Studies On Synthesis And Bioactivities Of Olaparib Analogs And In Silico Screening For PARP-1 & AChE Dual-targeted Inhibitors

Objective1.To obtain novel compounds with PARP-1 inhibitive activities and antitumor activities,aseriesofOlaparibanalogues,E-4-?3-?4-?3-argioacryloyl?piperazine-1-carbonyl?-4-fluorobenzyl?phthalazine-1?2H?-onederivatives,were designed and synthesized.PARP-1 inhibitive activities and antitumor activities were evaluated.Since PARP-1 was a new target for Alzheimer's Disease?AD?,AChE/BuChE inhibition were also evaluated in discovery for multi-target-directed ligands?MTDL?.2.A series of computational researches on the identification of PARP-1 and ACh E dual-targeted candidates against AD were conducted.The original PARP-1 inhibitors selected from Pubchem database were employed in the virtual screening.Rigid-flexible molecular docking and molecular dynamics?MD?simulations were applied to study on the receptor-ligand interactions and the stability.Methods1.Design and synethsis of target compounds:The main pharmcophore of Olaparib was unchanged,and the cyclopropane group was replaced with substituted aromatic propylene structures to obtain 16 noval Olaparib analogues.2-Formoxyl benzoic acid was used as a raw material,after cyclization,oxidation,reduction,2-fluoro-5-[?4-oxo-3,4-dihydropyridazin-1-yl?methyl]benzoic acid was generated.Substituted aromatic formaldehydes weae reacted with malonate to obtain substituted aromatic acrylics which then coupled with 1-tert-butoxycarbonylpiperazine to obtain amides.The tert-butoxycarbonyl group of the amides were removed by trifluoroacetic acidandtheaminogroupwasfutherreactedwith2-fluoro-5-[?4-oxo-3,4-dihydropyridazin-1-yl?methyl]benzoic acid to obtain a series of E-4-?3-?4-?3-argioacryloyl?piperazine-1-carbonyl?-4-fluorobenzyl?phthalazine-1?2H?-one derivatives.2.BioactivitiesofE-4-?3-?4-?3-argioacryloyl?piperazine-1-carbonyl?-4-fluorobenzyl?phthalazine-1?2H?-one derivatives:PARP-1 inhibition activities were evaluated by HT Fluorescent Homogeneous Inhibition PARP Assay Kit.Olaparib was selected as the positive control.AChE/BuChE inhibition activities were tested by Ellman's Method.Methylsulfate Neostigmine was selected as the positive control.Anticancer activities in vitro were evaluated by MTT assay against human breast cancer cell line?MDA-MB-436?and human colon cancer cell line?SW-620?.Olaparib was selected as the positive control.3.Virtual screening:The original 859 PARP-1 inhibitors were employed as the inputs in the virtual screening by AutoDock Vina to obtain the hits for AChE inhibition.Top-scored hits were employed in the molecular dynamics simulation by GROMACS to evaluate the stability of the AChE-ligand complexes.Results1.A total of 16 E-4-?3-?4-?3-argioacryloyl?piperazine-1-carbonyl?-4-fluorobenzyl?phthalazin-1?2H?-one derivatives were designed and synthesized,their structures were characterized by IR,¹H-NMR,¹³C-NMR and Mass spectrometry techniques.2.The tests on the inhibition of PARP-1 showed that compounds 44d,44e and 44n were the most potent agents in vitro with IC₅₀ of 41.50±2.65,37.90±1.89 and16.10±1.25 nM,IC₅₀ of Olaparib was 8.20±1.06 nM.Anticancer activities of target compounds evaluated by MTT assay showed that compounds 44d,44e and 44n were the most potent in the anti-proliferation against human breast cancer cell line?MDA-MB-436?with IC₅₀ of 10.25±2.20,13.95±1.28 and 11.62±2.15?M,respectively.IC₅₀ of Olaparib was 8.63±1.25?M.The least anti-proliferation against human colon cancer cell line?SW-620?were also observed on 44d,44e,44n.Results indicated the44d,44e,44n might be the PARP-1 targeted inhibitors with high selectivity.AChE/BuChE inhibition activities were tested by Ellman's Method.44d,44e,44n showed the AChE inhibitive ability with IC₅₀ of 27.44±0.46,12.24±0.49 and24.55±1.10?M,respectively.IC₅₀ of Methylsulfate Neostigmine was 0.04±0.01?M.BuChE IC₅₀ of 44d,44e,44n and Methylsulfate Neostigmine were 13.17±0.23,5.93±0.19,9.16±0.91 and 12.01±4.05?M,respectively.3.We reported the in silico identification of AChE and PARP-1 dual-targeted candidates against Alzheimer's disease.The original PARP-1 inhibitors were employed as the inputs in the virtual screening by AutoDock Vina to obtain the hits for AChE inhibition.Top-scored hits were employed in the molecular dynamics simulation by GROMACS to evaluate the stability of the AChE-ligand complexes.Pyrrolocarbazole structure might play an important role in the molecular recognition with the binding pocket of AChE.CID71605390 and CID57390505 were with the most stability with AChE.Conclusion and Highlights1.A total of 16 E-4-?3-?4-?3-argioacryloyl?piperazine-1-carbonyl?-4-fluorobenzyl?phthalazin-1?2H?-one derivatives were designed and synthesized.Their structures were characterized by IR,¹H-NMR,¹³C-NMR and Mass spectrometry techniques.2.Compounds 44d,44e and 44n were the most potent agents against PARP-1 in vitro.Moreover,these three compounds were the most potent in the anti-proliferation against human breast cancer cell line?MDA-MB-436?but the least against human colon cancer cell line?SW-620?,indicating that 44d,44e,44n might be the PARP-1 targeted inhibitors with high selectivity.Moreover,compound 44n was worth further research.BuChE inhibitive activities of 44d,44e,44n were more potent compared with Methylsulfate Neostigmine as well.3.We reported the in silico identification of AChE and PARP-1 dual-targeted candidates against Alzheimer's disease.The pyrrolocarbazole structure might play an important role in the molecular recognition with the binding pocket of AChE.CID71605390 and CID57390505 were found with AChE and PARP-1 dual-inhibitory activities in silico,which gave a clue for further optimization to search for more potent agents against AD.