Design,Synthesis Of Targeted Drugs Based On Anthraquinone Skeleton Structure And Their Antitumor Activity In Vitro

Objective:On the basis of anthraquinone keleton structure of rhein,targeting induction endoplasmic reticulum（ER）stress and regulation of EGFR and RAC1 protein transcription levels,design and synthesis of two major classes of novel rhein derivatives using the principle of pharmacophore combinatorial.Investigate their antitumor activity and mechanism in vitro,to provide new insights for the design and development of novel targeted antitumor drugs.Method:We utilized rhein as raw material and synthesized series of novel rhein derivatives using Schlenk technology.The synthesized compounds were characterized by HRMS,IR,¹H NMR and ¹³C NMR.The cell inhibitory of compounds on human hepatoma cells（SMMC-7721,Hep G2）,nasopharyngeal carcinoma cells（CNE-1,CNE-2）and lung cancer cells（A549）was determined by MTT and CCK-8 assay.After incubating cells with compounds,morphological images were obtained with HE staining by phase-contrast microscopy.The effects of nuclear and endoplasmic reticulum were observed by laser confocal microscopy with ER Tracker and Hoechst33342 staining,and the change of ultrastructure of cells was observed by transmission electron microscope.Flow cytometry detected apoptosis and cell cycle distribution.The expression levels of target proteins EGFR and RAC1,apoptosis protein Caspase-3,endoplasmic reticulum stress protein GRP78 and autophagy key proteins LC3 and p62 were detected by Western blotting.Results:（1）All the compounds were confirmed by HRMS,IR,¹H NMR and ¹³C NMR.They are 1,8-dibenzyloxy-9,10-anthraquinone-3-carboxamide derivatives（4a-b）,1,8-dibenzyloxy-9,10-anthraquinone-3-carboxylic acid ester derivatives（5a-d）,1,8-dibenzyloxy-9,10-anthraquinone-3-carboxylic acid alkyl ester derivatives（6a-g）and 1,8-dibenzyloxy-9,10-anthraquinone-N-[6-amine-（4-anilino）-quinazoline]-3-carboxamide derivatives（7a-j）,respectively.（2）The results of MTT and CCK-8 showed that the half inhibitory concentration of rhein IC₅₀>100μM.IC₅₀of 4a-b and 5a-d were lower than that of rhein,and 6a-g were similar to that of the lead compound rhein,after SMMC-7721,Hep G2,CNE-1 and CNE-2 cells were treated with different compounds for48h.The half inhibitory concentrations of all 10 new 1,8-dibenzyloxy-9,10-anthraquinone-N-[6-amine-（4-anilino）-quinazoline]-3-carboxamide derivatives inhibited CNE-1 cells were lower than that of rhein,only compound 7a-c,7f,and7j were lower than that of rhein for A549 cells.The IC₅₀ values of compound 4a was determined by CCK-8 assay in human nasopharyngeal carcinoma cells CNE-1,CNE-2 and human hepatocellular carcinoma Hep G2 and SMMC-7721 cells for48h were 4.7±1.2μM,2.1±0.2μM,3.2±0.9μM and 1.5±0.3μM,respectively.（3）Inverted light microscopy and HE staining showed that a large number of vacuoles appeared in the cytoplasm,and there was no cell with proliferation,after SMMC-7721 cells were treated with 4a（3μM）for 48h.Cellular vacuoles increased in size and number in a time-dependent manner eventually,the whole cytoplasm is filled with vacuoles;at the same time,the cell contents was lost and the nucleus was concentrated.（4）After SMMC-7721 cells was treated with 4a（3,5μM）48h,under laser confocal microscopy using ER Tracker and Hoechst33342 staining,vacuoles were observed at the perinuclear endoplasmic reticulum region and in the cytosol.With the concentration of 4a increased,the number of vacuoles gradually increased.The SMMC-7721 cells treated with 4a（5μM）after 48h by transmission electron microscopy were observed that the cytoplasm was filled with vacuoles of different sizes,the endoplasmic reticulum,mitochondria,and Golgi were swollen,vacuoles originated from the swelling and expansion of the endoplasmic reticulum.In addition,we also found that the nucleus was concentrated,the cell membrane remains intact and no apoptotic bodies are found.The characteristics of cell death induced by 4a included cytoplasmic vacuolation,induction of ER stress,fulfilling the criteria for paraptosis.（5）The results of apoptosis and cycle detected by flow cytometry showed that there was no apoptotic cells at all in 4a-induced vacuolization-like cells and there is no interfering effect on the cell cycle in concentration-dependent.（6）Compared with control,Western blotting immunoblot results showed that4a could upregulate endoplasmic reticulum stress protein GRP78,autophagy protein p62 and enhance the ratio of LC3-II/I after the SMMC-7721 cells treated with 4a（3μM）for 24h;but apoptotic protein cleaved-caspase-3 didn’t change compared with cisplatin.（7）After the same concentration（10μM）of rhein,1,8-dibenzyloxyrhein,4a and cisplatin treated with SMMC-7721 cells,the results showed that rhein has no effect on the expression of GRP78,p62 and the ratio of LC3-II/I;1,8-dibenzyloxyrhein could up-regulate the expression of GRP78 and p62,but had no effect on the ratio of LC3-II/I;cisplatin could activate apoptotic protein cleaved-caspase-3,and highly down-regulate the expression of GRP78 protein,and has no effect on the expression of p62 and the ratio of LC3-II/I.cleaved-caspase-3 was not found during the 4a-induced vacuolization-like cell death of SMMC-7721cells,but 4a could upregulate the ER stress protein GRP78 and increase the ratio of LC3-II/I and the expression level of p62.（8）We selected 7a,7b,7c,and 7f,their half-inhibition concentration lower than rhein,tested their effects on EGFR and RAC1 protein expression in A549cells.The results showed that 7a and 7c can significantly downregulate the expression of EGFR protein,which is significantly superior to the positive control drug Gefitinib,7c downregulated EGFR protein,but also upregulated RAC1protein expression,compared with the control.Conclusion:（1）We successfully designed and synthesized series of novel rhein derivatives with a certain targeting effect using pharmacophore assembly techniques.（2）The novel compound 1,8-dibenzyloxy-9,10-anthraquinone-N-（2-hydroxyethyl）-3-carboxamide（4a）is not accompanied by any other death mechanism occurs in the process of inducing paraptosis.（3）During the study of the structure-activity relationship and mechanism,the increase of LC3-II/I ratio can promote cytoplasmic vacuolization and accelerate the progression of paraptosis.（4）The growth inhibitory effect of new compound 1,8-dibenzyloxy-9,10-anthraquinone-N-[6-amine-4-（4-methylanilino）-quinazoline]-3-carboxamide（7f）on CNE1 cells was significantly superior to that of A549 cells.New compound 1,8-dibenzyloxy-9,10-anthraquinone-N-[6-amine-4-（3-methoxyanilino）-quinazoline]-3-carboxamide（7c）can both downregulate EGFR expression and upregulate RAC1 protein expression in the A549 cell radiosensitization pathway.