Rhodamine inhibitors of P-glycoprotein: an amide/thioamide "switch" for ATPase activity.

We have examined 46 tetramethylrosamine/rhodamine derivatives with structural diversity in the heteroatom of the xanthylium core, the amino substituents of the 3- and 6-positions, and the alkyl, aryl, or heteroaryl group at the 9-substituent. These compounds were examined for affinity and ATPase stimulation in isolated MDR3 CL P-gp and human P-gp-His(10), for their ability to promote uptake of calcein AM and vinblastine in multidrug-resistant MDCKII-MDR1 cells, and for transport in monolayers of MDCKII-MDR1 cells. Thioamide 31-S gave K(M) of 0.

Substituent control of DNA binding modes in a series of chalcogenoxanthylium photosensitizers as determined by isothermal titration calorimetry and topoisomerase I DNA unwinding assay.

The DNA binding efficacy and preferred mode of binding of a series of rhodamine-related chalcogenoxanthylium dyes was investigated by isothermal titration calorimetry (ITC) using ctDNA, [poly(dCdG)](2) and [poly(dAdT)](2), and by a topoisomerase I DNA unwinding (Topo I) assay. The dyes of this study showed tight binding to ctDNA with binding constants, K(b), on the order of 10(6)-10(7)M(-1). The ITC and Topo I assay studies suggested that the 9-substituent has a strong impact on binding modes ranging from an apparent preference for intercalation with a 9-2-thienyl substituent (similar binding to [poly(dCdG)](2) and [poly(dAdT)](2), re-supercoiling of DNA in the Topo I assay at <10(-5)M dye), to mixed binding modes with 9-phenyl derivatives (2- to 3-fold preference for binding to [poly(dAdT)](2), re-supercoiling of DNA in the Topo I assay at approximately 2 x 10(-5)M dye), to minor groove binding in a 9-(2-thienyl-5-diethylcarboxamide) derivative (strong preference for binding to [poly(dAdT)](2), did not show complete re-supercoiling in the Topo I assay).

ATP occlusion by P-glycoprotein as a surrogate measure for drug coupling.

The multidrug efflux pump P-glycoprotein (Pgp) couples drug transport to ATP hydrolysis. Previously, using a synthetic library of tetramethylrosamine ( TMR) analogues, we observed significant variation in ATPase stimulation ( V m (D)). Concentrations required for half-maximal ATPase stimulation ( K m (D)) correlated with ATP hydrolysis transition-state stabilization and ATP occlusion (EC 50 (D)) at a single site.

"Switched-on" flexible chalcogenopyrylium photosensitizers. Changes in photophysical properties upon binding to DNA.

2,4-Bis(4-dimethylaminophenyl)-6-alkylthiopyrylium and selenopyrylium dyes are essentially nonfluorescent (phi F < 0.001) and are poor generators of singlet oxygen in aqueous solution. However, upon complexation to calf thymus DNA, quantum yields for both fluorescence and generation of singlet oxygen increased dramatically.

A microwave-assisted synthesis of julolidine-9-carboxamide derivatives and their conversion to chalcogenoxanthones via directed metalation.

9-formyljulolidine was oxidized via a microwave-assisted Willgerodt-Kindler reaction to the N-piperidine or N-morpholine julolidine-9-thioamide. 9-formyl-1,1,7,7-tetramethyljulolidine gave the corresponding N-piperidine tetramethyljulolidine-9-thioamide. The thioamides were converted to the corresponding carboxamides with trifluoroacetic anhydride.

Generation of 3- and 5-lithiothiophene-2-carboxylates via metal-halogen exchange and their addition reactions to chalcogenoxanthones.

Deprotonation and lithium-bromine exchange in 5- or 3-bromothiophene-2-carboxylic acids with t-BuLi form the corresponding dianion, which reacts highly regioselectively in the presence of 0.25 equiv of tetramethyl-1,2-ethylenediamine with 3,6-bis(dimethylamino) chalcogenoxanthones to give S- and Se-containing rhodamines. Quenching studies with D2O indicate that an extra equivalent of t-BuLi is not necessary in these reactions.

Stimulation of P-glycoprotein ATPase by analogues of tetramethylrosamine: coupling of drug binding at the "R" site to the ATP hydrolysis transition state.

The multidrug resistance efflux pump P-glycoprotein (Pgp) couples drug export to ATP binding and hydrolysis. Details regarding drug trajectory, as well as the molecular basis for coupling, remain unknown. Nearly all drugs exported by Pgp have been assayed for competitive behavior with rhodamine123 transport at a canonical "R" drug binding site.