Design,Synthesis And Antitumor Activity Of Ligustrazine Dimers,Tetramers And Their Analogues

Cancer is one of the most common causes of death.Natural products and their derivatives play a vital role in the development of anticancer drugs.Ligustrazine?Tetramethylpyrazine,TMP?,an effective alkaloid monomer extracted from the Chinese traditional medicinal herb Chuanxiong?Ligusticum chuanxiong Hort?,has various pharmacological activities,such as dilating blood vessels,inhibiting platelet aggregation,enhancing immune function and anti-tumor etc.And it had been widely used in the treatment of cardiovascular,cerebrovascular,respiratory and digestive diseases,like coronary heart disease,cerebral thrombosis and cerebral embolism etc.Recently,TMP had been reported to possess anticancer activity,including apoptosis,invasion,metastasis,angiogenesis,drug resistance and tumor immunity.It is a good anti-tumor lead compound with simple structure.Usually,the combination of TMP with different anti-tumor groups was helpful to improve the anti-tumor activity.And many ligustrazine derivatives with potent anti-tumor activity had been developed in the past few years,like ligustrazine-triterpene conjugate,ligustrazine-curcumin conjugate and ligustrazine-podophyllotoxin conjugate etc.This had stimulated our interest in using TMP as the scaffold to synthesize new anti-tumor agents.Dimers and polymers could dramatically enhance the activity over the monomer molecules,and this phenomenon has attracted attention in drug design.Usually,two or more anti-tumor fragments were joined together with a linker to form a homo-dimer or homo-polymer.And many effective bivalent anti-cancer drugs were discovered,like artemisinin dimers,?-carboline dimers,apigenin dimers etc.More importantly,the linker was vital for the design of dimers and polymers.In the previous work of this field,several ligustrazine dimes,linked by curcumin,triterpenes,cyclohexanone or oxime,showed promising cytotoxicity on human cancer cells.It revealed that the dimerization of ligustrazine could obviously improve the anti-tumor potency over its monomer.In our continuous effort to develop new anti-tumor agents,seven ligustrazine tetramers and eight ligustrazine dimers were synthesized by using the flexible alkyl chains as connector.And their antiproliferation activities against five cancer cell lines?He La,Hep G2,MCF-7,Fa Du and A549?and normal cell line MCF 10A were screened by MTT method.The results showed that the anti-tumor activity of ligustrazine dimers was more potent than that of tetramers,and the alkyl linker chain with 8–12 carbon were vital to maintain its anti-tumor activity.In particular,ligustrazine dimer 8e linked with decane-1,10-diamine exhibited the highest cytotoxicity,IC₅₀ values were 1.42±0.71?M,0.037±0.001?M,0.00136±0.00035?M and 0.047±0.008?M respectively against He La,MCF-7,Fa Du,A549 and MCF10A.And it showed high selectivity to Fa Du cells,SI value(IC_{50 MCF 10A}/IC_{50 Fa Du})was34.56.Then,inspired by the importance of small molecular heterocyclic rings in drug discovery,thirty-six heterocyclic dimers were synthesized.And most of them showed potent anti-proliferative ability in Fa Du cell lines,but had nearly no toxicity in normal cells MCF 10A.SAR revealed that the introduction of heteroatoms was helpful to enhance the anti-tumor activity,and the two terminal amide bonds were the vital elements for these dimers.In addition,a series of biological experiments were conducted to investigate the antitumor efficacy and mechanism in vivo and in vitro.The anti-tumor activity in vitro of 8e was further confirmed by colony formation assay and Calcein AM/PI double staining.The Fa Du xenograft Balb/c nude mice model was also established to investigate the efficacy of compound 8e in vivo,the tumor inhibition rates of low,middle and high dose groups were 28.4%,54.8%and 70.3%,respectively,and there were no obvious side effects.Moreover,the ADMET calculation predicted that most of the potent compounds had good properties,especially,the compound 8e was expected to be an anti-tumor drug candidate.Hoechst 33342 staining,Annexin V/PI staining,JC-1 staining and PI staining suggested that 8e could induce apoptosis of Fa Du cells through the depolarization of mitochondrial membrane potential and S-phase cell cycle arrest.ELISA assay and tubulin polymerization assay showed that 8e could also inhibit the expression of EGFR and tubulin polymerization,and molecular docking study showed that 8e could be tightly embedded into the active pocket of EGFR and tubulin.Overall,this study revealed a class of ligustrazine tetramers,dimers and their analogues with potent anti-tumor activity,and preliminarily discussed the anti-tumor mechanism of the most potent compound 8e,which provided a scientific reference for further research.