Discovery of Highly Efficient Novel Antifungal Lead Compounds Targeting Succinate Dehydrogenase: Pyrazole-4-carboxamide Derivatives with an <i>N</i>-Phenyl Substituted Amide Fragment

Abstract

Developing environmentally friendly fungicides is crucial to tackle the issue of rising pesticide resistance. In this study, a series of novel pyrazole-4-carboxamide derivatives containing <i>N</i>-phenyl substituted amide fragments were designed and synthesized. The structures of target compounds were confirmed by ¹H NMR, ¹³C NMR, and HRMS, and the crystal structure of the most active compound <i>N</i>-(1-(4-(4-(<i>tert</i>-butyl)benzamido)phenyl)propan-2-yl)-3-(difluoromethyl)-<i>N</i>-methoxy-1-methyl-1<i>H</i>-pyrazole-4-carboxamide (<b>U22</b>) was further determined by X-ray single-crystal diffraction. The bioassay results indicated that the 26 target compounds possessed good in vitro antifungal activity against <i>Sclerotinia sclerotiorum</i> with EC₅₀ values for compounds <b>U12</b>, <b>U13</b>, <b>U15</b>, <b>U16</b>, <b>U18</b>, <b>U22</b>, and <b>U23</b> being 4.17 ± 0.46, 8.04 ± 0.71, 7.01 ± 0.71, 12.77 ± 1.00, 8.11 ± 0.70, 0.94 ± 0.11, and 9.48 ± 0.83 μg·mL^-1, respectively, which were the similar to controls bixafen (6.70 ± 0.47 μg·mL^-1), fluxapyroxad (0.71 ± 0.14 μg·mL^-1), and pydiflumetofen (0.06 ± 0.01 μg·mL^-1). Furthermore, in vivo antifungal activity results against <i>S. sclerotiorum</i> indicated that compounds <b>U12</b> (80.6%) and <b>U22</b> (89.9%) possessed excellent preventative efficacy at 200 μg·mL^-1, which was the same as the control pydiflumetofen (82.4%). Scanning electron microscopy and transmission electron microscopy studies found that the compound <b>U22</b> could destroy the hyphal morphology and damage mitochondria, cell membranes, and vacuoles. The results of molecular docking of compound <b>U22</b> and pydiflumetofen with succinate dehydrogenase (SDH) indicated they interact well with the active site of SDH. This study validated our approach and design strategy to produce compounds with an enhanced biological activity as compared to the parent structure.