Studies On Design,Synthesis And Antitumor Biological Activity Of New Pt(?) Prodrugs Based On Cyclooxygenase 2 Inhibitors

Cisplatin is referred to "penicillin" in anticancer drugs and has been widely used for a variety of cancers since it was approved for clinical treatment.The success of cisplatin has greatly promoted the development of anti-tumor drugs based on platinum(II).However,the clinical application of platinum(II)drugs was limited by their severe resistance and side effect.The latest study shows that platinum(?)compounds can significantly overcome the disadvantage of platinum(II)drugs by acting as prodrugs.The octahedral platinum(?)complexes are kinetically more inert in the substitution with good stability by reducing the reaction with the nucleophilic substances in vivo,decreasing inactivation before entering cancer cells.These advantages could overcome the pre-target resistance of platinum(II)drugs.The platinum(?)prodrugs can regain their cytoxicity by generating platinum(II)drugs under the aid of tion substance.More importantly,it is easy to modify platinum(?)complexes by introducing biologically active groups at their axial positions,in order to improve antitumor activities and alter metabolism parameters(such as increasing fat-solubility or offerring multi-targets).Cyclooxygenase(COX)is involved in tumorigenesis,development and metastasis.Inhibiting cyclooxygenase can improve the effects of some antitumor drugs.In this work,we hope to obtain high efficiency and low toxicity of new anti-tumor platinum(?)prodrug by combinating cyclooxygenase inhibitors and platinum(?)complexes.Object: Focused on the problems of toxicity and resistance of the classical platinum(II)anticancer drugs represented by cisplatin,we introduce COX-2 inhibitors to the design of platinum(?)drugs to develop novel platinum(?)prodrug.The antitumor activity,molecular mechanism and the structure-activity relationship would be investigated to overcome the disadvantages of the existing platinum(II)drugs.Methods: The active groups containing carboxyl were bonded to the "cisplatin"(cisplatin oxide)by a simple esterification reaction to generate new platinum(?)prodrugs.The cisplatin was first oxidized to the tetravalent hydroxypropoxide by hydrogen peroxide,and then the condensing agent TBTU(O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate)were added to make cyclooxygenase inhibitor with carboxyl group and tetravalent hydroxyl platinum with hydroxyl groups condensed to a series of novel tetravalent platinum compounds based on COX-2 inhibitors.Their structures were characterized by IR,1H-NMR,13C-NMR and mass spectrometry.The purity of these prodrugs were analyzed by high performance liquid chromatography(HPLC).The reduction reaction of prodrugs with ascorbic acid and glutathione were analyzed by HPLC.The anti-tumor effect and molecular mechanism of cyclooxygenase-containing platinum compounds were studied by means of MTT assay,ICP-MS,flow cytometry,comet assay,fluorescence staining,western blotting and scratching.Results: Three novel platinum prodrugs based on COX-2 inhibitors were successfully synthesized,including disubstituted etodolac platinum complexes,carprofen platinum complexes and sulindac platinum complexes.Varied methods were used to characterize the physical and chemical properties of the prodrugs.HPLC test showed the purity of these prodrugs were more than 95%.MTT assay showed that these novel tetravalent platinum prodrugs had a good inhibitory effect on tumor cells and induced apoptosis,which were better than those of cisplatin and combination of cisplatin and COX-2 inhibitors.ICP-MS showed that the uptake of new prodrugs was much higher than that of cisplatin.The results of comet assay,flow cytometry and fluorescence staining showed that the novel platinum prodrugs could induce the cell cycle arrest,leading to DNA repair.If the DNA repair failed,tumor cell undergoed apoptosis.Western blotting indicated that the expression of Bax was up-regulated,while,Bcl-2 and MDM2 down-regulated by these prodrugs.Scratch test results showed that the platinum prodrugs can significantly inhibit the invasion of tumor cells.Conclusion: In this study,we successfully designed and synthesized a number of novel cyclooxygenase platinum(?)prodrugs based on inhibitors(disubstituted etodolac platinum complex,disubstituted carprofen platinum complex and disubstituted sulindac platinum complex)by introducing lipophilic COX-2 inhibitors in the axial positions of the cisplatin oxide,in order to alter the pharmacokinetics of the original platinum drug and exert double targets effect of COX-2 and DNA.The results showed that cyclooxygenase platinum(?)prodrugs could release the original cyclooxygenase inhibitors and cisplatin under the action of ascorbic acid,and the cyclooxygenase inhibitor could inhibit the activity of cyclooxygenase by regulating downstream genes Bax,Bcl-2,MDM2,etc.and demonstrate synergistic anti-tumor effect with cisplatin.Cyclooxygenase inhibitors etodolac,carprofen,sulindac and cisplatin are all FDA-approved drugs for clinical use.Their mechanism of action is relatively clear and safety.Moreover,the preparation methods of the prodrugs based on COX-2 inhibitors and cisplatin are simple,effective and easy to produce on industrial scale with wide application prospect.