Design,Synthesis And Mechanisms Of Action Of Novel Harmine Derivatives As Potential Antitumor Agents

Harmine is a natural derived?-carboline alkaloid showing diverse pharmacological activities.Recent studies are focused on its anticancer activity.Here,31 novel harmine derivatives were designed and synthesized from harmine based on its structure-activity relationship?SAR?analysis including 23 novel N⁹-substituted?3,5a⁵n,8a⁸h?and eight N^2,9-disubstituted?10a¹0h?harmine derivatives.The chemical structures of all the compounds were characterized by mass spectroscopy and ¹H and ¹³C NMR spectroscopy.The CCK-8 assay was applied to evaluate the effects of all the compounds on proliferation of A549 lung adenocarcinoma,MDA-MB-231 triple-negative breast carcinoma,PANC-1 pancreatic cancer,and U2OS osteosarcoma cancer cells.The results showed that compounds 7,8g,10d,10e,and 10f exhibited strong inhibitory effects on the proliferation of the four cancer cell lines.Among them,compound 10f elicited the strongest cytotoxic effect against carcinoma cell lines.Preliminary SAR analysis revealed that introducing a benzyl group at the N² position for some N⁹-substituted harmine derivatives will dramatically increase their anticancer activities.The acyloxy substitution was better than acylamino substitution at the N⁹ position for N^2,9-disubstituted harmine derivatives.We determined the IC₅₀0 of 10f in A549 and MDA-MB-231 cancer cells to be³.2and 4.5 mM,respectively.On the other hand,10f showed only minor effect on the proliferation of a normal lung fibroblast HLF-?,suggesting that this compound could selectively inhibit cancer cell proliferation.Cell cycle analysis suggested that 10f only caused a moderate S phase accumulation of A549 cells after 24 hour treatment.Meanwhile,by determining the sub-G1 population that often indicates dead cells in flow cytometry;we found that 10f caused a significant increase in cell death.The cell death-inducing effect of 10f was already evident at a concentration as low as 100 nM after treatment of 24 h.To the best of our knowledge,100 nM is among the lowest concentrations of harmine analogs reported so far that induce significant cancer cell death.Anticancer mechanism of action?MOA?study suggested that harmine analogs failed to induce the DNA damage response as previously reported in in vitro studies.Our studies demonstrated that compound 10f could activate apoptosis while suppressing autophagy,leading to death of cancer cells.Since 10f contains an ester modification,a potential issue in future drug development is whether this compound is stable inside the cells.Our HPLC analysis showed that 10f is stable in vitro.Also,to determine its stability in cells,we assessed the concentration of 10f in cells by UPLC-Q-TOF-MS.Our study revealed that 10f is also stable in vivo.These results revealed previously unidentified insights into the anticancer effect of harmine derivatives,providing conceptual advance in future application of this compound class in the treatment of human cancers.