Discovery, Synthesis, and Optimization of Diarylisoxazole‐3‐carboxamides as Potent Inhibitors of the Mitochondrial Permeability Transition Pore

Abstract

The mitochondrial permeability transition pore (mtPTP) is a Ca2+-requiring mega-channel which, under pathological conditions, leads to the deregulated release of Ca2+ and mitochondrial dysfunction, ultimately resulting in cell death. Although the mtPTP is a potential therapeutic target for many human pathologies, its potential as a drug target is currently unrealized. Herein we describe an optimization effort initiated around hit 1, 5-(3-hydroxyphenyl)-N-(3,4,5-trimethoxyphenyl)isoxazole-3-carboxamide, which was found to possess promising inhibitory activity against mitochondrial swelling (EC50 < 0.39 µm) and showed no interference on the inner mitochondrial membrane potential (rhodamine 123 uptake EC50 > 100 µm). This enabled the construction of a series of picomolar mtPTP inhibitors that also potently increase the calcium retention capacity of the mitochondria. Finally, the therapeutic potential and in vivo efficacy of one of the most potent analogues, N-(3-chloro-2-methylphenyl)-5-(4-fluoro-3-hydroxyphenyl)isoxazole-3-carboxamide (60), was validated in a biologically relevant zebrafish model of collagen VI congenital muscular dystrophies.

Identification of ER-000444793, a Cyclophilin D-independent inhibitor of mitochondrial permeability transition, using a high-throughput screen in cryopreserved mitochondria

“…Much like ER-000444793, a final class of compounds has been identified which inhibited mPT across species with no effect on ATP synthesis and mitochondrial function. Isoxazoles inhibited mPTP opening with potency similar to CsA and improved motor function and muscle structure in zebrafish model of collagen VI congenital muscular dystrophy34.…”

“…More recently, a number of groups have also undertaken large compound library screens aimed at identifying novel inhibitors of mPTP opening343653. Following structure-activity relationship studies around the N -phenyl-benzamide scaffold, Roy et al .…”

“…Despite identification of small molecule inhibitors of mPT presenting an obvious therapeutic opportunity, the availability and development of such agents remains limited. A number of groups have identified novel molecules modulating mitochondrial propensity for permeability transition30323334353637. However, as yet, reports of positive clinical development are yet to emerge.…”

Shutting down the pore: The search for small molecule inhibitors of the mitochondrial permeability transition

“…Here, we screened over 350,000 compounds at a concentration of 10 μM in isolated mouse liver mitochondria using the screening assays outlined above 20,14 . The validated hits, along with CsA and a representative CA as positive controls, were subsequently assayed using the CRC test.…”

“…The restriction of cyclophilin inhibitors is that CyPD modulates the pore indirectly as shown by the fact that the PTP can still open when CyPD has been genetically ablated 12 . Therefore, a number of programs have been aimed at identifying novel PTP inhibitors through the unbiased high-throughput screening (HTS) of several small molecule libraries 131415 . Since our recognition of the molecular components forming the PTP is only beginning to be appreciated, each represents a “phenotypic screen”; a widely-used alternative to the target-centric approaches that have dominated since the molecular biology revolution in the 1980s 16 .…”