Synthesis, Screening And Activity Evaluation Of New Anti-tumor Benzothiazoles

Apoptosis is important in chemotherapy-induced tumor-cell killing,and many anticancer drugs induce restoration of apoptosis.Many tumors have been associated with the inhibition of apoptosis,and the disruption of apoptotic function which contribute substantially to the transformation of a normal cell into a tumor cell. Intracellular reactive oxygen species(ROS) is considered to be a death signal in apoptosis.ROS induces disruption of the mitochondrial membrane potential(MMP) and release of cell death signals to trig cell death.Accumulation of excessive ROS leads to lipid peroxidation,protein oxidation,enzyme inactivation,oxidative DNA damage.Cancer cells are more sensitive to ROS than normal cells;therefore,ROS is a hotpoint for anti-cancer drug research.Thiazole,an important heterocyclic ring,is widely used in anticancer drug development.Many natural chemotherapeutic agents containing thiazole moiety have been discovered and studied,like tiazofurin,distamycin,bleomycin and netropsin. Among the thiazole derivatives,benzothiazole is an important scaffold of drugs,and it has been studied extensively.Benzothiazole derivatives are known as inhibitors of topoisomeraseâ…¡,tyrosine kinase and ubiquitin proteasome system.Some studies reported the antioxidant properties of benzothiazole derivatives,and their biological activity was indicated to be strongly related to ROS.In this respect,a new serious of benzothiazole derivatives based on 2-(1,3-diphenyl-1H-pyrazol-4-yl)benzo[d]thiazole(DPB) were designed and synthesized.Next,we evaluated their anti-cancer effects and found 3-(4-(benzo[d]thiazol-2-yl)-1-phenyl-1H-pyrazol-3-yl) phenyl acetate(DPB-5). Further more the molecular mechanism of DPB-5 for its anticancer effect was studied.We found that DPB-5 induced apoptosis in human liver cancer cells through a caspase-dependent and ROS-mediated AIF pathway.The main results of this study are as follows:1.10 benzothiazole derivates based on DPB-5 were designed and synthesized, the structures was determined by NMR and MS.The optimum reaction conditions: solvent,ethanol;raw materials ratio pyrazole:2-aminophenyl mercaptan=1:1.1; p-toluene sulfonic acid as catalyst,with the concentration of 5%(mmol volume ratio). The reaction conditions can be at room temperature,with the yields at 80%～90%.2.The anti-cancer effects of benzothiazoles were evaluated by MTT method using Hep G2,MCF-7,Hep 2 cell lines.The results showed that DPB-5 was the highest cytotoxic to these cancer cell lines and Hep G2 was most sensitive to the cytotoxicity of benzothiazole.Hep G2 cells were shown to be much more sensitive to DPB-5-induced cytotoxicity with an IC₅₀ of 4.6μM while LO2 normal liver cells with an IC₅₀ of 9.6μM.The cytotoxic effect of DPB-5 caused apoptotic cell death,as determined by DNA fragmentation,morphological change of the nucleus,increase of pro-G1 DNA content,and externalization of phosphatidylserine.The apoptotic effect was associated with an early elevated level of ROS and significant decrease of GSH, however DPB-5 induced less change of ROS and GSH on normal liver cells LO2. The higher vulnerability to ROS of Hep G2 cells than LO2 cells lead to the higher cytotoxicity of Hep G2 cells on DPB-5 than that of LO2 cells.3.ROS burst was a prerequisite for mitochondrial membrane potential(MMP) collapse and cell death induced by DPB-5.Anti-oxidants(N-acetyl-L-cysteine and catalase) blocked the MMP depolarization,inhibited the cytotoxicity of DPB-5, decreased cell apoptosis and enhanced cell survival associating with suppressed ROS generation and GSH depletion.4.Considering the death signal trigged by MMP collapse,there was no significantly release of cytochrome C,which was supported by no activation of caspase-9 or caspase-3.However,apoptosis inducing factor(AIF,another death signal molecular) was clearly increased in cytoplasm.It is suggested DPB-5 induced Hep G2 cell apoptosis via AIF/Endo G pathway.Although no need of caspase-9 or caspase-3 activated,DPB-5 induced Hep G2 cells apoptosis in a caspase dependent pathway.Pan-caspase inhibitor zVADfmk significantly reduced cell death rate and inhibited cell apoptosis.Taken together,DPB-5 showed a good anti-cancer activity while low cytotoxicity to normal cells.And DPB-5 induced Hep G2 cell apoptosis in a ROS-mediated AIF pathway which was caspase dependent.These findings will contribute to develop of new serious of benzothiazole anti-cancer drugs.