Design, synthesis and characterization of new molecules which counteract drug resistance mechanisms

Multidrug Resistance (MDR) is a type of acquired resistance that cancer cells develop against structurally and mechanistically unrelated drugs to which they are initially sensitive. MDR is mainly due to the overexpression of proteins, as P-gp, MRP1 and BCRP, that work as efflux pumps, reducing the intracellular concentration of drugs below their active dose. P-gp is the most studied transporter, overexpressed in many blood and solid tumors. BCRP is often overexpressed in solid tumors and leukemia, together with P-gp. A possible approach to overcome MDR is to co-administer efflux pump inhibitors with anticancer drugs, to increase drugs’ intracellular concentration and restore their therapeutic effects. Interestingly, on the membrane of several resistant cancer cells, P-gp is co-localized and physically associated to the isoform XII of human carbonic anhydrase (hCA XII). Moreover, the pharmacological inhibition of hCA XII reduced the ATPase efflux activity of P-gp. So, compounds with dual P-gp/hCA XII inhibitory effects could be useful to target resistant cancer cells that overexpress both proteins. The aim of my PhD project was to design and synthesize new compounds able to reverse MDR in cancer cells. This PhD thesis consists in two main projects: 1. The first part focused on compounds with dual P-gp/hCA XII inhibitory effects. To maintain a high potency on P-gp and introduce a selective activity towards hCA XII, we designed hybrid inhibitors characterized by both P-gp and hCA XII binding moieties. In these three series of compounds, we introduced on the structure of our potent P-gp inhibitors, as the N,N-bis(alkanol)amine aryl diesters and the methoxy-substituted arylpiperazine scaffolds, two specific residues, the benzene sulfonamide or the coumarin moieties, to target hCA XII. As regards the activity on P-gp, all these molecules were able to enhance the intracellular accumulation of two P-gp substrates, Rhodamine-123 and Doxorubicin, in K562/DOX cells that overexpress only P-gp. Moreover, coumarin derivatives were selective inhibitors of the tumor-associated hCA IX and hCA XII isoforms. Interestingly, most of our compounds displayed the highest MDR reverser effects on the tested resistant cell lines (LoVo/DOX, HT29/DOX and A549/DOX), that overexpress both proteins, showing an interesting synergistic effect. 2. The second main project of this thesis is based on the design and synthesis of MDR reversers active as ABC modulators: Tariquidar analogues and quinazoline derivatives. The Tariquidar analogues derive structurally from two potent P-gp inhibitors, Tariquidar and Elacridar. First, we designed and synthesized a series of compounds, bearing the 6,7-dimethoxy-2-phenethyl-1,2,3,4-tetrahydroisoquinoline nucleus present in the lead compounds, linked to an aryl-substituted amide or ester group. Then, we modified the amide function, by introducing two bioisosteric heterocycles, the tetrazole and the oxadiazole ones, linked to methoxy-substituted aryl groups. Notably, we designed and synthesized both the 1,5- and the 2,5-disubstituted tetrazoles, and the 2,5-disubstituted-1,3,4-oxadiazoles. Otherwise, quinazoline derivatives maintain the quinazoline-4-amine scaffold of two tyrosine kinase inhibitors (TKIs), Gefitinib and Erlotinib, that have been identified as ABC transporters modulators. In this series of quinazoline derivatives, we introduced secondary or tertiary protonable amines in position 4 of the quinazoline scaffold, while in position 2 we inserted aromatic groups, such as anthracene or methoxy-substituted aryl moieties. All these MDR modulators 74-152 were studied on three different transfected cell lines (MDCK-MDR1, MDCK-MRP1 and MDCK-BCRP that overexpress P-gp, MRP1 and BCRP, respectively) to evaluate their activity on these ABC proteins, by measuring the inhibition of the transport of two fluorescent probes. In general, all these derivatives showed high inhibitory effects on P-gp: the most potent P-gp modulators were further studied in association with Doxorubicin on resistant cancer cells (MDCK-MDR1, HT29/DOX) that overexpressed P-gp. Moreover, some compounds also displayed a good or moderate activity on the other two transporters MRP1 and BCRP. Moreover, during my PhD thesis, I also performed a series of chemical stability tests on derivatives bearing liable ester groups: these experiments were carried out to evaluate the susceptibility of our ester molecules towards spontaneous and enzymatic hydrolysis. Stability analyses were performed by liquid chromatography coupled with mass spectrometry (LC-MS/MS) methods. First, I studied the chemical stability of the previously synthesized N,N-bis(alkanol)amine aryl diesters active as P-gp inhibitors; then, I performed these stability tests on all the other ester derivatives synthesized in this PhD project. Concerning the chiral P-gp inhibitors, we also developed a valid method to evaluate the enantiomeric excess of (R) and (S) enantiomers by enantioselective liquid chromatography coupled with diode array detector (LC-DAD) analysis.