A new series of Pgp-dependent MDR inhibitors having a N,N-bis(cyclohexanol)amine scaffold was designed on the basis of the frozen analogue approach. The scaffold chosen gives origin to different geometrical isomers. The new compounds showed a wide range of potencies and efficacies on doxorubicin-resistant erythroleukemia K562 cells in the pirarubicin uptake assay. The most interesting compounds (isomers of 3) were studied further evaluating their action on the ATPase activity present in rat small intestine membrane vesicles and doxorubicin cytotoxicity potentiation on K562 cells. The latter assay was performed also on the isomers of 4. The four isomers of each set present different behavior in each of these tests. Compound 3d shows the most promising properties as it was able to completely reverse Pgp-dependent pirarubicin extrusion at low nanomolar concentration, inhibited ATPase activity at 5 x 10(-9) and increased the cytotoxicity of doxorubicin with a reversal fold (RF) of 36.4 at 3 microM concentration.

The overexpression of permeability-glycoprotein (P-gp) and other drug transporters (ATP-binding cassette) confers a multidrug resistance (MDR) phenotype on cells in various diseases, including many forms of cancer. Development of MDR is one of the main reasons of failure in malignant tumour chemotherapy, as tumour cells, by increasing drug efflux, acquire cross-resistance to many structurally and functionally unrelated anticancer agents, which therefore never achieve effective intracellular concentrations. Endeavouring to find MDR-reverters is a crucial task for exploring new anti-cancer therapeutic intervention. Although many P-gp inhibitors have so far been identified, it is widely recognised that their interaction with P-gp is a complex process and, presently, the details of the mechanisms of action are still a matter of debate. These compounds turned out, however, to be of limited clinical usefulness owing to their inherent pharmacological activities (first generation compounds) and their accessory, inhibiting activity on CYP enzyme system (second generation compounds). Moreover, recent advances of the knowledge on P-gp structure and function and on the mechanisms of P-gp inhibition will prove fruitful for the development of novel therapeutically effective P-gp inhibitors. A dibenzoyl-1,4-dihydropyridine compound (DP7) has been shown to be a powerful P-gp inhibitor, almost devoid of cardiovascular effects, but capable of inhibiting liver CYP3A. DP7 is considered a lead compound for the development of novel dihydropyridines which do not affect CYP enzyme system but still retain the activity towards ABC-efflux transporters.