Synthesis and biological evaluation of novel Ani9 derivatives as potent and selective ANO1 inhibitors

Anoctamin 1 (ANO1), a calcium-activated chloride channel, is highly expressed and amplified in a number of carcinomas including breast, pancreatic and prostate cancers. Downregulation of ANO1 expression and function significantly inhibits cell proliferation, migration, and invasion of various cancer cell lines. Development of potent and selective ANO1 inhibitors is currently desirable, which may provide a new strategy for cancer treatment. Our previous study revealed a new class of ANO1 inhibitor, (E)-2-(4-chloro-2-methylphenoxy)-N'-(2-methoxybenzylidene)acetohydrazide (Ani9) and structural optimization via chemical modification of Ani9 basic skeleton was undertaken for the development of more potent and specific inhibitors of ANO1. Structure-activity relationship studies with newly synthesized derivatives revealed a number of potent ANO1 inhibitors, among which 5f is the most potent inhibitor with an IC₅₀ value of 22 nM. The selectivity analyses showed that 5f has excellent selectivity to ANO1 (>1000-fold over ANO2). In cellular assays, 5f significantly inhibited cell proliferation of PC3, MCF7, and BxPC3 cells expressing high levels of ANO1. In addition, 5f strongly reduced the protein levels of ANO1 in PC3 cells. This study will be useful in the development of ANO1 inhibitors for treatment of cancer and other ANO1-related diseases.