Design,Synthesis And Antitumor Activity Evaluation Of Glycoconjugates Derived From Natural Product Harmine

Harmine is a tricyclicβ-carboline alkaloid that can be extracted from the seeds of Peganum harmala L,which can exert antitumor effects through various mechanisms such as inducing cancer cells apoptosis,altering cell cycle distribution,and inhibiting epithelial-mesenchymal transition in cancer cells.However,due to the poor selectivity for cancer cells and low bioavailability of Harmine,its clinical application is limited and further optimization is needed.Glucose transporter 1（GLUT1）is the main channel for cell uptake of glucose,overexpressed on the surface of cancer cells.2-Amino-2-deoxy-D-glucose（2-DG）can be recognized by GLUT1,and its phosphorylated metabolites cannot further participate in glycolysis to provide energy for cancer cells,which is an effective tool for antitumor drug modification.Therefore,this project envisages conjugating Harmine with 2-DG to improve its selectivity and druggability.It has been reported that the C7,N9 and N2 of Harmine are the main positions of structural modification.In this project,a carboxyl group was introduced at the Harmine N9 position through a straight-chain alkane and coupled with methyl 2-amino-2-deoxy-β-D-glucoside.First,the glycosyl is conjugated by short straight-chain alkanes on Harmine position N9.The effects of substituents on the activity of conjugated compounds at C7 oxygen in Harmine were investigated,and the best groups of C7 were selected from methyl,isopentyl,cyclohexylmethyl and benzyl groups.Subsequently,the group of C7 was fixed,and the structure-activity relationship of the length of linker（1～9 straight-chain alkanes）was systematically investigated to determine the optimal linker.Finally,N2-alkylation was performed on the optimized conjugate in the above two steps to further enhance its antitumor activity.Following the above ideas,sixteen glycoconjugates of Harmine were synthesized in three steps,and their structures were confirmed by ¹H NMR,¹³C NMR and HRMS.In vitro anti-tumor activity screening and structure-activity relationship studies showed that cyclohexylmethyl is the optimal modification group on the oxygen atom of Harmine C7.The antitumor activity of the conjugates increased with the lengthening of the alkyl chain in the linker at N9.However,introducing chemical groups at N2 of Harmine did not enhance the antitumor activity.Compound 24c-9,which is modified with cyclohexylmethoxy at C7 and the longest linker at N9 showed the best antitumor activity.It exhibited significantly better proliferation inhibition activity against MDA-MB-231 breast cancer cells(IC₅₀=12.79μM)than Harmine(IC₅₀=28.30μM).As compared to Harmine,the introduction of 2-DG improved the water solubility of 24c-9,it was 4.5 times that of Harmine.Meanwhile,2-DG enhanced the cancer cell selectivity of 24c-9 through the Warburg effect,after treatment with 20μM of 24c-9,the survival rate of normal cells MCF-10A was 6.1 times higher than that of MDA-MB-231cells.Flow cytometry study revealed that 24c-9 induced apoptosis and G0/G1 cell cycle arrest in MDA-MB-231 cells.Western blot and would healing assays showed that 24c-9 could affect the expression of key proteins during epithelial-mesenchymal transformation and inhibit the migration of cancer cells.MMP-3 protein is closely related to EMT,migration and invasion of cancer cells.Protein simulation docking further suggested that the binding ability of the Harmine fragment in conjugate 24c-9 to the active site of MMP-3 might be one of the key factors in its inhibition of EMT and migration of cancer cells.In summary,this study designed N9 glycosyl conjugates of Harmine using the Warburg effect.By means of medicinal chemistry strategies,the C7,N9,and N2 positions of Harmine in the conjugate were modified and the structure-activity relationship was studied.Compound24c-9 with the best anti-tumor activity was screened out,and its activity on breast cancer cells was better than that of Harmine.Due to the introduction of the 2-DG moiety,24c-9 showed a higher selectivity toward tumor cells.This study provides a new approach for the development of Harmine-based antitumor drugs.