New pyrrole derivatives for the treatment of glioblastoma and chronic myeloid leukemia

Background Glioblastoma multiforme (GBM), is one of the most aggressive malignant brain tumors in humans. Long- term survivors of glioblastoma multiforme are at high risk of developing second primary neoplasms, including leukemia. Chronic myeloid leukemia (CML) and GBM patients are both treated with classic tyrosine kinase inhibitors (TKIs), such as imatinib mesylate. The use of imatinib mesylate, is strongly discouraged, since this treatment causes a tremendous increase of tumor and stem cell migration and invasion. Aims We aimed to develop agents useful for the treatment of patients with GBM and CML using an alternative mechanism of action from the TKIs, specifically based on the inhibition of tubulin polymerization. 3-Aroyl- 1,4-diarylpyrrole RS5156 (1) is a potent inhibitor of tubulin polymerization and cancer cell growth by binding the colchicine site of tubulin. The aim of the present research project is that to fully evaluate the potential of ARDAP derivatives in treatment of CML.1 Methods The study will be structured as follows:1. design of the new compounds and molecular modelling studies; 2. chemical synthesis of the most promising compounds; 3. biological evaluation of the newly prepared compounds as inhibitors of tubulin polymerization and [3H]colchicine binding; 4. biological evaluation of the most active compounds as inhibitors of TKI-sensitive or TKI-resistant CML cell lines as single agent or in combination with other TKIs, using normal blood cells as control; 5. further biological characterization of the selected compounds in leukemia cells in terms of cell morphological features, cell cycle distribution, apoptosis study, caspase-3 activity; 6. in vitro and in vivo pharmacokinetic characterization of the selected compounds. Results Starting from the ARDAP derivatives, characterized by the presence of one or two aminophenyl groups at position 1 and 4, we aimed to improve multiple parameters, such as potency, metabolic stability and blood- brain barrier (BBB) permeability. We thus replaced the 4-aminophenyl group with five- and six-membered bioisosteric heterocycles, while retaining the important methyl and fluoro substituents on the 1-phenyl ring.1 We synthesized compounds 2-25 which were shown to inhibit tubulin polymerization with IC50 values at submicromolar concentrations, as compared with combretastatin A-4 (CSA4, IC50 = 0.54-0.73 μM range), except for derivative 16. In particular, compounds 7 and 14 were found to be more potent inhibitors of tubulin polymerization, with IC50 values of 0.39 and 0.38 mM, respectively. Two human glioblastoma cell lines, U343MG and U87MG, were select to evaluate the potential of two of 7 and 25. The IC50 values obtained highlighted as the presence of the thiophen-3-yl ring at position 4 of the pyrole is important for inhibition of U343MG cells. Compounds 7 and 25 were thus compared with the TKIs imatinib (IM) and third-generation ponatinib (PN) in the Bcr-Abl-expressing KU812 cells, in the IM-sensitive KBM5 cells and in the IM-resistant KBM5-T315I cells expressing the threonine for isoleucine mutation at position 315 of the oncoprotein. The IC50 values of both compounds were at least one order of magnitude superior to imatinib. Furthermore, the combination of 7 with PN, in the KBM5-T315I cells yielded 70% apoptotic cells, as compared with 35% for PN alone. Finally, experiments on human topoisomerases I and II confirmed that 7 and 25 were able to block human topoisomerase II selectively and completely, at a concentration of 100 μM, but they were inactive against hTopoI. Taking into account these very promising data the activity of ARDAP 7 against the U87MG human glioma cell line was also tested in vivo, where significantly inhibited GBM cancer cell proliferation, tumorigenesis and tumor angiogenesis. In fact, the comparison of tumors formed on the backs of nude mice shown as the weight and volume of tumors following treatment with 7 for 35 days were significantly reduced as compared with those following treatment with saline. Conclusion Compared to the previously reported lead compound, 7 showed stronger inhibition of tubulin polymerization and cancer cell growth and had a calculated improvement of metabolic stability and higher lipophilicity. These results allowed us to confirm 7 has potential as a dual-target antitumor agent for the treatment of both GBM and CML. References 1. Puxeddu, M.; Shen, H.; Bai, R.; Coluccia, A.; Bufano, M.; Nalli, M.; Sebastiani, J. et al. Discovery of Pyrrole Derivatives for the Treatment of Glioblastoma and Chronic Myeloid Leukemia. Eur. J. Med. Chem. 2021, accepted.