Process Optimization Of NVP-BEZ235 And Design,Synthesis And Antitumor Activity Of Its Derivatives

NVP-BEZ235?dactolisib?is an anti-tumor inhibitor which developed by Novartis targeting PI3K-Akt-mTOR signaling pathway.It binds PI3K and mTOR kinase activity by binding to ATP binding groove.The IC₅₀0 values of NVP-BEZ235against p110?/?/?/?and mTOR?p70S6K?was 4,5,7,75 and 6 nM,respectively.Currently it has entered the clinical phase II study.The clinical results of stage?showed that it has a good inhibitory effect on solid tumors and hematological malignancies.Single administration of NVP-BEZ235 can inhibit the development of the disease and combined with other anti-tumor drug administration can significantly enhance the efficacy.In this paper,we describes the PI3K-Akt-mTOR pathway and the corresponding,and introduce the importance of PI3K/mTOR dual inhibitor NVP-BEZ235 in anti-tumor therapy.The synthetic route of PI3K/mTOR dual inhibitor NVP-BEZ235 was analyzed and optimized.Furthermore,the design,synthesis and anti-tumor activity of novel NVP-BEZ235 derivatives were also studied on the basis of the study of structure-activity relationship of NVP-BEZ-235.Based on the reported synthetic route of NVP-BEZ235,the synthetic route of NVP-BEZ235 was established through experimental investigation.The compound 3?3-nitro-4-chloro-6-bromoquinoline?wassynthesizedfrom2,2-dimethyl-1,3-dioxane-4,6-dione with triethoxy methane and 4-bromoanilines through condensation,cyclization,nitration,chlorination.Then,the compound 3 was reacted with 2-?4-aminophenyl?-2-methylpropionitrile?5?which were obtained by methylation and reduction of p-nitrophenylacetonitrile to give 2-[4-[?6-bromo-3-nitroyl?amino]phenyl]-2-methylpropanenitrile?4?.Finally,NVP-BEZ235 was prepared by reduction,cyclization,methylation and Suzuki coupling reaction.The process route was optimized from reaction temperature,reaction time,yield and so on.The optimized synthetic route has the advantages such as the raw materials are cheap,the operation is easy,reaction conditions are mild and the yield was significant improved.It provides us a reasonable way for large-scale pilot study and practical application.On the basis of the process research,we also carried out the structure modification of NVP-BEZ235.In this paper,we summarized the structure-activity relationship of NVP-BEZ235 and its analogues reported in the literature.On the basis of the retention of quinoline ring,different substituents were introduced to the quinoline ring at C-2,C-3,C-4 and C-6 postion according to the principle of skeleton transition and bioelectron isotope.Two series?imidazoles and sulfonylureas?of 61NVP-BEZ235 derivatives?L-1^L-61?which have not been reported in the literature were designed and synthesized through modification of NVP-BEZ235.The target compound were evaluated for anti-tumor activity against human lung cancer cell A549,human prostate cell PC-3,human hepatocellular carcinoma cell HepG2 by MTT assay using NVP-BEZ235 and Sorafenib as the positive controls.The results showed that the two series of compounds showed varying degrees of cytotoxicity against the four tumor cells.Most of the target compounds showed moderate activity against A549 and PC-3 cell lines and some of the compounds showed good and excellent cytotoxicity toward HepG2 and MCF-7 tumor cell lines.Among them,the imidazole compound L-1 showed the best activity against HepG2,A549 and PC-3 with IC₅₀0 values of 2.4?M,6.2?M and 5.1?M,resectively,which was0.61¹.68 times more active than sorafenib.The structure-activity relationship shows that the introduction of electron withdrawing groups such as Br and cyano groups can enhance the activity.The sulfonylurea derivatives L-49 showed the best activity against HepG2,A549 and MCF-7 with IC₅₀0 values of 2.7?M,7.4?M and 6.5?M which was 0.68 to 1.56 times more active than sorafenib.The introduction of methoxy group can increase the activity of the target compound,whereas the different substitutions on the aromatic ring in the arylsulfonylurea structure have no significant effect on the activity of the compound.In general,the overall cellular activity of the sulfonylurea derivatives was lower than that of the imidazole derivatives.Four selected compounds?L-1,L-6,L-26,L-49?were evaluated against PI3K/mTOR kinase by LANCE^?Ultra kinase assay to further examine the targets of target compounds,using NVP-BEZ235 and PI103 as the positive controls.The results showed that the activity of the tested compounds was lower than that of the positive control drugs.The IC₅₀0 values of compound L-26 against PI3K?and mTOR was 0.72?M,2.62?M.It has a certain gap with the positive control and may act as other targets.According to the results of the antitumor activity in vitro,the compounds?L-1,L-6,L-26,L-49?were docked with kinase protein by molecular docking software SYBLY.The activity of the target compound is related to the number of hydrogen bonds formed by the compound with the ligand protein and the number of linked amino acid residues through analyzing the results of molecular docking and in vitro antitumor activity.It provides a reference for the later study.We summarized the structure-activity relationship of the target compounds with the antitumor activity in vitro and molecular docking results.The introduction of imidazole had no effect on the activity of the compound.The activity of compounds bearing electron withdrawing groups was higher than that of the compounds bearing electron-donating group at the C-6 position of quinoline ring.The introduction of cyano groups can enhance the activity on.The introduction of sulfonylurea reduced the overall activity,and the introduction of different substituents on the arylsulfonylurea structure aryl has little effect on the activity,and the introduction of the methoxy group on the aniline makes the activity better than that of the other compounds.In general,the activity of imidazole derivatives is higher than that of sulfonylurea derivatives,and the activity of compounds which introducting electron withdrawing groups at the position of the quinoline ring C-6 is superior to that of electron-donating group.In summary,we synthesiz and optimize the process of NVP-BEZ-235 to establish a synthetic route with cheap raw materials,simple operation and mild reaction conditions.The compounds with better activity?L-1,L-6,L-26,L-49?were screened from the synthesized target compounds.The structure-activity relationship was analyzed,which provided the right direction for the later design and modification of new compounds.