Ametoctradin is a Potent Qo Site Inhibitor of the Mitochondrial

The cytochrome bc1 complex (EC 1.10.2.2, bc1, also known as complex III) is an essential component in the respiratory chain; it catalyzes electron tra...
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Ametoctradin is a Potent Qo Site Inhibitor of the Mitochondrial Respiration Complex III Xiaolei Zhu,† Mengmeng Zhang,† Jingjing Liu,† Jingming Ge,† and Guangfu Yang*,†,‡ †

Key Laboratory of Pesticide and Chemical Biology, College of Chemistry, Ministry of Education, Central China Normal University, Wuhan 430079, P.R. China ‡ Collaborative Innovation Center of Chemical Science and Engineering, Tianjing 30071, P.R.China S Supporting Information *

ABSTRACT: Ametoctradin is a new Oomycete-specific fungicide under development by BASF. It is a potent inhibitor of the bc1 complex in mitochondrial respiration. However, its detailed action mechanism remains unknown. In the present work, the binding mode of ametoctradin was first uncovered by integrating molecular docking, MD simulations, and MM/PBSA calculations, which showed that ametoctradin should be a Qo site inhibitor of bc1 complex. Subsequently, a series of new 1,2,4triazolo[1,5-a]pyrimidine derivatives were designed and synthesized to further understand the substituent effects on the 5- and 6position of 1,2,4-triazolo[1,5-a]pyrimidine. The calculated binding free energies (ΔGcal) of newly synthesized analogues as Qo site inhibitors correlated very well (R2 = 0.96) with their experimental binding free energies (ΔGexp). Two compounds (4a and 4c) with higher inhibitory activity against porcine SQR than ametoctradin were successfully identified. The structural and mechanistic insights obtained from the present study will provide a valuable clue for future designing of a new promising bc1 inhibitor. KEYWORDS: cytochrome bc1 complex, ametoctradin, molecular docking, molecular dynamics

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INTRODUCTION

that ametoctradin is an inhibitor of mitochondrial respiration bc1 complex with a new chemical scaffold, the pyrimidylamines. Field trials indicated that ametoctradin is a highly selective and effective fungicide that can be used in preventive spray applications against late blight and downy mildews in a wide range of special crops. It has an excellent toxicological and ecotoxicological profile and is highly suitable for use in integrated crop management programs. Since its discovery in 2004, an increasing interest in ametoctradin was not from its high potency but from its having no cross-resistance to the existing Qo inhibitors (e.g., strobilurins). However, it remains unclear whether ametoctradin is a Qo site inhibitor or a Qi site inhibitor of the bc1 complex. In order to uncover the detailed interaction mechanism between ametoctradin and bc1 complex, a series of 5,6disubstituted-7-amino-1,2,4-triazolo[1,5-a]pyrimidines 4a ∼ z (Figure 1) were designed and synthesized. Herein, we first described the synthetic chemistry, inhibition effects against porcine bc1 complex, and the structure−activity relationships of these newly synthesized compounds. Then, we carried out molecular docking of these compounds based on Qo and Qi sites, respectively, and followed by molecular dynamics (MD) simulations and the molecular mechanics Poisson−Boltzmann surface area (MM/PBSA) calculations. We believe that the detailed molecular mechanism of ametoctradin uncovered by the present study would put forward the structure-based design

Fungicides have become an integral part of efficient food production. In agrochemical research, the discovery of new fungicides with broad-spectrum fungal control and excellent crop selectivity is still remaining as a challenge. The cytochrome bc1 complex (EC 1.10.2.2, bc1, also known as complex III) is an essential component in the respiratory chain; it catalyzes electron transfer from quinol to cyt c1 and concomitantly translocates protons across membranes.1−4 The bc1 complex has a diheme cytochrome b, an iron−sulfur protein (ISP) with a Rieske-type Fe2S2 cluster, and cyt c1 that undergoes reduction and oxidation during the turnover of the enzyme.5 The bc1 complex has been found in the plasma membrane of bacteria and in the inner mitochondrial membrane of eukaryotes. Due to its critical roles in life processes, the bc1 complex has been identified as a promising action target for agricultural fungicides. So far, two separate catalytic sites, a quinone reduction site near the negative side of the membrane (Qi) and a quinol oxidation site close to the positive side of the membrane (Qo), have been identified and confirmed by X-ray crystallographic studies.6,7 The detailed action mechanism of a number of specific Qo and Qi sites inhibitors of cytochrome bc1 complex have been studied intensively.8,9 Among them, antimycin A, funiculosin, ilicicolin H, and dichlorophenyl dimethyl urea are well-known Qi site inhibitors, whereas the Qo site inhibitors include azoxystrobin, pyraclostrobin, kresoxim-methyl, stigmatellin, various hydroxyquinones and myxothiazol.10−14 Besides, some inhibitors, such as NQNO and ascochlorin, can bind both at Qo and Qi sites simultaneously.15 Ametoctradin (Initium)16,17 is an innovative new fungicide under development by BASF. The existing research has proved © XXXX American Chemical Society

Received: January 15, 2015 Revised: March 16, 2015 Accepted: March 16, 2015

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DOI: 10.1021/acs.jafc.5b00228 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Design of the title compounds.

reactive temperature was heated to 160 °C under reflux for 12− 16 h as monitored by TLC detection. The mixture was cooled to room temperature, and the product was precipitated in the form of colorless crystals. Water (5 mL) was added to the reaction mixture with pH equal to 8−9 adjusted by adding 20% triethylamine. Finally, the precipitate was purified by flash chromatography on silica gel to provide the desired product. Due to the complexity of reaction, the yield of some compound is lower than 10%. Our purpose was to obtain a certain compound to assay its activity. So, we did not to optimize the reaction condition to increase the yield. 5-Ethyl-6-octyl-1,2,4-triazolo[1,5-a]pyrimidin-7-amine (ametoctradin). Yield: 76%; mp 197.7−198.7 °C; 1H NMR (600 MHz, d6-DMSO) δ 8.33 (s, 1H), 7.71 (s, 2H), 2.74 (q, J = 7.2 Hz, 2H), 2.63−2.56 (m, 2H), 1.41 (d, J = 13.8 Hz, 4H), 1.33−1.19 (m, 11H), 0.86 (t, J = 6.6 Hz, 3H); MS: m/z = 275.33 (M+). 6-Octyl-5-(p-tolyl)-1,2,4-triazolo[1,5-a]pyrimidin-7amine (4a). Yield: 10%; mp 156−158 °C; 1H NMR (600 MHz, d6-DMSO): δ 8.44 (s, 1H), 8.01 (s, 2H), 7.34 (d, J = 7.2 Hz, 2H), 7.28 (d, J = 7.2 Hz, 2H), 2.56 (s, 2H), 2.38 (s, 3H), 1.33 (s, 2H), 1.25 (d, J = 12.0 Hz, 2H), 1.23−1.16 (m, 2H), 1.08 (dd, J = 42.6, 6.0 Hz, 6H), 0.83 (t, J = 7.2 Hz, 3H); MS: m/z = 337.27 (M+). Anal. Calcd for C20H27N5: C, 71.18; H, 8.06; N, 20.75. Found: C, 71.22; H, 8.102; N, 20.50. 6-Octyl-5-(3-(trifluoromethyl)phenyl)-1,2,4-triazolo[1,5-a]pyrimidin-7-amine (4b). Yield: 11%; mp 130−131 °C; 1H NMR (600 MHz, CDCl3): δ 8.42 (s, 1H), 7.78 (s, 1H), 7.73 (dd, J = 15.6, 7.2 Hz, 2H), 7.62 (t, J = 7.8 Hz, 1H), 6.00 (s, 2H), 2.60−2.55 (m, 2H), 1.58−1.46 (m, 2H), 1.23 (dd, J = 25.2, 19.8 Hz, 10H), 0.86 (t, J = 67.2 Hz, 3H). Anal. Calcd for C20H24F3N5: C, 61.37; H, 6.18; N, 17.89. Found: C,61.35; H, 6.070; N, 18.01. 5-(3-Chlorophenyl)-6-octyl-1,2,4-triazolo[1,5-a]pyrimidin-7-amine (4c). Yield: 7%; mp 154−155 °C; 1H NMR (600 MHz, d6-DMSO): δ 8.47 (s, 1H), 8.12 (s, 2H), 7.58−7.47 (m, 3H), 7.42 (d, J = 7.2 Hz, 1H), 2.53 (d, J = 7.8 Hz, 1H), 1.39- 1.28 (m, 2H), 1.21 (dd, J = 13.8, 7.2 Hz, 2H), 1.17- 0.99 (m, 8H), 0.83 (t, J = 7.2 Hz, 3H); MS: m/z = 357.26 (M+). Anal. Calcd for C19H24ClN5: C, 63.77; H, 6.76; N, 19.57. Found: C, 63.61; H, 6.783; N, 19.31. 5-Ethyl-6-propyl-1,2,4-triazolo[1,5-a]pyrimidin-7amine (4d). Yield: 64%; mp 179−181 °C; 1H NMR (400 MHz, d6-DMSO): δ 8.34 (s, 1H), 7.74 (s, 2H), 2.75 (d, J = 7.2 Hz, 2H), 2.58 (s, 2H), 1.45 (s, 2H), 1.23 (t, J = 7.2 Hz, 3H), 0.98 (t, J = 6.8 Hz, 3H); MS: m/z = 205.06 (M+). Anal. Calcd for C10H15N5: C, 58.51; H, 7.37; N, 34.12. Found: C, 58.32; H, 7.379; N, 34.22. 6-Benzyl-5-phenyl-1,2,4-triazolo[1,5-a]pyrimidin-7amine (4e). Yield: 33%; mp 249−251 °C; 1H NMR (400 MHz, d6-DMSO): δ 8.49 (s, 1H), 8.05 (s, 2H), 7.64−7.27 (m,

and discovery of novel ametoctradin analogues and fungicides targeting the bc1 complex.

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MATERIALS AND METHODS General Methods. Unless otherwise noted, all chemical reagents were commercially available from Acros Organics and/ or Sigma-Aldrich and treated with standard methods before used. 1H NMR spectra were recorded in CDCl3 or DMSO-d6 solution on a varian Mercury 600 or 400 MHz spectrometer. Chemical shifts were recorded in parts per million (ppm), with TMS as the internal reference. The following abbreviations were used to designate chemical shift multiplicities: s = singlet, d = doublet, t = triplet, m = multiplet, br = broad; MS spectra were determined with a Tracems2000 organic mass spectrometer, and signals were given in m/z. Melting points were measured on a Buchi B-545 melting point apparatus. Element analyses (EA) were measured on a Vario ELIII CHNSO elemental analyzer. The silica gel (200−300 mesh) for flash column chromatography was purchased from QingDao Marine Chemical Factory in China. General Procedure for the Preparation of Intermediate 2.18−20 Under a nitrogen atmosphere, THF (tetrahydrofuran, 30 mL) and anhydrous acetonitrile (25 mmol) was mixed and stirred for 5 min at −78 °C. BuLi (2.M) was added into the mixture and stirred for 1 h at −78 °C. Then, different substituent benzyl bromide was added dropwise slowly and then the mixture was stirred for 20 min and monitored by TLC detection. After 20 min, the reaction was complete. The resulting suspension was then diluted with water (15 mL) and extracted with ethyl acetate (2 × 25 mL). The combined organic extracts were washed with brine (2 × 25 mL), dried with Na2SO4, and concentrated, and the residue was purified by flash chromatography on silica gel to provide the desired product. General Procedure for the Preparation of Intermediate 3.18−20 A mixture of solid potassium methoxide (1.75g) and o-xylene (2.7 mL) was stirred and heated with reflux. When the mixture changed to light yellow, as monitored by HPLC detection, only 10% of 2 (total 10 mmol) and ester (total 20 mmol) were added in 5 min and then stirred for 15 min. The complete addition required another 10 min. The reaction mixture was refluxed 2.5 h and was allowed to warm room temperature. Water (3.3 mL) was added to the reaction mixture with pH equal to 5−6 adjusted by adding 20% hydrochloric acid. Then it was extracted with ethyl acetate (3 × 15 mL). The combined organic extracts were washed with brine 15 mL and dried with Na2SO4 to yield 2-propionyldecanonitrile. General Procedure for the Preparation of the Title Compounds 4a ∼ z. A mixture of 3 (3 mmol), 1,2,4-triazol-3amine (3 mmol), and o-xylene (2.5 mL) was quickly stirred, and then ClSO3H (0.6 mmol) was slowly added dropwise. The B

DOI: 10.1021/acs.jafc.5b00228 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry 5H), 7.27−7.19 (m, 2H), 7.18−7.09 (m, 1H), 6.96 (d, J = 7.2 Hz, 2H), 4.01 (s, 2H); MS: m/z = 300.09 (M+). Anal. Calcd for C18H15N5: C, 71.74; H, 5.02; N, 23.24. Found: C, 71.80; H, 5.169; N, 23.07. 6-Benzyl-5-(3,3,3-trifluoropropyl)-1,2,4-triazolo[1,5a]pyrimidin-7-amine (4f). Yield: 39%; mp 212−214 °C; 1H NMR (600 MHz, d6-DMSO): δ 8.44 (s, 1H), 8.06 (s, 2H), 7.29 (t, J = 7.2 Hz, 2H), 7.20 (t, J = 7.2 Hz, 1H), 7.12 (d, J = 7.2 Hz, 2H), 4.11 (s, 2H), 2.93−2.80 (m, 2H), 2.61 (dd, J = 16.2, 10.8 Hz, 2H); MS: m/z = 321.08 (M+). Anal. Calcd for C15H14F3N5: C, 56.07; H, 4.39; N, 21.80. Found: C, 56.24; H, 4.514; N, 21.65. 6-(4-Bromobenzyl)-5-ethyl-1,2,4-triazolo[1,5-a]pyrimidin-7-amine (4g). Yield: 53%; mp 239−241 °C; 1H NMR (600 MHz, d6-DMSO): δ 8.39 (s, 1H), 7.88 (s, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.4 Hz, 2H), 4.05 (s, 2H), 2.61 (q, J = 7.2 Hz, 2H), 1.09 (t, J = 7.2 Hz, 3H); MS: m/z = 332.96 (M+). Anal. Calcd for C14H14BrN5: C, 50.62; H, 4.25; N, 21.08. Found: C, 50.73; H, 4.103; N, 21.99. 5-Ethyl-6-(4-fluorobenzyl)-1,2,4-triazolo[1,5-a]pyrimidin-7-amine (4h). Yield: 85%; mp 216−218 °C; 1H NMR (600 MHz, d6-DMSO): δ 8.39 (s, 1H), 7.87 (s, 2H), 7.15 (t, J = 7.2 Hz, 2H), 7.09 (t, J = 9.0 Hz, 2H), 4.06 (s, 2H), 2.63 (q, J = 7.2 Hz, 2H), 1.09 (t, J = 7.2 Hz, 3H); MS: m/z = 271.06 (M+). Anal. Calcd for C14H14FN5: C, 61.98; H, 5.20; N, 25.81. Found: C, 62.15; H, 5.269; N, 25.56. 6-(3-Bromobenzyl)-5-ethyl-1,2,4-triazolo[1,5-a]pyrimidin-7-amine (4i). Yield: 52%; mp 211−212 °C; 1H NMR (600 MHz, d6-DMSO): δ 8.40 (s, 1H), 7.89 (s, 2H), 7.39 (d, J = 7.8 Hz, 1H), 7.34 (s, 1H), 7.24 (t, J = 7.8 Hz, 1H), 7.09 (d, J = 7.8 Hz, 1H), 4.09 (s, 2H), 2.63 (q, J = 7.2 Hz, 2H), 1.10 (t, J = 7.2 Hz, 3H); MS: m/z = 332.93 (M+). Anal. Calcd for C14H14BrN5: C, 50.62; H, 4.25; N, 21.08. Found: C, 50.73; H, 4.103; N, 20.99. 5-Ethyl-6-(2-fluorobenzyl)-1,2,4-triazolo[1,5-a]pyrimidin-7-amine (4j). Yield: 66%; mp 209−211 °C; 1H NMR (600 MHz, d6-DMSO): δ 8.40 (s, 1H), 7.89 (s, 2H), 7.26 (dd, J = 13.2, 6.6 Hz, 1H), 7.20 (t, J = 9.0 Hz, 1H), 7.05 (t, J = 7.2 Hz, 1H), 6.84 (t, J = 7.8 Hz, 1H), 4.06 (s, 2H), 4.47 (q, J = 7.2 Hz, 2H), 1.08 (t, J = 7.2 Hz, 3H); MS: m/z = 271.03 (M+). Anal. Calcd for C14H14FN5: C, 61.98; H, 5.20; N, 25.81. Found: C, 61.82; H, 5.347; N, 25.80. 6-(2-Bromobenzyl)-5-ethyl-1,2,4-triazolo[1,5-a]pyrimidin-7-amine (4k). Yield: 59%; mp 228−239 °C; 1H NMR (600 MHz, d6-DMSO): δ 8.40 (s, 1H), 7.90 (s, 2H), 7.67 (d, J = 7.8 Hz, 1H), 7.23 (t, J = 7.2 Hz, 1H), 7.17 (t, J = 7.8 Hz, 1H), 6.73 (d, J = 7.8 Hz, 1H), 4.06 (s, 2H), 2.48 (d, J = 7.5 Hz, 1H), 1.08 (t, J = 7.2 Hz, 3H); MS: m/z = 332.99 (M+). Anal. Calcd for C14H14BrN5: C, 50.62; H, 4.25; N, 21.08. Found: C, 50.75; H, 4.234; N, 21.29. 5-Ethyl-6-(3-fluorobenzyl)-1,2,4-triazolo[1,5-a]pyrimidin-7-amine (4l). Yield: 65%; mp 226−228 °C; 1H NMR (600 MHz, d6-DMSO): δ 8.39 (s, 1H), 7.88 (s, 2H), 7.31 (dd, J = 14.4, 7.8 Hz, 1H), 7.02 (t, J = 7.8 Hz, 1H), 6.95 (t, J = 10.2 Hz, 2H), 4.10 (s, 2H), 2.63 (q, J = 7.2 Hz, 2H), 1.10 (t, J = 7.2 Hz, 3H); MS: m/z = 271.08 (M+). Anal. Calcd for C14H14FN5: C, 61.98; H, 5.20; N, 25.81. Found: C, 61.92; H, 5.467; N, 25.71. 5-Ethyl-6-(3-iodobenzyl)-1,2,4-triazolo[1,5-a]pyrimidin-7-amine (4m). Yield: 87%; mp 214−215 °C; 1H NMR (600 MHz, d6-DMSO): δ 8.39 (s, 1H), 7.88 (s, 2H), 7.55 (d, J = 15.6 Hz, 2H), 7.08 (s, 2H), 4.06 (s, 2H), 2.62 (dd, J = 13.8, 6.5 Hz, 2H), 1.10 (t, J = 7.2 Hz, 3H); MS: m/z = 378.92

(M+). Anal. Calcd for C14H14IN5: C, 44.34; H, 3.72; N, 18.47. Found: C, 44.59; H, 3.550; N, 18.50. 6-(2-Chlorobenzyl)-5-ethyl-1,2,4-triazolo[1,5-a]pyrimidin-7-amine (4n). Yield: 59%; mp 225−227 °C; 1H NMR (600 MHz, d6-DMSO): δ 8.40 (s, 1H), 7.89 (s, 2H), 7.50 (d, J = 7.8 Hz, 1H), 7.25 (t, J = 7.8 Hz, 1H), 7.19 (t, J = 7.8 Hz, 1H), 6.75 (d, J = 7.8 Hz, 1H), 4.09 (s, 2H), 2.55−2.46 (m, 5H), 1.08 (t, J = 7.2 Hz, 3H); MS: m/z = 287.01 (M+). Anal. Calcd for C14H14ClN5: C, 58.44; H, 4.90; N, 24.34. Found: C, 58.17; H, 4.710; N, 24.21. 5-Ethyl-6-(4-(trifluoromethoxy)benzyl)-1,2,4-triazolo[1,5-a]pyrimidin-7-amine (4o). Yield: 69%; mp 212−213 °C; 1H NMR (600 MHz, d6-DMSO): δ 8.40 (s, 1H), 7.89 (s, 2H), 7.25 (dd, J = 23.4, 8.4 Hz, 4H), 4.11 (s, 2H), 2.63 (q, J = 7.2 Hz, 2H), 1.10 (t, J = 7.2 Hz, 3H); MS: m/z = 337.02 (M+). Anal. Calcd for C15H14F3N5O: C, 53.41; H, 4.18; N, 20.76. Found: C, 53.53; H, 4.336; N, 20.56. 6-(3-Chlorobenzyl)-5-ethyl-1,2,4-triazolo[1,5-a]pyrimidin-7-amine (4p). Yield: 80%; mp 217−218 °C; 1H NMR (600 MHz, d6-DMSO): δ 8.40 (s, 1H), 7.89 (s, 2H), 7.30 (t, J = 7.8 Hz, 1H), 7.26 (d, J = 8.4 Hz, 1H), 7.20 (s, 1H), 7.05 (d, J = 7.8 Hz, 1H), 4.10 (s, 2H), 2.63 (q, J = 7.2 Hz, 2H), 1.10 (t, J = 7.2 Hz, 3H); MS: m/z = 287.02 (M+). Anal. Calcd for C14H14ClN5: C, 58.44; H, 4.90; N, 24.34. 6-(3,4-Dichlorobenzyl)-5-ethyl-1,2,4-triazolo[1,5-a]pyrimidin-7-amine (4q). Yield: 48%; mp 256−257 °C; 1H NMR (600 MHz, d6-DMSO): δ 8.40 (s, 1H), 7.89 (s, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.41 (s, 1H), 7.06 (d, J = 7.8 Hz, 1H),4.08 (s, 2H), 2.62 (d, J = 7.8 Hz, 2H), 1.11 (t, J = 7.2 Hz, 3H); MS: m/z = 321.19 (M+). Anal. Calcd for C14H13Cl2N5: C, 52.19; H, 4.07; N, 21.74. Found: C, 52.23; H, 4.251; N, 21.49. 5-Benzyl-6-phenyl-1,2,4-triazolo[1,5-a]pyrimidin-7amine (4r). Yield: 17%; mp 251−252 °C; 1H NMR (600 MHz, d6-DMSO): δ 8.45 (s, 1H), 7.47 (ddd, J = 24.0, 15.6, 7.8 Hz, 4H), 7.33 (d, J = 4.2 Hz, 1H), 7.25 (d, J = 6.6 Hz, 2H), 7.17 (t, J = 7.2 Hz, 2H), 7.13 (d, J = 7.2 Hz, 1H), 6.94 (d, J = 7.2 Hz, 2H), 3.78 (s, 2H); MS: m/z = 300.07 (M+). Anal. Calcd for C18H15N5: C, 71.74; H, 5.02; N, 23.24. Found: C, 71.64; H, 5.270; N, 23.13. 5-Benzyl-6-(4-fluorophenyl)-1,2,4-triazolo[1,5-a]pyrimidin-7-amine (4s). Yield: 19%; mp 248−250 °C; 1H NMR (600 MHz, d6-DMSO): δ 8.44 (s, 1H), 7.48 (s, 2H), 7.31−7.24 (m, 4H), 7.18 (t, J = 7.2 Hz, 2H), 7.14 (d, J = 7.2 Hz, 1H), 6.94 (d, J = 7.2 Hz, 2H), 3.78 (s, 2H); MS: m/z = 318.07 (M+). Anal. Calcd for C18H14FN5: C, 67.70; H, 4.42; N, 21.93. Found: C, 67.74; H, 4.250; N, 21.88. 5-Benzyl-6-(4-chlorophenyl)-1,2,4-triazolo[1,5-a]pyrimidin-7-amine (4t). Yield: 5%; mp 262−263 °C; 1H NMR (600 MHz, d6-DMSO): δ 8.44 (s, 1H), 7.52 (d, J = 8.4 Hz, 4H), 7.25 (d, J = 8.4 Hz, 2H), 7.18 (t, J = 7.2 Hz, 2H), 7.14 (d, J = 7.2 Hz, 1H), 6.95 (d, J = 7.2 Hz, 2H), 3.78 (s, 2H); MS: m/z = 334.01 (M+). Anal. Calcd for C18H14ClN5: C, 64.38; H, 4.20; N, 20.86. Found: C, 64.17; H, 4.308; N, 20.67. 6-Benzyl-5-ethyl-1,2,4-triazolo[1,5-a]pyrimidin-7amine (4u). Yield: 80%; mp 229−230 °C; 1H NMR (400 MHz, CDCl3) δ 8.40 (s, 1H), 7.88 (s, 2H), 7.27 (d, J = 7.2 Hz, 2H), 7.19 (d, J = 6.4 Hz, 1H), 7.12 (d, J = 6.4 Hz, 2H), 4.08 (s, 2H), 2.80−2.57 (m, 2H), 1.09 (t, J = 7.2 Hz, 3H); MS: m/z = 253.09 (M+). Anal. Calcd for C14H15N5: C, 66.38; H, 5.97; N, 27.65. Found: C, 66.41; H, 5.842; N, 27.60. 5-Ethyl-6-phenyl-1,2,4-triazolo[1,5-a]pyrimidin-7amine (4v). Yield: 60%; mp 216−218 °C; 1H NMR (400 MHz, CDCl3) δ 8.43 (s, 1H), 7.66−7.50 (m, 2H), 7.47 (d, J = C

DOI: 10.1021/acs.jafc.5b00228 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry 7.2 Hz, 1H), 7.35 (s, 2H), 7.29 (s, 2H), 2.40 (dd, J = 14.8, 7.2 Hz, 2H), 1.06 (t, J = 7.6 Hz, 3H); MS: m/z = 238.09 (M+). Anal. Calcd for C13H13N5: C, 65.25; H, 5.48; N, 29.27. Found: C, 65.25; H, 5.48; N, 29.27. 5-Ethyl-6-(4-fluorophenyl)-1,2,4-triazolo[1,5-a]pyrimidin-7-amine (4w). Yield: 57%; mp 224−225 °C; 1H NMR (400 MHz, d6-DMSO): δ 8.43 (s, 1H), 7.35 (dd, J = 20.4, 8.0 Hz, 5H), 2.40 (dd, J = 14.8, 7.2 Hz, 2H), 1.07 (t, J = 7.6 Hz, 3H); MS: m/z = 256.07 (M+). Anal. Calcd for C13H12FN5: C, 60.69; H, 4.70; N, 27.22. Found: C, 60.72; H, 4.822; N, 27.41. 6-(4-Chlorophenyl)-5-ethyl-1,2,4-triazolo[1,5-a]pyrimidin-7-amine (4x). Yield: 47%; mp 235−236 °C; 1H NMR (400 MHz, d6-DMSO): δ 8.42 (s, 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.43 (s, 2H), 7.36 (d, J = 8.0 Hz, 2H), 2.40 (dd, J = 15.2, 7.6 Hz, 2H), 1.06 (t, J = 7.6 Hz, 3H); MS: m/z = 272.05 (M+). Anal. Calcd for C13H12ClN5: C, 57.04; H, 4.42; N, 25.59. Found: C, 57.01; H, 4.527; N, 25.31. 6-(4-Bromophenyl)-5-ethyl-1,2,4-triazolo[1,5-a]pyrimidin-7-amine (4y). Yield: 39%; mp 242−244 °C; 1H NMR (400 MHz, d6-DMSO): δ 8.42 (s, 1H), 7.70 (d, J = 8.0 Hz, 2H), 7.44 (s, 2H), 7.30 (d, J = 8.0 Hz, 2H), 2.47−2.23 (m, 2H), 1.07 (t, J = 7.6 Hz, 3H); MS: m/z = 316.01 (M+). Anal. Calcd for C13H12BrN5: C, 49.07; H, 3.80; N, 22.01. Found: C, 49.01; H, 3.926; N, 22.09. 6-Phenyl-5-(trifluoromethyl)-1,2,4-triazolo[1,5-a]pyrimidin-7-amine (4z). Yield: 11%; mp 279−280 °C; 1H NMR (400 MHz, d6-DMSO): δ 8.67 (s, 1H), 8.05 (s, 2H), 7.50 (d, J = 5.2 Hz, 3H), 7.36 (d, J = 7.2 Hz, 2H); MS: m/z = 279.03 (M+). Anal. Calcd for C12H8F3N5: C, 51.62; H, 2.89; N, 25.08. Found: C, 51.48; H, 3.128; N, 25.35. Enzyme Assays. The preparation of succinate−cytochrome c reductase (SCR, mixture of respiratory complex II and bc1 complex) from porcine heart was essentially as reported.21 The activity of SCR was measured by monitoring the increase in absorbance of cytochrome c1 at 550 nm, by using the extinction coefficient of 18.5 mM−1 cm−1. The succinate−ubiquinone reductase (complex II) activity was measured by monitoring the decrease in absorbance of 2,6-dichlorophenolindophenol (DCIP) at 600 nm, by using the extinction coefficient of 21 mM−1 cm−1. The reaction mixture may be scaled down to 1.8 mL with final concentrations of PBS (pH 7.4), 100 mM; EDTA, 0.3 mM; succinate, 20 mM; oxidized cytochrome c1, 60 uM (or DCIP, 53 uM); and appropriate amounts of enzyme to start the reaction. The ubiquinol−cytochrome c1 reductase (bc1 complex) activity in catalyzing the oxidation of DBH2 by cytochrome c1 was assayed in 100 mM PBS (pH 7.4), 0.3 mM EDTA, 750 uM lauryl maltoside (n-dodecyl-β-D-maltoside), 20−120 uM DBH2, 100 uM oxidized cytochrome c1, and an appropriate amount of SCR. The preparation of DBH2 from DB was carried out according to the procedure described in previous publications, and the concentration of DBH2 was determined by measuring the absorbance difference between 288 and 320 nm by using an extinction coefficient of 4.14 mM−1 cm−1 for the calculation. For the steady-state studies, the reaction was carried out in the absence or presence of various concentrations of the inhibitor.21 Data Analysis. The concentrations at 50% inhibition (absolute IC50 values) for experiments with SCR were obtained from a nonlinear regression of the activity data according to a four parameter logistic model. The absolute IC50 was calculated according to eq 1.

y = min +

max −min 1 + 10 x − logIC50

(1)

Sigma Plot software 9.0 was used to determine all kinetic constants. Computational Methods and Binding-Free-Energy Calculations. Molecular Docking. The three-dimensional (3D) structure of bc1 complex was obtained from PDB database (PDB ID: 1PPJ, from bovine). The AutoDock 4.2 program22 was applied to dock ametoctradin and its derivates into the Qo and or Qi site of the bc1 complex. The Gasteiger charges were used for these inhibitors. To select the best set of docking parameters and to test the reliability of the docking results, the antimycin and stigmatellin were first docked into the Qi and Qo site, respectively. In the docking process, a conformational search was performed for the antimycin/ stigmatellin molecule using the Solis and Wets local search method, and the Lamarckian genetic algorithm (LGA)23,24 was applied for the conformational search of the binding complex of antimycin/stigmatellin with bc1 complex. Among a series of docking parameters, the grid size was set to be 40 × 40 × 40, and the used grid space was the default value of 0.375 Å. The interaction energy that resulted from probing the cyt b with the antimycin/stigmatellin molecule was assessed by the standard AutoDock scoring function. Among a set of 100 candidates of the docked complex structures, the best one was first selected according to the interaction energy and was then compared with the conformation of antimycin/stigmatellin extracted from the crystal structure. By tuning the docking parameters, the final antimycin/stigmatellin-bc1 complex was obtained, based on the smallest root-mean-square deviation (rmsd) of the docked conformation of antimycin/stigmatellin from its conformation in the crystal structure. The RMSD between the selected conformation and previously noted in the X-ray crystal structure is 0.84 for stigmatellin and 0.89 for antimycin (data not shown), confirming the reliability of docking parameters. The same docking parameters were adopted to perform molecular docking of other compounds. Molecular Dynamics Simulations. All the complex structures derived from molecular docking were used as starting structures for further energy minimizations using the Sander module of the Amber8 program before the final binding structures were achieved. The Amber ff99 force field was used for amino acid and General Amber force field was used for ligands. For temperature regulation, the Langevin thermostat was used to maintain a temperature of 300 K. The bcc charges were used as the atomic charges for ligands. There are two stages for energy minimization. First, the ligand was minimized with the protein fixed. Second, the backbone atoms of the protein were fixed, and other atoms were relaxed. The final minimization was performed with both the ligand and the protein relaxed. In each step, the energy minimization was executed by using the steepest descent method for the first 1000 cycles, and the conjugated gradient method for the subsequent 3000 cycles, with a convergence criterion of 0.1 kcal mol−1 Å−1. The atomic coordinates were saved every picosecond. Then, an additional 10 ns MD simulation was performed for ametoctradin-Qo and ametoctradin-Qi complex. In order to reduce the computational cost, only 20 ps MD simulations were performed for 12 newly synthesized analogues. MM/PBSA Calculation. For ametoctradin-Qo and ametoctradin-Qi, 100 snapshots of the simulated structure within the D

DOI: 10.1021/acs.jafc.5b00228 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Scheme 1. Synthetic Route of the Title Compounds: (a) BunLi THF −78 °C Ice Bath; (b) CH3OK o-Xylene Reflux Stirred; (c) ClSO3H o-Xylene Stirred Reflux

Figure 2. X-ray crystal structure of ametoctradin.

molecule and surrounding solvent were set to 1 and 80, and the probe radius of the solvent was set to 1.4 Å. The entropy contribution to the binding free energy can be divided into two parts: the solvation free entropy (ΔSsolv) and the conformational free entropy (ΔSconf).

stable MD trajectory were selected and minimized to perform the MM/PBSA calculations (see below). For newly synthesized analogues, the last snapshot of the MD simulation was minimized to a convergence criterion of 0.1 kcal mol−1 Å−1. Then, the ΔH calculation was performed on the minimized complex, as described elsewhere.25,26 In this method, the binding free energy of the protein−ligand complex (ΔGbind) is obtained from the difference between the free energies of the protein−ligand complex (ΔGcpx), the unbound receptor (△Grec), and the free ligand (△Glig) as follows: ΔG bind = △Gcpx − △Grec − △G lig

ΔSsol = ΔSsolv + ΔSconf

The solvation free entropy is gained by the tendency of water molecules to minimize their contacts with hydrophobic groups in protein. The conformational free entropy is related to the change of the number of rotatable bonds during the binding process. The detailed computational procedure used to evaluate the entropy contribution (−TΔS) to the binding free energy was as previously described.26

(2)

■

The binding free energy (ΔGbind) can be evaluated as the sum of the changes in the molecular mechanical (MM) gasphase binding energy (ΔEMM), solvation free energy (ΔGsol), and entropic contribution (−TΔS). The ΔEMM was evaluated as a sum of electrostatic energy (ΔEele) and van der Waals interaction energy (ΔEvdw). ΔG bind = ΔEMM + ΔGsol − T ΔS

(3)

ΔEMM = ΔEele + ΔEvdw

(4)

RESULTS AND DISCUSSION Synthetic Chemistry of the Title Compounds. As shown in Scheme 1, the target compounds 4a ∼ z were smoothly prepared by a three-step synthetic route using bromide compound as starting materials. Compound 2, resulting from the reaction of bromide compound with acetonitrile, underwent an elimination reaction with substituent ester to afford the key intermediates β-ketonitriles (3). By performing the cyclization reaction with 1,2,4-triazol-3-amine, compound 3 was successfully transformed into the corresponding substituted 1,2,4-triazolo[1,5-a]pyrimidine derivates 4a ∼ z. The structures of all synthesized compounds were characterized by element analyses, 1H NMR, and GC-MS techniques. The crystal structure of ametoctradin was determined by X-ray diffraction analysis (CCDC 981965). As shown in Figure 2, the crystal structure showed that ametoctradin had an extended conformation. Inhibition Effect against bc1 Complex of Compounds 4a ∼ z. SCR is the mixture of respiratory complex II and the bc1 complex (complex III), which was also deemed to form complex II−complex III supercomplexes.27 Complex II (also

The solvation free energy ΔGsol was composed of two parts: ΔGsol = ΔG PB + ΔGnp

(5)

ΔGnp = γ SASA + β

(6)

(7)

The electrostatic contribution to the solvation free energy (ΔGPB) is evaluated by Poisson−Boltzmann (PB) methods. The nonpolar solvation energy, ΔGnp, can be estimated by an empirical relation of ΔGnp = γSASA + β, where SASA and is defined as the solvent-accessible surface area, and the solvation parameters γ and β were set to 0.00542 kcal·mol−1 Å−2 and 0.92 kcal·mol−1, respectively. The dielectric constant for the E

DOI: 10.1021/acs.jafc.5b00228 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry Table 1. Percent Inhibition and IC50 Value of the Title Compounds against Porcine SCR no. 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n 4o 4p 4q 4r 4s 4t 4u 4v 4w 4x 4y 4z ametoctradin a

R2

% inhibition (100 μM)

IC50 (μM)

4-CH3−Ph 3-CF3−Ph 3-Cl-Ph CH3CH2 Ph CF3CH2CH2 CH3CH2 CH3CH2 CH3CH2 CH3CH2 CH3CH2 CH3CH2 CH3CH2 CH3CH2 CH3CH2 CH3CH2 CH3CH2 PhCH2 PhCH2 PhCH2 CH3CH2 CH3CH2 CH3CH2 CH3CH2 CH3CH2 CF3 CH3CH2

82% 75.5% 77.6% 24.1%