Study On Synthesis Of Halogenated Indole Matrine Derivatives And Their Anti-Tumor Cytological Mechanism On Human Cervical Carcinoma Hela 229

In this paper,sophorine（natural derivative of matrine）was uesed as starting material to synthesize a series of halogenated indole matrine derivatives.The active groups of halogenated indole were introduced at C13 of sophorine through the Michael addition reaction.Whereafter anti-cervical cancer activity screening and anti-tumor mechanism research of new derivatives were carried out.The rusults were as follows:（1）A series of halogenated indole groups were introduced on the C13 of the sophorine core by Michael addition method and seven target compounds were synthesize:4-fluoroindole matrine（4-F-Ind-Mat）,4-bromoindole matrine（4-Br-Ind-Mat）,4-iodoindole matrine（4-I-Ind-Mat）,5-fluoroindole matrine（5-F-Ind-Mat）,5-chloroindole matrine（5-Cl-Ind-Mat）,5-bromoindole matrine（5-Br-Ind-Mat）,5-indomatrine（5-I-Ind-Mat）.At the same time,the purification conditions of 4-chloroindole matrine（4-Cl-Ind-Mat）were optimized.Finally,the structure of target products were confirmed by characterization with IR,MS,NMR,HPLC,XRD and so on.（2）Human cervical cell line（Hela 229）was as the active screening model,MTT detection and inverted phase contrast microscopic observation（IPCM）technology were used to analyze the effect of neomatrine derivatives on the proliferation and growth and cell morphology of Hela 229 cells.The results showed that the inhibitory activity of the new derivatives on Hela 229 was far superior to matrine,sophocarpine and indole matrine,and it showed obvious time and concentration dependence.Among them,the inhibitory effects of 5-I-Ind-Mat and 5-Br-Ind-Mat were pretty well,the inhibition rates of them were as high as 80.94%and 80.74%,which induced cells for 72 h at 0.04 and 0.05 mmol/L respectively.The IC₅₀ was 0.032 and 0.022 mmol/L.IPCM observation showed that the proliferation and growth of Hela 229 cells was inhibited,the number of adherent cells decreased,the cell density decreased,the overall cell shrinkage became smaller,the cell morphology was irregular,and the cells showed apoptosis or necrosis when Hela 229 induced by new derivatives,.（3）In order to explore the anti-tumor cytology mechanism of the new matrine derivatives,flow cytometry,fluorescence microscopy,transmission electron microscopy and other cell biology techniques were used to analyze the apoptosis rate,apoptosis morphology,mitochondrial membrane potential cell cycle and the shape of the main organelles and apoptosis of Hela 229 cells induced by the new derivatives for 48 h.The results showed that:compared with Mat,Sop and Ind-Mat（the test concentration were 3,3and 2 mmol/L,the apoptosis rate was 35.2%,32.3%and 39.3%at 48 h）,the apoptosis inducing effect of the 4-halogenated indole matrine derivatives were significantly enhanced.Well below 1.5 mmol/L of new derivatives,the apoptosis rate of them was close to or exceeded 60%;Induced by 5-halogenated indole matrine derivatives,the inhibition rate of Hela 229 cells were high while the apoptosis rate was low,the apoptosis rate of them were only 18.1%-28.6%.With the action of the new derivatives,most cell membranes were destroyed,their permeability increased,cell nucleus were stained by PI,phosphatidylserine exposed on the cell surfaces and early apoptosis occurred.It can be seen that 4-haloindole matrine derivatives,5-F-Ind-Mat and 5-I-Ind-Mat could inhibit the proliferation of Hela229 cells by inducing early cell apoptosis from the phenomenon of mitochondrial membrane potential（ΔΨm）decrease.At the same time,they can also inhibit cell replication in Hela 229 cells,triggering cell cycle G1 arrest and inhibiting cell proliferation and growth.Inducing by 5-Cl-Ind-Mat and 5-Br-Ind-Mat,there were no significant changes in theΔΨm in the cell.They might inhibited cell proliferation by blocking DNA replication in the cell and stalling G2 phase of the cycle.Ultrastructural observation revealed that the new halogenated indo matrine derivatives generally induce various death（apoptosis/autophagy/necrosis）morphological features of Hela 229 cells,such as nuclear shrinkage/pagination,lysosomal swelling and fullness Endogenous substances,mitochondrial condensation or swelling,and the phenomenon of co-existence of apoptosis and necrosis in DNA fragmen-tation detection.Therefore,the new derivatives inhibit tumor cell growth in multiple pathways.In conclusion,inducing by 4-haloindole matrine derivative,cell number,cell cycle G1and their morpholog were in an exception so that they activated the apoptotic mechanism and inhibited the cell proliferation.The mechanism of 5-halogenoindole matrine derivatives inhibited Hela 229 cell proliferation is more complicated.Apoptosis is not the main factor for cell proliferation inhibition.It might induce cell death by other pathways（autophagy/necrosis）to inhibit Hela 229 cells proliferation,while their specific cellular mechanism of proliferation needs to be further study.