Triterpenoid pyrazines and pyridines - Synthesis, cytotoxicity, mechanism of action, preparation of prodrugs

Abstract

A set of fifteen triterpenoid pyrazines and pyridines was prepared from parent triterpenoid 3-oxoderivatives (betulonic acid, dihydrobetulonic acid, oleanonic acid, moronic acid, ursonic acid, heterobetulonic acid, and allobetulone). Cytotoxicity of all compounds was tested in eight cancer and two non-cancer cell lines. Evaluation of the structure-activity relationships revealed that the triterpenoid core determined whether the final molecule is active or not, while the heterocycle is able to increase the activity and modulate the specificity. Five compounds (1b, 1c, 2b, 2c, and 8) were found to be preferentially and highly cytotoxic (IC₅₀ ≈ 1 μM) against leukemic cancer cell lines (CCRF-CEM, K562, CEM-DNR, or K562-TAX). Surprisingly, compounds 1c, 2b, and 2c are 10-fold more active in multidrug-resistant leukemia cells (CEM-DNR and K562-TAX) than in their non-resistant analogs (CCRF-CEM and K562). Pharmacological parameters were measured for the most promising candidates and two types of prodrugs were synthesized: 1) Sugar-containing conjugates, most of which had improved cell penetration and retained high cytotoxicity in the CCRF-CEM cell line, unfortunately, they lost the selectivity against resistant cells. 2) Medoxomil derivatives, among which compounds 26-28 gained activities of IC₅₀ 0.026-0.043 μM against K562 cells. Compounds 1b, 8, 21, 22, 23, and 24 were selected for the evaluation of the mechanism of action based on their highest cytotoxicity against CCRF-CEM cell line. Several experiments showed that the majority of them cause apoptosis via the mitochondrial pathway. Compounds 1b, 8, and 21 inhibit growth and disintegrate spheroid cultures of HCT116 and HeLa cells, which would be important for the treatment of solid tumors. In summary, compounds 1b, 1c, 2b, 2c, 24, and 26-28 are highly and selectively cytotoxic against cancer cell lines and were selected for future in vivo tests and further development of anticancer drugs.

Keywords: Apoptosis; Cancer; Cytotoxicity; Electron microscopy; Fluorescence; Heterocycle; Huisgen cycloaddition; MDR; Medoxomil; Mitochondria; Molecular target; Pharmacology; Prodrug; Pyrazine; Pyridine; Spheroid cultures; Triterpene.