Process Optimization Of Sorafenib And Design,Synthesis And Anti-tumor Activity Of Its Derivatives

Sorafenib?Nexavar?is the first multi-target anti-tumor drug which developed by Bayer and Onyx Pharmaceuticals.US Food and Drug Administration?FDA?approved Sorafenib as a first-line drug for the treatment of advanced kidney cancer in December 2005.It is a new type of diaryl urea derivatives and can inhibit the Ras/Raf/MEK signal transduction pathway,and also can block the formation of new blood vessels by suppressing the receptor protein of VEGFR and PDGFR.Thus,it inhibits the growth of tumor cells.This paper introducts tyrosine kinases and their signal transduction pathways.The recent research is summarized about the synthetic route of sorafenib.On this foundation,we choose appropriate route to optimize the process and carry out the design,synthesis and anti-tumor activity of the new sorafenib derivatives.In this paper,we first optimize the synthesis process of sorafenib.On the basis of the existing literature,we chose the optimum synthetic route and selected 2-picolinic acid as the starting material to get the intermediate N-methyl-4-?4-nitrophenoxy?pyridine-2-carboxamide through chlorination,amidation and etherification.Then the key intermediate N-methyl-4-?4-aminophenoxy?pyridine-2-carboxamide?1?was obtained by reduction.The isocyanate was formed by 4-chloro-3-?trifluoromethyl?aniline and phosgene.And then with the key intermediate?1?to obtained the target compound Sorafenib.The overall yield was 61.3%.In this paper,the adventages of the improved synthesis method are easy work-up,cheap materials,mild reaction conditions,little environmental pollution,fewer steps and the postprocessing is more convenient and more suitable than the original synthesis method for the industrial production.At the same time,Sorafenib was used as the lead compound.We summarized the structure-activity relationship and efficacy of sorafenib and its analogues which reported in the literature.Analyze its binding characteristics with the target and on the basis of preliminary research in the laboratory.We retained the structure of N-methyl-4-phenoxypyridine-2-carboxamide which was an important pharmacophore.And we studied the changes of antitumor activity after substituting the structure of urea.According to the literature,the structure of chalcone is a good Michael receptor and it can be combined with tyrosine residues.Therefore it has excellent antibacterial,antiviral and anti-tumor activity.Some researchers introduced this group into VEGFR-2 kinase inhibitors and and had good antitumor activity currently.In this paper,the structure of chalcone was introduced to replace the bis-aryl urea structure.And in order to investigating the effect of double bond position of chalcone on the activity of the compounds,we design twenty compounds of W1 series.In order to make the sorafenib derivatives more tightly to the receptor,it is proposed to increase the nitrogen atom in the??-unsaturated aldehydes.And on the basis of the literature,compounds containing pyrazole are widely used in food,pesticide,antibacterial,anti-tumor and other fields.And anti-tumor activity is good.Therefore,on the basis of chalcone derivatives,the compounds were reacted with hydrazine hydrate to construct active pyrazole pharmacophores.Thus we replaced the first series of chalcone structures by pyrazole.Similarly in order to investigating the position of carbon-nitrogen double bond on the activity of the compound,we designed seventeen compounds of W2 series.Secondly,on the basis of pyrazole structure,we induced the structure of hioacid amide.And then we designed six compounds of W3 series.It has been reported that the design of the anticancer agent containing 1,2,3-triazole is widely used.They showed good antitumor activity.Thus we replaced urea by1,2,3-triazole-phenyl-4-carboxamide-5-trifluoromethyl?methyl?and six compounds of W4 were designed.Through biological electron isosphere,instead 1,2,3-triazole with pyridazinone structure.Six compounds of W5 series containing4-methyl-6-oxo-1,6-dihydropyridazine-phenyl-5-carboxamide were designed.A total of fifty-five compounds have not been reported in literature.The structure was confirmed by MS and ¹H-NMR.MTT method was used.Human lung cancer cell line A549,human lung cancer cell HepG2,human breast cancer cell MCF-7 and human prostate cancer cell line PC-3 were used as test cell lines.Sorafenib was the positive control drug.A total of fifty-five compounds were synthesized and tested for in vitro antitumor activity.The results showed that eighteen compounds showed better inhibitory activity against one or more tumor cells,which was superior to that of sorafenib.Compound WM-3showed well cytotoxic activity against HepG2,MCF-7 and PC-3 cell lines with IC₅₀values of 0.56±0.83?M,3.88±1.03?M and 3.15±0.81?M,which were 1.03–6.14-fold more active than sorafenib?3.44±1.50?M,3.18±1.43?M,3.24±0.45?M?respectively.Compound WM-22 exhibited strong antitumor activity against each cell,IC₅₀?2.84±0.78?M,1.85±1.03?M and 1.96±1.28?M,respectively?for A549,HepG2,MCF-7 were sorafenib?2.92±0.68?M,3.44±1.50?M,3.18±1.43?M?1.02 to 1.86 times.In order to investigate the inhibitory activity,the method of LANCE?Ultra enzyme activity evaluation was adopted.Sorafenib was the positive control drug.Ten active compounds?WM-2,WM-3,WM-9,WM-17,WM-22,WM-28,WM-29,WM-35,WM-41 and WM-43?were tested the activities of VEGFR-2/KDR and B-Raf respectively.The results showed that the inhibitory activity of WM-2 and WM-22 on VEGFR-2/KDR enzyme was similar to that of sorafenib,indicating that the designed W1 and W3 series compounds might be VEGFR-2/KDR kinase inhibitors.The compounds of W1 series,the substitution of electron withdrawing groups[3-Br?WM-2?,4-F?WM-17?]and electron donating groups?C-3,4,5 or C-2,3,4?are heplful to the activity of the compounds.The compounds of W2 and W3series,the substituents 3-Br and the unsubstituted compounds have a certain increase in the activity of them;The compounds of W5 series,the electron withdrawing group?-F?of the benzene ring can enhance the activity of the compounds.To further explore the interaction between the target and VEGFR-2/KDR kinase,it was analyzed and discussed by the molecular docking module.The result showed that the introduction of chalcone and pyrazole structures improved the activity of the compounds,and that some of the compounds were comparable or superior to sorafenib.Finally,in order to understand the changes of cell morphology after the action of the compound on the tumor cells,the morphological changes of the cells were observed under fluorescence microscope by acridine orange?AO?single staining.It was found that the compound WM-22 could induce the apoptosis of tumor cells.Annexin V-FITC/PI double dyeing-flow cytometry was used to detect the apoptosis of cells.The target compound WM-22 can induce apoptosis in a concentration-dependent manner.In summary,firstly this project studied the process optimization of sorafenib.Secondly we designed,synthesized and screened some series compounds of sorafenib derivatives,which had strong anti-tumor activity and bearing the structure of chalcone,pyrazole,1,2,3-triazole and pyridazinone.These results of structure-activity relationship could provide ideas and give us new research directions for the further study.