N-4-aminobenzyl-N'-(2-(2-phenylpyrrolidin)-2-oxoethyl)urea-based inhibitors of cyclophilin D – Preparation of distinct stereoisomers and comparative analysis of their in vitro bioactivity

Cyclophilin D (CypD) is a mitochondrial enzyme and the key regulator of mitochondrial permeability transition pore (mPTP). Inhibition of CypD/mPTP holds promise as a therapeutic strategy for treatment of variety of diseases including ischemia-reperfusion injury, or neurodegeneration. Compounds based on the N-4-aminobenzyl-N'-(2-(2-phenylpyrrolidin)-2-oxoethyl)urea structural scaffold present the most potent class of small-molecule CypD inhibitors identified to date. Numerous independent studies on their synthesis and evaluation were published by different research groups. Unfortunately, the results of particular studies cannot be compared due to use of distinct methods of in vitro evaluation and, in most cases, unresolved stereochemistry of prepared chiral compounds. This did not allow for comprehensive analysis to identify the best inhibitors and their structural features. Therefore, we decided to synthesize the most potent inhibitors and their close analogues in form of pure stereoisomers to perform a side-by-side comparison of their inhibition potency and binding affinity to CypD as well as their ability to suppress mPTP opening. In addition, the selectivity of inhibition has been determined using CypA as an off-target. Compound 13(R) was found superior to other tested small molecule inhibitors in all the tested parameters and it was equipotent to the reference inhibitor cyclosporine A.