Design,Synthesis And Antitumor Activity Of Rosin Diterpenoids And Pentacyclic Triterpenoid Derivatives Based On Multi-target Strategy

Tumor metastasis and drug resistance are the main factors leading to treatment failure and death of cancer patients,and are two major challenges in current anti-tumor clinical practice.Mechanistic studies indicate that nuclear transcription factor NF-κB signaling involves in the overexpression of genes in tumorigenesis and progression,including immune response,cell survival,differentiation,proliferation,invasion and angiogenesis,and has emerged as a promising target for the research and development of anti-tumor drugs.Matrix metalloproteinases(MMPs)are downstream target genes of NF-κB and have been shown to be closely related to tumor invasion and metastasis.Based on the preliminary research results obtained,the dehydroabietic acid,ursolic acid and asiatic acid that with good anti-tumor activity and multi-signal mechanism were selected for optimiztion to obtain novel and highly potent anti-tumor drug targeting NF-κB or MMPs.(1)A series of dehydroabietic acid dipeptide derivatives(30 compounds)containing the sulfonamide moiety were synthesized and evaluated the effects of in vitro cell migration.These compounds exhibited relatively good inhibition activity against MMPs,wherein the inhibitory activity against MMP-3 was superior to that of MMP-8 and MMP-9.A docking study of the most active compound 8k revealed key interactions between 8k and MMP-3 that were important for improving activity,in which the sulfonamide moiety and the dipeptide group formed multiple hydrogen bond interactions with key amino acid residues of MMP-3 active site.In vitro antitumor activity screening showed that some compounds exhibited better inhibitory activity than the clinical anticancer drug 5-FU.In particular,compound 8k showed the best inhibitory activity against HepG2 cell line,IC50=4.2 ± 1.1 μM.The treatment of HepG2 cells with compound 8k resulted in inhibition of in vitro cell migration through wound healing assay and induction of apoptosis in a dose-dependent manner.In addition,cell cycle analysis indicated that compound 8k arrested Hep G2 cell growth at G1 phase.(2)A series of dehydroabietic acid thiourea derivatives(11 compounds)containing bisphosphonate moiety were synthesized and the growth inhibitory activities of the target compounds against SK-OV-3,BEL-7404,A549,HCT-116 and NCI-H460 tumor cell lines were studied.Especially,compound 6e(IC50=1.79± 0.43 m M)exhibited the best anticancer activity against SK-OV-3 cell line.Its role as an inducer of apoptosis was investigated in this cell line by Annexin-V/PI binding assay and by following its capability for ROS generation,depolarization of mitochondrial transmembrane potential,activation of caspases and expression of pro-and anti-apoptotic proteins.Elevated level of ROS generation,activation of caspase-3,caspase-8,caspase-9,and Fas,higher expression of Bax,lower expression of Bcl-2,and increased level of Bax/Bcl-2 ratio identified 6e as a promising inducer of apoptosis that follows both of the mitochondria dependent pathway and the death receptor-mediated pathway.In addition,the cell cycle analysis indicated that compound 6e caused cell cycle arrest at G1 phase.Furthermore,molecular docking studies showed that 6e could bind to the ATP pocket site of CDK2(3)A series of inhibitors of NF-κB based on ursolic acid derivatives(27 compounds)containing side chain functionalized aniline or amide side chains were synthesized and evaluated for inhibition of NF-κB as well as their antitumor effects.In vitro activity assay showed that these compounds exhibited significant inhibitory activity toward TNFα-induced NF-κB in NCI-H460 cells with IC50 values in the micromolar range.The structure-activity relationship analysis revealed that small and strong electron-withdrawing groups in the side chain could increase the inactivation of NF-κB,and the C-3 acetyl group had a certain effect on the NF-κB inhibitory activity.Molecular docking studies further revealed that the side chain of dimethyl propylamine of compound 5Y8 formed a hydrogen bond with NF-κB,which fully demonstrated the importance of the small and the strong electrophilic functionalized side chain at C-28 for improving activity.In particular,compound 5Y8 appeared to be the most potent compound against the NCI-H460 cell line,even stronger than HCPT.In addition,selected compounds displayed efficiency in reversing adriamycin-resistant cancer cell lines,at least in part by blocking the NF-κB signaling pathway and inducing apoptosis.Mechanistically,compound 5Y8 may inhibit the proliferation of NCI-H460 cells and trigger apoptosis by blocking the NF-κB signaling pathway.At the same time,cell cycle analysis indicated that compound 5Y8 arrested NCI-H460 cell growth at G1 phase(4)A series of asiatic acid based 1,2,3-triazoles derivatives(20 compounds)were synthesized as NF-κB inhibitors.Compound 6k was determined to have significant inhibitory activity against TNF-α-induced NF-κB activation by a cell-based luciferase assay with IC50 values in the lower micromolar range.Molecular docking studies revealed a pattern of interaction between 6k and NF-κB,in which the 1,2,3-triazole moiety and the hydroxyl group of the asiatic acid skeleton formed multiple hydrogen bonding interactions internet with key amino acid residues of NF-κB.Surface plasmon resonance analysis validated the high binding affinity between compound 6k and NF-κB protein with an equilibrium dissociation constant value of 0.36 μM.Further mechanism of action studies showed that compound 6k observably inhibited NF-κB/DNA binding,nuclear translocation and IκBα phosphorylation.In vitro anti-tumor activity screening indicated that compound 6k exhibited the best activity against A549 cells(IC50=2.67±0.06 μM),but slightly weaker than DOX inhibitory activity.The apoptosis induction potency of compound 6k in A549 cells was confirmed by flow cytometry and AO/EB staining.Additionally,transwell migration assays indicated that compound 6k inhibited cell migration in vitro by blocking the NF-κB signaling pathway.In this paper,a total of 88 compounds were synthesized in four series.Mechanism studies showed that representative compounds could inhibit tumor cell migration and reverse tumor resistance by targeting NF-κB or MMPs.It provides a certain research basis for the further development of rosin diterpene and pentacyclic triterpene derivatives as highly potential antitumor agents.