Design,Synthesis And Activity Evaluation Of Novel Antitumor Compounds With Phenothiazine As The Skeleton

Objective:According to the latest statistics from the journal of CA,the incidence and mortality of cancer are still rising worldwide.The incidence and mortality rate of cancer in China are both in the first place.Cancer is still a major disease affecting human health.In addition,breast cancer has replaced lung cancer in the world as the cancer with highest incidence,so there is still an urgent need to develop new and effective breast cancer treatment drugs.Heterocyclic structure is always an important structure in drug discovery,and more than half of the drugs on the market contain heterocyclic structure.In various heterocyclic structure,phenothiazine is a kind of important heterocyclic,based on phenothiazine compounds with unique nearly flat structure to fundamental form stacking interaction with the amino acid residues of targets,sulfur as hydrogen bond acceptor atoms can form hydrogen bonds,after oxidation can also generate sulfoxide and sulfone,nitrogen atom on 10 position can be connected with alkyl chain and can also form amide bonds and urea groups,etc.Phenothiazine provides a framework for designing drug molecules.Phenothiazine compounds have also shown a variety of bioactivity,including antipsychotic,anti-microbial,anti-tumor and so on.Phenothiazines with different structures have been reported as antitumor lead compounds.Therefore,in this work,we also selected phenothiazine as the basic skeleton to obtain new compounds against cancer,especially breast cancer.Methods:Based on the literature review,a reasonable and feasible synthetic route was designed,and 10 novel compounds with phenothiazine as the nucleus structure were successfully synthesized.The purity and structure of these 10 compounds were determined by TLC,chromogenic agent,1H NMR and MS.A series of in vitro antitumor bioactivity tests were performed on the candidate compound,including:in vitro antitumor cell proliferation assay（MTT assay）,promoting tumor cell apoptosis assay,cell cycle inhibition assay,transwell assay,western blot assay and computer docking simulation assay.Results:Through organic synthesis and activity screening,the author finally obtained a leading compound TTi-2 from 10 compounds,which met the requirements of Lipinski’s role of five in terms of physical and chemical properties.Activity:the compounds under 50micro molar concentration,TTi-2 inhibited the proliferation of PC-3 prostate cancer cells with IC₅₀ value of 12μM.In further testing,TTi-2 also showed strong antiproliferative activity to 22more human tumor cells,the IC₅₀ values were all within the range ofμM,comparable to the positive control drug cisplatin.In particular,the IC₅₀ value of TTi-2against breast cancer cells MDA-MB-231 was 9.33μM.TTi-2 can effectively induce apoptosis of MDA-MB-231 cells in a concentration dependent manner.In cell cycle inhibition experiment,TTi-2 could block MDA-MB-231 cells in G0/G1 phase.Transwell assay result showed that TTi-2 could effectively inhibit the invasion and metastasis of MDA-MB-231 cells.Western blot results further verified that TTi-2 can effectively induce apoptosis of MDA-MB-231 cells.Finally,through computer docking simulation experiment,we proved that the possible target of TTi-2 is topoisomerase II,but this conclusion still needs to be verified by subsequent activity experiments.Conclusion:Ten novel antitumor compounds with phenothiazine scaffold have been designed and synthesized.Their structure and purity are verified using 1H NMR and MS.The representative compound TTi-2 showed micromole inhibitory activity against 23 kinds of human tumor cells,indicating that it has a broad spectrum of antitumor effect.It also has a significant effect on the proliferation,migration,cell cycle and apoptosis of MDA-MB-231 cells,which could be regarded as a potential lead compound in the treatment of breast cancer.The inhibitory rate of TTi-2 on normal cells was much lower than that of cisplatin,demonstrating its good safety profile.