Thirty-four new pyrido[4,3-]pyrimidine analogs were designed, synthesized, and characterized. The crystal structures for compounds and were measured by means of X-ray diffraction of single crystals. The bioassay results showed that most target compounds exhibited good fungicidal activities against , , , and at 16 μg/mL. Compounds , , , and possessed better fungicidal activities than the commercial fungicide epoxiconazole against . Their half maximal effective concentration (EC) values were 0.191, 0.487, 0.369, 0.586, and 0.670 μg/mL, respectively. Furthermore, the inhibitory activities of the bioactive compounds were determined against sterol 14α-demethylase (CYP51). The results displayed that they had prominent activities. Compounds , , , and also showed better inhibitory activities than epoxiconazole against CYP51. Their half maximal inhibitory concentration (IC) values were 0.219, 0.602, 0.422, 0.726, and 0.802 μg/mL, respectively. The results of molecular dynamics (MD) simulations exhibited that compounds and possessed a stronger affinity to CYP51 than epoxiconazole.
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