Design, Synthesis, Biological Activity Exploration On Tubulin Assembling Inhibitors And Human NNOS Inhibitors

Microtubules are cytoskeletal structures and critical elements in various fundamental cellular processes such as cell division, formation, and maintenance of cell shape, motility, cell signaling, secretion, and intracellular transport. This is one of the reasons why microtubules are an attractive target for anticancer agents. So far, microtubule-targeting agents could be simply classified into microtubule stabilizer and microtubule distabilizer according to the mechanism by interfering with microtubule dynamics. We mainly focus on design, synthesis, biological activity exploration on tubulin assembling Inhibitors.(1) A series of novel benzozyl [1,3] dioxol-containing pyrazoline scaffold derivatives (A1-A32) has been designed, synthesized and evaluated for their biological activities. Among them, compound A4 displayed the most potent antiproliferative activity against A549, MCF-7, and HepG-2 cells line (IC50= 0.07 μM,0.05μM,0.03μM, respectively) and the tubulin polymerization inhibitory activity (IC50= 1.88μM), being comparable to CA-4. Furthermore, compound A4 was a potent inducer of apoptosis in HepG-2 cells and it had cellular effects typical for microtubule interacting agents, causing accumulation of cells in the G2/M phase of the cell cycle.(2) According to the result of above, further structural modification was performed. We synthesized a series of indole-containing pyrazoline scaffold derivatives as novel tubulin polymerization inhibitors (C1-C25), and tested their biological activity. Among them, compound C24 displayed the most potent tubulin polymerization inhibitory activity (IC50=1.6μM), and also showed good antiproliferative activity against A549, MCF-7 and HepG-2 cells line (IC50= 0.09 μM, IC50=0.59 pM, IC50=0.029 μM respectively). Furthermore, compound C24 was a potent inducer of apoptosis in HepG-2 cells and it had cellular effects typical for microtubule interacting agents, causing accumulation of cells in the G2/M phase of the cell cycle. Next, C24 could inhibit HepG2 cells in the immunofluorescence assay and had an effect on tubulin and the cytoskeleton. Molecular docking results showed that C24 could make interaction with amino acid residues of colchicine binding site. That should play an important role on the activity of C24.Nitric oxide (NO) is an essential second messenger in mammals, which regulates a variety of biological processes, such as vasodilation, smooth muscle relaxation, neurotransmission, and immune response. NO is generated by nitric oxide synthases (NOSs), of which there are three mammalian NOS isoforms:inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS) which regulates the release of neurotransmitters and is involved in neuronal communication. Overproduction of NO in the central nervous system (CNS) has been reported to be associated with chronic neurodegenerative pathogenesis, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s diseases (HD), as well as amyotrophic lateral sclerosis (ALS). Therefore, inhibition of nNOS is a viable therapeutic strategy for treating neurodegenerative disorders. A major challenge in the design of nNOS inhibitors focuses on potency in humans and selectivity over other NOS isoforms.We reported potent and selective human nNOS inhibitors based on the 2-aminopyridine scaffold with a central pyridine linker. Synthesized inhibitors were assayed against purified rat nNOS, murine macrophage iNOS, bovine eNOS and selected compounds were tested with human nNOS and eNOS using the hemoglobin capture assay. We also cultured and tested a lot of protein crystal structures of NOS complex with inhibitors. Compound D9, the most promising inhibitor in this study, exhibited excellent potency for rat nNOS (Ki=16 nM) with 828-fold n/e and 118-fold n/i, selectivity, with a Ki value of 13 nM against human nNOS with 1761-fold human n/e selectivity. X-ray crystal structures of D9 bound to both rat and human nNOS showed that the N of the pyridine linker formed two H-bonds (Tyr567 and Tyr593) in human nNOS but only one H-bond in rat nNOS. Compound D9 also displayed good metabolic stability in human liver microsomes, low plasma protein binding, and minimal binding to cytochromes P450 (CYPs).