster heads was determined by displacement of 3 nM [3H]IMI binding (26). Binding of 10 nM [3H]nicotine to the vertebrate R4β2 nAChR subtype expressed in mouse fibroblast M10 cells was performed according to the method of Tomizawa et al. (6). IC50 values (molar concentrations of test compounds for 50% inhibition of specific radioligand binding) were calculated by iterative least-squares regression using Sigmaplot (Jandel Scientific Software, San Rafael, CA).

Acknowledgment. This project was supported by Grant R01 ES08424 from the National Institute of Environmental Health Sciences (NIEHS), NIH. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NIH. We thank Gary B. Quistad and Kevin A. Ford of this laboratory and Otto Klein of Bayer CropScience AG (Monheim, Germany) for advice. Supporting Information Available: Proposed mechanisms of neonicotinoid reactions with hydrazine. This material is available free of charge via the Internet at http://pubs.acs.org.

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