Purines and 9-deazapurines as Modulators of Multidrug Resistance-associated Protein 1 (MRP1/ABCC1)-mediated Transport

Abstract

Expression of ABC-transport proteins is a major obstacle in cancer chemotherapy. Especially MRP1, P-gp and BCRP are expressed in different malignancies like lung cancer, colon carcinomas, leukemias, bladder cancer, myeloma, ovarian carcinomas or breast cancer, often simultaneously. These key proteins extrude a variety of structurally diverse antineoplastic agents out of cells, which results in the phenomenon called multidrug resistance (MDR). Many compounds of different origin were found to inhibit different ABC-transporters, e.g. pharmacological drugs, natural compounds, intrinsic substrates and synthetic compounds derived from screening of huge compound libraries. But most of these inhibitors lacked the necessary potency and selectivity, and often led to severe side effects in clinical evaluations. Especially with respect to MRP1, only rare reports have been made for compounds in the submicromolar concentration range. Hence, the development of new compounds is desirable to obtain more potent inhibitors for this transport protein.<br /> The aim of the thesis was to develop new, potent, nontoxic and selective inhibitors of MRP1, based on earlier reports of pyrrolo- and indolopyrimidines. The compounds have been evaluated in two different assays, the calcein AM and daunorubicin assay.<br /> The substituents of the starting compound 74 were varied to elucidate the preferences with regard to MRP1 inhibition. It was found that the phenethylpiperazine side chain at position 6 was superior in comparison to shortened alkyl linker or abolishing of the piperazine partial structure. Additionally, alkyl, aryl or arylalkyl substituents could be introduced at position 7 leading to potent compounds in submicromolar range. Compound 92 with its cyclopropyl residue was shown to be the best inhibitor of the newly synthesized compounds. It was equally potent in comparison to the standard inhibitor and starting compound 74. But in contrast to the latter, the former had no inhibitory activity against P-gp and BCRP, which makes this compound a very selective inhibitor of MRP1. Compound 92 was able to reduce MDR of H69AR cells by half in submicromolar concentration range, reversed MRP1-mediated MDR completely at 10 µmol ∙ L-1 and possessed no noteworthy toxicity at this concentration. Compound 92 is only six times less effective than the best known inhibitor of MRP1, and in contrast to the latter, it is absolutely selective, which makes it a great aspirant for clinical evaluation in cancer cell lines that overexpress MRP1.<br /> Purine analogs of 9-deazapurines showed only inhibitory power in upper double-digit concentration range, but were able to stimulate MRP1-mediated transport. This was found for calcein AM and daunorubicin in two different MRP1 expressing cell lines, H69AR and MDCK II MRP1. Several 9-deazapurines also stimulated MRP1-mediated transport, although long side chains at position 6 also gave compounds with moderate to good inhibitory power. These compounds gave biphasic concentration-effect curves. Most of the compounds were selective inhibitors of MRP1, only compound 104 affected all three transporters in an inhibitory way. It was the first herein reported triple inhibitor, although the inhibitory power toward P-gp and BCRP was rather low. The purine and 9-deazapurine activators were biologically evaluated with respect to their half-maximal activating concentration and activation ratio, which could only be observed with respect to MRP1. Analyzation of the type of activation of compound 101 as representative of the MRP1 activators showed that these compounds are nonessential activators, most likely “mixed-type” according to Segel’s “Enzyme Kinetics”. Although the compounds could reduce the intracellular concentration of the cytotoxic agent daunorubicin in the daunorubicin assay, this activating power could not be transferred to the MDR reversal-efficacy assay. Except for compound 110, none of the compounds enhanced MDR, on the contrary compounds with good inhibitory power rather reversed resistance in MRP1 overexpressing cancer cell lines (e.g. 104).<br /> Since pyrrolo- und indolopyrimidines were shown before to inhibit P-gp, but also BCRP, some 9-deazapurines were evaluated regarding their capability to inhibit all three transporters reported in this thesis. Besides some dual inhibitors, compound 125 was found as the best triple inhibitor ever reported until now. It was able to inhibit MRP1 in submicromolar range and P-gp as well as BCRP at low micromolar concentrations. It is a noncompetitive inhibitor of daunorubicin, calcein AM and pheophorbide A transport mediated by MRP1, P-gp and BCRP, respectively. Finally, the compound could reduce MDR in several cancer cell lines. The effectiveness of this MDR reversal was in submicromolar range, comparable to the data obtained for the standard inhibitor 74 and the best compound of this thesis, 92. This makes compound 125 also a great aspirant for clinical evaluation for use in cancers with multiple expression of ABC-transport proteins.<br /> Finally, the H69AR lung cancer cell line could be established as test system and compounds 74 and 126 were established as standard inhibitors for the calcein AM and daunorubicin assay, superseding the former standard inhibitors indomethacin, cyclosporine A, MK571 and ONO-1078, which all lacked either the necessary selectivity, potency or reliability.<br /> The results of this thesis give new insights in the field of MRP1-mediated MDR and the compound classes of purines and 9-deazapurines. These contain compounds that inhibit MRP1 with great potency and selectivity, which has rarely been reported. But also broad-spectrum inhibitors have been found, which is a good starting point for clinical evaluation in oncology. Finally, the activating property of several representatives might be a good tool for further experiments to elucidate the mechanistic aspects of ABC-transport proteins.