Synthesis and biological evaluation of new saccharin-based inhibitors of cancer-related carbonic anhydrase IX and XII isoforms & Benzo[b]tiophen-3-ol derivatives as effective inhibitors of hMAOs: design, synthesis and biological activity

Synthesis and biological evaluation of new saccharin-based inhibitors of cancer-related carbonic anhydrase IX and XII isoforms Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread metalloenzymes implicated in many cellular and physiological processes requiring bicarbonate as substrate (e.g. electrolyte secretion, pH homoeostasis, respiration etc.). The fifteen human isoforms of carbonic anhydrase (hCAs) differ in cellular localization (cytosol, mitochondria or cell membrane), sensibility to inhibitors and catalytic activity. hCAs are well established therapeutic targets to treat a wide range of disorders. In particular, hCA IX and XII have been recognized as tumor-related isoforms, participating in the complex machinery which regulate the pH of cells in hypoxic tumors. In our previously reported studies we showed that N-alkylated saccharin derivatives effectively inhibited the two cancer-related isoforms. Here we report the design, synthesis and the inhibitory activity of novel compounds based on saccharin scaffold. We used two different approaches in order to obtain two series of new compounds. In the first approach, we performed a reductive ring opening of N-alkylated saccharin derivatives, obtaining molecules endowed with secondary sulfonamide and primary alcohol. In the second approach, a series of tertiary sulfonamides (saccharin/isoxazole and saccharin/isoxazoline derivatives) were obtained by the insertion of isoxazole or isoxazoline moiety as spacer between the saccharin nitrogen and (un)substituted aryl or heteroaryl rings. All the synthesized compounds were tested to evaluate their inhibitory activity towards the ubiquitous off-target isoforms, hCA I and II, and the cancer-related ones, hCA IX and XII, by a stopped-flow, CO2 hydrase assay. All the compounds were inactive against the two cytosolic off-target hCA I and II (Kis > 10 µM), while showed inhibitory activity in the low nanomolar range against the two cancer-related isoforms hCA IX and XII. Computational approach has been performed in order to better understand the binding mode of these inhibitors. Benzo[b]tiophen-3-ol derivatives as effective inhibitors of hMAOs: design, synthesis and biological activity Monoamine oxidases (MAOs; EC 1.4.3.4) are mitochondrial bound flavoenzymes, which catalyse the oxidative degradation of amines. They have been widely recognised as important pharmacological targets for the treatment of mood disorders (anxiety, depression) and neurodegeneration (Parkinson’s disease, PD) as the result of their effects on monoamine metabolism and level. Furthermore, evidences show the probable implication of these enzymes in some tumors (e.g prostatic cancer) and cardiomyopathies. Based on tissue localization, structural homology, active site differences, substrate/inhibitor selectivity and catalytic efficiency, two isoforms have been characterized (MAO-A and MAO-B). Both of them could be the target of selective inhibitors acting as reversible or irreversible agents. An extensive number of natural and synthetic compounds have shown effective inhibition of human MAOs. Indole analogues, aurones and indanone derivatives are examples of MAO inhibitors. They show a common structural feature that is the chalcone moiety, whose ability to bind hMAO enzymes has been reported by our research group. With the aim to expand our knowledge about hMAO inhibtion, we performed the design, synthesis, characterization and in vitro biological activity evaluation of some novel benzo[b]thiophen-3-ol derivatives. This scaffold retained the above cited chalcone system, differing from the already studied inhibitors in some features like the isosteric replacement of aurones’ oxygen with sulphur one, the presence of 1,3-diketone system giving keto-enol tautomerism and the presence of a carbonyl group instead of methylylidene one, bound at position 2 of the bicyclic system. These compounds have been synthesised through a new simple synthetic approach consisting in a one-step reaction, which led to moderate to high yields. The obtained compounds showed activity in the micromolar/low micromolar range against hMAO-B with the best inhibition profile observed for compounds containing phenyl ring substituted in the meta- position with halogens. These compounds have also been tested with cortex synaptosomes in both basal and LPS-induced inflammatory conditions to evaluate their ability to affect the DOPAC/DA ratio as well as LDH activity. DOPAC/DA ratio is an indirect index of MAO-B activity, and all the tested compounds are effective in reducing this value in cortex synaptosomes challenged with LPS, showing outcomes better than the reference drug deprenyl. Furthermore, all the tested molecules inhibited LDH activity in the concentration range 0.1-1 µM, showing potential activity as neuroprotective agents.