Design, Synthesis And Preliminary Antitumor Activity Evaluation Of Novel GSK-3? Small Molecule Inhibitors

Glycogen synthase kinase-3?GSK-3?is a versatile serine/threonine protein kinase with two subtypes of GSK-3??51 KD?and GSK-3??47 KD?,which are commonly found in most eukaryotic organisms.Related studies have shown that GSK-3 plays an important role in various physiological processes such as cell proliferation and differentiation,apoptosis,and glycogen metabolism.Many human diseases such as Alzheimer's disease,diabetes,human colorectal cancer and other tumors are closely related to the occurrence and development.Because the GSK-3?subtype is relatively stable,the current research on GSK-3 is mainly focused on GSK-3?.With the development of society,people's living standards have gradually improved,and their living habits have also been quietly changing,the incidence of malignant tumors has been increasing,the study of small molecule inhibitor compounds for malignant tumors has subsequently become a hot spot and difficulty in research.After consulting the literature,we found that GSK-3?plays a key regulatory role in many tumor cell signaling pathways such as PI3K/AKT?Wnt/?-catenin?NF-?B.For example,GSK-3?act as a substrate for the PI3K/AKT signaling pathway,which can directly interact with AKT,and can be inactivated by phosphorylation of AKT.Thereby reducing the formation of the GSK3?-LKB1-Axin complex and leading to increased accumulation of?-catenin,thus accelerating transcription of cells.The process also upregulates the expression of c-Jun and cyclinD1 and promotes tumor cell proliferation.Therefore,GSK-3?has attracted more and more researchers'attention as a popular target for antitumor drug research.The development of computer technology makes computer-aided drug design a new and rational drug molecular design method.Using computer technology to simulate the interaction between drug molecules and target proteins,explore the influencing factors of drug activity.It has important guiding significance for the design and synthesis of new drug compounds.Compared with traditional drug development methods,it has the obvious advantages of fast screening and high efficiency,and gradually plays a key role in modern drug development.In view of the above research background,in order to seek a new safe and efficient GSK-3?small molecule inhibitor,our group has designed and synthesized3,5-diamino-N-?3-?trifluoromethyl?phenyl?benzamide compounds through in-depth analysis based on the structures of some GSK-3?inhibitors that have been reported so far.And through the molecular docking experiment to verify its ability to bind to the active site of GSK-3?,on this basis,a series of derivatives with3,5-diamino-N-substituted benzamide as the mother nucleus were designed and synthesized.A preliminary in vitro anti-tumor activity evaluation was performed.The main research contents of this paper are as follows:1.Design of target compounds 4a-qThis paper has first synthesized 3,5-diamino-N-?3-?trifluoromethyl?phenyl?benzamide compounds?compound 4d?by comprehensively analyzing the structures of some of the GSK-3?small molecule inhibitors that have been reported so far.In addition,the binding ability of compound 4d to GSK-3?active site was verified by molecular docking experiments.It was found that it can directly generate hydrogen bonds with the isoleucine residue at position 62?ILE 62?and the proline residue at position 136 of recognition site?PRO 136?at the recognition site of the crystal protein structure of GSK-3?.Preliminary anti-tumor experiments also proved that it has a certain anti-tumor activity?IC50=12.5?M?.In view of this,our group has designed and synthesized a series of benzamide derivatives with 3,5-diamino-N-substitution as the parent nucleus,which will lay a foundation for the development of relevant GSK-3?small molecule inhibitors.2.Synthesis and structure identification of the target compounds 4a-qThis paper used 3,5-dinitrobenzoic acid and amines substituted with different substituents as raw materials to obtain a series of 3,5-dinitro-N-substituted benzoylamide compounds through the nucleophilic attack reaction of amines and acid chlorides,and then reduced by hydrazine hydrate to obtain 17 3,5-diamino-N-substituted benzoylamide compounds,which have not been reported in the literature.The substituted benzamides were confirmed by high resolution mass spectrometry?ESI-MS?,nuclear magnetic resonance?¹H-NMR?and nuclear magnetic carbon(¹³C-NMR)spectra.3.Preliminary anti-tumor activity screening of target compounds 4a-qThis paper used the MTT assay?thiazolyl blue colorimetry?to determine the antitumor activity of target compounds for human metastatic pancreatic adenocarcinoma cells?AsPC-1?,human colorectal cancer cells?HCT-116?,and human glial cell tumor cells?U251?.The preliminary anti-tumor activity screening results showed that compound 4d has good proliferation inhibitory effect on different types of tumor cells,its IC50 on pancreatic cancer cells is 12.5?M.And the activities of compounds 4f and 4c are second only to the best active compound 4d,and compounds 4e,4p,4k and 4b have moderate inhibitory activity.Compounds 4o,4a,and 4g had certain tumor suppressive effects on only one type of tumor cell,while compounds 4h,4j,4l,4m,4n,and 4q did not show significant inhibitory activity on the three types of tumor cells.In addition,based on the results of the preliminary anti-tumor activity of 17target compounds,and based on their structure,this paper undertook a related structure-activity relationship study.We have found that the difference in substituent R,the difference in tumor inhibitory activity between the target compounds is also greater.When R is replaced by a large group,its activity is significantly higher than that of a small group;When the R-nucleus of the substituent is the same,the substituents on the R-nucleus have a great difference in the inhibitory effect on tumor cells.The activity of the meta substituent is higher than that of the o-para-position substitution,and the electron-withdrawing group substitution is stronger than the electron-donating group substitution compound.