Structure-Based Discovery of Potential Fungicides as Succinate

Jan 22, 2017 - Fisher , N.; Brown , A. C.; Sexton , G.; Cook , A.; Windass , J.; Meunier , B. Modeling the Qo site of crop pathogens in Saccharomyces ...
0 downloads 0 Views 2MB Size
Article pubs.acs.org/JAFC

Structure-Based Discovery of Potential Fungicides as Succinate Ubiquinone Oxidoreductase Inhibitors Li Xiong,† Hua Li,† Li-Na Jiang,† Jing-Ming Ge,† Wen-Chao Yang,† Xiao Lei Zhu,*,† and Guang-Fu Yang*,†,§ †

Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, People’s Republic of China § Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300071, People’s Republic of China S Supporting Information *

ABSTRACT: A series of diphenyl ether-containing pyrazole-carboxamide derivatives was designed and synthesized as new succinate ubiquinone oxidoreductase (SQR) inhibitors. This highly potent molecular scaffold was developed from a moderately activie hit 3, obtained from our previous pharmacophore-linked fragment virtual screening (PFVS) method. The results of greenhouse tests indicated that some analogues showed good SQR inhibitory activity, with promising fungicidal activity against Rhizoctonia solani and Sphaerotheca fuliginea at a dosage of 200 mg/L. Most surprisingly, compound 62 showed the highest SQR inhibitory activity with a Ki value of 0.081 μM, about 4-fold more potent than penthiopyrad (Ki = 0.307 μM). In addition, compounds 43 and 62 displayed excellent fungicidal activity even at a dosage as low as 6.25 mg/L, which was superior to thifluzamide. Moreover, compound 62 exhibited excellent protection effect against R. solani and provided about 81.2% protective control efficancy after 21 days with two sprayings. The present work indicated that these two compounds could be used as potential agricultural fungicides targeting SQR. KEYWORDS: succinate ubiquinone oxidoreductase, complex II, diphenyl ether, structure−activity relationship, molecular docking



benzamides (flutolanil, benodanil, mepronil).11 Due to their long use and high application rates, many pathogens have developed a serious resistance toward carboxamide fungicides, such as Alternaria alternata,12,13 Botrytis cinerea,14,15 Podosphaera xanthii,16 and Mycosphaerella graminicola.17−19 Therefore, there is still demand to discover new SQR inhibitors with simpler structures and superior performance.20−24 In a previous study,25 we reported the computational discovery of six hit compounds (Figure 1) as potential SQR inhibitors using the pharmacophore-linked fragment virtual screening (PFVS) method. Among them, the indole-containing pyrazole-carboxamide hit compound 6 was selected for further structural optimization, yielding several sub-micromolar and nanomolar SQR inhibitors with good fungicidal activity against Rhizoctonia solani. Herein, to continue our previous work, the structural optimization of hit compound 3 containing the diphenyl-ether fragment was reported. Very fortunately, two compounds, 43 and 62, were finally identified and showed excellent fungicidal potency toward R. solani and Sphaerotheca fuliginea. Further computational simulations revealed that, compared to the commercial fungicide thifluzamide, the diphenyl-ether fragment in compound 62 exhibited greater flexibility upon binding with SQR, forming T−π interaction with C_W35 in porcine SQR and π−π interaction with D_Y91 in R. solani SQR. The present study provided an interesting

INTRODUCTION Succinate ubiquinone oxidoreductase (SQR; EC 1.3.5.1), one of the components of mithochondrial respiration, contains four chains: hydrophilic subunits flavoprotein (FP, chian A), iron− sulfur protein (Ip, chain B), and two membrane anchor proteins (CybL, chain C; CybS, chain D).1−6 Many biological experiments have extablished that SQR could transfer an electron from succinate to fumarate, accompanied with the reduction of ubiquinone to ubiquinol. In general, SQR was identified as one fungicidal targets and its inhibitors would block the electron transfer, leading to the death of plant pathogen. More recently, some research groups had pointed out that the chains B, C, and D could also act as tumor suppressors and then lead to many physiological disorders.7−10 Therefore, SQR has been considered as a promising target not only in agriculture but also in medicine. The SQR agricultural fungicides are always called carboxamide fungicides due to their common chemical structure. Among this family, carboxin was the earlist commericial product launched in 1966.11 Since then, many new fungicides were discovered with higher potency and broader biological spectrum. Till now, there are 19 commercial SQR fungicides divided into 9 categories, including pyrazole-4-carboxamides (fluxapyroxad, furametpyr, penflufen, penthiopyrad, bixafen, isopyrazam, sedaxane, benzovindiflupyr), thiazole-carboxamides (thifluzamide), phenyl-oxo-ethyl-thiophene amide (isofetamid), pyridinyl-ethyl benzamides (fluopyram), N-methoxy-(penylethyl)-pyrazole-carboxamides (pydiflumetofen), pyridine carboxamides (boscalid), oxathilin carboxamides (carboxin, oxycarboxin), furan carboxamides (fenfuram), and phenyl© XXXX American Chemical Society

Received: Revised: Accepted: Published: A

November 15, 2016 January 16, 2017 January 21, 2017 January 22, 2017 DOI: 10.1021/acs.jafc.6b05134 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of six hit candidates identified by PFVS.25 The fragments determined by PFVS are shown in blue. homology models using the AutoDock 4.249 program according to our previously published method.50 Calculation of Binding Free Energies. The molecular mechanics−Possion−Boltzmann surface area (MM-PBSA) method51,52 was used to calculate the binding free energies of the title compounds.

example of application of computational design in pesticide discovery.



MATERIALS AND METHODS

Chemistry. Reagents and Equipment. All commercial materials were directly used without further purification. Solvents, analytical reagents, would be dried using a routine procedure and redistilled for extraction, washing, and chromatography. With TMS as an internal reference, a Varian Mercury-Plus 600 and or a 400 spectrometer was used to record 13C NMR and 1H NMR spectra in CDCl3 or DMSOd6. The Thermo Fisher Mass platform DSQII was used to give mass spectra with low resolution, whereas for mass spectra with high resolution, a Waters MALDI Synapt G2 HDMS was used. A Büchi B545 was used to give melting points for titled compounds. To save time and experimetal cost, we did not optimize the chemical yields, which represent just a single experiment result. Synthetic Chemistry. Compounds 16−55 and 59−62 were synthesized by following published methods.26−36 Detailed synthetic procedures and characterization data for all of the newly synthesized compounds are given in the Supporting Information. Enzymatic Kinetics.37,38 As described in our previous studies,24,25 the SCR (succinate cytochrome c reductase, the mixture of SQR and complex III) was obtained from porcine heart. To assay the biological activity of compounds, different substrates were used for SQR (succinate and dichlorophenolindophenol (DCIP) as substrate), complex II (decylubiquinol (DBH2) and cytochrome c as substrate), and SCR (succinate and cytochrome c as substrates), respectively. In addition, the detailed mechanism of inhibition kinetics for some compounds was also studied using a method similar to that described previously.39,40 Data Analysis.24 On the basis of the extinction coefficient of the measured substance, the product concentration variation was converted from the absorbance change and then made a linear fit on time. Here, the slope would stand for the enzymatic reaction velocity. The inhibition rates of the title compounds could be obtained by comparison with the control. In Vivo Fungicidal Activity. R. solani and S. fuliginea were selected as representative plant pathogens for the compound screening using the assay method as described in ref 41. The results are summarized in Table 1. Field Trial. A rice field naturally infected by R. solani was selected to perform the field trial by using the standard method.42−44 Homology Model. The homology model is one of the most popular methods to build the 3D structure of protein using the crystal structures of some homology proteins as template. Until now, the 3D structure of SQR from R. solani is still unavailable. Therefore, MODELER 9v1445,46 was used to build homology models of R. solani SQR based on the crystal structure of 2WQY. Then, Amber 1447,48 was used to do further energy minimization for selected models. Finally, compound 62 and thifluzamide were docked into the



RESULT AND DISCUSSION Binding Mode of Hit 3. As described previously,25 hit 3 was identified as the most promising inhibitor with an IC50 of 19.79 μM against porcine SCR. To better understand the relevant interactions between hit 3 and the target protein, we carried out a molecular docking study using AutoDock. The binding mode of hit 3 was nearly the same as that of commercial fungicides. As shown in Figure 2A, two hydrogen bonds were formed between the carbonyl oxygen atom in hit 3 and the residues of D_Y91 and B_W173. Meanwhile, the pyrazole ring in hit 3 also showed cation−π interaction with the residue of C_R46. Different from our previous study, the terminal phenyl ring in hit 3 took an extended conformation to form a π−π stacking interaction with C_W35, which will be discussed in the following section. Scalliet et al.18 had pointed out that the hydrophobic rest of amide side in the chemical structure of SQR inhibitors had an important effect on the potency and fungicidal spectrum. On the basis of the computational simulation results, the inhibitory activity could be improved by optimizing the interaction between the amideside groups with SQR. Enzyme Inhibition and Structure−Activity Relationships. As shown in Table 1, a series of biphenyl ethercontaining pyrazole-carboxamides was synthesized. The introduced substituents include halogen, methyl, methoxyl, methylthio, cyano, trifluoromethoxyl, and so on. Compounds 17 (2Cl, IC50 = 1.98 μM), 18 (2-Br, IC50 = 4.20 μM), and 19 (2SCH3, IC50 = 1.78 μM) with 2-position substituents showed significantly improved potency, whereas compound 16 (2-F, IC50 = 22.41 μM) showed almost the same level of activity as the initial hit 3. Compound 20 with 3-chloro showed a slightly lower activity (IC50 = 36.9 μM), whereas compound 22 with 4chloro showed an approximately 4-fold improved potency (IC50 = 4.90 μM) compared with hit 3. In addition, compounds 59 (4-CF3, IC50 = 3.17 μM) and 24 (4-OCF3, IC50 = 11.69 μM) also exhibited higher potency than hit 3. However, compounds 21 (4-F) and 23 (4-CN) displayed a total loss of activity. Therefore, we can conclude that a 3-position substituent is B

DOI: 10.1021/acs.jafc.6b05134 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Table 1. Chemical Structures, Fungicidal Activities (Inhibition Ratings 1−100) of Titled Compounds, and Their Inhibitory Activities against Porcine SCR

compd hit 3 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 59 60 61 62 penthiopyrad thifluzamide a

R1

R2

dosage (mg/L)

R. solania

S. fuligineaa

CF3 CF3 CF3 CF3 CF3 CF3 CF3 CF3 CF3 CF3 CF3 CF3 CF3 CF3 CF3 CF3 CF3 CF3 CHF2 CHF2 CHF2 CHF2 CHF2 CHF2 CHF2 CHF2 CHF2 CHF2 CHF2 CHF2 CHF2 CHF2 CHF2 CHF2 CHF2 CHF2 CHF2 CHF2 CHF2 CHF2 CHF2 CF3 CF3 CHF2 CHF2

H 2-F 2-Cl 2-Br 2-SMe 3-Cl 4-F 4-Cl 4-CN 4-OCF3 2,4-Cl2 2,3-Me2 2,3-F2 2-Me-5-F 3,4-Me2 3,5-Me2 3,4,5-OMe3 3,4,5-F3 2-F 2-Cl 2-Br 2-SMe 3-Cl 4-F 4-Cl 4-OMe 4-CN 4-OCF3 2,4-Cl2 2,3-Me2 2,3-F2 2-Me-5-F 3,4-Me2 3,5-Me2 2,5-Cl2 2-Cl-4-F 2,4-F2 2-Me-5-iPr 2-Me-4-Cl 3,4,5-OMe3 3,4,5-F3 4-CF3 2-Cl-4-CF3 4-CF3 2-Cl-4-CF3

200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200

76 100 82 100 0 69 57 66 30 60 0 63 90 73 0 90 0 100 95 100 100 90 100 100 68 59 45 52 100 66 100 95 0 90 95 100 90 74 58 0 100 56 100 72 100 NTd 100

100 70 56 95 0 22 15 44 0 40 72 0 40 50 0 0 0 100 100 100 100 0 0 44 0 60 0 95 80 0 0 35 0 0 30 90 30 20 70 0 80 0 100 44 100 NT 100

100

IC50b (μM)

ΔGcal

ΔGexpc

± ± ± ± ± ±

1.07 1.23 1.39 1.12 1.11 1.47

−19.05 −19.35 −20.95 −20.47 −21.13 −18.66

−6.42 −6.35 −7.78 −7.34 −7.85 −6.05

± 1.28

−20.63

−7.25

± ± ± ± ± ± ±

1.29 1.37 1.27 1.22 1.05 1.11 1.23

−20.13 −21.09 −19.83 −19.73 −20.28 −20.94 −20.65

−6.73 −7.60 −6.83 −6.86 −6.92 −7.25 −7.43

± ± ± ± ± ± ± ± ±

1.17 1.12 1.32 1.07 1.14 1.28 1.17 1.15 2.36

−21.04 −19.34 −21.99 −21.82 −20.87 −20.16 −19.30 −21.12 −18.51

−7.62 −6.49 −8.12 −7.94 −7.62 −7.25 −6.60 −7.77 −6.12

± ± ± ± ± ± ± ± ± ± ± ±

1.12 0.01 1.33 1.17 1.17 1.17 1.19 0.13 1.15 1.14 0.12 1.10

−20.98 −22.42 −21.16 −21.19 −20.70 −20.78 −20.18 −22.03 −21.52 −19.71 −22.15 −21.15

−7.41 −8.73 −7.50 −7.73 −7.28 −7.50 −7.17 −8.06 −7.99 −6.67 −8.44 −7.74

± ± ± ± ± ±

0.12 1.12 0.02 1.35 0.012 0.117

−22.60 −21.16 −22.98 −21.39 −23.86 −21.76

−8.49 −7.50 −8.95 −7.90 −9.48 −8.04

19.79 22.41 1.98 4.20 1.78 36.9 >100 4.90 >100 11.69 2.70 9.86 9.37 8.47 4.87 3.59 >100 2.62 17.66 1.13 1.52 2.59 4.90 14.52 2.02 32.76 >100 3.72 0.40 3.22 2.17 4.67 3.19 5.62 1.25 1.40 12.93 0.65 2.13 >100 0.60 3.17 0.28 1.62 0.11 1.29 NT

In vivo assay. bIn vitro assay. cΔGexp = −RT Ln IC50. dNot tested.

unfavorable for retaining high activity, but 4-position substitutions have a complicated effect on the activities. Furthermore, disubstituted compounds 25−30 were also synthesized to investigate their structure−activity relationship. Interestingly, all of these compounds showed better inhibitory

activity than hit 3. For example, the 2,4-dichloro compound 25 displayed a noticeable improvement in activity with an IC50 value of 2.70 μM. Similar results were obtained for the 2,3dimethyl analogue 26 and 3,4-dimethyl analogue 29, with IC50 values of 9.86 and 4.87 μM, respectively. Meanwhile, other C

DOI: 10.1021/acs.jafc.6b05134 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 2. Binding modes of (A) hit 3 (yellow sticks) with porcine SQR, (B) compound 62 (yellow sticks) with porcine SQR, (C) flutolanil derivatives with porcine SQR in crystal structure 3ABV, and (D) compound 62 (orange sticks) with R. solani SQR. (E) Overlay of compound 62 bonding with porcine SQR (represented by magenta and yellow sticks) and R. solani SQR (represented by gray and green sticks). (F) Overlay of thifluzamide bonding with porcine SQR (represented by magenta and yellow sticks) and R. solani SQR (represented by gray and green sticks). C_M39/W, C_W35/P, and C_I30/F respectively indicate C_M39, C_W35, and C_I30 in porcine SQR and C_W39, C_P35, and C_F30 in R. solani SQR. For clarity, just some key residues are shown.

derivatives, 27 (2,3-F2, IC50 = 9.37 μM), 28 (2-Me-5-F, IC50 = 8.47 μM), and 30 (3,5-Me2, IC50 = 3.59 μM), all exhibited good activities. The introduction of a 2-Cl-4-CF3 substitution to the phenyl ring resulted in compound 60 with a dramatic increase of activity (IC50 = 0.277 μM), about 71-fold higher than that of hit 3. This demonstrated the importance of 2,4-disubstitution for high potency. Meanwhile, we also explored the effect of trisubstituted phenyl rings. As a result, compound 31 (R2 = 3,4,5-OMe3) produced a dramatic loss of activity. On the contrary, compound 32 (R2 = 3,4,5-F3) showed about 8-fold increased potency compared with hit 3. All of these results indicated that 2- and 4-position substitutions are favorable to the potency, especially 2-Cl and 4-CF3 substituents, always producing higher potency than other groups. Having optimal substitutions at the phenyl ring in hand, we then tried to optimize the pyrazole scaffold. In our previous study,24 the replacement of trifluoromethyl with difluoromethyl on the pyrazole ring could increase the activity of SQR inhibitors. Therefore, on the basis of the above results, a series of compounds with difluoromethyl on the pyrazole ring were also designed and synthesized. In contrast to the corresponding trifluoromethyl-substituted derivatives, they exhibited significantly enhanced potency against porcine SCR. 2-F, 4-F, 4OCF3, and 4-CF3 analogues 33, 38, 42, and 61 exhibited higher potency than hit 3. Similar to the above studies, some analogues containing di- or tri-substituted groups also were investigated. For example, the 2-Cl-4-F compound (50, IC50 = 1.40 μM) was synthesized and exhibited a slight decrease in activity compared to the 2,4-dichloro compound 43, whereas other substituents (2-Me-5-F 46 and 2,4-difluoro 51) tended to be slightly less active. Inspired by the results from the 2,4disubstituted designs, compound 62 with 2-Cl-4-CF3 was prepared and synthesized, and its IC50 value was 0.11 μM,

leading to a distinct boost with a nearly 180-fold improvement in activity relative to hit 3. Also, 3,4,5-trifluoro analogue 55 (IC50 = 0.60 μM) was generated, and a similar activity was observed. Inhibitory Kinetics of Compound 62 and Penthiopyrad. To further figure out the molecular mechanism, compound 62 was selected as an example to study the mechanism of SQR and SCR inhibition. The results showed that compound 62 was a noncompetitive inhibitor upon SCR and SQR, whether the substrate was cytochrome c (Figure 3A) or DCIP (Figure 3B). Nearly the same thing occurred for the control of commercial fungicide penthiopyrad (Figure 3C for SCR and Figure 3D for SQR). All of these things indicated that compound 62 had the same binding target (SQR) and the same binding site (Q-site) as penthiopyrad. In addition, as shown in Table 2, the Ki values of compound 62 were 0.107 μM in SCR and 0.081 μM in SQR, whereas those of penthiopyrad would decrease to 1.45 μM in SCR and 0.307 μM in SQR, respectively. The IC50 values also took on the same trend between compound 62 and penthiopyrad. These results indicate compound 62 showed 1.3 times (1.8 times indicated by IC50) higher inhibition against SQR than SCR indicated by Ki values, whereas penthiopyrad showed 4.7 times (2.5 times indicated by IC50) higher inhibition against porcine SQR than SCR. We also noted that both compounds did not exhibit significant inhibitory activity against complex III even at 10 mM concentrations (data not shown). Moreover, the IC50 and Ki values of both compounds against the SQR system have nearly the same magnitude as in the SCR system. This phenomenon was consistent with our expection because the title compounds were designed to be SQR inhibitors. The above results showed compound 62 should be an SQR inhibitor with a novel scaffold. As a result, SCR could be also D

DOI: 10.1021/acs.jafc.6b05134 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 3. Kinetic analysis of inhibition by compound 62 (A, SCR; B, SQR) and penthiopyrad (C, SCR; D, SQR) against porcine SCR and SQR and inhibition of porcine SCR by (A) compound 62 (1, 0 nM; 2, 300 nM; 3, 500 nM; 4, 1000 nM) and (C) penthiopyrad (1, 0 nM; 2, 300 nM; 3, 500 nM; 4, 1000 nM). Each reaction mixture contains 100 mM PBS (pH 7.4), 0.3 mM EDTA, 20 mM succinate, 0.1 nM enzyme, 0.29−10.36 μM cytochrome c, and the indicated penthiopyrad or compound 62. Ki was estimated to be 1.450 ± 0.069 μM for penthiopyrad and 0.107 ± 0.043 μM for compound 62 by assuming noncompetitive inhibition. Inhibition of porcine SQR by (B) compound 62 (1, 0 nM; 2, 20 nM; 3, 40 nM; 4, 70 nM) and (D) penthiopyrad (1, 0 nM; 2, 100 nM; 3, 200 nM; 4, 300 nM). Each reaction mixture contains 100 mM PBS (pH 7.4), 0.3 mM EDTA, 20 mM succinate, 2 nM SCR, 1.18−41.22 μM DCIP, and the indicated amount of penthiopyrad or compound 62. Ki was estimated to be 0.307 ± 0.008 μM for penthiopyrad and 0.081 ± 0.004 μM for compound 62 by assuming noncompetitive inhibition.

Table 2. Inhibitory Effect of Some Inhibitors against SCR and SQR SCR (succinate−cyt c system 23 °C)

a

SQR (DCIP system 23 °C)

inhibitor

IC50 (μM)

inhibition type

Ki (μM)

IC50 (μM)

inhibition type

Ki (μM)

62 penthiopyrad penthiopyrada

0.113 ± 0.012 1.294 ± 0.117 1.321 ± 0.110

noncompetitive noncompetitive noncompetitive

0.107 ± 0.043 1.450 ± 0.069 1.393 ± 0.087

0.062 ± 0.013 0.511 ± 0.126 0.527 ± 0.111

noncompetitive noncompetitive noncompetitive

0.081 ± 0.04 0.307 ± 0.008 0.327 ± 0.008

Obtained from ref 25.

procedure.25,53 Thifluzamide, which is one commercial fungicide targeting SQR, was selected as positive control. As shown in Table 1, most of the compounds displayed promising fungicidal activities against R. solani and S. fuliginea at a dosage of 200 mg/L. Very promisingly, compounds 18, 32, 33, 34, 35, 50, 60, and 62 exhibited >90% control against the two tested fungi, and compounds 16, 27, 30, 37, 38, 43, 45, 46, 48, 49, 51, and 55 showed >90% control against R. solani, whereas compound 42 exhibited >90% control against S. fuliginea. After careful analysis of the chemical structures of compounds with high fungicidal activity, one interesting finding

used as a testing system to assay the inhibitors’ relative inhibitory activity opon SQR. In Vivo Fungicidal Activity. R. solani has developed as the “number one” pathogen in rice (Oryza sativa L.) fields because it can cause heavy losses in rice yield worldwide. Meanwhile, S. fuliginea is also another important plant pathogen, limiting the production of cucurbits throughout the world. Therefore, in this study, the fungicidal activities of all of the title compounds were evaluated against these two important pathogens in a greenhouse environment according to a previously reported E

DOI: 10.1021/acs.jafc.6b05134 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry Table 3. Further Fungicidal Activities (Inhibition Rates 0−100) and EC50 Values of Titled Compounds in Vivo compd

dosage (mg/L)

R. solani

S. fuliginea

EC50a (mg/L)

compd

dosage (mg/L)

R. solani

S. f uliginea

EC50a (mg/L)

100 50 25 12.5 6.25 100 50 25 100 50 25 12.5 6.25 3.125 100 50 25 12.5 6.25 100 50 25

100 100 80 72 75 96 68 64 100 98 95 82 69 65 100 75 70 58 49 100 100 82

100 83 41 7 0 0 0 0 0 0 0 0 0 0 100 89 41 0 0 100 63 22

5.29

35

38

2.07

49

81 78 66 51 47 100 62 43 74 65 47 37 36

95 60 30 10 0 0 0 0 0 0 0 0 0

4.31

21.91

50 25 12.5 6.25 3.125 100 50 25 50 25 12.5 6.25 3.125

6.66

62

6.08

thifluzamide

100 50 25 12.5 6.25 12.5 6.25

100 100 99 95 70 61 54

100 100 94 67 0 0 0

34

37

43

60

thifluzamide

a

33.08

10.95

1.57

EC50 valus for R. solani.

Table 4. Relative Control Effect of Compounds 43 and 62 against R. solani in Field Trials 7 days after two sprayingsa treatment

concentration (g ai/ha)

disease index

protection effect (%)

disease index

protection effect (%)

43 (5%, EC)b

112.5 75 37.5

2.68 3.13 4.56

61.5 55.0 34.5

2.56 3.33 3.97

70.4 61.3 54.0

62 (5%, EC)

112.5 75 37.5

1.59 2.12 2.40

77.2 69.6 65.5

0.99 1.62 1.87

88.5 81.2 78.3

75

3.59

48.4

2.12

75.4

0

6.97

0

8.63

0

thifluzamide (24%, SC)c control a

21 days after two sprayingsa

Average of three tests. bEmulsifiable concentrate. cAqueous suspension concentrate.

was that compounds substituted with a halogen in the R2 postition generally displayed higher fungicidal activity than its corresponding compound with an electron-donating group. One possible reason for this phenomenon may be that halogens favor the interaction between ligand and protein. In addition, as shown in Table 1, compounds bearing −CF2H generally showed better fungicidal activity than their corresponding −CF3-substituted analogues not only in vitro but also in vivo. For example, the inhibition rate of compound 34 (R1 = CHF2, IC50 = 1.13 μM) was 100% for both tested fungi, whereas it decreased to 82% against R. solani and to 56% against S. fuliginea for compound 17 (IC50 = 1.98 μM) bearing −CF3 in the R1 position. The same thing occurred between compounds 38 (R1 = CHF2, IC50 = 14.52 μM, 100% inhibitory rate for R. solani) and 21 (R1 = CF3, IC50 > 100 μM, 57% inhibitory rate for R. solani), between compounds 43 (R1 = CHF2, IC50 = 0.401 μM, 100% inhibitory rate for R. solani) and 25 (R1 = CF3, IC50 = 2.7 μM, no inhibitory rate for R. solani), and between compounds 36 (R1 = CHF2, IC50 = 4.90 μM,

100% inhibitory rate for R. solani) and 20 (R1 = CF3, IC50 = 36.9 μM, 69% inhibitory rate for R. solani). Therefore, some compounds with promising fungicidal activities (inhibition >90%) were selected for further testing at lower dosages. The inhibitory rates for compounds 16, 18, 32, 33, 45, 50, and 55 are shown in Table 3S (Supporting Information), which did not show good fungicidal activities against both tested plant pathogens. However, as shown in Table 3, at a lower dosage of 50 mg/L, compounds 34, 35, and 62 still displayed >80% control against both tested plant pathogens. Surprisingly, compound 60 can effectively control S. fuliginea (>80% inhibition) at a dosage of 50 mg/L, which was superior to commercial control thifluzamide. In addition, compound 43 showed more promising fungicidal activities against R. solani than commercial control thifluzamide at all tested dosages (Table 4). A most promising outcome was that compound 62 exhibited higher control against R. solani and S. fuliginea than commercial control thifluzamide at all tested dosages. F

DOI: 10.1021/acs.jafc.6b05134 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

bonds with the residues of D_Y91 and B_W173, and then its pyrazole ring showed cation−π interaction with the residue of C_R46. The terminal phenyl ring in compound 62 pointed to C_W35, forming T−π interaction with the indole ring of C_W35 (Figure 2B). This binding mode was similar to the flutolani derivative bound with SQR (PDB ID 3ABV, Figure 2C), which further confirmed our simulated binding mode. Nearly the same thing occurred for other compounds. The binding mode of thifluzamide had been discussed in our previous study.50 For clarity, the residue in R. solani SQR was numbered according to crystal structure 1ZOY (nearly the same residue number with 2WQY). The biggest difference in the SQR Q-site might be C_M39, C_I30, and C_W35 in porcine SQR, while changing to C_W39, C_30F, and C_P35 in R. solani SQR. Just as we expected, compound 62 and thifluzamide showed nearly the same position when bound with both species SQR, forming hydrogen bonds with the residues of B_W173 and D_Y91 and cation−π interaction with the residue of C_R46. The interesting thing was that, compared to binding mode in porcine SQR, compound 62 bound more deeply with R. solani SQR than with porcine SQR, and the pyrazole and diphenylether in it turned about 180° (Figure 2D,E) to avoid clashing with C_W39 in R. solani SQR. As a consequence, the terminal phenyl ring in compound 62 formed a π−π interaction with D_Y91 in R. solani SQR. Compared to thifluzamide bound with procine SQR, its thiazole ring also showed about 180° turnover, whereas trifluoromethoxy-substituted phenyl just deviated about 2 Å to avoid clashing with C_F30 when bound with R. solani SQR (Figure 2F). The binding free energies were respectively −18.86 and −19.17 kcal/mol for thifluzamide and compound 62 bound with R. solani SQR, which correlate well with the experimental results of the in vivo assay. All of these results indicated that the fragment of diphenyl-ether in compound 62 could randomly regulate its conformation to fit better with both species SQR than thifluzamide by forming T−π interaction with C_W35 in porcine SQR and π−π interaction with D_Y91 in R. solani SQR. On the contrary, nearly the same conformation for thifluzamide when bound with different species SQR might be used to explain its narrow biological spectra. In summary, a series of highly potent novel diphenyl-ether containing pyrazole-carboxamide inhibitors were discovered through hit-to-lead optimization. Several potent compounds from this class showed very good fungicidal activity not only in vitro but also in vivo. Among these compounds, compounds 18, 32, 33, 34, 35, 50, 60, and 62 exhibited >90% control against the two tested fungus, and compounds 16, 27, 30, 37, 38, 43, 45, 46, 48, 49, 51, and 55 showed >90% control against R. solani, whereas compound 42 exhibited >90% control against S. fuliginea. More importantly, compounds 62 and 43 showed better fungicidal activity against both fungi than commercial fungicide thifluzamide even in lower dosages, such as 6.25 mg/ L. The results obtained from molecular modeling indicated compound 62 could induce its conformation to match different species SQR. On the contrary, the same thing did not occur for thifluzamide, resulting in its lower activity. Therefore, compared with the chemical structure of thifluzamide, a more flexible fragment should be introduced into the amine side for carboxamide fungicide, such as diphenyl-ether. The methods and insights from this work will inform the design of new novel SQR inhibitors. Compound 62 (named flubenetheram) has

To further study the potential of compounds 43 and 62 against sheath blight, field experiments were carried out during the rice-growing season. Rice plants at fruiting stage naturally infected by R. solani were used. Foliar applications of compounds 43 and 62 at 112.5 g ai/ha recorded 61.5 and 77.2% protection effect over control after two sprayings at the seventh day, respectively. Then, the protection effect would respectively go up to 70.4 and 88.5% after 21 days for compounds 43 and 62. Then, almost no damage was observed on the leaves of rice plants. If the treatment concentration of compounds decreased to 75 g ai/ha, especially for compound 62, lesions on the treated plant leaves expanded very slowly, even ceased expanding, which demonstrated that compound 62 has a curative activity against the expansion of damage by R. solani with a protection effect of approximately 81.2% after 21 days. Lesions on the control plant leaves, however, broadened quickly, and intense fungal hyphae could be observed. The protection effects are about 48.4 and 75.4% for thifluzamide-treated (75 g ai/ha) rice plants at the 7th and 21st days, respectively. In addition, if we still lower the treatment concentation of both compounds to 37.5 g ai/ha, compound 62 continued to show a higher protection effect than thifluzamide control. As shown in Table 3, compounds 43 and 62 showed excellent fungicidal activities against R. solani with EC50 values of 2.07 and 1.57 mg/L, respectively. Moreover, the field experiments show that the protection effect of compound 62 is better than that of thifluzamide as a treatment of sheath blight. Molecular Modeling. As we know, the structure−activity relationship plays an important role in understanding the molecular mechanism of drug molecules. As shown in Table 1, the binding free energies (ΔGcal) for all of the title compounds (except compounds with IC50 > 100 μM) were calculated. The range of ΔGcal was from −18.51 to −23.86 kcal/mol, whereas ΔGexp (ΔGexp = −RT Ln IC50) ranged from −6.05 to −9.48 kcal/mol. We noted that MM/PBSA calculations systematically overvalued the binding free energies between ligand and protein. However, the value of ΔGcal in Table 1 was qualitatively consistent with ΔGexp. Most importantly, the correlation coefficient (r2) between ΔGcal and ΔGexp was 0.96 (Figure 3S, Supporting Information), further confirming the credibility of the computational models obtained from this study. As shown in Tables 2 and 3, compound 62 was identified as the highest potent inhibitor among the titled compounds toward both R. solani and S. fuliginea SQR. All of these results forced us to rethink profoundly the reason for these phenomena. In general, there exist two reasons: good ADME (absorption, distribution, metabolism, excretion) characters and higher binding affinity when bound with target. As we know, the ADME characters of one compound just like one black box, and now we cannot accurately predict it on the basis of our present laboratory condition. On the contrary, the binding free energy could be accurately calculated on the basis of the binding mode of compound with SQR. Therefor, the 3D structure of R. solani SQR was built on the basis of 2WQY template by using a homology model method. Then, compound 62 and thifluzamide were docked into the binding site (Q-site) of both R. solani and porcine SQR. The binding mode of compound 62 with porcine SQR is shown in Figure 2B. Compared with the binding mode of commercial carboxamide fungicides published in our previous study, the carboxyl oxygen in compound 62 also formed two hydrogen G

DOI: 10.1021/acs.jafc.6b05134 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

anion overproduction and abnormal energy metabolism in Caenorhabditis elegans. J. Biol. Chem. 2001, 276, 41553−41558. (9) Rustin, P.; Rǒig, A. Inborn errors of complex IIunusual human mitochondrial diseases. Biochim. Biophys. Acta, Bioenerg. 2002, 1553, 117−122. (10) Carvalho, L.; Juan, R. L. O.; Carmen, L. M.; Santiago, C.; Luis, R.; Francisco, G. The 8-aminoquinoline analogue sitamaquine causes oxidative stress in Leishmania donovani promastigotes by targeting succinate dehydrogenase. Antimicrob. Agents Chemother. 2011, 55, 4204−4210. (11) Mode of action of fungicides. FRAC Classification on Mode of Action, Crop Life, 2015; http://www.frac.info/. (12) Avenot, H. F.; Sellam, A.; Karaoglanidis, G.; Michailides, T. J. Characterization of mutations in the iron−sulphur subunit of succinate dehydrogenase correlating with boscalid resistance in Alternaria alternata from California pistachio. Phytopathology 2008, 98, 736−742. (13) Avenot, H.; Sellam, A.; Michailides, T. Characterization of mutations in the membrane-anchored subunits AaSDHC and AaSDHD of succinate dehydrogenase from Alternaria alternata isolates conferring field resistance to the fungicide boscalid. Plant Pathol. 2009, 58, 1134−1143. (14) Veloukas, T.; Leroch, M.; Hahn, M.; Karaoglanidis, G. S. Detection and molecular characterization of boscalid resistant Botrytis cinerea isolates from strawberry. Plant Dis. 2011, 95, 1302−1307. (15) Angelini, R. M. D.; Habib, W.; Rotolo, C.; Pollastro, S.; Faretra, F. Selection, characterization and genetic analysis of laboratory mutants of Botryotinia fuckeliana (Botrytis cinerea) resistant to the fungicide boscalid. Eur. J. Plant Pathol. 2010, 128, 185−199. (16) Avenot, H. F.; Thomas, A.; Gitaitis, R. D.; Langston, D. B., Jr.; Stevenson, K. L. Molecular characterization of boscalid- and penthiopyrad-resistant isolates of Didymella bryoniae and assessment of their sensitivity to fluopyram. Pest Manage. Sci. 2012, 68, 645−651. (17) Fraaije, B. A.; Bayon, C.; Atkins, S.; Cools, H. J.; Lucas, J. A.; Fraaije, M. W. Risk assessment studies on succinate dehydrogenase inhibitors, the new weapons in the battle to control Septoria leaf blotch in wheat. Mol. Plant Pathol. 2012, 13, 263−275. (18) Scalliet, G.; Bowler, J.; Luksch, T.; Kirchhofer-Allan, L.; Steinhauer, D.; Ward, K.; Niklaus, M.; Verras, A.; Csukai, M.; Fonné-Pfister, R. Mutagenesis and functional studies with succinate dehydrogenase inhibitors in the wheat pathogen Mycosphaerella graminicola. PLoS One 2012, 7, e35429. (19) Skinner, W.; Bailey, A.; Renwick, A.; Keon, J.; Gurr, S.; Hargreaves, J. A single amino-acid substitution in the ironsulphur protein subunit of succinate dehydrogenase determines resistance to carboxin in Mycosphaerella graminicola. Curr. Genet. 1998, 34, 393− 398. (20) Charvolin, D.; Picard, M.; Huang, L. S.; Berry, E. A.; Popot, J. L. Solution behavior and crystallization of cytochrome bc1 in the presence of amphipols. J. Membr. Biol. 2014, 247, 981−996. (21) Hao, G. F.; Wang, F.; Li, H.; Zhu, X. L.; Yang, W. C.; Huang, L. S.; Wu, J. W.; Berry, E. A.; Yang, G. F. Computational discovery of picomolar Q(o) site inhibitors of cytochrome bc1 complex. J. Am. Chem. Soc. 2012, 134, 11168−11176. (22) Wang, Z. J.; Gao, Y.; Hou, Y. L.; Zhang, C.; Yu, S. J.; Bian, Q.; Li, Z. M.; Zhao, W. G. Design, synthesis, and fungicidal evaluation of a series of novel 5-methyl-1H-1,2,3-trizole-4-carboxyl amide and ester analogues. Eur. J. Med. Chem. 2014, 86, 87−94. (23) Ye, Y. H.; Ma, L.; Dai, Z. C.; Xiao, Y.; Zhang, Y. Y.; Li, D. D.; Wang, J. X.; Zhu, H. L. Synthesis and antifungal activity of nicotinamide derivatives as succinate dehydrogenase inhibitors. J. Agric. Food Chem. 2014, 62, 4063−4071. (24) Xiong, L.; Zhu, X. L.; Shen, Y. Q.; Wishwajith, W. K. W. M.; Yang, G. F.; Li, K. Discovery of N-benzoxazol-5-yl-pyrazole-4carboxamides as nanomolar SQR inhibitors. Eur. J. Med. Chem. 2015, 95, 424−434. (25) Xiong, L.; Zhu, X. L.; Gao, H. W.; Fu, Y.; Hu, S. Q.; Jiang, L. N.; Yang, W. C.; Yang, G. F. Discovery of potent succinate-ubiquinone oxidoreductase inhibitors via pharmacophore-linked fragment virtual screening approach. J. Agric. Food Chem. 2016, 64, 4830−4837.

progressed to industry development as an agricultural fungicide in China.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b05134. Figure 1S: IC50 curve of penthiopyrad and compound 62. Table 1S: binding energy contribution for titled compounds. Table 2S: sequence identities between different species SQR. Table 3S: fungicidal activity of some compounds. Table 4S: EC50 values. Figure 2S: alignment between target and template protein. Figure 3S: relationship between ΔGexp and ΔGcal. 1H and 13C NMR spectra of representative title compounds (PDF)



AUTHOR INFORMATION

Corresponding Authors

*(X.-L.Z.) College of Chemistry Central China Normal University Luoyu Road 152, Wuhan 430079, China. Phone: 86-27-67867800. Fax: 86-27-67867141. E-mail: xlzhu@mail. ccnu.edu.cn. *(G.-F.Y.) College of Chemistry Central China Normal University, Luoyu Road 152, Wuhan 430079, China. Phone: 86-27-67867800. Fax: 86-27-67867141. E-mail: gfyang@mail. ccnu.edu.cn. ORCID

Guang-Fu Yang: 0000-0003-4384-2593 Funding

The research was supported in part by the National Key Technologies R&D Program (2014BAD23B00) and the National Natural Science Foundation of China (No. 21332004 and 21272092). Notes

The authors declare no competing financial interest.



REFERENCES

(1) Cecchini, G. Function and structure of complex II of the respiratory chain. Annu. Rev. Biochem. 2003, 72, 77−109. (2) Maklashina, E.; Cecchini, G. The quinone-binding and catalytic site of complex II. Biochim. Biophys. Acta, Bioenerg. 2010, 1797, 1877− 1882. (3) Yankovskaya, V.; Horsefield, R.; Törnroth, S.; Luna-Chavez, C.; Miyoshi, H.; Léger, C.; Byrne, B.; Cecchini, G.; Iwata, S. Architecture of succinate dehydrogenase and reactive oxygen species generation. Science 2003, 299, 700−704. (4) Hägerhäll, C. Succinate: quinone oxidoreductases. Variations on a conserved theme. Biochim. Biophys. Acta, Bioenerg. 1997, 1320, 107− 141. (5) Sun, F.; Huo, X.; Zhai, Y.; Wang, A.; Xu, J.; Su, D.; Bartlam, M.; Rao, Z. Crystal structure of mitochondrial respiratory membrane protein complex II. Cell 2005, 121, 1043−1057. (6) Huang, L. S.; Shen, J. T.; Wang, A. C.; Berry, E. A. Crystallographic studies of the binding of ligands to the dicarboxylate site of complex II, and the identity of the ligand in the “oxaloacetate inhibited” state. Biochim. Biophys. Acta, Bioenerg. 2006, 1757, 1073− 1083. (7) Ishii, N.; Fujii, M.; Hartman, P. S.; Tsuda, M.; Yasuda, K.; SenooMatsuda, N.; Yanase, S.; Ayusawa, D.; Suzuki, K. A mutation in succinate dehydrogenase cytochrome b causes oxidative stress and ageing in nematodes. Nature 1998, 394, 694−697. (8) Senoo-Matsuda, N.; Yasuda, K.; Tsuda, M.; Ohkubo, T.; Yoshimura, S.; Nakazawa, H.; Hartman, P. S.; Ishii, N. A defect in the cytochrome b large subunit in complex II causes both superoxide H

DOI: 10.1021/acs.jafc.6b05134 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry (26) Yoshikawa, Y.; Katsuta, H.; Kishi, J.; Yanase, Y. Structure-activity relationship of carboxin-related carboxamides as fungicide. J. Pestic. Sci. 2011, 36, 347−356. (27) Hwang, J.; Li, P.; Carroll, W. R.; Smith, M. D.; Pellechia, P. J.; Shimizu, K. D. Additivity of substituent effects in aromatic stacking interactions. J. Am. Chem. Soc. 2014, 136, 14060−14067. (28) Sanz, R.; Fernández, Y.; Castroviejo, M. P.; Pérez, A.; Fañanás, F. J. A route to regioselectively functionalized carbazoles, dibenzofurans, and dibenzothiophenes through anionic cyclization of benzynetethered aryllithiums. J. Org. Chem. 2006, 71, 6291−6294. (29) Wang, Y.; Cai, W.; Liu, Q.; Meng, Q.; Cheng, Y.; Yang, T.; Zhang, G.; Xiang, J.; Wu, C. Novel compounds. WO 2013029338, 2013. (30) Gao, K.; Yu, C.-B.; Li, W.; Zhou, Y.-G.; Zhang, X. Synthesis and enantioselective hydrogenation of seven-membered cyclic imines: substituted dibenzo[b,f ][1,4]oxazepines. Chem. Commun. 2011, 47, 7845−7847. (31) Chesworth, R.; Shapiro, G.; Beaulieu, P.; Chantigny, Y.; Mancuso, J.; Deziel, R.; Leit, S.; Tessier, P.; Smil, D. Inhibiteurs de l’histone désacétylase. WO 2009137499, 2009. (32) Vicker, N.; Day, J. M.; Bailey, H. V.; Heaton, W.; Gonzalez, A. M. R.; Sharland, C. M.; Reed, M. J.; Purohit, A.; Potter, B. V. L. 17βHydroxysteroid dehydrogenase type 3 (17β-hsd3) inhibitors. WO 2007003934, 2007. (33) Schmidt, D. M.; Bonvicino, G. E. The halogen-activated Smiles rearrangement. 2. J. Org. Chem. 1984, 49, 1664−1666. (34) Beke, G.; Bozo, E.; Eles, J.; Farkas, S.; Hornok, K.; Keserue, G.; Schmidt, E.; Szentirmay, E.; Vago, I.; Vastag, M. New sulfonamide derivatives as bradykinin antagonists. WO 2008068540, 2009. (35) Polisetti, D. R.; Kodra, J. T.; Lau, J.; Bloch, P.; Valcarce-Lopez, M. C.; Blume, N.; Guzel, M.; Santhosh, K. C.; Mjalli, A. M. M.; Andrews, R. C. Aryl carbonyl derivatives as therapeutic agents. WO 2004002481, 2004. (36) Olmsted, M. P.; Craig, P. N.; Lafferty, J. J.; Pavloff, A. M.; Zirkle, C. L. Analogs of phenothiazines. II.1 Phenoxazine and phenoselenazine analogs of phenothiazine drugs. J. Org. Chem. 1961, 26, 1901− 1907. (37) Zhao, P. L.; Wang, L.; Zhu, X. L.; Huang, X.; Zhan, C. G.; Wu, J. W.; Yang, G. F. Subnanomolar inhibitor of cytochrome bc1 complex designed by optimizing interaction with conformationally flexible residues. J. Am. Chem. Soc. 2010, 132, 185−194. (38) King, T. E. Preparations of succinate-cytochrome c reductase and the cytochrome bc1 particle, and reconstitution of succinatecytochrome c reductase. Methods Enzymol. 1967, 10, 216−225. (39) Fisher, N.; Bourges, I.; Hill, P.; Brasseur, G.; Meunier, B. Disruption of the interaction between the Rieske iron-sulfur protein and cytochrome b in the yeast bc1 complex owing to a human diseaseassociated mutation within cytochrome b. Eur. J. Biochem. 2004, 271, 1292−1298. (40) Fisher, N.; Brown, A. C.; Sexton, G.; Cook, A.; Windass, J.; Meunier, B. Modeling the Qo site of crop pathogens in Saccharomyces cerevisiae cytochrome b. Eur. J. Biochem. 2004, 271, 2264−2271. (41) Zhao, P. L.; Wang, F.; Zhang, M. Z.; Liu, Z. M.; Huang, W.; Yang, G. F. Synthesis, fungicidal, and insecticidal activities of βmethoxyacrylate-containing N-acetyl pyrazoline derivatives. J. Agric. Food Chem. 2008, 56, 10767−10773. (42) Wang, B. L.; Shi, Y. X.; Ma, Y.; Liu, X. H.; Li, Y. H.; Song, H. B.; Li, B. J.; Li, Z. M. Synthesis and biological activity of some novel trifluoromethyl-substituted 1,2,4-triazole and bis(1,2,4-triazole) Mannich bases containing piperazine rings. J. Agric. Food Chem. 2010, 58, 5515−5522. (43) Wang, L.; Li, B. J.; Xiang, W. S.; Shi, T. X.; Liu, C. L. Control effects of pyraoxystrobin on cucumber powdery mildew. Agrochemicals 2008, 47, 378−380. (44) Chen, Y.; Yao, J.; Yang, X.; Zhang, A. F.; Gao, T. C. Sensitivity of Rhizoctonia solani causing rice sheath blight to fluxapyroxad in China. Eur. J. Plant Pathol. 2014, 140, 419−428.

(45) Marti-Renom, M. A.; Stuart, A.; Fiser, A.; Sánchez, R.; Melo, F.; Sali, A. Comparative protein structure modeling of genes and genomes. Annu. Rev. Biophys. Biomol. Struct. 2000, 29, 291−325. (46) Sali, A.; Blundell, T. L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 1993, 234, 779−815. (47) Case, D. A.; Cheatham, T. E., III.; Darden, T.; Gohlke, H.; Luo, R.; Merz, K. M., Jr.; Onufriev, A.; Simmerling, C.; Wang, B.; Woods, R. The Amber biomolecular simulation programs. J. Comput. Chem. 2005, 26, 1668−1688. (48) Ponder, J. W.; Case, D. A. Force fields for protein simulations. Adv. Protein Chem. 2003, 66, 27−85. (49) Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.; Olson, A. J. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J. Comput. Chem. 1998, 19, 1639−1662. (50) Zhu, X. L.; Xiong, L.; Li, H.; Song, X. Y.; Liu, J. J.; Yang, G. F. Computational and experimental insight into the molecular mechanism of carboxamide inhibitors of succinate-ubquinone oxidoreductase. ChemMedChem 2014, 9, 1512−1521. (51) Sitkoff, D.; Sharp, K. A.; Honig, B. Accurate calculation of hydration free energies using macroscopic solvent models. J. Phys. Chem. 1994, 98, 1978−1988. (52) Connolly, M. L. Analytical molecular surface calculation. J. Appl. Crystallogr. 1983, 16, 548−558. (53) Hao, G. F.; Yang, S. G.; Huang, W.; Wang, L.; Shen, Y. Q.; Tu, W. L.; Li, H.; Huang, L. S.; Wu, J. W.; Berry, E. A.; Yang, G. F. Rational design of highly potent and slow-binding cytochrome bc1 inhibitor as fungicide by computational substitution optimization. Sci. Rep. 2015, 5, 13471−13479.

I

DOI: 10.1021/acs.jafc.6b05134 J. Agric. Food Chem. XXXX, XXX, XXX−XXX