Semisynthesis, cytotoxicity, antiviral activity, and drug interaction liability of 7-O-methylated analogues of flavonolignans from milk thistle

Silymarin, an extract of the seeds of milk thistle (Silybum marianum), is used as an herbal remedy, particularly for hepatoprotection. The main chemical constituents in silymarin are seven flavonolignans. Recent studies explored the non-selective methylation of one flavonolignan, silybin B, and then tested those analogues for cytotoxicity and inhibition of both cytochrome P450 (CYP) 2C9 activity in human liver microsomes and hepatitis C virus infection in a human hepatoma (Huh7.5.1) cell line. In general, enhanced bioactivity was observed with the analogues. To further probe the biological consequences of methylation of the seven major flavonolignans, a series of 7-O-methylflavonolignans were generated. Optimization of the reaction conditions permitted selective methylation at the phenol in the 7-position in the presence of each metabolite's 4-5 other phenolic and/or alcoholic positions without the use of protecting groups. These 7-O-methylated analogues, in parallel with the corresponding parent compounds, were evaluated for cytotoxicity against Huh7.5.1 cells; in all cases the monomethylated analogues were more cytotoxic than the parent compounds. Moreover, parent compounds that were relatively non-toxic and inactive or weak inhibitors of hepatitis C virus infection had enhanced cytotoxicity and anti-HCV activity upon 7-O-methylation. Also, the compounds were tested for inhibition of major drug metabolizing enzymes (CYP2C9, CYP3A4/5, UDP-glucuronsyltransferases) in pooled human liver or intestinal microsomes. Methylation of flavonolignans differentially modified inhibitory potency, with compounds demonstrating both increased and decreased potency depending upon the compound tested and the enzyme system investigated. In total, these data indicated that monomethylation modulates the cytotoxic, antiviral, and drug interaction potential of silymarin flavonolignans.