Synthesis of Novel 3,4-Chloroisothiazole-Based Imidazoles as

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Article Cite This: J. Agric. Food Chem. 2018, 66, 7319−7327

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Synthesis of Novel 3,4-Chloroisothiazole-Based Imidazoles as Fungicides and Evaluation of Their Mode of Action Lai Chen,† Bin Zhao,*,† Zhijin Fan,*,†,‡ Xiumei Liu,† Qifan Wu,† Hongpeng Li,† and Haixia Wang† †

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State Key Laboratory of Elemento-Organic Chemistry, College of Chemistry, Nankai University, No. 94, Weijin Road, Nankai District, Tianjin 300071, P. R. China ‡ Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, No. 94, Weijin Road, Nankai District, Tianjin 300071, P. R. China S Supporting Information *

ABSTRACT: A molecular design approach was used in our laboratory to guide the development of imidazole-based fungicides. Based on homology modeling and molecular docking studies targeting the cytochrome P450-dependent sterol 14αdemethylase, 3,4-dichloroisothiazole-based imidazoles showed great potential. Several such compounds were then rationally designed, synthesized, characterized, and their antifungal activities were evaluated. Bioassay results showed that compounds such as (R)-11, (R)-12, and (S)-11 have commendable, broad-spectrum antifungal activities that are comparable to those of commercial products. Based on Q-PCR testing and microscopy observations, the imidazole derivatives affect fungal cell wall formation through the inhibition of the BcCYP51 expression system. These findings strongly suggest that the mode of action of these imidazole compounds is similar to that of tioconazole and imazalil. This report indicates that this molecular design strategy is not only practical but productive. KEYWORDS: BcCYP51, fungicide, 3,4-dichloroisothiazole, imidazole, mode of action

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INTRODUCTION Producing enough food for our growing population and keeping up with the increasingly strict safety regulations for consumers and the environment are a great challenge for modern agriculture.1−3 Agrochemicals have been one of the most effective tools for increasing both crop quality and quantity while reducing labor costs.4 Smarter approaches are needed for identifying more effective agrochemicals that can meet the increasing market demands and environment protection requirements.5 Starting with heterocyclic compounds that have previously played important roles in agrochemicals is a logical approach.6−9 Over a dozen imidazole-based compounds have been widely used as antifungal agents (imazalil) for plant protection and for the protection of animal health (tioconazole, econazole, and miconazole) (Figure 1).10−12 It is commonly believed that imidazole-based compounds can

inhibit ergosterol biosynthesis, thus limiting fungal growth by affecting their cytochrome P450 sterol 14α-demethylase. Further studies have suggested that the imidazole ring efficiently binds with the pro-heme iron in the enzyme.13 Heterocycles have also been widely used to improve biological activity in the optimization of lead compounds in agrochemical studies.14,15 Five-membered aromatic frameworks, such as 3,4-dichloroisothiazole, which contains N and S atoms, have potentially inducing plant defense responses, antifungal, antiviral, and antitumor activities.16,17 Therefore, it could be a potential bioactive scaffold. In fact, several heterocyclic analogues such as isotianil and dichlobentiazox were successfully developed as novel fungicides for rice blast management by inducing plant defense responses.17,18 We have designed and synthesized a series of novel 3,4dichloroisothazoles to obtain novel fungicides for controlling plant pathogens such as Botrytis cinerea and to conveniently study their modes of action by homology modeling and molecular docking using B. cinerea cytochrome P450-dependent sterol 14α-demethylase as the target and by combining 2,4dichlorobenzene imidazole fungicide lead compounds with 3,4dichloroisothiazole, a substructure with systemic acquired resistance activity. Specific bioassays were used to verify the validity of our molecular design models (Figure 1). Received: Revised: Accepted: Published:

Figure 1. Design of target compounds. © 2018 American Chemical Society

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May 3, 2018 June 13, 2018 June 18, 2018 June 18, 2018 DOI: 10.1021/acs.jafc.8b02332 J. Agric. Food Chem. 2018, 66, 7319−7327

Article

Journal of Agricultural and Food Chemistry

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The preparation of compound 4b followed the same steps as described above, except compound 3b was used as the starting material. 4b: colorless oil; yield, 50−60%; [α]24 D = −23.7 (c 1.00, CH3OH); 1H NMR (400 MHz, CDCl3) δ 4.11 (dd, J = 3.9, 2.4 Hz, 1H, CH), 3.23 (dd, J = 5.3, 3.9 Hz, 1H, CH2), 2.87 (dd, J = 5.3, 2.4 Hz, 1H, CH2); 13C NMR (101 MHz, CDCl3) δ 161.26 (s), 148.55 (s), 120.85 (s), 52.19 (s), 47.44 (s); HRMS (m/z) calcd for C5H3Cl2NOS (M − H)+: 193.9312, found 193.9242. General Procedure for the Synthesis of Compounds 5a and 5b. Potassium carbonate (0.36 g, 2.62 mmol) and imidazole (0.18 g, 2.62 mmol) were added to a solution of compound 4a (0.42 g, 1.75 mmol) in 5 mL of N,N-dimethylformamide. The mixture was stirred at room temperature for 20 h and then washed with water; the organic layer was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated, and the residue was then purified on a silica gel column (203 mm × 26 mm) eluted with ethyl acetate/petroleum ether (bp 60−90 °C) (1:20, v/v) and afforded compound 5a in a yield 80− 86%. 5a: colorless crystal; m.p., 156−157 °C; [α]24 D = +45.4 (c 1.00, CH3OH); 1H NMR (400 MHz, DMSO-d6) δ 7.55 (s, 1H, imidazoleH), 7.09 (s, 1H, imidazole-H), 6.88 (s, 1H, imidazole-H), 5.28 (dd, J = 7.1, 3.0 Hz, 1H, OCH), 4.39−4.31 (m, 2H, CH2); 13C NMR (101 MHz, DMSO-d6) δ 167.84 (s), 147.21 (s), 138.39 (s), 128.50 (s), 120.76 (s), 116.63 (s), 68.20 (s), 50.83 (s); HRMS (m/z) calcd for C8H7Cl2N3OS (M + H)+: 263.9687, found 263.9755. The preparation of the compound 5b followed the same steps as described above, except compound 4b was used as the starting material. 5b: colorless crystal; yield, 50−60%; m.p., 156−157 °C; 1 [α]24 D = −44.9 (c 1.00, CH3OH); H NMR (400 MHz, DMSO-d6) δ 7.53 (s, 1H, imidazole-H), 7.08 (s, 1H, imidazole-H), 6.86 (s, 1H, imidazole-H), 5.25 (s, 1H, OCH), 4.40−4.32 (m, 1H, NCH2), 4.28 (dd, J = 14.3, 5.9 Hz, 1H, NCH2); 13C NMR (101 MHz, DMSO-d6) δ 167.02 (s), 147.56 (s), 137.44 (s), 127.70 (s), 121.07 (s), 116.94 (s), 67.93 (s), 50.75 (s); HRMS (m/z) calcd for C8H7Cl2N3OS (M + H)+: 263.9687, found 263.9755. General Procedure for the Synthesis of Compounds (R)-1− 12 and (S)-1, (S)-11−12. Sodium hydroxide (1 mol/L, 0.5 mL) and the desired amount of tetrabutylammonium bromide (TBAB) catalyst was added to a solution of R1CH2Br (0.33 mmol) and compounds 5 (0.08 g, 0.30 mmol) in tetrahydrofuran. The solution was stirred at room temperature for 3 h. The solvent was evaporated, and the residue was then purified on a silica gel column (203 mm × 26 mm) eluted with ethyl acetate/petroleum ether (bp 60−90 °C) (1:20, v/v) and afforded compounds R and S (Figure 3). The yields, physical properties, 1H NMR, 13C NMR, and HRMS data of the target compounds (R)-1−12 and (S-1), (S)-11−12 were as follows. Data for (R)-1. White solid; yield, 89%; m.p., 40−41 °C; [α]24 D = +6.5 (c 1.00, CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.42 (s, 1H, imidazole-H), 6.97 (s, 1H, imidazole-H), 6.87 (s, 1H, imidazole-H), 5.72−5.58 (m, 1H, CHCH2), 5.16 (s, 1H, CHCH2) 5.13 (d, J = 3.9 Hz, 1H, CHCH2), 4.83 (dd, J = 4.7, 2.4 Hz, 1H, OCH), 4.26 (dd, J = 5.9, 2.9 Hz, 1H, OCH2), 4.13 (dd, J = 14.5, 7.2 Hz, 1H, OCH2), 4.02 (dd, J = 11.9 Hz, J = 2.9 Hz, 1H, NCH2), 3.86 (dd, J = 11.9, 5.9 Hz, 1H, NCH2); 13C NMR (101 MHz, CDCl3) δ 161.82 (s), 148.56 (s), 137.79 (s), 132.49 (s), 129.43 (s), 119.79 (s), 119.26 (s), 118.86 (s), 75.20 (s), 71.91 (s), 50.35 (s); UV (CH3OH): λmax (log ε) 280 (2.67), 239 (2.00) nm; HRMS (m/z) calcd for C11H11Cl2N3OS (M + H)+: 304.0000, found 304.0074. Data for (R)-2. White solid; yield, 67%; m.p., 49−50 °C; [α]24 D = +12.9 (c 0.67, CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.24 (s, 1H, imidazole-H), 6.79 (s, 1H, imidazole-H), 6.69 (s, 1H, imidazole-H), 4.94 (dd, J = 6.6, 2.9 Hz, 1H, OCH), 4.14 (dd, J = 14.7, 2.8 Hz, 1H, OCH2), 4.06−3.96 (m, 2H, OCH2 and NCH2), 3.90 (dd, J = 16.1, 2.3 Hz, 1H, NCH2), 2.23 (t, J = 2.2 Hz, 1H, CCH); 13C NMR (101 MHz, CDCl3) δ 160.62 (s), 148.70 (s), 137.79 (s), 129.54 (s), 119.88 (s), 119.25 (s), 77.27 (s), 76.75 (s), 74.86 (s), 58.15 (s), 50.24 (s); UV (CH3OH): λmax (log ε) 277 (2.75), 242 (2.78), 239 (2.60) nm; HRMS (m/z) calcd for C11H9Cl2N3OS (M + H)+: 301.9843, found 301.9919.

MATERIALS AND METHODS

Equipment and Materials. Melting points of the new compounds were determined in an X-4 melting point apparatus (Beijing Tech Instruments Co., Beijing, China). 1H NMR and 13C NMR spectra were taken on an Avance 400 spectrometer at 400 and 101 MHz (Bruker, Switzerland). High-resolution mass spectra (HRMS) were obtained by using a 7.0 T Fouurier transform-ion cyclotron resonance-mass spectrometry (FTICR-MS) instrument (Varian, Palo, Alto, CA). Crystal structure was recorded on a Saturn 724 CCD diffractometer (Rigaku, Tokyo, Japan). UV spectra were determined with a Cary 5000 UV spectrophotometer (Agilent, Australia). Optical rotations were measured on a 341MC polarimeter (PerkinElmer, Norwalk, CT). General Procedure for the Synthesis of Compound 2. Compound 1 was synthesized as described by Chen et al.19 Compound 2 was prepared from corresponding compound 1. To a stirred solution of compound 2 (0.60 g, 3.06 mmol) in a solution of 33% hydrogen bromide in acetic acid (5 mL) was added pyridinium tribromide (1.08 g, 3.37 mmol).20 The mixture was stirred at room temperature for 3 h and then poured into ice-cold water; sodium bicarbonate was added until the mixture became clear and colorless. The organic layer was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated, and the residue was then purified on a silica gel column (203 mm × 26 mm) eluted with ethyl acetate/petroleum ether (bp 60−90 °C) (1:20, v/v) and afforded compound 2 in a 95% yield. 2: white solid; m.p., 52−53 °C; 1H NMR (400 MHz, CDCl3) δ 4.47 (s, 2H, CH2); 13C NMR (101 MHz, CDCl3) δ 182.30 (s), 156.43 (s), 150.49 (s), 123.25 (s), 32.26 (s); HRMS (m/z) calcd for C5H2BrCl2NOS (M − H)+, 271.8418, found 271.8344. General Procedure for the Synthesis of Compounds 3a and 3b.21 (+)-Diisopinocampheyl chloroborane (DIP-Cl) (3.40 mmol, dissolved in 2 mL of dry heptane) was added to compound 2 (0.47 g, 1.70 mmol, dissolved in 15 mL of dry tetrahydrofuran) under argon at −20 °C for 2 h. The solution was stirred for 16 h at room temperature. The tetrahydrofuran and heptane were removed in vacuo, and the (+)-α-pinene was removed under a high vacuum at room temperature overnight. The resulting viscous colorless oil was then purified on a silica gel column (203 mm × 26 mm) eluted with ethyl acetate/petroleum ether (bp 60−90 °C) (1:20, v/v) and afforded compound 3a in a 50−60% yield. 3a: white solid; m.p., 57− 1 58 °C; [α]24 D = +38.9 (c 1.00, CH3OH); H NMR (400 MHz, CDCl3) δ 5.21 (dd, J = 7.8 Hz, 3.0 Hz, 1H, CH), 3.82 (dd, J = 10.8, 3.0 Hz, 1H, CH2), 3.53 (dd, J = 10.8, 7.8 Hz, 1H, CH2); 13C NMR (101 MHz, CDCl3) δ 163.23 (s), 148.55 (s), 118.08 (s), 69.06 (s), 36.29 (s); HRMS (m/z) calcd for C5H4BrCl2NOS (M − H)+: 273.8574, found 273.8501. The preparation of compound 3b followed the same steps as the above procedure, except the reducing agent was (−)-DIP-Cl. 3b: white solid; yield, 50−60%; m.p., 57−58 °C; [α]24 D = −38.2 (c 0.90, CH3OH); 1H NMR (400 MHz, CDCl3) δ 5.28 (dd, J = 7.8, 3.0 Hz, 1H, OCH), 3.88 (dd, J = 10.8, 3.0 Hz, 1H, CH2), 3.60 (dd, J = 10.8, 7.8 Hz, 1H, CH2); 13C NMR (101 MHz, CDCl3) δ 163.87 (s), 148.57 (s), 118.05 (s), 68.90 (s), 36.07 (s); HRMS (m/z) calcd for C5H4BrCl2NOS (M − H)+: 273.8574, found 273.8501. General Procedure for the Synthesis of Compounds 4a and 4b. Potassium carbonate (0.36 g, 2.62 mmol) was added to a solution of compound 3a (0.61 g, 2.19 mmol) in acetone. The mixture was stirred at room temperature for 20 h and then filtered. The solvent was evaporated, and the residue was then purified on a silica gel column (203 mm × 26 mm) eluted with ethyl acetate/petroleum ether (bp 60−90 °C) (1:20, v/v) and afforded compound 4a in a 50− 1 60% yield. 4a: colorless oil; [α]24 D = +23.3 (c 1.00, CH3OH); H NMR (400 MHz, CDCl3) δ 4.20 (dd, J = 3.9, 2.3 Hz, 1H, CH), 3.33 (dd, J = 5.3, 3.9 Hz, 1H, CH2), 2.96 (dd, J = 5.3, 2.3 Hz, 1H, CH2); 13 C NMR (101 MHz, CDCl3) δ 161.30 (s), 148.41 (s), 120.77 (s), 52.10 (s), 47.36 (s); HRMS (m/z) calcd for C5H3Cl2NOS (M − H)+: 193.9312, found 193.9242. 7320

DOI: 10.1021/acs.jafc.8b02332 J. Agric. Food Chem. 2018, 66, 7319−7327

Article

Journal of Agricultural and Food Chemistry Data for (R)-3. White solid; yield, 67%; m.p., 78−79 °C; [α]24 D = +37.5 (c 0.13, CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.42 (s, 1H, imidazole-H), 7.30−7.23 (s, 3H, Ph-H), 7.11 (d, J = 2.9 Hz, 2H, PhH), 6.98 (s, 1H, imidazole-H), 6.83 (s, 1H, imidazole-H), 4.86 (dd, J = 7.2, 2.0 Hz, 1H, OCH), 4.56 (d, J = 11.4, 1H, OCH2), 4.34 (d, J = 11.4, 1H, OCH2), 4.24 (dd, J = 14.6 Hz, 2.0 Hz, 1H, NCH2), 4.13 (dd, J = 14.4, 7.2 Hz, 1H, NCH2); 13C NMR (101 MHz, CDCl3) δ 161.57 (s), 148.69 (s), 137.89 (s), 135.72 (s), 129.70 (s), 128.69 (s), 128.10 (s), 119.77 (s), 119.21 (s), 75.28 (s), 73.04 (s), 50.48 (s); UV (CH3OH): λmax (log ε) 279 (2.84), 242 (2.60) nm; HRMS (m/z) calcd for C15H13Cl2N3OS (M + H)+: 354.0156 found 354.0227. Data for (R)-4. White solid; yield, 68%; m.p., 56−57 °C; [α]24 D = +24.4 (c 0.50, CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.72 (s, 1H, imidazole-H), 7.37 (dd, J = 8.2, 5.5 Hz, 2H, Ph-H), 7.27 (s, 1H, imidazole-H), 7.24 (t, J = 8.6 Hz, 2H, Ph-H), 7.13 (s, 1H, imidazoleH), 5.14 (dd, J = 7.5, 2.9 Hz, 1H, OCH), 4.81 (d, J = 11.4 Hz, 1H, OCH2), 4.59 (d, J = 11.4 Hz, 1H, OCH2), 4.54 (dd, J = 14.6, 2.9 Hz, 1H, NCH2), 4.44 (dd, J = 14.6, 7.5 Hz, 1H, NCH2); 13C NMR (101 MHz, CDCl3) δ 162.74 (d, J = 247.4 Hz), 161.34 (s), 148.78 (s), 137.85 (s), 131.56 (d, J = 3.1 Hz), 129.95 (d, J = 8.4 Hz), 129.71 (s), 119.66 (s), 119.33 (s), 115.70 (d, J = 21.7 Hz), 75.24 (s), 72.26 (s), 50.49 (s); UV (CH3OH): λmax (log ε) 277 (2.94), 242 (2.99) nm, 239 (2.96); HRMS (m/z) calcd for C15H12Cl2FN3OS (M + H)+: 372.0062, found 372.0133. Data for (R)-5. Colorless crystal; yield, 85%; m.p., 68−69 °C; [α]24 D = +60.5 (c 0.50, CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.63 (s, 1H, imidazole-H), 7.43 (d, J = 7.3 Hz, 2H, Ph-H), 7.23 (d, J = 7.5 Hz, 2H, Ph-H), 7.18 (s, 1H, imidazole-H), 7.04 (s, 1H, imidazole-H), 5.06 (d, J = 7.0 Hz, 1H, OCH), 4.73 (d, J = 11.5 Hz, 1H, OCH2), 4.53−4.43 (m, 2H, OCH2 and NCH2), 4.35 (dd, J = 14.5, 7.3 Hz, 1H, NCH2); 13C NMR (101 MHz, CDCl3) δ 161.19 (s), 148.78 (s), 137.84 (s), 134.38 (s), 134.24 (s), 129.69 (s), 129.33 (s), 128.92 (s), 119.71 (s), 119.40 (s), 75.36 (s), 72.13 (s), 50.45 (s); UV (CH3OH): λmax (log ε) 277 (2.95), 242 (2.97) nm; HRMS (m/z) calcd for C15H12Cl3N3OS (M + 3H)+: 389.9767, found 389.9813. Data for (R)-6. Colorless crystal; yield, 69%; m.p., 81−82 °C; [α]24 D = +16.8 (c 1.00, CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.54 (d, J = 7.7 Hz, 2H, Ph-H), 7.47 (s, 1H, imidazole-H), 7.23 (d, J = 7.5 Hz, 2H, Ph-H), 7.00 (s, 1H, imidazole-H), 6.86 (s, 1H, imidazole-H), 4.90 (d, J = 7.1 Hz, 1H, OCH), 4.63 (d, J = 11.9 Hz, 1H, OCH2), 4.42 (d, J = 11.9 Hz, 1H, OCH2), 4.31 (d, J = 14.5 Hz, 1H, NCH2), 4.19 (dd, J = 14.5, 7.4 Hz, 1H, NCH2); 13C NMR (101 MHz, CDCl3) δ 160.86 (s), 148.90 (s), 139.76 (s), 137.84 (s), 130.76 (s), 130.43 (s), 129.67 (s), 127.91 (s), 125.69 (q, J = 3.6 Hz), 119.69 (s), 119.58 (s), 75.76 (s), 72.07 (s), 50.48 (s); UV (CH3OH): λmax (log ε) 279 (2.78), 239 (2.52) nm; HRMS (m/z) calcd for C16H12Cl2F3N3OS (M + H)+: 422.0030, found 422.0101. Data for (R)-7. Colorless crystal; yield, 64%; m.p., 44−45 °C; [α]24 D = +8.5 (c 0.40, CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.38 (s, 1H, imidazole-H), 7.25 (d, J = 7.0 Hz, 2H, Ph-H), 7.02 (d, J = 7.2 Hz, 2H, Ph-H), 6.92 (s, 1H, imidazole-H), 6.79 (s, 1H, imidazole-H), 4.83 (d, J = 6.9 Hz, 1H, OCH), 4.48 (d, J = 11.1 Hz, 1H, OCH2), 4.28 (d, J = 11.1 Hz, 1H, OCH2), 4.19 (d, J = 14.2 Hz, 1H, NCH2), 4.08 (dd, J = 14.2, 6.9 Hz, 1H, NCH2), 1.20 (s, 9H, t-Bu-H); 13C NMR (101 MHz, CDCl3) δ 161.79 (s), 151.70 (s), 148.62 (s), 137.86 (s), 132.72 (s), 129.63 (s), 127.99 (s), 125.67 (s), 119.73 (s), 119.08 (s), 75.35 (s), 72.98 (s), 50.49 (s), 31.30 (s); UV (CH3OH): λmax (log ε) 279 (2.70), 242 (2.29) nm; HRMS (m/z) calcd for C19H21Cl2N3OS (M + H)+: 410.0782, found 410.0862. Data for (R)-8. White solid; yield, 91%; m.p., 88−89 °C; [α]24 D = +29.0 (c 0.90, CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.48 (s, 1H, imidazole-H), 7.38 (d, J = 1.8 Hz, 1H, Ph-H), 7.25 (dd, J = 8.3, 1.9 Hz, 1H, Ph-H), 7.18 (d, J = 8.3 Hz, 1H, Ph-H), 7.04 (s, 1H, imidazole-H), 6.91 (s, 1H, imidazole-H), 5.01 (dd, J = 7.1, 3.0 Hz, 1H, OCH), 4.66 (d, J = 12.2 Hz, 1H, OCH2), 4.54 (d, J = 12.2 Hz, 1H, OCH2), 4.37 (dd, J = 14.6, 3.0 Hz, 1H, NCH2), 4.25 (dd, J = 14.6, 7.1 Hz, 1H, NCH2); 13C NMR (101 MHz, CDCl3) δ 160.92 (s), 148.74 (s), 137.81 (s), 134.96 (s), 134.07 (s), 132.26 (s), 130.43 (s), 129.76 (s), 129.44 (s), 127.50 (s), 119.63 (s), 119.33 (s), 76.23 (s), 69.58 (s), 50.36 (s); UV (CH3OH): λmax (log ε) 278 (2.72), 242

(2.56) nm; HRMS (m/z) calcd for C15H11Cl4N3OS (M + H)+: 421.9377, found 421.9451. Data for (R)-9. Colorless crystal; yield, 61%; m.p., 64−65 °C; [α]24 D = +53.2 (c 0.50, CH3OH); 1H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 1.3 Hz, 1H, pyridine-H), 7.44−7.35 (m, 2H, pyridine-H and imidazole-H), 7.23 (d, J = 8.3 Hz, 1H, pyridine-H), 6.96 (s, 1H, imidazole-H), 6.84 (s, 1H, imidazole-H), 4.89 (dd, J = 7.5, 2.8 Hz, 1H, OCH), 4.55 (d, J = 11.9 Hz, 1H, OCH2), 4.34 (d, J = 11.9 Hz, 1H, OCH2), 4.29 (dd, J = 14.6, 2.8 Hz,1H, NCH2), 4.17 (dd, J = 14.6, 7.5 Hz, 1H, NCH2); 13C NMR (101 MHz, CDCl3) δ 160.49 (s), 151.63 (s), 148.95 (s), 148.89 (s), 138.39 (s), 137.78 (s), 130.44 (s), 129.79 (s), 124.56 (s), 119.74 (s), 119.65 (s), 75.89 (s), 69.53 (s), 50.39 (s); UV (CH3OH): λmax (log ε) 281 (2.25), 239 (1.42) nm; HRMS (m/z) calcd for C14H11Cl3N4OS (M + H)+: 388.9719, found 388.9790. Data for (R)-10. White solid; yield, 92%; m.p., 147−149 °C; [α]24 D = +15.7 (c 0.66, CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 8.6 Hz, 1H, Ar−H), 7.67 (d, J = 1.8 Hz, 1H, Ar−H), 7.44 (s, 1H, imidazole-H), 7.35 (dd, J = 9.7, 2.8 Hz, 2H, Ar−H), 7.03 (s, 1H, imidazole-H), 6.87 (s, 1H, imidazole-H), 4.98 (dd, J = 7.2, 3.1 Hz, 1H, OCH), 4.81 (d, J = 12.2 Hz, 1H, OCH2), 4.63 (d, J = 12.2 Hz, 1H, OCH2), 4.29 (dd, J = 14.6, 3.1 Hz, 1H, NCH2), 4.19 (dd, J = 14.6, 7.2 Hz, 1H, NCH2); 13C NMR (101 MHz, CDCl3) δ 160.97 (s), 148.71 (s), 138.73 (s), 138.63 (s), 137.62 (s), 130.90 (s), 130.41 (s), 129.44 (s), 128.45 (s), 125.34 (s), 123.91 (s), 121.48 (s), 119.74 (s), 119.43 (s), 75.18 (s), 66.61 (s), 50.42 (s); UV (CH3OH): λmax (log ε) 278 (2.79), 249 (2.52) nm; HRMS (m/z) calcd for C17H12Cl3N3OS2 (M + H)+: 443.9487, found 443.9559. Data for (R)-11. White solid; yield, 44%; m.p., 106−107 °C; [α]24 D = +4.0 (c 1.00, CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.42 (s, 1H, imidazole-H), 7.04−6.95 (m, 2H, thiophene-H and imidazole-H), 6.82 (s, 1H, imidazole-H), 6.70 (d, J = 5.3 Hz, 1H, thiophene-H), 4.84 (d, J = 7.3 Hz, 1H, OCH), 4.49 (d, J = 11.8 Hz, 1H, OCH2), 4.37 (d, J = 11.8 Hz, 1H, OCH2), 4.22 (d, J = 14.2 Hz, 1H, NCH2), 4.11 (dd, J = 14.2, 7.3 Hz, 1H, NCH2); 13C NMR (101 MHz, CDCl3) δ 161.30 (s), 148.68 (s), 137.79 (s), 132.95 (s), 129.67 (s), 129.23 (s), 127.46 (s), 123.76 (s), 119.61 (s), 119.21 (s), 75.38 (s), 65.03 (s), 50.43 (s); UV (CH3OH): λmax (log ε) 278 (2.99) nm; HRMS (m/z) calcd for C13H10Cl3N3OS2 (M + H)+: 393.9331, found 393.9403. Data for (R)-12. Colorless crystal; yield, 99%; m.p., 100−101 °C; 1 [α]24 D = +48.3 (c 0.40, CH3OH); H NMR (400 MHz, CDCl3) δ 7.71 (t, J = 7.9 Hz, 3H,), 7.47 (s, 1H, imidazole-H), 7.45−7.36 (m, 3H, naphthalene-H), 7.15 (d, J = 8.4 Hz, 1H, naphthalene-H), 6.95 (s, 1H, imidazole-H), 6.79 (s, 1H, imidazole-H), 4.86 (dd, J = 7.2, 3.0 Hz, 1H, OCH), 4.68 (d, J = 11.7 Hz, 1H, OCH2), 4.44 (d, J = 11.7 Hz, 1H, OCH2), 4.17 (dd, J = 14.6, 3.0 Hz, 1H, NCH2), 4.08 (dd, J = 14.6, 7.2 Hz, 1H, NCH2); 13C NMR (101 MHz, CDCl3) δ 161.51 (s), 148.72 (s), 137.86 (s), 133.21 (s), 133.14 (s), 133.07 (s), 129.47 (s), 128.74 (s), 128.01 (s), 127.75 (s), 127.20 (s), 126.48 (s), 125.47 (s), 119.83 (s), 119.31 (s), 75.02 (s), 67.16 (s), 50.47 (s); UV (CH3OH): λmax (log ε) 277 (2.74), 243 (2.98) nm; HRMS (m/z) calcd for C19H15Cl2N3OS (M + H)+: 404.0313, found 404.0388. Data for (S)-1: White solid; yield, 90%; m.p., 40−41 °C; [α]24 D = −6.1 (c 0.90, CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.50 (s, 1H, imidazole-H), 7.05 (s, 1H, imidazole-H), 6.95 (s, 1H, imidazole-H), 5.71 (m, 1H, CHCH2), 5.24 (s, 1H, CHCH2), 5.21 (d, J = 3.7 Hz, 1H, CHCH2), 4.91 (d, J = 5.8 Hz, 1H, OCH), 4.35 (d, J = 14.5 Hz, 1H, OCH2), 4.21 (dd, J = 14.5, 7.1 Hz, 1H, OCH2), 4.10 (d, J = 11.8, 1H, NCH2), 3.94 (dd, J = 11.8, 5.8 Hz, 1H, NCH2); 13C NMR (101 MHz, CDCl3) δ 161.82 (s), 148.56 (s), 137.79 (s), 132.49 (s), 129.43 (s), 119.79 (s), 119.26 (s), 118.86 (s), 75.20 (s), 71.91 (s), 50.35 (s); UV (CH3OH): λmax (log ε) 280 (2.57), 239 (1.77) nm; HRMS (m/z) calcd for C11H11Cl2N3OS (M + H)+: 304.0000, found 304.0074. Data for (S)-11: White solid; yield, 37%; m.p., 106−107 °C; [α]24 D = −4.3 (c 1.00, CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.39 (s, 1H, imidazole-H), 7.01 (d, J = 5.6 Hz, 1H, thiophene-H), 6.96 (s, 1H, imidazole-H), 6.82 (s, 1H, imidazole-H), 6.70 (d, J = 5.7 Hz, 1H, thiophene-H), 4.84 (dd, J = 7.3, 4.7 Hz, 1H, OCH), 4.48 (d, J = 11.9 7321

DOI: 10.1021/acs.jafc.8b02332 J. Agric. Food Chem. 2018, 66, 7319−7327

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Journal of Agricultural and Food Chemistry

Figure 2. (A) Docking model display in Pymol; Tioconazole, (S)-11, (R)-12, and (R)-7. (B) Binding model in Ligplot. Hz, 1H, OCH2), 4.37 (d, J = 11.9 Hz, 1H, OCH2), 4.21 (dd, J = 14.6, 4.7 Hz, 1H, NCH2), 4.11 (dd, J = 14.6, 7.3 Hz, 1H, NCH2); 13C NMR (101 MHz, CDCl3) δ 161.29 (s), 148.70 (s), 137.80 (s), 132.94 (s), 129.70 (s), 129.25 (s), 127.45 (s), 123.75 (s), 119.59 (s), 119.22 (s), 75.40 (s), 65.05 (s), 50.46 (s); UV (CH3OH): λmax (log ε) 277 (2.96) nm; HRMS (m/z) calcd for C13H10Cl3N3OS2 (M + H)+: 393.9331, found 393.9403. Data for (S)-12. Colorless crystal; yield, 91%; m.p., 100−101 °C; 1 [α]24 D = −49.0 (c 0.40, CH3OH); H NMR (400 MHz, CDCl3) δ 7.83 (t, J = 8.9 Hz, 3H, naphthalene-H), 7.59 (s, 1H, imidazole-H), 7.55− 7.48 (m, 3H, naphthalene-H), 7.27−7.25 (s, 1H, naphthalene-H), 7.07 (s, 1H, imidazole-H), 6.91 (s, 1H, imidazole-H), 4.97 (dd, J = 7.2, 2.7 Hz, 1H, OCH), 4.81 (d, J = 11.7 Hz, 1H, OCH2), 4.57 (d, J = 11.7 Hz, 1H, OCH2), 4.31 (dd, J = 14.5, 2.7 Hz, 1H, NCH2), 4.22 (dd, J = 14.6, 7.4 Hz, 1H, NCH2); 13C NMR (101 MHz, CDCl3) δ 161.48 (s), 148.78 (s), 137.89 (s), 133.26 (s), 133.10 (s), 129.66 (s), 128.75 (s), 128.00 (s), 127.75 (s), 127.24 (s), 126.48 (s), 125.45 (s), 119.77 (s), 119.34 (s), 75.10 (s), 59.19 (s), 50.57 (s); UV (CH3OH): λmax (log ε) 278 (2.61), 243 (2.86) nm; HRMS (m/z) calcd for C19H15Cl2N3OS (M + H)+: 404.0313, found 404.0388. Crystal Structure Determination of Compound (S)-12. A crystal of compound (S)-12 was obtained from methanol (Figure 4). All measurements were made on a Saturn 724 CCD diffractometer (Rigaku, Tokyo, Japan) with Mo Kα radiation (λ = 0.71073 Å). The crystal was kept at 113 K during data collection. A total of 9788 integrated reflections were collected, and of those 4280 were unique in the range of 1.771° ≤ θ ≤ 27.934° with Rint = 0.0489 and Rsigma = 0.0457, and 3637 with I > 2σ(I) were used in the subsequent refinements. The structure was solved by using the Olex2 software and the ShelXL-97 program according to reported methods.22 All non-hydrogen atoms were refined anisotropically by full-matrix leastsquares to give the final values of R = 0.0446 and wR = 0.1185 (w = 1/ [σ2((F02) + (0.0741P)2 + 0.2676P], where P = (F02 + 2Fc2)/3 with (Δ/σ)max = 0.998 and S = 1.009. The hydrogen atoms were added according to theoretical models. The X-ray crystal structure data of (S)-12 was submitted to Cambridge Crystallographic Data Centre (CCDC 1838941). Molecular Docking. The protein sequence of the cytochrome P450 14α-demethylase enzyme from B. cinerea (BcCYP51) was obtained from the National Center for Biotechnology Information

(NCBI). The 3D structure of BcCYP51 was established based on the crystal structure of the Saccharomyces cerevisiae CYP51 (PDB, 4LXJ) by homologous modeling using Modeler 9.13 software.23 The modeled structure was evaluated by using Procheck,24 Verify3d,25 and Errat procedures.26 Under these three evaluation systems, the modeled structure of BcCYP51 met all the criteria. The docking of the receptor (BcCYP51) and ligands (designed target compounds, tioconazole, and imazalil) was analyzed using the AutoDockVina program.27 We followed the format used by Pymol and Ligpolt to show the docking results (see Figure 2).28 Fungicidal Activity. The fungicidal activities of the test compounds against Alternaria solani, Botrytis cinerea, Rhizoctonia cerealis, Cercospora arachidicola, Pellicularia sasakii, Gibberella zeae, Sclerotinia sclerotiorum, Physalospora piricola, Phytophthora infestans (Mont) de Bary were evaluated in vitro at 50 μg/mL according to established procedures.29 Any compounds that exhibited 90% or better inhibition at this concentration were further evaluated, and their median effective concentration (EC50) values were established following reported procedures.29 In addition, the disease preventive activity of the selected compounds against B. cinerea on cucumber were also tested at 100 μg/mL using the following procedure.30 Briefly, the selected compound (10 mg) was dissolved in 0.5 mL of N,N-dimethylformamide and then diluted with distilled water containing 0.1% Tween 80 to a final concentration of 100 μg/mL. This test solution was sprayed onto the cucumber plants and allowed to runoff. Then, the plants were allowed to dry for 2 h. Control plants were sprayed with a blank solution without any test compound. After 24 h, the cucumbers were inoculated with B. cinerea spores. Diseased plants were recorded after inoculation. The results are relative to the percentage of disease in the diseased control plants compared with that in the healthy control plants, wherein the disease control is set as 100 and the healthy control as 0. Imazalil and tioconazole were used as the positive controls in this disease prevention experiment. Microscopy Observations. Optical Microscopy. (R)-12 was selected as a representative test compound in this study. The B. cinerea spores were treated with 1 μg/mL (R)-12 for 24 h, and then the spore germination, mycelium growth, and degree of infection of B. cinerea were observed. These effects were observed by using an optical microscope. 7322

DOI: 10.1021/acs.jafc.8b02332 J. Agric. Food Chem. 2018, 66, 7319−7327

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Journal of Agricultural and Food Chemistry

Figure 3. General synthetic procedure for the target compounds including its isomers R and S. Reagents and conditions: (i) pyridiniumtribromide, 33% wtHBr in HOAc, r.t. 3 h; (ii) (+)-DIP-Cl, THF, −20 °C, 16 h; (iii) (−)-DIP-Cl, THF, −20 °C, 16 h; (iv) K2CO3, acetone, 20 h; (v) imidazole, K2CO3, N,N-dimethylformamide, 24 h; (vi) 1 mol/L NaOH, R1Br, TBAB, THF, 2 h. Transmission Electron Microscopy (TEM). Ultrastructures on the growing mycelium were prepared (24 h post treatment) for TEM analysis according to the standard protocols.31 The effect was examined with an H7650 transmission electron microscope (Hitachi, Tokyo, Japan) at an accelerating voltage of 60 kV. RNA Extraction and Q-PCR Analysis. The B. cinerea was treated with compounds (S)-11, (R)-7, and (R)-12 at 1 μg/mL for 24 h. Imazalil and tioconazole were used as positive controls in this study. The RNA extract was then isolated by using an E.Z.N.A. fungal RNA Kit (Omega, Norcross, GA), and mRNA was reverse transcribed into cDNA. Q-PCR was performed with Top Green Q-PCR Super Mix (TransStart, Beijing, China). BcCYP51 (5′-TATGTGGCAGTTGATGCG-3′ and 5′-TCTGATGGAGAGGGAGTTTG-3′) and tubulin (5′-TCTCCGTCAAGAGTGGGTTG-3′ and 5′ACTGTGGCTACAGGGTACATT-3′) were used for the fluorescence quantitative PCR as references.32

tioconazole, (R)-7, and (R)-12 had the highest affinities between the ligands and BcCYP51, suggesting these three compounds may have good antifungal activities. Their docking models, performed by Pymol tools, are shown in Figure 2. No H-bonding was predicted between the compounds and the protein, and all tested compounds were surrounded by residues (e.g., Phe130, Leu125, Val124, and Tyr122) due to van der Waals interactions. Moreover, a nitrogen atom in imidazole can bind to the Fe2+ of the heme of BcCYP51 (Figure 2B). In particular, the naphthalene ring at R1 in (R)-12 formed a π−π stacking interaction with Tyr122, which effectively enhanced its binding strength. These results suggested (R)-12 would be a good fungicide, as it can block sterol biosynthesis by targeting the BcCYP51 site. A synthesis of (R)-12 and its analogues was designed as shown in Figure 3. Starting material 1 was synthesized according to a reported procedure.19 Intermediate 2 was prepared by α-bromination of the acetyl group of compound 1

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RESULTS AND DISCUSSION Molecular Design and Chemistry. The affinity values determined in the docking analysis demonstrated that 7323

DOI: 10.1021/acs.jafc.8b02332 J. Agric. Food Chem. 2018, 66, 7319−7327

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Journal of Agricultural and Food Chemistry

R. cerealis, P. sasakii, S. sclerotiorum, and P. piricola. (R)-3, (R)4, and (R)-6 could also completely inhibit R. cerealis growth, but they did not show this level of inhibition against other species. (R)-8 exhibited total inhibition of S. sclerotiorum and R. cerealis but did not inhibit other species. These results indicated that the introduction of 2-chlorothiophene-3-yl and 1-naphthyl moieties at the R1 showed good and broader activities, respectively, against 5−6 different kinds of the plant fungi tested. The bioassay data suggested that our molecular design targeting specific enzymes could lead to potent fungicide candidates. The molecular docking analysis predicted (R)-7 and (R)-12 would be the most potent fungicides, and this speculation was verified by the antifungal bioassays. In this study, we also noticed that there was a chiral carbon atom in these structures; therefore, all the target compounds synthesized were the R isomers. We were interested to see if there was any significant difference in activities between the different isomers; thus, we chose two active compounds (R)11 and (R)-12 and synthesized their S isomers for comparison. We also decided to construct the S isomer of (R)-1 for comparison because (R)-1 showed no fungicidal activity. The results of the in vitro antifungal evaluation showed that the S isomers exhibited fungicidal activities similar to the corresponding R isomers, while the (S)-1 isomer showed poor antifungal activity. To better compare the antifungal activities of these active compounds, their EC50 values were calculated and are shown in Table 2. The result indicated that (R)-12 showed excellent activities against B. cinerea, R. cerealis, P. sasakii, S. sclerotiorum, and P. piricola with EC50 values of 3.14, 0.31, 2.84, 0.33, and 0.64 μg/mL, respectively. In the single species evaluation, the EC50 values of (R)-6−8 and (R)-11−12 against R. cerealis were in general better than that of the positive control tioconazole. It is worth pointing out that (R)-7 showed the best inhibition with an EC50 value of 0.02 μg/mL, which was 10 times better than that of imazalil and 80 times better than that of tioconazole. In addition, (S)-11 was twice as active against B. cinerea, R. cerealis, and S. sclerotiorum than was tioconazole and

Figure 4. Crystal structure for compound (S)-12 by X-ray diffraction determination.

in 90−95% yield. Intermediate 2 reacted with (+)-DIP-Cl or (−)-DIP-Cl in anhydrous tetrahydrofuran to give compounds 3 in 50−60% yield.21 Intermediate epoxide 4, obtained by removing hydrogen bromide from compounds 3 using potassium carbonate in acetone, underwent subsequent nucleophilic addition with imidazole in N,N-dimethylformamide to give compounds 5 in 40−50% yield. Target compounds (R)-1−12, (S)-1, and (S)-11−12 were obtained by etherification of 5 and R1Br in 36−99% yield. Fungicidal Activity. To validate our efficient molecular design method, an in vitro fungicidal activity study on several target compounds was carried out. Their antifungal activities were evaluated at 50 μg/mL, and the results are shown in Table 1. Our data indicated that (R)-5 showed completed growth inhibition of R. cerealis, P. sasakii, and P. piricola, which is the same as the positive controls, imazalil and tioconazole. (R)-7, which has a 4-butylbenzyl moiety at the R1 position, also showed 100% inhibition of R. cerealis and S. sclerotiorum, and this analogue showed better inhibition of C. arachidicola (100% inhibition) than that of the positive controls, imazalil (50%) and tioconazole (88%). (R)-11, which has a 2chlorothiophen-3-yl moiety at the R1 position, showed 100% inhibition of B. cinerea, R. cerealis, and P. sasakii. (R)-12, with a 1-naphthyl moiety at the R1 position, seemed to have a broader spectrum of activity; it provided 100% inhibition of B. cinerea,

Table 1. In Vitro Fungicidal Activity of Compounds (R)-1−12 and (S)-1, (S)-11−12a fungicidal activity (%) ± SD at 50 μg/mL cmpd

A. s

B. c

R. c

C. a

P. s

G. z

S. s

P. p

P. i

(R)-1 (R)-2 (R)-3 (R)-4 (R)-5 (R)-6 (R)-7 (R)-8 (R)-9 (R)-10 (R)-11 (R)-12 (S)-1 (S)-11 (S)-12 imazalil tioconazole

14.3 ± 1.1 36.2 ± 0.8 53.8 ± 1.0 22.3 ± 1.2 58.8 ± 0.0 65.0 ± 1.1 65.6 ± 0.2 55.6 ± 2.0 6.4 ± 1.0 8.8 ± 0.0 73.8 ± 0.0 73.0 ± 1.5 20.0 ± 1.0 72.3 ± 2.1 71.5 ± 1.0 100 ± 0.0 80.0 ± 0.9

33.5 ± 0.6 19.7 ± 1.9 53.2 ± 0.5 68.6 ± 0.8 38.9 ± 0.6 82.3 ± 0.0 49.6 ± 1.0 79.6 ± 0.7 24.2 ± 1.5 26.4 ± 2.0 100 ± 0.0 100 ± 0.0 32.1 ± 1.2 100 ± 0.0 65.1 ± 0.8 100 ± 0.0 100 ± 0.0

86.2 ± 1.3 50.0 ± 0.9 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 63.2 ± 2.3 46.1 ± 0.3 100 ± 0.0 100 ± 0.0 50.4 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0

19.6 ± 0.4 40.5 ± 1.0 48.9 ± 0.3 39.5 ± 1.1 87.4 ± 0.3 68.5 ± 0.6 100 ± 0.0 78.4 ± 0.4 28.1 ± 0.6 33.1 ± 0.8 96.5 ± 0.3 90.1 ± 0.0 22.6 ± 0.7 92.5 ± 1.2 63.5 ± 0.0 50.4 ± 1.0 88.2 ± 0.6

49.4 ± 1.6 36.1 ± 0.7 68.3 ± 1.2 72.8 ± 0.0 100 ± 0.0 80.5 ± 0.8 88.0 ± 0.3 68.5 ± 0.0 30.9 ± 1.0 10.0 ± 2.0 100 ± 0.0 100 ± 0.0 50.2 ± 0.3 100 ± 0.0 88.2 ± 0.6 100 ± 0.0 100 ± 0.0

12.5 ± 1.3 18.8 ± 0.5 20.0 ± 0.3 40.5 ± 0.4 33.5 ± 0.9 19.0 ± 1.2 46.2 ± 1.3 30.0 ± 0.5 57.4 ± 0.1 20.2 ± 1.5 76.6 ± 0.4 25.3 ± 0.7 24.5 ± 0.6 71.2 ± 0.1 70.5 ± 1.0 100 ± 0.0 52.6 ± 4.6

67.5 ± 1.3 57.9 ± 0.0 80.5 ± 2.2 87.6 ± 0.1 78.2 ± 0.0 83.0 ± 1.4 100 ± 0.0 100 ± 0.0 36.2 ± 0.2 11.7 ± 0.6 92.2 ± 0.0 100 ± 0.0 73.6 ± 1.2 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0

56.2 ± 1.2 31.8 ± 0.2 78.0 ± 1.4 70.0 ± 0.0 100 ± 0.0 82.0 ± 0.0 80.5 ± 0.4 66.5 ± 1.8 19.4 ± 0.6 25.0 ± 0.0 85.2 ± 0.5 100 ± 0.0 60.0 ± 1.8 80.2 ± 0.7 88.0 ± 0.1 100 ± 0.0 100 ± 0.0

18.2 ± 0.5 39.0 ± 0.2 39.7 ± 2.0 78.2 ± 0.9 19.3 ± 0.0 59.4 ± 0.6 85.1 ± 0.0 84.1 ± 0.7 26.5 ± 0.3 2.4 ± 0.9 50.4 ± 0.3 32.6 ± 2.0 40.0 ± 0.1 64.7 ± 0.9 35.7 ± 2.1 100 ± 0.0 66.1 ± 0.9

a

A. s: Alternaria solani; B. c: Botrytis cinerea; R. c: Rhizoctonia cerealis; C. a: Cercospora arachidicola; P. s: Pellicularia sasakii; G. z: Gibberella zeae; S. s: Sclerotinia sclerotiorum; P. p: Physalospora piricola; P. i: Phytophthora infestans (Mont) de Bary. 7324

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Journal of Agricultural and Food Chemistry Table 2. EC50 Values of Compounds R and S fungi B. cinerea

R. cerealis

C. arachidicola

P. sasakii

S. sclerotiorum

P. piricola

cmpd (R)-11 (R)-12 (S)-11 imazalil tioconazole azoxystrobina (R)-3 (R)-4 (R)-5 (R)-6 (R)-7 (R)-8 (R)-11 (R)-12 (S)-11 (S)-12 imazalil tioconazole azoxystrobina (R)-7 (R)-11 (R)-12 (S)-11 imazalil tioconazole azoxystrobinb (R)-5 (R)-11 (R)-12 (S)-11 imazalil tioconazole azoxystrobina (R)-7 (R)-8 (R)-11 (R)-12 (S)-11 (S)-12 imazalil tioconazole azoxystrobina (R)-5 (R)-12 imazalil tioconazole azoxystrobin

regression equation y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y

= = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = =

3.9260 4.2479 4.7762 5.4712 2.6388 4.1911 4.1403 3.0235 4.6254 4.9686 7.2408 4.3870 5.4039 5.7183 5.1814 4.5270 7.9444 4.5563 5.3684 3.7609 3.7960 3.7445 3.5401 3.7188 4.8716 4.8911 3.1646 4.1336 4.2363 4.4979 4.3188 3.7985 4.6174 3.7355 3.8383 4.1308 5.6634 5.3552 5.4824 5.0936 4.4859 4.6795 4.5048 5.2487 5.2813 2.0174 4.7465

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

1. 6416x 1.5338x 0.7986x 0.5906x 3.2756x 0.6860x 1.0779x 3.7172x 1.0493x 1.2068x 1.2685x 2.8551x 1.3027x 1.3754x 0.9896x 1.2212x 5.2994x 1.8667x 0.2997x 1.9061x 1.0199x 1.6143x 1.7117x 0.7647x 1.0333x 0.2738x 1.9175x 1.2902x 1.7104x 1.1234x 0.8985x 2.5757x 0.6765x 1.6334x 1.8955x 1.4602x 1.3847x 1.5789x 1.7045x 1.2175x 1.4506 x 0.5283x 1.1843x 1.2850x 1.0448x 4.3348x 0.2720x

R2

EC50 (μg/mL)

0.9927 0.9785 0.9716 0.9848 0.9869 0.9549 0.9993 0.9479 0.9905 0.9976 0.9983 0.9930 0.9847 0.9974 0.9877 0.9936 0.9633 0.9463 0.9785 0.9956 0.9864 0.9370 0.9896 0.9977 0.9863 0.9987 0.9913 0.9899 0.9949 0.9954 0.9559 0.9387 0.9235 0.9967 0.9913 0.9904 0.9854 0.9979 0.9885 0.9979 0.9939 0.8375 0.9879 0.9895 0.9581 0.9144 0.9896

4.65 3.14 1.96 0.18 5.26 15.11 6.44 3.40 2.36 1.11 0.02 1.63 0.50 0.31 0.67 2.56 0.29 1.76 0.06 4.76 16.21 6.10 7.44 47.92 1.36 2.50 9.16 5.08 2.84 2.90 6.07 3.06 3.68 6.52 4.21 3.99 0.33 0.63 0.57 0.87 2.28 4.04 2.62 0.64 0.57 5.09 14.48

a

Azoxystrobin, the data cited from ref 17. bAzoxystrobin, the data cited from ref 19. All these data were determined by our group at the same conditions as in this study.

compounds with a 2-chlorothiophen-3-yl moiety at R1 showed better activity than compounds without that substituent, and (S)-11 provided complete protection at 100 μg/mL as did imazalil and tioconazole; however, the spatial arrangement of substituents on tioconazole and (S)-11 is different (Figure 2). The data also suggested that by introducing a 3,4dichloroisothiazole ring, the target compounds showed fungicidal activity against several fungi species. Morphology and Ultrastructure Transformation: Effects of (R)-12 on B. cinerea. Optical microscopy

6 times more active against C. arachidicola than was imazalil. Azoxystrobin, another positive control used in this study, exhibited good fungicidal activity with EC50 values of 0.06 μg/ mL against R. cerealis and 15.11 μg/mL against B. cinerea.17 In summary, (R)-12 and (S)-11 exhibited excellent and relatively broad-spectrum fungicidal activities. Their fungicidal activities were in accord with the predictions of the molecular docking studies. The results of the evaluation of the in vivo protective activities are listed in Table 3. The data suggested that any 7325

DOI: 10.1021/acs.jafc.8b02332 J. Agric. Food Chem. 2018, 66, 7319−7327

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Journal of Agricultural and Food Chemistry Table 3. In Vivo Fungicidal Activity of Compounds (R)-1− 12 and (S)-1, (S)-11−12 Against Botrytis cinerea fungicidal activity (%) ± SD at 100 μg/mL cmpd

B. cinerea

cmpd

(R)-1 (R)-2 (R)-3 (R)-4 (R)-5 (R)-6

50 ± 2 30 ± 3 40 ± 1 60 ± 2 10 ± 0 0±0

(R)-7 (R)-8 (R)-9 (R)-10 (R)-11 (R)-12

B. cinerea

cmpd

B. cinerea

± ± ± ± ± ±

(S)-1 (S)-11 (S)-12 imazalil tioconazole

50 ± 2 100 ± 0 60 ± 3 100 ± 0 100 ± 0

20 70 55 40 95 60

2 1 2 1 4 0

Figure 6. Gene expression of BcCYP51 influenced by active compounds.

observations of B. cinerea treated with 1 μg/mL (R)-12 revealed that the mycelium growth, spore germination, and infection were significantly affected by the treatment (Figure 5A,D). Transmission electron microscopy (TEM) observation of the B. cinerea cell structure indicated that after exposure to 1 μg/mL (R)-12 for 24 h, most of the cell structure was damaged, and cell wall formation was seriously affected. They become thinner, and wrinkled cell walls were observed (Figure 5E,F). These results showed that (R)-12 affected cell wall formation in B. cinerea. For comparison, in untreated cells, the cell walls and various organelles grew normally. Good adhesion between the cytoplasmic membrane and the outer wall as well as normal, functional cytoplasm could clearly be observed (Figure 5B,C). Effects of BcCYP51 Gene Expression. To further explore the mode of action of these compounds, (R)-7, (R)-12, and (S)-11 were chosen as representatives for this study. The CYP51 gene displayed a feedback regulation of sterol biosynthesis.33 Any antifungal compounds with CYP51 as the target would affect the CYP51 expression level. The QPCR results, shown in Figure 6, showed that the BcCYP51 expression level was significantly affected, and the cytochrome P450-dependent sterol 14α-demethylase was inhibited by these three compounds. Among these representative compounds, (R)-12 showed the strongest effect as a sterol biosynthesis inhibitor. Proposed Mode of Action. Based on the result of this gene express study plus the microcopy observations and the QPCR data, it is reasonable to conclude that the mode of action of (R)-12 and its analogues is similar to that of tioconazole and

imazalil; they function as sterol biosynthesis inhibitors and target 14α-demethylase.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b02332. CheckCIF PLATON report of crystal data of (S)-12 (PDF) Crystal data of (S)-12 (CIF) Score of molecular docking; amino acid sequence homology of CYP51; 1H NMR, 13C NMR, and UV spectra for the target compounds (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Phone: +86-23499464. E-mail: [email protected]. *Phone: +86-23499464. E-mail: [email protected]. ORCID

Lai Chen: 0000-0002-0722-1514 Zhijin Fan: 0000-0001-5565-0949 Funding

This work was supported in part by the National Key Research & Development Plan (Grant No. 2017YFD0200900), the National Natural Science Foundation of China (Grant No. 31571991), the Fundamental Research Funds for the Central Universities (Grant No. 020/63171311), and the China Postdoctoral Science Foundation (Grant No.

Figure 5. (A,D) Optical microscopy observations of mycelium and spore germination of B. cinerea. (B,C) Transmission electron microscopy observations of cell structure of B. cinerea. Sections of B. cinerea cell were grown in the absence of (R)-12 (control). (E,F) Sections of B. cinerea cell were grown containing 1 μg/mL (R)-12. C, cytoplasm; CW, cell wall; ehy, empty hyphae; N, nucleus. 7326

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Journal of Agricultural and Food Chemistry

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2017M611156). The authors also thank Dr. Jiaxing Huang for providing pyrazole compounds (synthesized in the Key Technologies R&D Program of China, Grant 2015BAK45B01, CAU) for model reaction. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED EC50, median effective concentration; TBAB, tetrabutylammonium bromide; DIP-Cl, diisopinocampheyl chloroborane; TEM, transmission electron microscopy

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