A potent and selective cis-amide inhibitor of ryanodine receptor 2 as a candidate for cardiac arrhythmia treatment.

Ryanodine receptor 2 (RyR2) is a Ca release channel mainly located on the sarcoplasmic reticulum (SR) membrane of heart muscle cells and regulates the concentration of Ca in the cytosol. RyR2 overactivation causes potentially lethal cardiac arrhythmias, but no specific inhibitor is yet available. Herein we developed the first highly potent and selective RyR2 inhibitor, TMDJ-035, containing 3,5-difluoro substituents on the A ring and a 4-fluoro substituent on the B ring, based on a comprehensive structure-activity relationship (SAR) study of tetrazole compound 1. The SAR study also showed that the amide conformation is critical for inhibitory potency. Single-crystal X-ray diffraction analysis and variable-temperature H NMR revealed that TMDJ-035 strongly favors cis-amide configuration, while the inactive analogue TMDJ-011 with a secondary amide takes trans-amide configuration. Examination of the selectivity among RyRs indicated that TMDJ-035 displayed high selectivity for RyR2. TMDJ-035 suppressed abnormal Ca waves and transients in isolated cardiomyocytes from RyR2-mutated mice. It appears to be a promising candidate drug for treating cardiac arrhythmias due to RyR2 overactivation, as well as a tool for studying the mechanism and dynamics of RyR2 channel gating.