Metabolite profiling in human and mouse liver microsomes of the aminosteroid RM-581, a new orally active anticancer agent

To progress towards clinical phase studies with orally active aminosteroid RM-581, it was necessary to address its metabolic stability. Analysis by ultra high-pressure liquid chromatography coupled with quadrupole time-of-flight-mass spectrometry (MS) showed the gradual disappearance of RM-581, making it possible to determine a half-life of 14 and 15 min in mouse and human liver microsomes, respectively, and the presence of 6 metabolites in varying proportions (M1 to M6). The MS/MS spectra of metabolites were investigated to assign sub-structures to fragment ions. These analyses made it possible to conclude that the amino acid proline in the C2-side chain of the steroid mestranol core was the main site of metabolism, and the following mass shift relative to the parent compound were observed for each metabolite relative to RM-581: M1(+34), M2(−2), M3(+16), M4(+18), M5(+32) and M6(+16). Suspecting the formation of RM-581 analogs whose pyrrolidine ring of proline has been opened, compound 8 (M4) was synthesized and characterized. Its chromatographic and MS properties made it possible to formally identify the metabolite M4 (M + H⁺ = 665.3684; Rt = 9.11 min) as a primary alcohol derivative. This result also indirectly supports the proposed structures of the other metabolites M6 (aldehyde), M5 (carboxylic acid) and M1 (dihydroxylated quinoline ring). M4 (IC₅₀ = 12–454 μM) was found to be much less potent than RM-581 (IC₅₀ = 0.78–7.55 μM) when tested on 9 human pancreas and leukemia cancer cell lines. M4 (63 %) was also more present in plasma than RM-581 (37 %) when RM-581 was orally administered to rats.