Biological activities of nitromethylene analogues of imidacloprid with a 4,5-dimethylated imidazolidine ring

Abstract

Four nitromethylene analogues of imidacloprid (CH-IMIs) having a 4,5-dimethylated (diMe) imidazolidine ring were stereospecifically synthesized to evaluate their affinity for the nicotinic acetylcholine receptors of the housefly Musca domestica. Among the analogues, the 4S,5R-diMe analogue showed the highest receptor affinity (K_i=0.39 nM). The insecticidal activity against M. domestica of the synthesized compounds was also measured under synergistic and nonsynergistic conditions. Under nonsynergistic conditions, the insecticidal activity of the 4S,5R-diMe analogue was the highest. The order of the insecticidal potency of the four diMe-CH-IMIs (4S,5R->4R,5S-=4R,5R->4S,5S-diMe analogues) was the same as that of the receptor affinity. Piperonyl butoxide (PBO) did not synergize with the test compounds, but both PBO and NIA16388 applications strengthened the activity of analogues other than the 4S,5S-diMe analogue. This suggests that the configuration of the substituents on the imidazolidine ring should influence the metabolism process of CH-IMI in houseflies.

Introduction

Neonicotinoid insecticides such as imidacloprid (IMI, 1 in Fig. 1) are widely employed for regulating various crop pests, such as aphids, as well as sanitary pests, including flies.¹⁾ Neonicotinoids induce acute toxicity against pests by acting on their nicotinic acetylcholine receptors (nAChRs), but adverse effects of some neonicotinoids on beneficial insects, such as honey and bumble bees, have been reported. In this situation, the development of novel insecticides targeting nAChRs that are ineffective against beneficial insects has continued, and analyses of the structure–activity relationship of nAChR-targeting compounds are still important.

Fig. 1. Chemical structures of imidacloprid (IMI, 1), its nitromethylene analogue (CH-IMI, 2), methylated derivatives 3–6, and dimethylated derivatives 7–12.

To elucidate the interaction between the insect nAChRs and their neonicotinoid ligands, we quantitatively analysed the structure–activity relationship for various series of derivatives of IMI and its nitromethylene analogue (CH-IMI, 2 in Fig. 1).^2–7) Furthermore, an in silico docking study using the CH-IMI derivatives and housefly receptor model was performed, suggesting that a space accepting a C1–C4 sized alkyl, alkoxyl, or alkylthio group attached at the 5 position of the imidazolidine ring should exist in the ligand binding region of the receptor.^6,7) The space is surrounded by several aromatic amino acid residues such as tryptophan and tyrosine, and it is expected that these amino acid residues should interact with the substituents at the 5 position of the imidazolidine ring via CH-π interaction.

On the other hand, IMI has been reportedly metabolized in the housefly, Musca domestica, through oxidative metabolism, in which the imidazolidine ring is hydroxylated, followed by olefination.⁸⁾ We then hypothesized that modification of the imidazolidine ring by introducing substituents should influence the insecticidal activity, and four CH-IMI derivatives containing a methylated imidazolidine ring (R-5-, R-4-, S-5- and S-4-Me–CH-IMIs, 3–6) were prepared to measure their insecticidal activity against houseflies under synergistic and nonsynergistic conditions.⁵⁾ The synergistic effect of piperonyl butoxide (PBO) on the insecticidal activity of 4-methylated Compounds 5 and 6 was suppressed, suggesting that the substituent on the imidazolidine ring should influence the metabolism system by cytochrome P450 monooxygenases in houseflies. The receptor affinity of Compounds 3–6 was also evaluated, and the CH-IMI derivative 3 containing the R-5-methylated imidazolidine ring was found to be almost equipotent to the unsubstituted CH-IMI.⁵⁾ These results suggest that the proper combination of substituents on the imidazolidine ring could give high receptor affinity as well as suppress monooxygenase-related metabolism.

We have already evaluated the biological activities of CH-IMI derivatives 7 and 8 containing 4,4- and 5,5-dimethylated imidazolidine rings, respectively, and reported that the introduction of dimethyl groups at one position of the imidazolidine ring should be unfavourable to the activities,⁴⁾ but it remains unknown whether the CH-IMI derivatives containing the 4,5-dimethylated imidazolidine ring show high biological activities.

In the present study, we synthesized CH-IMI derivatives containing the 4,5-dimethylated imidazolidine ring (9–12 in Fig. 1) to elucidate the effect of substituents on the imidazolidine ring on their biological activities, such as insecticidal activity against houseflies and M. domestica and receptor affinity to housefly nAChR.

Materials and methods

1. Insects and chemicals

An insecticide-susceptible strain of the housefly (M. domestica L, Takatsuki strain) was reared at 25°C in our laboratory. Compounds 9–12 (Fig. 1) were newly synthesized through Schemes 1 and 2. Reagents used for the syntheses were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), Nacalai Tesque, Inc. (Kyoto, Japan), Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), and Sigma-Aldrich Japan K.K. (Tokyo, Japan). The metabolic inhibitor, NIA16388 (NIA; propargyl propyl phenylphosphonate), was our stock sample.⁷⁾ The ¹H and ¹³C NMR analyses were performed using a JEOL ECS-400 NMR spectrometer in deuterochloroform (CDCl₃) with tetramethylsilane as the internal standard. The chemical shift values are expressed in parts per million (ppm) relative to tetramethylsilane. The authenticity of the final compounds was also confirmed by HRMS using Xevo Q-TOFMS (Waters, UK). Melting points of the compounds were measured with a Yanaco melting point apparatus (Kyoto, Japan) and were uncorrected. Optical rotation values (unit degrees; c is the concentration in grams per 100 mL of solution) were evaluated by using a P-2100 polarimeter (Jasco, Tokyo, Japan). The values of enantiomer excess were determined by HPLC analyses using Prominence UFLC system (Shimadzu) with the CHIRALPAK AD-H column chromatography (0.46 cm×25 cm (DAICEL), 80% hexane +20% 2-propanol, flow rate 1 mL/min, UV 254 nm).

Scheme 1. Preparation of Compounds 9 and 10.

Scheme 2. Preparation of Compounds 11 and 12.

2. Synthesis of chemicals

2.1. (R)-4-Benzyl-3-propionyloxazolidin-2-one (13) and (S)-4-benzyl-3- propionyloxazolidin-2-one (ent-13)

To a solution of R-(+)-4-benzyl-2-oxazolidinone (5.3 g, 0.030 mol) in dry THF (50 mL) at −78°C under nitrogen was added n-BuLi (ca. 2.8 M, 19.1 mL). The mixture was stirred at −78°C for 1 hr. Propionyl chloride (3.0 mL, 0.034 mol) was added dropwise, and the mixture was stirred for 1 hr at −78°C and then for an additional 30 min at room temperature. The reaction mixture was quenched with sat. NH₄Cl aq. and extracted with ethyl acetate. The organic phase was washed with H₂O, dried over sodium sulfate and evaporated. The residue was purified by column chromatography (hexane : ethyl acetate=1 : 1) to afford 13 (7.1 g, quant.). ¹H-NMR (400 MHz, CDCl₃) δ: 1.21 (3H, t, J=6.4 Hz, CH₃–), 2.78 (1H, dd, J=10 Hz, 13 Hz, one of Ph–CH₂–), 2.86–3.03 (2H, m, CH₃–CH₂–), 3.30 (1H, dd, J=3 Hz, 13 Hz, one of Ph–CH₂–), 4.14–4.23 (2H, m, –O–CH₂–), 4.62–4.70 (1H, m, –CH₂–CH–N), 7.20–7.35 (5H, m, C₆H₅–), ¹³C-NMR (100 MHz, CDCl₃) δ: 8.2, 29.1, 37.8, 55.1, 66.1, 127.2, 128.9, 129.3, 135.2, 153.4, 174.0. [α]²⁵_D −56 (c 1.05, CHCl₃). (S)-4-Benzyl-3-propionyloxazolidin-2-one (ent-13) was synthesized from S-(−)-4-benzyl-2-oxazolidinone according to the same scheme. [α]²⁵_D +58 (c 1.07, CHCl₃).

2.2. (R)-4-Benzyl-3-((2R,3S)-3-hydroxy-2-methylbutanoyl)oxazolidin-2-one (14) and (S)-4-benzyl-3-((2S,3R)-3-hydroxy-2-methylbutanoyl)oxazolidin-2-one (ent-14)

To a solution of 13 (4.22 g, 0.031 mol) in dry CH₂Cl₂ (100 mL) at 0°C under nitrogen was added dropwise dibutyl trifluoromethanesulfonate solution (1.0 M in methylene chloride, 36.27 mL, 0.036 mol) and triethylamine (5.66 mL, 0.040 mol). Then, a mixture of CH₂Cl₂ (5 mL) and acetaldehyde (abt. 90%) (4.58 g) was added dropwise at −78°C, and the mixture was stirred at −78°C for 1 hr and then at 0°C for another 2 hr. Methanol : 30% H₂O₂=2 : 1 (150 mL) was added dropwise and stirred at 0°C for 2 hr and then at room temperature overnight. CH₂Cl₂ (100 mL) and H₂O (100 mL) were added to the reaction mixture and extracted with CH₂Cl₂. The organic phase was washed with sat. NaHCO₃ aq. and brine, dried and evaporated. The residue was purified by column chromatography (hexane : ethyl acetate=1 : 1) to afford 14 (6.19 g, 72%). ¹H-NMR (400 MHz, CDCl₃) δ: 1.22 (3H, d, J=7.2 Hz, CH₃–), 1.27 (3H, d, J=7.2 Hz, CH₃–), 2.80 (1H, dd, J=9.2 Hz, 13.2 Hz, one of –CH₂–Ph), 3.06 (1H, br, –OH), 3.25 (1H, dd, J=3.2 Hz, 13.2 Hz, one of –CH₂–Ph), 3.75 (1H, dq, 3.2 Hz, 7.2 Hz, –CH–CH₃), 4.16–4.28 (3H, m, –O–CH₂–, –CH–), 4.65–4.78 (1H, m, –CH–N), 7.16–7.38 (5H, m, C₆H₅–) ¹³C-NMR (100 MHz, CDCl₃) δ: 10.5, 19.5, 37.8, 43.0, 55.1, 66.2, 67.7, 127.4, 128.9, 129.4, 134.9, 153.2, 177.3. [α]²⁵_D −49 (c 1.03, CHCl₃). (S)-4-Benzyl-3-((2S,3R)-3-hydroxy-2- methylbutanoyl)oxazolidin-2-one (ent-14) was prepared from ent-13 using the same procedures. [α]²⁵_D +48 (c 1.20, CHCl₃).

2.3. (R)-4-Benzyl-3-((2R,3S)-3-((tert-butyldimethylsilyl)oxy)-2-methylbutanoyl) oxazolidin-2-one (15) and (S)-4-benzyl-3-((2S,3R)-3-((tert-butyldimethylsilyl)oxy)-2- methylbutanoyl)oxazolidin-2-one (ent-15)

To a solution of 14 (4.37 g, 0.158 mol) in CH₂Cl₂ was added dropwise 2,6-lutidine (4.4 mL, 0.019 mol) at room temperature and tert-butyldimethylsilyl trifluoromethanesulfonate (4.4 mL, 0.038 mol) at 0°C. The mixture was stirred at room temperature for 1 hr, washed with sat. CuSO₄ aq. (2 times), dried and evaporated to afford 15 (5.40 g, 87%). ¹H-NMR (400 MHz, CDCl₃) δ: 0.01 (3H, s, –Si–CH₃), 0.03 (3H, s, –Si–CH₃), 0.86 (9H, s, –Si–C(CH₃)₃), 1.17 (3H, d, J=6.0 Hz, –CH₃), 1.21 (3H, d, J=7.2 Hz, –CH₃), 2.76 (1H, dd, J=9.8 Hz, 13.2 Hz, one of –CH₂–Ph), 3.26 (1H, dd, J=3.0 Hz, 13.2 Hz, one of –CH₂–Ph), 3.76–3.87 (1H, m, –CH–CH₃), 4.03–4.11 (1H, m, –CH–CH₃), 4.12–4.18 (2H, m, –O–CH₂–), 4.55–4.67 (1H, m, –CH–N), 7.14–7.36 (5H, m, C₆H₅–). ¹³C-NMR (100 MHz, CDCl₃) δ: −5.1, −4.5, 12.5, 17.9, 21.6, 25.7, 37.7, 44.9, 55.6, 65.9, 69.5, 127.3, 128.9, 129.4, 135.3, 153.1, 175.2. [α]²⁵_D −45 (c 1.09, CHCl₃). (S)-4-Benzyl-3-((2S,3R)-3-((tert-butyldimethylsilyl)oxy)-2-methylbutanoyl) oxazolidin-2-one (ent-15) was prepared from ent-14 using the same procedures. [α]²⁵_D +47 (c 0.99, CHCl₃).

2.4. (2R,3S)-3-((tert-Butyldimethylsilyl)oxy)-2-methylbutanoic acid (16) and (2S,3R)-3-((tert-Butyldimethylsilyl)oxy)-2-methylbutanoic acid (ent-16)

To a solution of 15 (5.40 g, 0.0138 mol) in THF : H₂O=5 : 4 (90 mL) was added 30% H₂O₂ (14 mL) and LiOH·H₂O (2.31 g, 0.055 mol) at 0°C. The mixture was stirred at room temperature for 18 hr, decomposed with 1.5 M Na₂SO₄ aq. (100 mL) at 0°C and washed with CH₂Cl₂. To the aqueous phase, was added conc. HCl (10 mL) at 0°C to acidify, and the mixture was stirred for 3 min. The resulting mixture was extracted with ethyl acetate, dried and evaporated to afford 16 (2.67 g, 83%). ¹H-NMR (400 MHz, CDCl₃) δ: 0.08 (3H, s, –Si–CH₃), 0.09 (3H, s, –Si–CH₃), 0.89 (9H, s, –Si–C(CH₃)₃), 1.17 (3H, d, J=6.8 Hz, –CH₃), 1.19 (3H, d, J=6.0 Hz, –CH₃), 2.45–2.54 (1H, m, –CH–C=O), 4.08–4.18 (1H, m, –CH–O), 10.53 (1H, br, –COOH). ¹³C-NMR (100 MHz, CDCl₃) δ: −5.2, −4.5, 11.6, 17.9, 20.9, 25.7, 46.8, 69.6, 179.7. [α]²⁵_D −15 (c 1.03, CHCl₃). (2S,3R)-3-((tert-Butyldimethylsilyl)oxy)-2-methylbutanoic acid (ent-16) was prepared from ent-15 using the same procedures. [α]²⁵_D +20 (c 0.99, CHCl₃).

2.5. tert-Butyl ((2R,3S)-3-hydroxybutan-2-yl)carbamate (17) and tert-butyl ((2S,3R)-3-hydroxybutan-2-yl)carbamate (ent-17)

To 16 (2.67 g, 0.0115 mol) was added tert-butyl alcohol (20 mL), triethylamine (1.92 mL, 0.0138 mol) and diphenylphosphonyl azide (3 mL, 0.0138 mol), and the mixture was refluxed for 16 hr. The solvent was evaporated, and the resulting residue was purified by column chromatography (hexane : ethyl acetate=3 : 1) to afford tert-butyl ((2R,3S)-3-((tert-butyldimethylsilyl)oxy)butan-2-yl)carbamate (2.60 g, 74%), which was found to contain some impurities and employed without further purification. To this compound (1.64 g, 0.0054 mol), tetrabutylammonium fluoride ca. 1 mol/L in THF (16.2 mL, 0.0162 mol), and the mixture was stirred at room temperature for 3 hr. To the mixture, CaCO₃ (3.24 g), Dowex^® 50 WX8 hydrogen form hydrogen form, 200–400 mesh (6.0 g) and MeOH (16 mL) were added and stirred for 1 hr. Dowex was removed by filtration over Celite and evaporated. The resulting residue was purified by column chromatography (hexane : ethyl acetate=1 : 4) to afford 17 (0.69 g, 68%). ¹H-NMR (400 MHz, CDCl₃) δ: 1.09 (3H, d, J=7.6 Hz, –CH₃), 1.14 (3H, d, J=6.4 Hz, –CH₃), 1.45 (9H, s, –C(CH₃)₃), 2.65 (1H, br, –OH), 3.71 (1H, br, –CH–CH₃), 3.81–3.89 (1H, m, –CH–CH₃), 4.72 (1H, br, –NH). ¹³C-NMR (100 MHz, CDCl₃) δ: 14.9, 18.5, 28.4, 51.5, 70.6, 79.6, 156.3. [α]²⁵_D +20 (c 0.99, CHCl₃). tert-Butyl ((2S,3R)-3-hydroxybutan-2-yl)carbamate (ent-17) was prepared from ent-16 using the same procedures. [α]²⁵_D −12 (c 1.01, CHCl₃).

2.6. tert-Butyl ((2R,3R)-3-(1,3-dioxoisoindolin-2-yl)butan-2-yl)carbamate (18) and tert-butyl ((2S,3S)-3-(1,3-dioxoisoindolin-2-yl)butan-2-yl)carbamate (ent-18)

To a solution of 17 (0.93 g, 0.0049 mol), phthalimide (1.44 g, 0.0098 mol), and triphenylphosphine (1.93 g, 0.00735 mol) in dry THF at 0°C was added dropwise diethyl azodicarboxylate 40% in toluene, ca. 2.2 mol/L (4.0 mL, 0.00882 mol). The mixture was stirred at room temperature for 23 hr and evaporated. The resulting residue was purified by column chromatography (hexane : ethyl acetate=3 : 1) and dissolved in ethyl acetate. The organic phase was washed with 1 M NaOH aq., dried and evaporated to afford 18 (1.29 g, 82%). ¹H-NMR (400 MHz, CDCl₃) δ: 1.14 (3H, d, J=6.4 Hz, –CH₃), 1.26 (9H, s, –C(CH₃)₃), 1.48 (3H, d, J=7.6 Hz, –CH₃), 4.15–4.25 (1H, m, –CH–CH₃), 4.26–4.37 (1H, m, –CH–CH₃), 5.26 (1H, d, J=9.2 Hz, –NH), 7.68–7.74 (2H, m, Phthaloyl-H), 7.81–7.86 (2H, m, Phthaloyl-H). ¹³C-NMR (100 MHz, CDCl₃) δ: 15.7, 18.8, 28.1, 48.8, 51.7, 79.0, 123.3, 131.9, 134.0, 155.5, 168.9. [α]²⁵_D −12 (c 1.03, CHCl₃). tert-Butyl ((2R,3R)-3-(1,3-dioxoisoindolin-2-yl)butan-2-yl)carbamate (ent-18) was prepared from ent-17 using the same procedures. [α]²⁵_D +18 (c 0.97, CHCl₃).

2.7. 2-((2R,3R)-3-(((6-Chloropyridin-3-yl)methyl)amino)butan-2-yl)isoindoline-1,3-dione (19) and 2-((2S,3S)-3-(((6-chloropyridin-3-yl)methyl)amino)butan-2-yl)isoindoline-1,3-dione (ent-19)

To a solution of 18 (1.29 g, 0.0040 mol) in THF was added conc. HCl (5 mL). The mixture was stirred at room temperature for 19 hr. Toluene was added and azeotroped to remove the solvent. The resulting residue was filtered with a Kiriyama funnel and washed with hexane to afford the deprotected amine (0.97 g, 96%). Triethylamine (1.59 mL, 0.0114 mol) was added to a solution containing the amine (0.97 g, 0.0038 mol) in acetonitrile, and the mixture was stirred for 10 min. A solution of 2-chloro-5-(chloromethyl)pyridine (0.62 g, 0.0038 mol) in acetonitrile was added dropwise, and the mixture was refluxed for 16 hr. The crystals were removed by filtration and washed with ethyl acetate, and the filtrate was evaporated. The resulting residue was purified by column chromatography (hexane : ethyl acetate=1 : 1) to afford 19 (0.46 g, 35%). ¹H-NMR (400 MHz, CDCl₃) δ: 1.22 (3H, d, J=6.4 Hz, –CH₃), 1.48 (3H, d, J=6.8 Hz, –CH₃), 3.15–3.30 (1H, m, –CH–CH₃), 3.56 (1H, d, J=13.6 Hz, one of –CH₂–Pyridine), 3.81 (1H, d, J=13.6 Hz, one of –CH₂–Pyridine), 4.06–4.18 (1H, m, –CH–CH₃), 6.98 (1H, d, J=8.4 Hz, –Pyridine–H), 7.33 (1H, dd, J=8.4 Hz, 2.4 Hz, –Pyridine–H), 7.70–7.76 (2H, m, Phthaloyl–H), 7.77–7.83 (2H, m, Phthaloyl–H), 8.08 (1H, d, J=2.4 Hz, –Pyridine–H), ¹³C-NMR (100 MHz, CDCl₃) δ: 15.9, 18.0, 47.3, 52.4, 54.3, 123.1, 123.7, 131.9, 134.0, 135.2, 138.6, 149.2, 149.7, 168.8. [α]²⁵_D −41 (c 1.06, CHCl₃). 2-((2S,3S)-3-(((6-Chloropyridin-3-yl)methyl)amino)butan-2-yl)isoindoline-1,3-dione (ent-19) was prepared from ent-18 using the same procedures. [α]²⁵_D +38 (c 1.02, CHCl₃).

2.8. 2-Chloro-5-(((4R,5R,E)-4,5-dimethyl-2-(nitromethylene)imidazolidin-1-yl) methyl)pyridine (9) and 2-chloro-5-(((4S,5S,E)-4,5-dimethyl-2-(nitromethylene)imidazolidin-1-yl)methyl)pyridine (10)

To a solution of 19 (0.46 g, 1.2 mmol) in ethanol was added hydrazine monohydrate (0.12 mL, 2.4 mmol) at 0°C, and the mixture was refluxed for 4 hr. After cooling with ice, the precipitated crystals were removed by filtration and washed with chloroform. The solvent was evaporated to afford the deprotected diamine (0.35 g, quant.). This compound was employed in the next reaction without further purification. To a solution containing diamine (0.35 g, 1.6 mmol) in ethanol (100 mL) was added 1,1-bis(methylthio)-2-nitroethylene (0.26 g, 1.6 mmol) and K₂CO₃ (0.22 g, 1.6 mmol), and the mixture was refluxed for 13 hr. The solvent was evaporated, and the resulting residue was purified by column chromatography (ethyl acetate : ethanol=9 : 1) to afford 9 (0.22 g, 49%). White solid, mp. 164–165°C, ¹H-NMR (400 MHz, CDCl₃) δ: 1.30 (3H, d, J=6.0 Hz, –CH₃), 1.36 (3H, d, J=6.4 Hz, –CH₃), 3.33–3.43 (1H, m, –CH–CH₃), 3.67–3.77 (1H, m, –CH–CH₃), 4.30 (2H, d, J=17 Hz, one of –CH₂–Pyridine), 4.38 (2H, d, J=17 Hz, one of –CH₂–Pyridine), 6.55 (1H, s, –CHNO₂), 7.36 (1H, d, J=8.4 Hz, –Pyridine–H), 7.57 (1H, dd, J=2.4 Hz, 8.4 Hz, –Pyridine–H), 8.28 (1H, d, J=2.4 Hz, –Pyridine–H), 8.77 (1H, br, NH). ¹³C-NMR (100 MHz, CDCl₃) δ: 17.2, 19.6, 44.2, 58.1, 63.1, 96.5, 124.8, 129.8, 137.6, 148.3, 151.5, 158.5. [α]²⁵_D +88 (c 0.21, CHCl₃). 100% ee (R_t 18 min). HRMS m/z [M+H]⁺: calcd for C₁₂H₁₆N₄O₂Cl, 283.0962, found, 283.0966. 2-Chloro-5-(((4S,5S,E)-4,5-dimethyl-2-(nitromethylene)imidazolidin-1-yl)methyl)pyridine (10) was prepared from ent-19 using the same procedures. White solid, mp. 143–145°C, [α]²⁵_D −93 (c 0.21, CHCl₃). 100% ee (R_t 22 min). HRMS m/z [M+H]⁺: calcd for C₁₂H₁₆N₄O₂Cl, 283.0962, found, 283.0951.

2.9. tert-butyl ((S)-3-oxo-4-((R)-p-tolylsulfinyl)butan-2-yl)carbamate (20)

The synthetic scheme reported previously was modified.⁹⁾ To a solution of L-alanine methyl ester hydrochloride (3.00 g, 21.5 mmol) in dry MeOH (30 mL) was added triethylamine (4.5 mL, 32.3 mmol) and di-tert-butyl dicarbonate (7.04 g, 32.3 mmol), and the mixture was stirred for 24 hr. The solvent was evaporated, and the resulting residue was dissolved in chloroform and washed with 10% citric acid aq. The organic phase was dried and evaporated to afford (S)-methyl 2-((tert-butoxycarbonyl)amino)propanoate 21 (5.47 g, quant.). Mg (1.59 g), a small amount of iodine and 1,2-dibromoethane were added to dry Et₂O (40 mL), and the mixture was heated and stirred under nitrogen. Methyl iodide (3.47 mL, 0.0558 mol) dissolved in dry Et₂O was added dropwise to afford methylmagnesium iodide. To a solution of (1R,2S,5R)-(−)-menthyl (S)-p-toluenesulfinate (6.0 g, 0.020 mol) in dry Et₂O (50 mL) and dry THF (20 mL) at 0°C under nitrogen was added dropwise over 30 min a solution of methylmagnesium iodide. After the addition was completed, the mixture was stirred at room temperature for 2 hr and then decomposed by the addition of sat. NH₄Cl aq. The solution was extracted with Et₂O, dried and evaporated. The resulting residue was purified by column chromatography (hexane : ethyl acetate=1 : 1) to afford (R)-1-methyl-4-(methylsulfinyl)benzene 22 (2.63 g, 85%). To a solution of diisopropylamine (5.60 mL, 0.02002 mol) in dry THF (60 mL) at −78°C under nitrogen was added n-BuLi ca. 2.8 M (14.14 mL), and the mixture was stirred at −78°C for 30 min. Then, a solution of 22 (1.68 g, 0.01 mol) in dry THF (10 mL) was added dropwise, and the mixture was stirred at −78°C for 1.5 hr. A solution of 21 (2.01 g, 0.01 mol) in dry THF was added dropwise, and the mixture was stirred at −25°C for 3.5 hr. After sat. NH₄Cl aq was added to the reaction mixture, and it was extracted with CH₂Cl₂. The organic phase was washed with brine, dried over Na₂SO₄ and evaporated. The resulting residue was purified by column chromatography (hexane : ethyl acetate=1 : 1) to afford 20 (0.82 g, 25%). ¹H-NMR and ¹³C-NMR data were same as the previously reported. [α]²⁵_D +170 (c 0.96, CHCl₃) ([α]_D +183.9 (c 1, CHCl₃)⁹⁾

2.10. tert-butyl((2S,3R)-3-(1,3-dioxoisoindolin-2-yl)butan-2-yl)carbamate (23)

The synthetic scheme of 23 reported before was modified.¹⁰⁾ To a cooled solution of ZnBr₂ in dry THF (100 mL) was added a solution of 20 (1.66 g, 0.0051 mol) in dry THF at 0°C under nitrogen. The mixture was stirred at this temperature for 1.5 hr. Then, the solution was cooled at −78°C, and diisobutylaluminium hydride 17% in toluene ca. 1.0 mol/L (15.3 mL) was added dropwise. The reaction mixture was stirred at −78°C for 1 hr, and the excess diisobutylaluminium hydride was decomposed by adding MeOH. After the solvent was evaporated, the residue was treated with sat. NH₄Cl aq. and extracted with CH₂Cl₂. The organic phase was washed with brine, dried over Na₂SO₄ and evaporated. The resulting residue was purified by column chromatography (hexane : ethyl acetate=1 : 3) to afford tert-butyl(3-hydroxy-4-(p-tolylsulfinyl)butan-2-yl)carbamate. To MeOH (20 mL) containing the activated Raney Ni (5.0 g) was added a MeOH solution of the carbamate, and the mixture was stirred at room temperature for 1 hr. The catalyst was removed by filtration over Celite and then evaporated to afford tert-butyl(3-hydroxybutan-2-yl)carbamate. To a dry THF solution (60 mL) of tert-butyl(3-hydroxybutan-2-yl)carbamate (0.78 g, 4.12 mmol), phthalimide (1.21 g, 8.24 mmol), and triphenylphosphine (1.62 g, 6.18 mmol) was added dropwise diethyl azodicarboxylate 40% in toluene (3.37 mL, 7.42 mmol) at 0°C. The mixture was stirred at room temperature for 20.5 hr and evaporated. The resulting residue was purified by column chromatography (hexane : ethyl acetate=4 : 1) and dissolved in ethyl acetate. The organic phase was washed with 1 M NaOH aq., dried over Na₂SO₄ and evaporated to afford 23 (0.99 g, 75%). The ¹H and ¹³C-NMR data demonstrated that the cis and trans diastereomers were contained at a ratio of ca. 7 : 3, but the mixtures were employed without further purification because of the difficulty of isolation. ¹³C-NMR (100 MHz, CDCl₃) δ: 14.1, 18.2, 28.3, 49.0, 51.7, 79.3, 123.2, 131.8, 133.9, 155.3, 168.5. These NMR data of cis forms were elucidated by comparing them with those of their trans diastereomers 18 and ent-18.

2.11. 2-Chloro-5-(((4R,5S,E)-4,5-dimethyl-2-(nitromethylene)imidazolidin-1-yl)methyl) pyridine (11)

To a solution of 23 (0.95 g, 3 mmol) in THF was added conc. HCl (8 mL), and the mixture was stirred at room temperature for 23 hr. The solvent was evaporated in vacuo, and the resulting crystalline residue was filtered, followed by washing with hexane to afford 2-((2R,3S)-3-aminobutan-2-yl)isoindoline-1,3-dione hydrochloride salt (0.83 g, quant.). Triethylamine (1.36 mL, 9.78 mmol) was added to a solution of the hydrochloride salt (0.83 g, 0.00326 mol) in acetonitrile, and the mixture was stirred for 10 min. A solution of 2-chloro-5-(chloromethyl)pyridine (0.53 g, 3.26 mmol) in acetonitrile was added dropwise, and the mixture was refluxed for 16 hr. After the crystals were removed by filtration, the filtrate was washed with ethyl acetate and evaporated. The resulting residue was purified by column chromatography (hexane : ethyl acetate=1 : 1) to afford 2-((2R,3S)-3-(((6-chloropyridin-3-yl)methyl)amino)butan-2-yl)isoindoline-1,3-dione (0.40 g, 36%). To a solution of this compound (0.40 g, 1.66 mmol) in ethanol was added hydrazine monohydrate (0.11 mL, 2.32 mmol) at 0°C, and the mixture was refluxed for 15.5 hr. After cooling with ice, the precipitated crystals were removed by filtration and washed with chloroform. The solvent was evaporated to afford (2S,3R)-N²-((6-chloropyridin-3-yl)methyl)butane-2,3-diamine (0.26 g, quant.). To a solution of diamine (0.26 g, 1.22 mmol) in ethanol (100 mL) was added 1,1-bis(methylthio)-2-nitroethylene (0.20 g, 1.22 mmol) and K₂CO₃ (0.17 g, 1.22 mmol), and the mixture was refluxed for 20 hr. The solvent was evaporated, and the resulting residue was purified by column chromatography (ethyl acetate : ethanol=9 : 1) to afford 11 (0.19 g, 53%). amorphous, ¹H-NMR (400 MHz, CDCl₃) δ: 1.20 (3H, d, J=6.8 Hz, –CH₃) 1.27 (3H, d, J=7.2 Hz, –CH₃), 3.83–3.95 (1H, m, –CH–CH₃), 4.14–4.25 (1H, m, –CH–CH₃), 4.28 (1H, d, J=17 Hz, one of –CH₂–Pyridine), 4.43 (1H, d, J=17 Hz, one of –CH₂–Pyridine), 6.56 (1H, s, –CHNO₂), 7.35 (1H, d, J=8.4 Hz, –Pyridine–H), 7.59 (1H, dd, J=8.4 Hz, 2.4 Hz, –Pyridine–H), 8.29 (1H, d, J=2.4 Hz, –Pyridine–H), 8.71 (1H, br, –NH). ¹³C-NMR (100 MHz, CDCl₃) δ: 12.0, 15.0, 43.6, 53.6, 58.2, 96.5, 124.6, 129.9, 137.6, 148.2, 151.2, 158.2. [α]²⁵_D −115 (c 0.24, CHCl₃). 94% ee (R_t 19.5 min). HRMS m/z [M+H]⁺: calcd for C₁₂H₁₆N₄O₂Cl, 283.0962, found, 283.0970.

2.12. 2-chloro-5-(((4S,5R,E)-4,5-dimethyl-2-(nitromethylene)imidazolidin-1-yl)methyl) pyridine (12)

To a solution of 23 (2.13 g, 6.69 mmol) in ethanol was added hydrazine monohydrate (0.65 mL, 13.4 mmol) at 0°C, and the mixture was refluxed for 4 hr. After cooling with ice, the precipitated crystals were removed by filtration and washed with chloroform. The solvent was evaporated to afford tert-butyl ((2S,3R)-3-aminobutan-2-yl)carbamate (1.53 g, quant.). To a solution of this carbamate (1.53 g, 8.18 mmol) in acetonitrile was added triethylamine (2.80 mL, 0.02 mol), and the mixture was stirred for 10 min. A solution of 2-chloro-5-(iodomethyl)pyridine (1.73 g, 0.0067 mol), which was prepared from 2-chloro-5-(chloromethyl)pyridine and sodium iodide, in acetonitrile was added dropwise, and the mixture was refluxed for 15 hr. The crystals were removed by filtration and washed with ethyl acetate, and the organic fraction combined with filtrate was evaporated. The resulting residue was purified by column chromatography (hexane : ethyl acetate=1 : 1) to obtain tert-butyl ((2S,3R)-3-(((6-chloropyridin-3-yl)methyl)amino)butan-2-yl)carbamate (0.46 g, 17%). To a solution of this carbamate (0.46 g, 1.46 mmol) in THF was added conc. HCl (2 mL). The mixture was stirred at room temperature for 17.5 hr. To the resulting mixture was added 2 M NaOH aq. until pH 8, and then the mixture was extracted with chloroform, dried over Na₂SO₄ and evaporated to afford (2R,3S)-N²-((6-chloropyridin-3-yl)methyl)butane-2,3-diamine (0.31 g, 99%). To a solution of diamine (0.31 g, 1.45 mmol) in ethanol (100 mL) was added 1,1-bis(methylthio)-2-nitroethylene (0.24 g, 1.45 mmol) and K₂CO₃ (0.20 g, 1.45 mmol), and the mixture was refluxed for 12.5 hr. The solvent was evaporated, and the resulting residue was purified by column chromatography (ethyl acetate : ethanol=9 : 1) to afford 12 (0.22 g, 53%). amorphous, ¹H- and ¹³C-NMR data were coincided with those of 11. [α]²⁵_D +131 (c 0.40, CHCl₃). 97% ee (R_t 23 min). HRMS m/z [M+H]⁺: calcd for C₁₂H₁₆N₄O₂Cl, 283.0962, found, 283.0962.

3. Evaluation of receptor binding affinity and insecticidal activity

The method of the competitive binding inhibition assay was essentially the same as in our previous reports.⁷⁾ In brief, the housefly head membrane fraction was incubated with various concentrations of the test compound and [³H]IMI (20 nM) 1 hr before filtering through a glass filter (GF/B; Whatman International Ltd., Maidstone, UK) pretreated with 0.1% polyethyleneimine. Radioactivity was measured using a liquid scintillation counter (ALOKA-1000; Aloka Co., Ltd., Tokyo, Japan), and the K_i value was calculated using PRISM software. The K_i values of the test compounds were obtained from three separate assays performed in duplicate and are listed in Table 1.

Table 1. Receptor affinity and insecticidal activity of the CH-IMI analogues against houseflies.

No.	Config.a	Ki (nM)b	ED50 (pmol/insect)c
			None (A)	PBO (B)	Ratio (A)/(B)	NIA (C)	Ratio (A)/(C)	PBO+NIA (D)	Ratio (A)/(D)
2	H	0.0367d	4.18d	1.00d	4.2d	0.128d	33d	0.136d	31d
9	4R5R	2.72±1.27	62.0±9.23 (3)	65.1±11.8 (3)	1.0	3.80±1.84 (3)	16	6.87±3.89 (3)	9.0
10	4S5S	51.7±19.6	275±65.2 (4)	343±70.0 (3)	0.8	10.9±1.86 (3)	25	200±56.9 (3)	1.4
11	4R5S	2.14±0.45	54.1±5.56 (3)	42.4±15.3 (3)	1.3	0.495±0.0471 (3)	109	7.68±2.68 (3)	7.0
12	4S5R	0.394±0.179	5.49±1.67 (3)	4.09±1.45 (3)	1.3	0.747±0.238 (3)	7.3	0.103±0.0328 (3)	53
3	5R	0.0428d	5.82d	1.72d	3.4d	0.474d	12d	0.0854d	68d
4	5S	0.313d	21.0d	4.85d	4.3d	0.829d	25d	0.643d	33d
5	4R	2.07d	6.30d	7.99d	0.8d	1.12d	5.6d	0.774d	8d
6	4S	5.64d	16.1d	11.4d	1.4d	0.943d	17d	1.00d	16d
7	4,4-	1550e	nd	nd	—	nd	—	6.34e	—
8	5,5-	1.05e	nd	nd	—	nd	—	2.15e	—
1	IMI	3.72f	nd	nd	—	nd	—	0.429f	—

^aPosition of the imidazolidine ring attached by the methyl groups and its configuration for Compounds 3–12. Compound 2 is unsubstituted. ^bThe inhibition constant of test chemicals to [³H]imidacloprid binding to nAChR. K_i values of the test compounds were obtained in six separate experiments. Data are represented as the mean and standard error of the mean. See detailed methods in Ref. 7. ^cThe effective dose for inducing paralysis or death 1 hr after injection in 50% of female adult houseflies. The value was calculated using the probit transformation. Data are represented as the mean and standard error of the mean (the figure in parentheses is the number of experiments). ^dCited in Ref. 5. ^eCalculated from the data cited in Ref. 12. ^fCalculated from the data cited in Ref. 6.

For evaluating the insecticidal activity, female houseflies anesthetized using carbon dioxide were topically treated with methanol containing synergists piperonyl butoxide and/or NIA16388 [0.2% (w/v)]. After 1 hr, 0.22 µL of 50% ethanol solutions containing a test chemical at various concentrations were injected in the dorsal side of the thorax of reanesthetized flies. Insecticidal activity was evaluated 1 hr after injection. The ED₅₀ values (effective dose for inducing paralysis or death in 50% of the houseflies) were calculated using probit transformation and are listed in Table 1.

Results and discussion

1. Receptor affinity

The inhibition constant, K_i (nM), was evaluated as an indicator of the affinity to the receptor (Table 1). In our previous studies, R-5-CH-IMI 3 showed the highest affinity among monomethylated CH-IMIs 3–6, and 3 was equipotent to CH-IMI 2.⁵⁾ Comparing the receptor affinity of 5,5-diMe-CH-IMI 8 with that of 4,4-diMe-CH-IMI 7, dimethylation at the 4 position of the imidazolidine ring was more unfavourable than dimethylation at the 5 position.⁴⁾ Among 4,5-dimethylated CH-IMIs 9–12, 4S,5R-diMe-CH-IMI 12 was found to show the highest affinity (0.394 nM). The 4R,5R- and 4R,5S-diMe-CH-IMI derivatives 9 and 11 were approximately 6-fold weaker than 12. 4S,5S-diMe-CH-IMI 10 showed the lowest affinity, which was approximately 130-fold weaker than 12.

Our previous study suggests that alkyl groups such as methyl or ethyl groups attached at the R-5-position should interact with the aromatic residues of the receptor via CH-π interactions. In addition, it is also suggested that the ligand binding region of the receptor surrounding the imidazolidine ring, especially around the R-5 and S-4 positions, would expand by the movement of some amino acid residues of the receptor subunit when interacting with bulky ligands.⁶⁾ This movement would allow the receptor to accept larger compounds, even dimethylated CH-IMI derivatives. The R-5- and S-4-monomethylated Compounds 3 and 6 were the highest and lowest among the monomethylated compounds, respectively, whereas Compound 12, having the syn-substituted S4,R5-dimethyl groups, showed the highest potency. As the receptors have the flexibility to accept the ligands, the ligand binding regions may change their shapes depending on the bulkiness of the ligands. This suggests that the R-5-methyl group could positively interact with both of the receptor forms (extended and nonextended forms), whereas the S-4-methyl group would exist without positively interacting with the receptor, even though in the extended ligand binding region, because the receptor affinity of Compound 6 was not as high.

2. Insecticidal activity and correlation between the biological activities

The ED₅₀ values were evaluated as an indicator of the insecticidal activity (Table 1). Among Compounds 9–12 under nonsynergistic conditions, 4S,5R-dimethylated Compound 12 showed the highest activity (5.49 pmol/fly), which was equipotent to unsubstituted CH-IMI 2. The 4R,5R- and 4R,5S-dimethylated Compounds 9 and 11 were approximately 10-fold less potent than 12, whereas the 4S,5S-dimethylated Compound 10 showed the lowest potency, 50-fold less potent than 12. The order of the insecticidal potency of the four diMe-CH-IMIs (4S,5R->4R,5S-=4R,5R->4S,5S-diMe analogues) was the same as that of the receptor affinity.

Under PBO-treated conditions, the insecticidal activity of dimethylated Compounds 9–12 did not synergize (the values of the synergistic ratio were 0.8–1.3). In our previous study, 4-monomethylated Compounds 5 and 6 also did not synergize under PBO treatment conditions, demonstrating that the compounds containing the methyl group at the 4-position of the imidazolidine ring could not be synergized by PBO. PBO is reported to inhibit kinds of oxidative enzymes, cytochrome P450s, and these cytochrome P450s seem less metabolize these 4-methylated compounds, and the other enzymes might be involved in metabolizing them. Alternatively, the metabolites produced by cytochrome P450s may exert the same insecticidal activity as the original compounds, and the metabolites may be transformed into more active compounds in houseflies, as seen in the example of olefin IMI, which is the high biological active metabolite in houseflies.⁸⁾ On the other hand, all 4,5-dimethylated compounds synergized 7–109-fold under NIA treatment conditions. NIA is reported to act as both an oxidase and esterase inhibitor,¹¹⁾ and our previous study showed that NIA suppressed the oxidative metabolism of IMI in houseflies.⁸⁾ Taking them into consideration, PBO and NIA should suppress the different metabolic enzymes. Under both synergist-treated conditions, the synergistic effects of NIA on Compounds 10 and 11 were largely nullified, especially 4S,5S-dimethylated Compound 10, which was not synergized (the synergistic ratio was 1.4). The reason remains unknown, but the metabolites may show insecticidal activity. To clear them, the analyses of structure of the metabolites and the evaluation of their biological activities should be performed.

Conclusion

We found that 4S,5R-dimethylated Compound 12 showed high biological activities, suggesting that the bulkiness of the imidazolidine moiety to some degree should be acceptable for receptor recognition, which is consistent with the high biological activities of derivatives having hexahydropyrimidine instead of the imidazolidine of CH-IMIs. A more detailed SAR study of this moiety will be conducted, and the present analyses may provide useful information for designing new insecticides.

Acknowledgements

This study, in part, was supported by Pesticide Science Society of Japan (HN). A part of this study was performed at the ADRES (Tarumi station) of Ehime University.

Electronic supplementary materials

The online version of this article contains supplementary materials, which are available at https://www.jstage.jst.go.jp/browse/jpestics/.

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