SAR And Structure Optimization Of STAT3 Signaling Inhibitor Angoline Targeting Cancer (Stem) Cells

ObjectiveLead compound Angoline is a select STAT3 pathway inhibitor isolated from Zanthoxylum nitidum. In the present research, a series of Angoline derivatives were designed, synthesized, and evaluated for their inhibitor activities against human liver cancer cells, and study the structure-activity relationship; And then, the effect of Angoline on self-renewal of human liver cancer stem cells (HLCSCs) were test and primary mechanisms of Angoline on HLCSCs apoptosis were clarified. Last, the mode of Angoline bingding to STAT3 was further elucidated.Methods①Silica gel column chromatography techniques was used to isolate the chelerythrine from the MeOH extract of rhizome from Zanthoxylum nitidum. ②based on chelerythrine, a series of Angoline derivatives were synthesized by Nucleophilic addition and redox reaction. ③NMR and MS spectroscopy data were used to characterized the chemical structures; ④MTT assay was used to detect the inhibitory effect of series derivatives on human cancer cells; ⑤Angoline induced apoptosis and cell cycle in human liver cancer stem cells were test by flow cytometry; ⑥Molecular docking technology was used to clarify the molecular mode of action of Angoline binding to STAT3 protein.Results①200mg of chelerythrine was isolated from the MeOH extract of rhizome fromZanthoxylum nitidum and its yield was 4% approximately. ②Twelve Angoline derivatives were designed, synthesized and identified in this research. Among them, four better compounds like DY30105 (IC50=0.45μM), DY20101 (IC50=0.55μM), nitidine chloride (IC50=5.44μM) and sanguinarine (IC50=6.80μM) we obtained, but their activities were weaker than Angoline; ③SAR studies demonstrated that Methoxy was the key pharmacophore and Methoxy at the C-8 and C-9 position played an important role on inhibitory effects toward human liver cancer cell, the introduction of the alkyl and niteogen heterocyclic significantly weaken the inhibitory efficiency on human liver cancer cells; ④Angoline induced apoptosis of HLCSCs in a dose-pependent manner and blocked cell cycle of HLCSCs at phase S; ⑤Molecular docking suggested Angoline formed arene-H bonds with the GIn635, Lys591 IIe634 residues and arene-cation with ionized Arg595 residue of the SH2 domain of STAT3.ConclusionIn this paper, SAR and structural optimization of STAT3 pathway inhibitor Angoline have been study. Methoxy at the C-8 and C-9 position were key pharmocophores and obtained four good activity small-molecule STAT3 signaling inhibitor, like DY30105, DY20101, nitidine chloride and sanguinarine, but their activities weaker than Angoline. Angoline induced apoptosis of HLCSCs in a dose-pependent manner and blocked cell cycle of HLCSCs at phase S. Molecular docking suggested Angoline formed arene-H bonds with the GIn635, Lys591 IIe634 residues and arene-cation with ionized Arg595 residue of the SH2 domain of STAT3.The results of this paper provide the scientific basis for further development of molecularly targeted anticancer agents.