Novel 1,4-benzothiazepin-2-one modulators of NCX3 as a promising strategy for neuroprotection in cerebral ischemia

The Na⁺ /Ca²⁺ exchanger plays a key role in the regulation of calcium homeostasis in most excitable cells. Three mammalian isoforms, NCX1, NCX2, and NCX3, are described, and with their splice variant, they are expressed in a tissue-specific manner and regulated by Ca²⁺ binding domains CBD1 and CBD2. Among the three isoforms, NCX3 is mainly expressed in the brain and skeletal muscle. The occurrence of [Na⁺]_i and [Ca²⁺]_i dyshomeostasis has been reported in several neurodegenerative diseases either at neuronal or glial levels. Although the role of each isoform is still under investigation, many lines of evidence point to a neuroprotective effect of NCX3 activation in several neurodegenerative diseases, including brain ischemia. On this light, we have designed, synthesized, and characterized novel 1,4-benzothiazepinonic derivatives structurally related to CGP37157, which is already described as a mitochondrial NCX (mNCX) blocker with a poor selectivity, where a cyclic amine has been linked in position 1 via an acetyl spacer. The newly synthesized compounds were screened for NCX3 activity by Fluo-4 single-cell microfluorimetry and patch-clamp electrophysiology in BHK cells singly expressing this isoform. Identification of the newly synthesized compounds modulating NCX3 activity was obtained by measuring Na⁺-free-dependent Ca²⁺ level changes above basal values and/or by electrophysiological detected NCX3 currents. Among the several newly synthesized pharmacological modulators of NCX3, compound 10 has shown the greatest capability of increasing the NCX3 activity in an in vivo model of focal brain ischemia.