Discovery of [1,2,4]triazolo[4,3-a]pyridines as potent Smoothened inhibitors targeting the Hedgehog pathway with improved antitumor activity in vivo.

•A series of 8-chloro-[1,2,4]triazolo[4,3-a]pyridine derivatives were designed and synthesized.•Compounds TPB3, TPB14, TPB15 and TPB17 showed significant inhibition of Hedgehog pathway activation (IC50 < 0.100 µM).•TPB15 not only suppressed Hh signaling by blocking Smo translocation into the cilia but also reduced the expression of Smo.•TPB15 demonstrated greater anti-tumor activity in our animal models than Vismodegib with significantly lower toxicity.