Methoxylation of 3′,4′-aromatic side chains improves P-glycoprotein inhibitory and multidrug resistance reversal activities of 7,8-pyranocoumarin against cancer cells

The overexpression of P-glycoprotein (Pgp), an ATP-driven membrane exporter of hydrophobic xenobiotics, is one of the major causes of multidrug resistance (MDR) in cancer cells. Through extensive screening we have found that the extracts of Peucedanum praeruptorum Dunn. and one of the major components (±)-praeruptorin A (PA) may reverse Pgp-mediated multidrug resistance. Studies on novel PA derivatives have shown that (±)-3′-O,4′-O-dicinnamoyl-cis-khellactone (DCK) is more active than PA or verapamil and is a non-competitive inhibitor of Pgp. Here, we report that methoxylation of the cinnamoyl groups on DCK may further enhance its bioactivity. The structure-activity relationship is demonstrated by comparing two new pyranocoumarins (±)-3′-O,4′-O-bis(3,4-dimethoxycinnamoyl)-cis-khellactone (DMDCK) and (±)-3′-O,4′-O-bis(4-methoxycinnamoyl)-cis-khellactone (MMDCK). While the co-existence of 3- and 4-methoxy groups on cinnamoyl remarkably enhanced the Pgp-inhibitory activity, the lone existence of the 4-methoxy group on cinnamoyl reduced the activity. Contrary to DCK, DMDCK promoted the binding of UIC2 antibody to Pgp which signifies a conformational change of Pgp similar to that induced by transport substrates. While DCK moderately stimulated the basal Pgp-ATPase activity, DMDCK inhibited the activity. A pharmacophore search with verapamil-based template revealed that four functional groups of DMDCK could be simultaneously involved in interaction with Pgp whereas for DCK or MMDCK only three groups were involved. It is speculated that the additional 3-methoxy group on cinnamoyl allows DMDCK to interact more efficiently with Pgp substrate site(s). If DMDCK was tightly bind to Pgp substrate site(s) the complexes could be inactive with regard to transportation and ATP hydrolysis could also be inhibited.