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Design, Synthesis, and Fungicidal Evaluation of Novel Pyrazole-furan and Pyrazole-pyrrole Carboxamide as Succinate Dehydrogenase Inhibitors Ting-Ting Yao,†,§ Dou-Xin Xiao,†,§ Zhong-Shan Li,† Jing-Li Cheng,† Shao-Wei Fang,† Yong-Jun Du,† Jin-Hao Zhao,*,† Xiao-Wu Dong,*,‡ and Guo-Nian Zhu† †

Institute of Pesticide and Environmental Toxicology, Ministry of Agriculture Key Lab of Molecular Biology of Crop Pathogens and Insects, Zhejiang University, Hangzhou 310029, P. R. China ‡ ZJU-ENS Joint Laboratory of Medicinal Chemistry, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, P. R. China S Supporting Information *

ABSTRACT: The identification of novel succinate dehydrogenase (SDH) inhibitors represents one of the most attractive directions in the field of fungicide research and development. During our continuous efforts to pursue inhibitors belonging to this class, some structurally novel pyrazole-furan carboxamide and pyrazole-pyrrole carboxamide derivatives have been discovered via the introduction of scaffold hopping and bioisosterism to compound 1, a remarkably potent lead obtained by pharmacophorebased virtual screening. As a result of the evaluation against three destructive fungi, including Sclerotinia sclerotiorum, Rhizoctonia solani, and Pyricularia grisea, a majority of them displayed potent fungicidal activities. In particular, compounds 12I-i, 12III-f, and 12III-o exhibited excellent fungicidal activity against S. sclerotiorum and R. solani comparable to that of commercial SDHI thifluzamide and 1. KEYWORDS: succinate dehydrogenase inhibitors, scaffold hopping, molecular docking, synthesis, fungicidal activity

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INTRODUCTION Fungicides are of value for global food security, attributed to its important role in controlling the growth and reproduction of plant pathogens. Among the 55 classes of fungicides listed by the Fungicide Resistance Action Committee (FRAC), succinate dehydrogenase inhibitors (SDHIs) have been extensively employed to combat destructive plant fungi, such as Sclerotinia sclerotiorum, Rhizoctonia solani, and Botrytis cinerea.1−3 The SDHI exert their biological activities by disrupting the mitochondrial respiration chain and the Krebs cycle.4,5 Benefiting from its unique mode of action, SDHIs have no cross-resistance with other commercial fungicides and have aroused a considerable research interest in exploiting fungicides of this class.6−10 To date, 19 commercial SDHI fungicides have been approved for plant protection since the first launch of carboxin in 1966. However, the unrestricted usage of SDHI fungicides over several decades has resulted in the development of resistance in fungi.11−14 Therefore, novel SDHIs with improved fungicidal potency are still in clear demand. As a well established lead optimization strategy, scaffold hopping is frequently utilized for discovering compounds with structural novelty, enhanced potency, improved physicochemical properties, or more favorable absorption, distribution, metabolism, and excretion (ADME) profiles.15−19 With respect to pesticide design, scaffold hopping is also widely introduced to modify the lead compounds. For example, scaffold hopping of fipronil and human adrenergic ligand clonidine contributed to the successful identification of a novel 7-pyrazolopyridine GABA/GluCl insecticide along with the thiourea and isothiourea insecticides.20,21 In addition to the rationality, another © 2017 American Chemical Society

factor that should be noted is the synthetic accessibility when employing a scaffold hopping strategy in compound design. In this article, we report a computer-aided scaffold hopping approach to a novel structural series of pyrazole-furan and pyrazole-pyrrole carboxamides as SDHIs. We recently disclosed a novel lead structure of a SDHI by pharmacophore based virtual screening. Our previous work in pharmacophore based virtual screening culminated in the discovery of a promising lead, the pyrazole-phenyl carboxamide derivative 1 (Figure 1), which displayed excellent activities against S. sclerotiorum (EC50 = 1.9 mg/L), R. solani (EC50 = 3.8 mg/L), and P. grisea (EC50 = 4.1 mg/L), as well as potent enzymatic inhibition of porcine SDH (IC50 = 1.07 μM).22 As a part of our continuous work in pursuit of SDHIs with novel structure and high efficacy, we initiated a structural optimization campaign focusing on introducing scaffold hopping to 1 in silico. Taking the molecular docking results and synthetic accessibility into consideration, a series of pyrazole-furan carboxamide and pyrazole-pyrrole carboxamide derivatives were identified as the target compound for chemical synthesis (Scheme 1). Their fungicidal activities were evaluated against S. sclerotiorum, R. solani, and P. grisea. Furthermore, docking analysis and structure−activity relationship (SAR) studies were extensively performed on all of the derivatives to identify key structural features responsible for their fungicidal potency. Received: Revised: Accepted: Published: 5397

March 20, 2017 June 14, 2017 June 15, 2017 June 15, 2017 DOI: 10.1021/acs.jafc.7b01251 J. Agric. Food Chem. 2017, 65, 5397−5403

Article

Journal of Agricultural and Food Chemistry

Figure 1. Lead structure 1 and its binding mode.

Scheme 1. Design of the Target Scaffolds

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1 H NMR (500 MHz, CDCl3) δ 7.58 (d, J = 0.8 Hz, 1H, furan-H), 7.18 (d, J = 0.8 Hz, 1H, furan-H), 3.91 (s, 3H, OCH3). 13C NMR (125 MHz, CDCl3) δ 158.04, 143.77, 141.15, 119.96, 108.89, 52.05. Synthesis of 2,2,2-Trichloro-1-(1-methyl-1H-pyrrol-2-yl)ethan-1one (6).24 A solution of 5 (30 g, 0.37 mol) in dichloromethane (100 mL) was purged with nitrogen and cooled to −10 °C. 2,2,2-Trichloroacetyl chloride (66 g, 0.37 mol) was dissolved in dichloromethane (100 mL) and added dropwise, and the resulting mixture was maintained at 23 °C overnight. The solvent was evaporated under vacuum, and the residue was purified by a silica column to afford 6 as a yellow solid. Yield 85%; mp = 60−61 °C. 1H NMR (500 MHz, CDCl3) δ 7.52 (dd, J = 4.4, 1.6 Hz, 1H, pyrrole-H), 7.10−6.81 (m, 1H, pyrrole-H), 6.24 (dd, J = 4.4, 2.4 Hz, 1H, pyrrole-H), 3.99 (s, 1H, CH3). 13C NMR (125 MHz, CDCl3) δ 172.81, 133.62, 123.97, 121.77, 108.86, 96.30, 38.48. Synthesis of 1-(4-Bromo-1-methyl-1H-pyrrol-2-yl)-2,2,2-trichloroethan-1-one (7). NBS (14.4 g, 0.09 mol) was added portion-wise to a solution of 6 (18 g, 0.08 mol) in anhydrous tetrahydrofuran (160 mL) at −10 °C. The resulting mixture was warmed to 23 °C and stirred overnight. The reaction mixture was concentrated under reduced pressure, and the crude residue was dissolved in ethyl acetate (150 mL). The solution was washed sequentially with saturated aqueous sodium carbonate (50 mL × 3) and brine (50 mL × 2). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford the title compound as an off-white solid. Yield 77%; mp = 97−98 °C. 1H NMR (500 MHz, CDCl3) δ 7.47 (d, J = 1.6 Hz, 1H, pyrrole-H), 6.97 (d, J = 1.4 Hz, 1H, pyrrole-H), 3.96 (s, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ 172.45, 132.84, 124.62, 122.15, 96.15, 95.72, 38.76. Synthesis of Methyl 4-Bromo-1-methyl-1H-pyrrole-2-carboxylate (8). The sodium methoxide (5.2 g, 0.09 mol) was added to an icecooled solution of 7 (20.0 g, 0.06 mol) in anhydrous methanol (320 mL). The resulting mixture was warmed to 23 °C and stirred for 1 h. The reaction mixture was quenched with HCl (1 N, 30 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layer washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford the title compound as a white solid. Yield 90%; mp = 63−64 °C. 1H NMR (500 MHz, CDCl3) δ 6.91 (d, J = 1.9 Hz, 1H, pyrrole-H), 6.78 (d, J = 1.9 Hz, 1H, pyrrole-H),

MATERIALS AND METHODS

Instruments and Chemicals. All reagents and solvents were commercially available and used directly without further purification. Reactions were monitored by thin-layer chromatography (TLC). Target compounds were purified by column chromatography using silica gel. 1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were recorded with a Bruker Avance III spectrometer in CDCl3 or DMSO-d6 solution, with SiMe4 (TMS) serving as the internal standard. Chemical shift values (δ) were listed in parts per million (ppm). ESI mass spectral data were obtained from a Accurate-Mass Q-TOF spectrometer and a Quattro Premier XE. The melting points were determined on an X-4 binocular microscope melting point apparatus (Beijing Tech Instruments Co., Beijing, China) and are uncorrected. Synthesis of Methyl 4,5-dibromofuran-2-carboxylate (3).23 Aluminum chloride (70.5 g, 0.53 mol) was added to a solution of 2 (31.5 g, 0.25 mol) in chloroform (250 mL) at −10 °C under nitrogen. Br2 (79.5 g, 0.1 mol) was then added dropwise over 1 h, and the resulting mixture was maintained at 23 °C for an additional 3 h. The reaction mixture was poured into water (200 mL). The mixture was extracted with dichloromethane (100 mL × 3), and the combined organic phases were further washed with 10% aqueous sodium thiosulfate, saturated sodium bicarbonate, and brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Purification by crystallization with hexane afforded the title compound as a paleyellow solid. Yield 78%; mp = 39−40 °C. 1H NMR (500 MHz, CDCl3) δ 7.19 (s, 1H, furan-H), 3.90 (s, 3H, OCH3). 13C NMR (125 MHz, CDCl3) δ 157.31, 143.33, 140.07, 121.93, 98.62, 52.35. Synthesis of Methyl 4,5-Dibromofuran-2-carboxylate (4). A solution of 3 (28.4 g, 0.1 mol) in anhydrous THF (200 mL) was purged with nitrogen and cooled to −40 °C. Isopropylmagnesium chloride (2 N in THF, 76 mL) was added dropwise, and the resulting mixture was stirred for 2 h. The reaction mixture was quenched with water (100 mL), and the precipitates were filtered off. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, and purified on silica gel to afford 4 as a yellow solid. Yield 89%; mp = 39−40 °C. 5398

DOI: 10.1021/acs.jafc.7b01251 J. Agric. Food Chem. 2017, 65, 5397−5403

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Journal of Agricultural and Food Chemistry 3.91 (s, 3H, CH3), 3.82 (s, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ 160.82, 128.76, 122.88, 119.22, 95.06, 51.27, 36.91. General Procedure for the Synthesis of 9I and 9II. A solution of 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)-1H-pyrazole (7.5 g, 0.036 mol), K3PO4·3H2O (12.0 g, 0.045 mmol), and bromide 4 or 8 (0.03 mol) in N,N-dimethylformamide (100 mL) was stirred under nitrogen. Tetrakis(triphenylphosphine)palladium (3.5 g, 0.003 mol) was added, and the mixture was heated at 100 °C for 10 h. The reaction mixture was dissolved in water (300 mL) and extracted with ethyl acetate (150 mL × 3). The combined organic layer was washed with brine (50 mL × 2), dried over anhydrous sodium sulfate, and concentrated under vacuum to afford the title compounds. The crude product was purified by flash silica chromatography to obtain the title compound. Data for 9I: Yellow solid; yield 67%; mp = 62−64 °C. 1H NMR (500 MHz, CDCl3) δ 7.43 (d, J = 2.0 Hz, 1H, furan-H), 7.20 (d, J = 3.6 Hz, 1H, pyrazole-H), 6.60 (d, J = 3.6 Hz, 1H, pyrazole-H), 6.56 (d, J = 2.0 Hz, 1H, furan-H), 4.07 (s, 3H, NCH3), 3.86 (s, 3H, OCH3). 13 C NMR (125 MHz, CDCl3) δ 158.75, 148.21, 143.93, 138.49, 132.59, 119.26, 109.60, 106.41, 77.29, 77.03, 76.78, 51.90, 38.84. Data for 9II: white solid; mp = 52−53 °C. 1H NMR (500 MHz, CDCl3) δ 7.44 (d, J = 1.9 Hz, 1H, furan-H), 7.04 (d, J = 2.0 Hz, 1H, pyrazole-H), 6.95 (d, J = 1.9 Hz, 1H, pyrazole-H), 6.23 (d, J = 1.9 Hz, 1H, furan-H), 3.97 (s, 3H, pyrazole-CH3), 3.93 (s, 3H, pyrrole-CH3), 3.84 (s, 3H, OCH3). 13C NMR (125 MHz, CDCl3) δ 161.37, 138.33, 137.35, 127.73, 123.09, 116.57, 112.97, 104.64, 51.25, 37.57, 36.96. General Procedure for the Synthesis of 10I and 10II. Aqueous sodium hydroxide (3 N, 65 mL) was added to a solution of 9I or 9II (0.02 mol) in methanol (20 mL). The reaction mixture was stirred at 23 °C for 5 h. The solvent was removed by rotary evaporation, and the crude residue was redissolved in 20 mL of water. The solution was further acidified with hydrochloric acid (3 N) until pH 1 and extracted with ethyl acetate (10 mL × 3). The collected organics were further washed with brine, dried over anhydrous sodium sulfate, and then concentrated to yield the title acid. Data for 10I: yellow solid; yield 60%; mp = 172−174 °C. 1H NMR (500 MHz, DMSO) δ 7.59 (s, 1H, furan-H), 7.33 (d, J = 3.6 Hz, 1H, pyrazole-H), 7.05 (d, J = 3.6 Hz, 1H, furan-H), 4.05 (s, 3H, CH3). 13 C NMR (125 MHz, CDCl3) δ 159.03, 145.15, 144.30, 137.11, 137.04, 128.91, 118.88, 112.63, 108.52. Data for 10II: yellow solid; yield 68%; mp = 207−208 °C. 1H NMR (500 MHz, DMSO) δ 7.44 (d, J = 2.0 Hz, 1H, pyrazole-H), 7.35 (d, J = 1.9 Hz, 1H, pyrrole-H), 7.05 (d, J = 2.1 Hz, 1H, pyrrole-H), 6.33 (d, J = 1.9 Hz, 1H, pyrazole-H), 3.89 (s, 6H, pyrazole-CH3, pyrrole-CH3). 13C NMR (125 MHz, CDCl3) δ 161.82, 137.62, 137.09, 128.11, 123.38, 115.84, 111.97, 103.99, 37.68, 36.55. General Procedure for the Synthesis of 11I and 11II. NCS (1.5 g, 0.011 mol) was added to an ice-cooled solution of 10I or 10II (0.01 mol) in dry THF/DMF (6/9 mL). The resulting mixture was warmed to 80 °C and stirred for 2 h. The reaction mixture was then diluted with ethyl acetate (20 mL), washed with saturated aqueous sodium bicarbonate and brine, dried over anhydrous sodium sulfate, and then concentrated under vacuum. Purification by flash silica chromatography afforded the title compound. Data for 11I: white solid; yield 85%; mp >200 °C. 1H NMR (500 MHz, DMSO) δ 7.59 (s, 1H, pyrazole-H), 7.33 (d, J = 3.6 Hz, 1H, furan-H), 7.05 (d, J = 3.6 Hz, 1H, furan-H), 4.05 (s, 3H, CH3). 13 C NMR (125 MHz, CDCl3) δ 159.03, 145.15, 144.30, 137.11, 137.04, 128.91, 118.88, 112.63, 108.52. Data for 11II: white solid; yield 87%; mp = 187−188 °C. 1H NMR (500 MHz, DMSO) δ 7.55 (s, 1H, pyrazole-H), 7.50 (d, J = 2.0 Hz, 1H, pyrrole-H), 7.11 (d, J = 2.0 Hz, 1H, pyrrole-H), 3.93 (s, 3H, pyrazole-CH3), 3.85 (s, 3H, pyrrole-CH3). 13C NMR (125 MHz, DMSO) δ 161.70, 136.21, 133.70, 129.31, 123.54, 116.45, 108.49, 106.16, 38.64, 36.68. General Procedure for the Synthesis of 12-I−12-IV.25 A mixture of 10I, 10II, 11I, or 11II (0.5 mmol), HATU (209 mg, 0.55 mmol), N,N-diisopropylethylamine (129 mg, 1 mmol), and corresponding amine (0.75 mmol) in acetonitrile (20 mL) was warmed to 60 °C and stirred overnight. The reaction mixture was concentrated under reduced

pressure. The crude residue was diluted with dichloromethane (20 mL) and washed with water (10 mL × 3). The organic layer was dried over anhydrous sodium sulfate and then concentrated under vacuum. Purification by flash silica chromatography afforded the title compounds. 1H NMR, 13C NMR, and HRMS spectral data are supplied in the Supporting Information. Fungicidal Assay. Sclerotinia sclerotiorum, Rhizoctonia solani, Pyricularia grisea, and susceptible cole leaves were provided by the Institute of Pesticide and Environment Toxicology, Zhejiang University. The fungicidal activities of the synthetic compounds were tested in vitro against the above-mentioned fungi according to the reported mycelia growth inhibition method.9 Each compound was dissolved in DMSO to prepare the 10 mg/mL stock solution. Compounds were first tested at a concentration of 100 mg/L for primary screening. Percentage inhibition was calculated by the following equation: percentage inhibition (%) = (1 − PT/CK) × 100, where PT is the mean colony diameter with compounds, and CK is the mean colony diameter without tested compounds. The commercial fungicide thifluzamide was employed as the positive control. Each treatment was performed three times. The inhibition rate of the potent compounds was further tested at the concentration of 100, 50, 25, 12.5, 6.25, 3.12, and 1.56 μg/mL, and the corresponding EC50 values were calculated using SPSS Statistics v17.0. The in vivo fungicidal activity of the target compounds against S. sclerotiorum was carried out on cole leaves.9 Healthy cole leaves were sprayed with target compounds (200 μg/mL) and then cultivated at 25 °C for 24 h before inoculation with S. sclerotiorum. Results were observed as diameters of the lesion after cultivation at 25 °C for 36 h. Thifluzamide was employed as the positive control. Each treatment was performed three times. The preventative rate was expressed as (1 − c/d) × 100, where c is the diameter of the treatment, and d is the diameter of the negative control. Molecular Docking. Because of the absence of the crystal structure of SDH from fungi, a homology model of Rhizoctonia solani SDH (RsSDH) was built and validated in our previous study. The constructed homology model of RsSDH was used for the subsequent docking study. The structures of small molecules were optimized with the ligand minimization protocol. A molecular docking study was performed by the Libdock module implemented in Discovery Studio 2.5.26 The active site of SDH was derived from the copied ligand flutolanil in the RsSDH model. Ten random conformations were generated for each ligand. The rest of the parameters were set to the default values. The optimal pose was selected in terms of docking score and visual inspection.

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RESULTS AND DISCUSSION Rational Design. According to our previous study, the pyrazole-benzoic “core” of lead compound 1 entered deep into the Q-site of the receptor protein, contributing preliminarily to its binding affinity. To explore novel SDHIs and make better interaction with the Q-site, herein we introduced scaffold hopping to the pyrazole-benzoic “core” of lead compound 1. It is well known that pyrazole was a favorable pharmacophore for SDHI fungicides.4−7 Thus, we retained the pyrazole group and replaced the phenyl linkage with five- or other six-membered ring systems (Scheme 1). From our assessment, pyrrole (L1) and furan (L2) isomers can maintain a binding pose similar to that of lead compound 1. Considering the favorable binding mode and synthetic tractability, pyrazole-pyrrole and pyrazole-furan acid cores were selected for chemical synthesis and structure−activity relationship studies. Chemical Synthesis. The synthetic route for 12I-a−12IV-e is shown in Figure 1−3. For the synthesis of bromide 4 (Figure 2), commercial furoate (2) was first brominated to provide dibromo-furoate 3, which was further treated with isopropylmagnesium chloride to afford monobromide 4. For the synthesis of bromide 8 (Figure 3), commercial methylpyrazole 5399

DOI: 10.1021/acs.jafc.7b01251 J. Agric. Food Chem. 2017, 65, 5397−5403

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Journal of Agricultural and Food Chemistry

(i.e., 12III-a, 12III-e, 12III-f, 12III-i, 12III-l, 12III-o, 12III-p, and 12III-q) exhibited improved fungicidal activity against R. solani in comparison to that of lead compound 1 (EC50 = 11.0 μM), with EC50 values ranging from 0.5 to 10.6 μM. However, replacement of the phenyl core by pyrrole or the furan group led to the unexpected decrease in bioactivity against P. grisea. As shown in Table 1, substitution on the pyrazole ring at the A site was detrimental to fungicidal activity of the synthesized compounds. Notably, 12I and 12III with chlorine substituted pyrazole displayed remarkably better activities than the corresponding unsubstituted ones (12II and 12IV). As for the chlorine substituted pyrazole-furan series 12I, the influence of substituents and their positions on the aniline moiety was investigated. Modification at the ortho-, meta-, and para-positions of aniline with electron-withdrawing or donating groups (12I-b−r) did not significantly augment the fungicidal activity as compared to 12I-a. Furthermore, among the single substituted aniline analogues, substituents in the ortho-positions were more favorable for their fungicidal activities compared with those of the meta- and para-substituted ones. For example, 12I-b, 12I-e, and 12I-i possessed better efficacy than corresponding metaand para-substituted ones. As for the chlorine-substituted pyrazole-pyrrole series 12III, a similar phenomenon was observed for the monosubstituted aniline derivatives. In particular, the ortho-methoxy substituted derivative (12III-f) displayed the most potent fungicidal activities, with EC50 values of 3.5, 1.4, and 129.3 μM against S. sclerotiorum, R. solani, and P. grisea, respectively. Meanwhile, replacing the aniline moiety with a flexible or rigid amine group was also investigated. Intriguingly, the introduction of a flexible 4-(tert-butyl) benzylamine moiety (12III-o) displayed significantly enhanced effects compared with that of 12III-a. Considering the above SAR results, these findings suggested that the fungicidal potency of designed compounds could be ascribed to a combination of factors, such as chlorine substitution at the pyrazole ring, replacement of the phenyl core, and variation of the amine part. In Vivo Fungicidal Activities. From the results of the in vitro test, compound 12III-f was identified as the most promising candidate for further study, and its in vivo fungicidal activity was determined in the greenhouse for the control of S. sclerotiorum infected cole. As shown in Figure 5, the untreated negative control (pathogen only) resulted in 100% disease incidence. To our delight, 12III-f afforded a preventative rate of 60.4% against S. sclerotiorum at the concentration of 200 mg/L. In contrast, leaves treated with thifluzamide at the same concentration only resulted in a preventative rate of 32.6%. The results further demonstrated the practical potential of this novel compound for crop protection. Docking Analysis. In order to elucidate the mechanism of newly designed SDH inhibitors and explain the SAR in details, all of the derivatives were docked into the active site of RsSDH. The detailed interactions of two representative derivatives 12III-f and 12I-g with RsSDH are provided in Figure 6. In the docked complex of compound 12III-f and RsSDH (Figure 6A), the pyrazole-pyrrole “core” of 12III-f was buried deep in the Q-site and mainly interacted with Arg358 through a π-cation interaction. Moreover, the amide function of 12III-f formed a hydrogen bond with the side chain of Trp206, which was crucial to the binding of inhibitors and SDH. The orthomethoxy-substituted phenyl moiety was oriented toward the entrance of the Q-site and interacted with nearby amino acids through hydrophobic interactions. All of these favorable

Figure 2. Synthetic route for intermediate 4. Reagents and conditions: (a) AlCl3, Br2, CHCl3, −10−23 °C, 4 h; (b) isopropylmagnesium chloride, THF, −40 °C, 2 h.

Figure 3. Synthetic route for intermediate 8. Reagents and conditions: (a) 2,2,2-trichloroacetyl chloride, DCM, −10−23 °C, overnight; (b) NBS, THF,-10−23 °C, 10 h; (c) sodium methoxide, MeOH, 0−23 °C, 1 h.

(5) was coupled with 2,2,2-trichloroacetyl chloride to generate 6, and this was followed by bromination using NBS, leading to bromide 7 in good yield. Treatment of intermediate 7 with sodium methoxide in MeOH furnished the target bromide 8. The bromides 4 and 8 were subsequently coupled with pyrazoleborate to provide 9I and 9II. Hydrolysis of 9I and 9II under basic conditions provided the corresponding acids 10I and 10II. The exposure of 10I and 10II to NCS resulted in the smooth chlorination of the reactive pyrazole ring to afford 11I and 11II. The chlorine substituted acids were further coupled with different amines to afford desired derivatives 12I-a−12IV-e (Figure 4). The chemical structures of all prepared compounds were confirmed by 1H NMR, 13C NMR, and HRMS.

Figure 4. Synthetic route for target compounds 12I−IV. Reagents and conditions: (a) 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)-1H-pyrazole, Pd(PPh3)4, H3PO4·3H2O, DMF, 100 °C, 10 h; (b) NaOH, MeOH/H2O, 23 °C, 5 h; (c) NCS, THF/DMF, 0−80 °C, 2 h; (d) HATU, N,N-diisopropylethylamine, MeCN, 60 °C, overnight.

Fungicidal Activities and SAR Discussion. The fungicidal activities of all the target compounds were evaluated against three destructive fungi (S. sclerotiorum, R. solani, and P. grisea). The commercial SDHI fungicide thifluzamide was used as the positive control, and the results are provided in Table 1. Most of the derivatives exhibited moderate to potent fungicidal activities against S. sclerotiorum, R. solani, and P. grisea, demonstrating the rationality of our scaffold hopping strategy. Among them, five newly synthesized derivatives (i.e., 12I-b, 12I-i, 12III-g, 12III-h, and 12III-q) were found to display improved fungicidal activities against S. sclerotiorum compared with that of thifluzamide (EC50 = 33.2 μM) and lead structure 1 (EC50 = 5.5 μM), with EC50 values ranging from 2.2 to 5.1 μM. In particular, compound 12I-i, identified as the most potent inhibitor against S. sclerotiorum with an EC50 value of 2.2 μM, showed 15-fold higher inhibitory activity than thifluzamide. Furthermore, eight derivatives 5400

DOI: 10.1021/acs.jafc.7b01251 J. Agric. Food Chem. 2017, 65, 5397−5403

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Journal of Agricultural and Food Chemistry

Table 1. Chemical Structures of Compounds 12I−IV and Their EC50 Values against S. sclerotiorum, R. solani, and P. grisea

EC50 (μM)a no.

X

A

R

Ssb

Rsb

Pgb

12I-a 12I-b 12I-c 12I-d 12I-e 12I-f 12I-g 12I-h 12I-i 12I-j 12I-k 12I-l 12I-m 12I-n 12I-o 12I-p 12I-q 12I-r 12II-a 12II-b 12II-c 12II-d 12III-a 12III-b 12III-c 12III-d 12III-e 12III-f 12III-g 12III-h 12III-i 12III-j 12III-k 12III-l 12III-m 12III-n 12III-o 12III-p 12III-q 12III-r 12IV-a 12IV-b 12IV-c 12IV-d 12IV-e THc

O O O O O O O O O O O O O O O O O O O O O O NMe NMe NMe NMe NMe NMe NMe NMe NMe NMe NMe NMe NMe NMe NMe NMe NMe NMe NMe NMe NMe NMe NMe

Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl H H H H Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl H H H H H

Ph 2-Cl-Ph 3-Cl-Ph 4-Cl-Ph 2-MeO-Ph 3-MeO-Ph 4-MeO-Ph 3,4-MeO2-Ph 2-CF3O-Ph 4-CF3O-Ph 2-CF3-Ph 4-CF3-Ph 3-Me-Ph 4-Me-Ph 2,5-Me2-Ph 3-NO2-Ph 4-cyano-Ph 4-cyanomethyl-Ph Ph 3-Cl-Ph 4-Cl-Ph 2-MeO-Ph Ph 2-Cl-Ph 3-Cl-Ph 4-Cl-Ph 3,5-Cl2-Ph 2-MeO-Ph 3-MeO-Ph 4-MeO-Ph 3,4-MeO2-Ph 2-CF3-Ph 4-CF3-Ph 4-cyanomethyl-Ph 2-(4′-Cl-phenyl)-Ph PhCH2 4-t-Bu-PhCH2 6-Cl-pyridin-3-yl-CH2 4-Cl-PhCH2CH2 1-naphthalen Ph 2-MeO-Ph 2-(4′-Cl-phenyl)-Ph PhCH2 4-t-Bu-PhCH2

5.6 ± 0.1 5.1 ± 1.5 17.3 ± 1.0 8.3 ± 0.6 14.5 ± 2.3 6.0 ± 1.4 >600 151.7 ± 2.5 2.2 ± 1.6 13.8 ± 3.2 17.2 ± 1.6 16.9 ± 1.7 88.4 ± 25.2 16.2 ± 2.2 13.9 ± 1.2 8.9 ± 0.6 12.9 ± 1.7 13.0 ± 2.8 298.2 ± 7.2 141.5 ± 1.7 36.1 ± 1.1 170.8 ± 2.8 10.8 ± 0.8 5.7 ± 1.0 14.6 ± 0.1 34.1 ± 0.3 106.6 ± 0.5 3.5 ± 0.1 81.8 ± 1.9 4.9 ± 1.2 8.8 ± 1.1 68.3 ± 3.4 130.6 ± 9.3 >600 275.3 ± 14.1 9.4 ± 1.0 7.5 ± 1.6 9.9 ± 0.1 2.9 ± 1.5 76.7 ± 1.9 431.9 ± 23.9 56.4 ± 2.6 84.9 ± 3.0 118.6 ± 6.2 132.4 ± 3.2 33.2 ± 0.8

37.8 ± 0.5 33.9 ± 0.4 27.4 ± 0.2 36.6 ± 0.5 23.8 ± 0.4 34.7 ± 0.3 417.1 ± 10.8 253.7 ± 27.0 27.4 ± 1.6 146.3 ± 4.5 35.1 ± 1.3 44.7 ± 1.1 150.1 ± 2.7 41.4 ± 0.3 36.1 ± 0.5 22.9 ± 1.1 21.8 ± 0.1 31.7 ± 1.2 102.5 ± 2.9 398.3 ± 20.3 85.2 ± 0.2 88.8 ± 1.6 5.4 ± 1.0 12.3 ± 1.8 37.2 ± 3.9 91.1 ± 7.8 7.3 ± 0.4 1.4 ± 3.4 37.1 ± 0.5 20.9 ± 2.1 8.3 ± 1.5 11.1 ± 1.6 285.3 ± 6.9 10.6 ± 1.1 77.1 ± 14.5 43.5 ± 4.2 0.5 ± 0.4 9.6 ± 0.5 9.3 ± 1.0 274.4 ± 0.9 59.9 ± 1.6 88.9 ± 1.4 82.9 ± 4.9 86.7 ± 7.9 45.9 ± 1.7 0.09 ± 0.01

109.0 ± 1.8 >600 100.5 ± 0.7 323.3 ± 14.2 365.0 ± 25.0 >600 261.0 ± 9.4 199.3 ± 0.7 >600 175.3 ± 2.6 302.6 ± 8.8 253.5 ± 8.3 80.1 ± 1.8 225.0 ± 13.3 >600 >600 380.2 ± 5.6 >600 191.5 ± 10.7 >600 37.8 ± 0.7 >600 334.2 ± 22.8 313.8 ± 21.8 254.6 ± 14.6 294.9 ± 8.7 210.6 ± 7.3 129.3 ± 2.9 82.9 ± 5.5 209.4 ± 6.4 140.3 ± 3.5 141.0 ± 5.3 129.0 ± 3.8 389.6 ± 4.8 192.3 ± 10.1 157.2 ± 7.4 196.4 ± 4.4 145.5 ± 3.0 109.2 ± 5.0 81.9 ± 4.2 >600 27.4 ± 2.3 273.2 ± 7.7 339.6 ± 14.5 285.6 ± 0.2 33.4 ± 4.2

a Values are the mean ± standard deviation (SD) of three replicates. bAbbreviations: Ss, Sclerotinia sclerotiorum; Rs, Rhizoctonia solani; Pg, Pyricularia grisea. cThifluzamide.

with Arg358. However, the introduction of a methoxy group to the para-position of the phenyl moiety resulted in the repulsion interaction with Phe342. Thus, the amide function of 12I-g failed to form a H-bond with Tyr206, which may account for its

interactions may result in the potent fungicidal activity of 12III-f. As shown in Figure 6B, the binding mode of 12I-g was similar to that of 12III-f. The pyrazole-furan “core” of 12I-g embedded deep into the Q-site, forming a π-cation interaction 5401

DOI: 10.1021/acs.jafc.7b01251 J. Agric. Food Chem. 2017, 65, 5397−5403

Journal of Agricultural and Food Chemistry

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Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b01251. 1 H NMR, 13C NMR, and HRMS for the target compounds (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(J.-H.Z.) Institute of Pesticide and Environmental Toxicology, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, P. R. China. Tel: 086-571-86971923. Fax: 086-571-86971923. E-mail: [email protected]. *(X.-W.D.) College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, P. R. China. Tel/Fax: 086-57188981051. E-mail: [email protected].

Figure 5. In vivo activity of 12III-f against S. sclerotiorum infected cole leaves.

remarkable decrease in bioactivity. Moreover, it was observed that the pyrazole moiety had adopted a noncoplanar conformation with the linkage group (pyrrole or furan). Thus, we speculated that the introduction of chlorine favored the active conformation and thus led to the enhanced bioactivity of 12I and 12III. Furthermore, the relative weaker fungicidal potency against P. grisea of these newly designed compounds may be attributed to the sequential differences of SDH from specific plant pathogens. In summary, we described the rational scaffold hopping and bioisosteric modification of lead structure 1. Facilitated by molecular docking analysis, a series of pyrazole-furan and pyrazole-pyrrole carboxamide derivatives were designed and subsequently synthesized on the basis of a systemic SAR study. Furthermore, their fungicidal activities were evaluated against S. sclerotiorum, R. solani, and P. grisea, demonstrating that most of these compounds displayed moderate to good fungicidal activities. Most surprisingly, compound 12I-i, 12III-f, and 12III-o exhibited improved fungicidal activity against S. sclerotiorum and R. solani comparable to that of commercial SDHI fungicide thifluzamide and lead structure 1. SAR analysis and molecular docking simulations revealed that the promising fungicidal potency of our designed compounds could be ascribed to structural alteration, including the chlorine substitution at the pyrazole ring, replacement of the phenyl core, and modification of the amine part of lead structure 1. Therefore, the rationality and efficiency of our molecular docking assisted scaffold hopping approach in the development of novel fungicidal agents was fully supported in our study, and the finding will lay the foundation for further structural modification and development of novel SDHIs.

ORCID

Jin-Hao Zhao: 0000-0002-9198-5399 Author Contributions §

T.-T.Y and D.-X.X. contributed equally to this study.

Funding

This work was supported financially by the National Key Research and Development Program of China (2017YFD0200505, 2017YFD0201805 and SQ2017ZY060059). Notes

The authors declare no competing financial interest.

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REFERENCES

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