Synthesis,Antitumor Activity And Mechanism Of Action Of Novel Allosecurinine Derivatives

Objective:Cancer is a major public health problem in the world today.It is the second leading cause of death in the global population.The survival rate is low,so effective antitumor drugs are urgently needed.The search for antitumor chemical constituents with high efficiency and low toxicity from natural products has always been a hot spot in the research of modern tumor drugs.Natural product allosecurinine is a bioactive indolicidine alkaloid derived from plants.At present,there are few reports related to allosecurinine,and only a few total synthesis studies.The aim of this study was to investigate the antitumor activity and mechanism of the natural product allosecurinine and its synthetic derivatives.Methods:（1）Compound synthesis:Under the catalytic action of titanium tetrachloride,Baylis-Hillman reaction occurs between allosecurinine and aromatic aldehydes（including electron-absorbing,electron-donating and heterocyclic group substitution）,and benzyl alcohol-substituted furanone structure is introduced at C-12 site to obtain a series of novel allosecurinine derivatives.In order to investigate the effect of newly generated chiral carbon on biological activity,the most active compounds were chiral separated to obtain optical pure isomers.（2）Studies on activity and mechanism:Firstly,seven leukemia cell lines NB4,U937,K562,HL-60,KG-1,THP-1,SUDHL-2 and two ovarian cancer cell lines SKVO-3 and SKVO-3/Taxol were selected,and camptothecin was used as positive control.MTT method and CCK8 method were used to determine the inhibitory activity of the synthetic derivatives on tumor cell proliferation,and the best compounds were selected for cytotoxicity study on human normal liver cells（WRL-68）to study the selectivity of the compounds.At the same time,the inhibitory activity of tumor cell proliferation of pure isomers obtained from chiral separation of the most active compounds was also studied,and the diastereoidal mixture was compared to determine whether the newly generated chiral carbon could affect the antitumor activity.Secondly,the most active compounds were selected to treat leukemia cells（NB4,HL-60,KG-1and THP-1）at low concentration（0.5μM）for 72 h.Flow cytometry was used to detect the expression of CD11b,a marker of granulocyte differentiation antigen,to preliminatively investigate the differentiation induction activity of the compounds on leukemia cells.Leukemia cells（NB4,HL-60,THP-1,KG-1,U937 and K562）were treated with the compound at high concentration（2.5μM）for 48 h.The apoptosis,DNA content and cell cycle arrest were detected by flow cytometry.THP-1,KG-1 and U937were treated with the compound at concentrations of 1μM,2.5μM and 5μM for 48 h.The changes of mitochondrial membrane potential and reactive oxygen species were detected by flow cytometry,and the accumulation of reactive oxygen species in cells was detected by laser confocal scanning.Finally,Western Blot assay was used to detect the expression of key proteins closely related to cell proliferation in leukemia cells（NB4,THP-1,KG-1,U937 and K562）treated with compounds at concentrations of1μM,2.5μM and 5μM for 48 h.The mechanism of antitumor activity of the compound was preliminarily discussed.Results:In this study,23 allosecurinine derivatives with novel chemical structures were synthesized.These derivatives showed better anti-proliferation activity against 7leukemia cell lines and 2 ovarian cancer cell lines than the parent of allosecurinine.The most active compound BA-3 had an IC₅₀ of about 1μM（NB4:1.88±0.18μM,KG-1:1.06±0.16μM,U937:1.53±0.11μM,K562:1.78±0.15μM）,and showed good selectivity to human normal hepatocytes.The activity of the two pure isomers BA-3-a and BA-3-b obtained by the chiral separation of BA-3 on all tumor cell lines was comparable to that of BA-3 itself,indicating that the newly generated chiral carbon had no significant effect on the antitumor activity of these derivatives.Flow cytometry showed that compound BA-3 had dual anti-leukemia effects in inducing leukemic cell differentiation at low concentration and apoptotic pathway at high concentration.Flow cytometry results also showed that BA-3 induced cell cycle arrest,mitochondrial membrane potential change and reactive oxygen species level increase.The results of laser confocal scanning also showed that BA-3 induced accumulation of reactive oxygen species in cells.Western blot analysis showed that BA-3 increased the expression of Bax and decreased the expression of Bcl-2,XIAP,PARP,YAP1,caspase3 and caspase 9.BA-3 also reduces the expression of STAT3 and p-STAT3,resulting in the downregulation of c-Myc and the up-regulation of p21,two key downstream proteins in the STAT3 pathway.Conclusion:In this study,BA-3,an allosecurinine derivative,was found to have the best anti-leukemia activity at low concentrations（<1μM）can induce differentiation of leukemia cells,and high concentration（1-5μM）can induce apoptosis,showing a dual anti-leukemia effect.Further mechanism studies showed that BA-3 induced cell cycle arrest,mitochondrial pathway mediated apoptosis,and increased intracellular reactive oxygen species（ROS）levels.The newly synthesized allosecurinine derivatives exhibit antitumor activity at least in part through the STAT3 pathway.This study will we believe lay a scientific foundation for the study of antitumor activity and mechanism of action of allosecurinine derivatives.