Abstract 5748: Metabolites of the orally potent anticancer agent and endoplasmic reticulum stress inducer RM-581

Abstract The development of new and more effective chemotherapy agents is a biomedical research priority, especially for cancer, still one of the deadliest diseases. To progress towards clinical phase studies with RM-581, an aminosteroid derivative producing promising in vitro and in vivo results against several types of cancers, including some with a poor prognosis such as pancreatic, prostate and breast cancers, it was necessary to address its metabolic stability. Analysis by ultra high-pressure liquid chromatography (UPLC) quadrupole time-of-flight (QTof)-mass spectrometry (MS) showed the gradual disappearance of RM-581, making it possible to determine a half-life of 14 and 15 min in concentrated preparations of 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 side chain at position C2 of the steroid mestranol 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, we synthesized an aminosteroid where the proline was replaced by a 2-amino-5-hydroxypentanoic acid. Starting from steroid estrone, 4 steps were needed to introduce a piperazine at position C2, an ethynyl at C17α and to methoxylate the OH at C3. The key Fmoc and TBS-diprotected amino acid was then added by a coupling reaction, the Fmoc protecting group removed before adding the quinaldic acid. A cleavage of the TBS protecting group then allowed to obtain the targeted RM-581 analog. After a full chemical characterization of this compound, these chromatographic and MS properties made it possible to formally identify the major 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 (primary alcohol or oxygen on the quinoline ring). When determining the concentration inhibiting 50% of cell proliferation (IC50) on 8 human leukemia and pancreas cancer cell lines, M4 (IC50 = 17-454 μM) was found to be much less potent than RM-581 (IC50 = 0.78-7.55 μM). The loss of efficiency ranged from 16 to 111-fold, according to cell lines. Furthermore, only M4 was observed when RM-581 was administered by gavage to mice at a 30 mg/kg dose with the plasma analyzed 2 h post-administration. The glucuronidated form of M4 was also detected at 2 h, suggesting the RM-581 elimination pathway and the fact that it does not accumulate in the blood. Citation Format: Donald Poirier, Martin Jutras, René Maltais, Jenny Roy, Maude Fleury, Camille Gagné. Metabolites of the orally potent anticancer agent and endoplasmic reticulum stress inducer RM-581 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5748.