Studies On The Discovery And Evaluation Of Pyrazolo[3,4-b]pyridin-6-one Scaffold And Its Derivatives As Novel Anticancer Lead Compounds

Drug research and development?R&D?include four parts,namely lead compound discovery,lead compound optimization,in vivo animal experiments?preclinical studies?,and clinical studies.Drug innovation starts with the discovery of lead compounds.The discovery of lead compounds with novel scaffold and good druggability plays a key role in drug innovation projects.Lead compounds are mainly identified by using High Throughput Screening?HTS?and Virtual Screening?VS?.Compared with VS,as we all know,HTS-based lead compounds discovery has the following features:high cost,long period,and low average hits rate in industry??0.3%?.In contrast,before the screening of biological activity,VS including computational algorithms and/or models are employed to predict whether the compounds will own the desired biological activities,which will results in reducing the number of compounds that actually need to be screened and increasing the discovery efficiency of lead compounds.Therefore,the discovery of lead compounds based on VS has attracted extensive attention from industry and academia.The virtual screening of anticancer lead compounds can be devided into two categories:structure-based VS?e.g.,molecular docking?and ligand-based VS?e.g.,pharmacophores,quantitative structure-activity relationships,molecularshape-and fingerprint-based similarity,etc.?.In the post-genome era,VS can be used to discover ligands for specific targets for cancer treatment.Undoubtedly,VS based on molecular docking relies on three-dimensional structure of the cancer target.For ligand-based VS methods,the majority computational models are also constructed based on biological assays data from a given cancer target to predict compound libraries.Currently,pharmacophores and quantitative structure-activity relationships?QSAR?are commonly methods to be used in ligand-based VS campaigns.This situation is primarily due to the overwhelming focus on targeted therapies.However,most of the origins of hits identified from in silico target-based screening campaigns do not show anticancer activity against tumor cells.Therefore,in previous study we reported on the development of computational cell-based bioactivity prediction models?termed as in silico phenotype-based screening?to enable the identification of novel anticancer agents,an approach that is in contrast to that of in silico target-based screening.The target of the best anticancer agent(G03,IC₅₀=2.82?M,human breast cancer cells MDA-MB-231)is then validated by a series of biochemical assays.Based on the NCI-60 anticancer activity prediction models developed in our group,this study aims to further expand its application to discover novel anticancer agents.First,a large-scale VS campaign based on the NCI-60 anticancer activity prediction models was designed to screen commercial Chemdiv compound libraries??1,738,238 compounds?.Using clustering analysis,scaffold diversity analysis,and viusal inspective,68 compounds were selected for purchase and in vitro antitumor biological screening.Biological experiment results confirmed that 17 compounds exhibited activities against human cancer cell lines.Among these compounds,compound h2 with pyrazolo[3,4-b]pyridin-6-one scaffold showed better antitumor activity.Second,using h2 compound as a query template,a molecular fingerprint-based similarity search method was used to find h2 analoges in the Chemdiv compound library.A total of 61 h2 structural derivatives were selected and purchased.In vitro antitumor tests were performed on the 61 h2 derivatives,and structure-activity relationship analysis was carried out.Biological assays results deomonstrated that compound B6 and I2were identified as anticancer lead compounds with anticancer activities against MDA-MB-231 cells(IC₅₀=4.27±0.73?M and IC₅₀=3.30±0.58?M).Fingerprint-based scaffold analysis results deomonstrated I2 is a new anticancer agent with novel scaffold.Meanwhile,compound I2 has high activity against drug-resistant tumor cell lines,e.g.,for amycin-resistant human hepatocellular carcinoma cell line HEPG2/ADR,the IC₅₀is 3.57±0.45?M.Therefore,we further explored the anticancer mechanisms of lead compound I2using computational and biological assay methods.Theoretical predictions and experimental verification results showed that I2 is a destabilizing agent for tubulin.In addition,I2 can inhibit cell migration,arrest cell cycle in G2/M phase,and induce apoptosis for MDA-MB-231.