LC/Q-TOF MS and LC/QQQ MS based bioanalysis of a new ferrocene derivative as a potential anticancer lead with promising drug-like characteristics

Abstract

Pyrazolopyrimidine ring present in various approved drugs is reported to target the tyrosine kinase receptor. A new pyrazolopyrimidine ferrocene derivative, which targets tumor pyruvate kinase M2 showed an impressive antiproliferative profile against human oral squamous cell carcinoma cell line CAL27 assessed using Alamar blue assay. In line with the lead optimization process, the molecule was studied for physicochemical properties where a bioanalytical method has been developed in plasma on liquid chromatography-mass spectrometry and validated following the USFDA bioanalytical method validation guideline. Plasma stability and plasma protein binding potential of the molecule have been evaluated. All the major metabolites of the compound have been identified through in vitro metabolite study employing rat liver microsome, human liver microsome, and human S9 fractions. The in silico toxicity profile of the metabolites was assessed using ProTox II software. Log P, Log D, and pKa of the molecule were found to be 4.5, 5, and 12, respectively. The molecule was found to be quite stable in plasma and have a moderate affinity towards plasma proteins (about 75 % binding). Four major metabolites have been identified and characterized by UHPLCQ-TOF-MS. The metabolites were found to have a moderate safety profile. The validated bioanalytical method and the metabolic pathway will be useful for future clinical studies and to assess the safety profile of the molecule. The finding of this study may also be useful in analyzing the desired drug-like properties through bioanalysis while designing new chemical entities based on metallocenes.