Research On Multi-Target-Directed Ligands To Central Nervous System Disorder

The pathogenic mechanism of neurodegenerative diseases are poly-etiological in origin and the processes leading to neuronal death are multifactorial, as indicated by transcriptomics and proteomic. The pathology of many disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD), and stroke, includes an array of different pathways, and many of pathways involved in etiology of these diseases overlap. The traditional drug design "one-target, one-disease" in earlier and in many current researches has not met with the expected success for Central Nervous System (CNS) disorders. As increasing understanding of the entire complex pharmacological picture associated with the disease, a concept the multi-target-directed ligands that are rationally designed to modulate multiple mechanisms of a specific disease emerges. It will broaden our outlook in the treatment of neurodegenerative diseases.The ligands of some key targets related to the pathogenic mechanism of AD were chosen to be research objects. The pharmacophore models of Acetylcholinesterase (AChE) inhibitors,Beta-site APP cleaving enzymes 1 (BACE-1) inhibitors,Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors,and Cysteine Asp-specific proteases-3 (Caspase-3) inhibitors were respectively established by using the HypoGen module of catalyst software. Firstly, molecules with 2-aminothiazole framework were chosen as a scaffold for further chemical modification by phamacophore model to explore PARP-1 inhibitors with multi-function neuroprotection (1-7). Secondary, because targets which resemble in ligands will exhibit similarity in pharmacophore models, on the basis of the designed PARP-1 inhibitors, multi-target-directed compounds (8-10) were designed by overlapping pharmacophores. As pharmacophore model predicted, compounds 1-10 with drug-likeness had good estimate activity to inhibit AChE and PARP-1. After synthesis, the biological activity screening was carried to validate the design method of multi-target-directed drugs based on the pharmacophore models. This work will provide theory guidance and experimental data for further design of multi-target neuroprotective agents.In this study, we prepared 2-aminothiazole analogs in which the application of microwave technology was used.The cyclization step, carried by microwave, was performed in only a few minutes compared to the conventional thermal heating for 12h. Then they were reacted with different acid or acyl chloride respectively at RT. Finally, ten 4, 5-substituted-2-aminothiazole derivatives were synthesized. They were all characterized and analyzed by UV,IR,1H-NMR,13C-NMR,EI-MS,and HPLC.After biological activity screening, the results included as follows:1. Enzyme inhibition screeningAssay for PARP-1 inhibition in vitro:Compounds 1-3 and 7 almost showed more than 50% PARP-1 inhibition at a concentration of 1μM. They were more potent (IC50 240,753, 224, and 682 nM, respectively).Assay for AChE inhibition in vitro:Compound 10 show potent AChE inhibition (IC50=2511nM).Preliminary screening for Caspase-3 inhibitory activity in vitro:Compound 3 showed 54.99±2.42% inhibitory activity of Caspase-3 in the concentration of 65.3μM.Preliminary screening for PTP-sigma inhibitory activity in vitro:In the concertration of 272.9μM, compound 4 showed 93.22±3.08%inhibitory activity.2. Assays for cytoprotective action in PC12 cellH2O2-induced PC12 cell injury model:In MTT assay, compounds 3 and 7 were highly protective (EC50 129, and 324 nM, respectively), and compounds 1,2, and 4 were moderately protective.oxygen-glucose deprivation (OGD) induced PC12 cell death model:In MTT assay, compounds 1,2, and 7 were more protective (EC50 143,191, and 361 nM, respectively). In LDH release assay, compounds 1-4 and 7 (3μM) significantly reduced the OGD-induced LDH release by 34.6%,20.1%,33.1%,24.0%, and 28.4% respectively. We also performed a biparametric cytofluorimetric analysis using fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI) double staining. A concentration of 3μM of compounds 1-3 significantly reduced OGD-induced total apoptotic cells (early and late apoptotic cells) by 70.7%,45.1%, and 64.4% respectively.3. Preliminary screening for cytoprotective action in SH-SY5Y cell Preliminary screening of cytoprotection against Aβinduced SH-SY5Y cell death: Compound 1 had 45.54% protection in the concentration of 32.9μM.In conclusion, compounds 1-3 possessed expecting neuroprotection against H2O2, OGD, and Aβinduced cell death, and compounds 10 had potent multi-target-directed activities with both PARP-1 and AChE inhibiton, which validated method of muliti-target-directed ligands design.The characters and innovation points:1. The muliti-target-directed ligands design is reasonable. The key targets related to the pathogenic mechanism of AD were chosen to be research objects, and targets which resemble in ligands were selected to use to multi-target-directed ligands design. A series of compounds that have potent multi-target-directed activities with both PARP-1 and AChE inhibiton were designed.2. The method of overlapping pharmacophores used to multi-target-directed ligands design is original.3. The results of biological activity screening validated the feasibility of the concept and method of muliti-target-directed ligands design.