Nrf2 is recognised as an attractive therapeutic target for oxidative stress-related disorders through its regulation of antioxidant gene transcription. Direct inhibition of Keap1-Nrf2 protein-protein interaction represents a promising strategy to modulate Nrf2 activity. Herein, we report the discovery of meta-substituted bis(arylsulfonamido)benzene derivatives using a molecular hybridisation strategy based onpotent inhibitors 2a and 3a. Among the initial hybrids, 7a demonstrated good potency in the FP assay, making it a suitable lead for SAR optimisation. Our study found 13b was the most potent analog, showing IC50 values of 183.4 nM in the FP assay and 107.5 nM in the TR-FRET assay. It also demonstrated excellent metabolic stability, with 93.9% remaining after a 30 minute-incubation in human liver microsomes. Collectively, these results highlight 13b as a non-covalent Keap1-Nrf2PPI inhibitor, with balanced potency and metabolic stability, supporting its potential as a tractable scaffold for further optimisation to modulate the Nrf2 pathway.

Non-targeted screening of alprazolam and flualprazolam metabolites in in vitro metabolism of different species by high-resolution mass spectrometry.

Pharmacokinetics, tissue distribution, in vivo metabolism and liver microsomal metabolism of GK-A, GK-2 and Eucomic acid in rats were studied by liquid chromatography-tandem mass spectrometry and mass spectrometry imaging of Ginkgo semen extract.

Synthesis of novel 6-azacytidine prodrugs as potent influenza A inhibitors.

The pharmacokinetic profile of a potential drug is largely determined by its metabolic stability, which reflects its susceptibility to biotransformation. Metabolic stability data allow one to assess the therapeutic value of a compound and its toxicological risk. This assesment relies primarily on pharmacokinetic parameters, particularly half-life (t1/2) and clearance (CL), which are typically determined using in vitro systems including hepatocytes and liver microsomal fractions. Using the publicly available ChEMBL v. 35 and PubChem databases, we collected over 8000 chemical compounds with experimental intrinsic CL and/or half-life data from liver microsome assays obtained in mice, rats, and humans. Different thresholds were applied to differentiate the stable and unstable molecules. The Naive Bayesian classifier with MNA (Multilevel Neighborhoods of Atoms) descriptors and Self-Consistent Extreme Classifier (SCEC) with QNA (Quantitative Neighborhoods of Atoms) descriptors were used for creating classification models. The accuracy (AUC) of most classification models exceeded 0.85. Self-Consistent Regression was used to create quantitative models. The coefficient of determination of the regression models varied from 0.35 (rat, t1/2) to 0.7 (human, CLint). These models were integrated into the freely available web application MetaStab-Analyzer, which provides a unique combination of qualitative (stable/unstable/moderate) and quantitative predictions for three species. A key feature of the application is the providing of numerical metrics for each prediction, which increases its interpretability. This combination of innovative algorithms (SCR and SCEC), dual qualitative-quantitative assessment, and a user-friendly interface is not available in any existing tool. MetaStab-Analyzer is freely available at https://www.way2drug.com/metastab/.

Metabolic stability assessment and metabolite profiling of gunagratinib, a novel FGFR inhibitor, in rat, monkey and human liver microsomes by an integrated analysis method based on HPLC-MS/MS and HPLC-Orbitrap-HRMS.

New insights into metabolism of desogestrel in humans using liver microsomes and an HPLC/UV method

Polynitrogen-containing compounds as multi-target sEH/FAAH inhibitors: Structure-activity relationship and pharmacological studies.

Assessing the hepatic metabolism of six sunscreen ultraviolet filters: In vitro-in vivo extrapolation approach using human liver microsomes and hepatic parameter predictions for exposure risk assessment.

The safety profile of two newly synthesized series of hydrazone derivatives (7 a–e and 8 a–e ) was studied in different liver-derived cell and subcellular culture models, i.e., isolated rat liver hepatocytes and microsomes, and the HepG2 cell line. The hydrazones, which have previously been reported to possess neuroprotective and antioxidant activity, were further assessed for possible hepatoprotective and antioxidant properties in several in vitro models of hepatotoxicity: non-enzymatic lipid peroxidation in isolated rat liver microsomes; metabolic bioactivation induced by carbon tetrachloride (CCl 4 , 86 µM) in rat hepatocytes; and oxidative stress models induced by tert-butyl hydroperoxide (t-BuOOH, 75 µM) in rat hepatocytes and hydrogen peroxide (H 2 O 2 , 0.1 mM) in HepG2 cells. Compounds 7d and 8d demonstrated the lowest hepatotoxic potential in isolated rat liver microsomes and hepatocytes. In HepG2 cells, all tested hydrazones exhibited low or negligible in vitro toxicity, with IC₅₀ values ranging from 64.28 to 205.10 µM. Compound 7c showed the lowest cytotoxicity, followed by 7a and 8d. All hydrazones exhibited significant antioxidant activity under conditions of non-enzymatic lipid peroxidation, with 7d and 8d displaying the most pronounced effects. In the CCl₄-induced hepatotoxicity model, all compounds demonstrated statistically significant hepatoprotective and antioxidant activity, as evidenced by improved hepatocyte viability, reduced lactate dehydrogenase (LDH) leakage, preserved intracellular glutathione (GSH) levels, and decreased malondialdehyde (MDA) production. Similar protective trends were observed in the model of t-BuOOH-induced oxidative stress, again with 7d and 8d showing superior effects. In the H 2 O 2 -induced oxidative stress model in HepG2 cells, compounds 7c, 7d, 7e, 8d, and 8e exhibited protective activity, with 7e and 8d providing the strongest protection. Overall, compounds 7d and 8d emerged as the most promising candidates, combining low hepatotoxicity with pronounced hepatoprotective and antioxidant effects, supporting their potential for further pharmacological development.

Buparlisib (BLB) is an oral pan-phosphoinositide 3 kinase (PI3K) inhibitor that selectively targets all class I PI3K isoforms. No prior UPLC-MS/MS method with combined in silico metabolism analysis and green metrics for BLB has been reported, so this research sought to establish a precise, ultra-fast, sustainable, and dependable UPLC-MS/MS approach for estimating BLB in the matrix of human liver microsomes (HLMs), which is employed for determining the metabolic stability of BLB. The UPLC-MS/MS method exhibited a good degree of greenness as approved by the ComplexMoGAPI (69.0) and the AGREEprep tool score (0.68). Ripretinib (RPB) was selected as an internal standard (IS) for BLB quantification in the HLMs matrix. The validated method exhibited a linearity over an extensive concentration span of 1 to 4000 ng mL-1. The accuracy and precision of interday and intraday measurements varied from -2.92% and 10.11% and 3.11 and 9.78%, respectively. The MS/MS analysis was performed by employing the positive ESI ionization mode, and chromatography using an Eclipse Plus C8 column with a run time of one minute. BLB had a moderate extraction ratio, with a clearance rate (Clint) of 25.15 mL min-1 kg-1 and an in vitro half-life (t1/2) of 32.24 min. In silico assessments (P450 and DEREK modules) indicate that minor structural changes in the 2-aminopyridine moiety (lability: 58%) and morpholine groups (lability: 42%) during drug design could increase the metabolic stability and safety profile of BLB. The integrated in vitro technique (metabolic incubation) and in silico tools (ADME, DEREK and WhichP450) provide a resource-effective strategy for preliminary metabolic screening and advancing new therapeutic development aimed at enhancing metabolic stability of new BLB derivatives.

ABSTRACTRationaleEstrogen‐related receptor (ERR) agonists such as the drug candidates SLU‐PP‐332 and SLU‐PP‐915 are currently being investigated as exercise mimetics, given their ability to trigger human physiological processes similar to those initiated by actual physical activity. This capability prompted the consideration of these compounds as drugs potentially relevant for sports drug testing programs.MethodsThe two pan‐ERR agonists SLU‐PP‐332 and SLU‐PP‐915 were characterized using liquid chromatography‐high resolution (tandem) mass spectrometry (LC–HRMS/MS). Furthermore, the in vitro metabolic transformation products of both compounds prepared by means of human liver S9 fraction (S9 fraction) and human liver microsomes (HLMs) were analyzed. In addition, selected metabolites of SLU‐PP‐915 were synthesized and their structures were analyzed by nuclear magnetic resonance (NMR) spectroscopy.ResultsA total of nine metabolites were identified for SLU‐PP‐332, consisting of six Phase‐I metabolites and three Phase‐II conjugates. Conversely, the analysis of SLU‐PP‐915 yielded only Phase‐I transformation products, with a total of seven metabolites identified. In both cases, an in‐depth structural elucidation was conducted to obtain a comprehensive overview of the detected metabolites. Furthermore, three metabolites of SLU‐PP‐915 were confirmed through chemical synthesis and NMR.ConclusionThe results obtained in this study gave an in‐depth view into the analysis and in vitro metabolism of the newly developed pan‐ERR agonists SLU‐PP‐332 and SLU‐PP‐915. This may help to uncover the illicit use of these novel compounds as potential performance‐enhancing substances.

Background/Objectives: Flavonoids are widely used as lead structures in drug discovery, and their pharmacological and metabolic properties are strongly influenced by structural features such as positional isomerism. This study aimed to compare the metabolic profiles and underlying mechanisms of two isoflavone-based positional isomers, ACF-02 (2-(4-hydroxy-3-methoxyphenyl)-6,7-dimethoxy-3-(4-methoxyphenyl)-4H-chromen-4-one) and ACF-03 (2-(3-hydroxy-4-methoxyphenyl)-6,7-dimethoxy-3-(4-methoxyphenyl)-4H-chromen-4-one). Methods: The metabolic pathways of synthetically prepared ACF-02 and ACF-03 were investigated using an in vitro incubation system with human liver microsomes (HLMs) supplemented with an NADPH-regenerating system, followed by liquid chromatography–high-resolution tandem mass spectrometry (LC–HRMS/MS) analysis. Metabolites were identified based on LC–HRMS/MS data and molecular networking-based node connectivity with the parent compounds. Major metabolites were further characterized by CYP phenotyping using recombinant CYP450 isoforms, and the potential for drug–drug interactions of ACF-03 was evaluated using a CYP probe substrate cocktail approach. Results: HLM incubation of ACF-02 and ACF-03 produced both hydroxylated and O-demethylated metabolites, with O-demethylation as the predominant pathway; notably, the most abundant O-demethylated metabolite differed in an isomer-dependent manner, occurring at the B2 ring for ACF-02 and at the A ring for ACF-03, with distinct CYP isoform involvement. Molecular networking supported the relationships between the parent compounds and their metabolites, and both compounds exhibited relatively high metabolic stability with limited CYP inhibition. Conclusions: Despite differing only in the position of a single methyl substituent, ACF-02 and ACF-03 exhibited distinct isomer-dependent metabolic profiles. These findings demonstrate that even subtle positional isomerism can significantly influence metabolic behavior and should be carefully considered during lead optimization and drug design.

TPD354 is a novel Proteolysis Targeting Chimera (PROTAC) drug candidate that targets the degradation of cellular mesenchymal-epithelial transforming factor (c-Met) kinase. It showed significant therapeutic efficacy in vivo gastric cancer models. We have developed a robust and sensitive analytical method for evaluating the pharmacokinetic properties of TPD354, using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) to quantify drug concentrations in diverse biological matrices. The method was validated and applied to pharmacokinetic studies of the compound. The method exhibited excellent specificity in the linear range of 6.995-6995.000ng/mL. Moreover, the method met or exceeded established criteria for precision, accuracy, recovery, and matrix effects, ensuring its suitability for in vivo analysis. The method was applied to the study of drug pharmacokinetics in rats, stability in different types of liver microsomes, and protein binding in plasma of different species. Our studies have demonstrated that TPD354 exhibits metabolic stability in liver microsomes and is characterized by high plasma protein binding, with a half-life of approximately 16h in rats. These findings suggest that TPD354 is a promising PROTAC drug candidate, with strong potential for the treatment of c-Met targeted cancer.

Hepatic clearance is crucial as it directly impacts drug exposure, efficacy, and safety. Cytochrome P450 (CYP450) enzymes play a pivotal role in drug metabolism and exhibit differences based on sex, species, and commercial liver microsome vendors. These variables can directly influence translational accuracy when preclinical data are applied to human drug development. In this study, we evaluated metabolic stability of isozyme-selective compounds across human, rat, and mouse liver microsomes, incorporating both male and female microsomes and multiple vendors. Our analysis revealed three layers of variability: (1) sex-specific differences consistent with prior clinical observations, where certain substrates displayed markedly faster clearance in one sex; (2) interspecies divergence, such as male-predominant isoforms in rodents without direct human orthologs; and (3) vendor-related discrepancies, where the same species-sex pool yielded divergent stability outcomes depending on microsome source. Together, these findings illustrate the combined effects of sex, species, and vendor source that contribute to variability in CYP450-mediated metabolism. By systematically comparing these factors, our work underscores the importance of considering these variables during early preclinical studies. Accounting for these sources of variability may improve the translational reliability of in vitro assays, reduce costly late-stage failures, and better support the development of safe and effective therapeutics.

Pharmaceutical excipients have traditionally been regarded as pharmacologically inert carriers, serving merely as vehicles for active pharmaceutical ingredients. However, this conventional paradigm is increasingly challenged by emerging evidence revealing clinically significant drug-excipient interactions that can profoundly influence therapeutic outcomes. This study investigates a previously unrecognized pharmacokinetic interaction between nimodipine, a dihydropyridine calcium channel blocker, and polysorbate 80, a commonly used solubilizing excipient in nimodipine formulations. A novel HPLC-MS/MS method for simultaneous quantification of nimodipine and polysorbate 80 was developed and validated, achieving excellent analytical performance with linearity (r > 0.995), precision (RSD < 15%), and accuracy (RE < 15%). Pharmacokinetic studies in rats revealed that polysorbate 80 co-administration dramatically enhanced nimodipine systemic exposure, increasing AUC by 51.1%, decreasing clearance by 36.5%, and prolonging half-life by 16.1%. Mechanistic investigations using rat liver microsomes demonstrated that polysorbate 80 competitively inhibited nimodipine metabolism with an IC50 of 103.30 μmol/L and Ki of 94.35 μmol/L, while remaining minimally metabolized itself. These findings provide the first evidence of a clinically significant pharmacokinetic interaction between nimodipine and polysorbate 80, challenging the assumption of excipient inertness and highlighting the critical importance of evaluating excipient-drug interactions in pharmaceutical development and clinical practice.

Synthetic cannabinoids (SCs) are a rapidly developing kind of novel psychoactive substance, frequently associated with acute intoxication and public health concerns. This study aimed to elucidate and compare the phase I metabolic pathways of two structurally related SCs, 5F-ADB-PINACA and 5F-ADBICA, using in vitro and in vivo models. Temporal metabolic profiling was performed to identify potential signature metabolites. Temporal abundance patterns and correlation cluster analysis of metabolites were analyzed to determine metabolite biomarkers. The two SCs were incubated with pooled human liver microsomes for 24 h and were also evaluated in vivo in zebrafish. Metabolite profiles were characterized using UHPLC-QE Orbitrap-MS. HLM analysis identified 21 5F-ADB-PINACA metabolites and 28 5F-ADBICA metabolites. Metabolites of 5F-ADBICA were detected for the first time in vitro and in a zebrafish model. Zebrafish studies confirmed the presence of all key metabolites observed in HLM. Comparative analysis of their metabolic pathways revealed differences in metabolism driven by structural differences between the indazole and indole cores. This is the first time that correlation analysis has been used in the temporal metabolic profiling of SCs. This study comprehensively characterized the metabolism of 5F-ADB-PINACA and 5F-ADBICA, identifying M13 (hydrolytic defluorination) as a potential metabolite biomarker for 5F-ADB-PINACA and M19 (hydrolytic defluorination) as a potential metabolite biomarker for 5F-ADBICA. The metabolic reactions of the main metabolites of the two synthetic cannabinoids are consistent. However, their metabolic processes (i.e., the overall metabolic pathways and temporal progression of these reactions) are different, which illustrates the metabolic similarity of structurally similar synthetic cannabinoids and the impact of different structures on the metabolic processes.

4-Methyl-5-Acetylthiazole (Q11), a novel CYP2E1 inhibitor, has a protective effect on the hepatotoxicity induced by acetaminophen.

A new class of benzoxaboroles with a phenylglycine appendage was found to display in vitro blood stage activity against the human malaria parasite Plasmodium falciparum (Pf). Structure-activity relationship studies of the starting hit compound 3 resulted in compounds active against PfNF54 drug-sensitive and PfK1 drug-resistant strains with an in vitro antiplasmodium IC50 < 0.4 μM, selectivity over mammalian cell-lines (selectivity index > 47) and high aqueous solubility (160 to >200 μM). Selected compounds showed good in vitro metabolic stability when incubated with human, rat, and mouse liver microsomes and showed no cross-resistance against barcoded mutant lines. Two frontrunner compounds, 6 and 7, were dosed orally at 50 mg·kg-1 using a standard quadrupole dosing regimen in a P. berghei mouse infection model and showed encouraging in vivo efficacy. This work identifies a promising new class of phenylglycine-based benzoxaboroles, which warrants further medicinal chemistry optimization.

Methyl 2-{[1-(5-fluoropentyl)-1H-indazole-3-carbonyl]amino}-3,3-dimethylbutanoate (5F-ADB), a potent synthetic cannabinoid, induces intense euphoria, hallucinations, and addiction, posing significant risks to human health. Current drug surveillance efforts lack data to identify drug abuse, and the environmental impacts of 5F-ADB entering aquatic systems via synthesis or use remain uncharacterized. To address these gaps, a multi-level assessment system (in vitro-invertebrate-vertebrate) was established to elucidate 5F-ADB metabolic pathways and identify robust biomarkers. Human liver microsomes (HLM), Daphnia magna, and zebrafish were exposed to 5F-ADB, with metabolites profiled using high performance liquid chromatography coupled with mass spectrometry (HPLC-MS). Metabolic pathways were inferred, and metabolite toxicity was evaluated. Results revealed 9, 11, and 22 metabolites in HLM, D. magna, and zebrafish models, respectively. Dominant pathways in HLM and zebrafish included ester hydrolysis, defluorinated-hydroxylation, and combined ester hydrolysis/defluorinated-hydroxylation. D. magna metabolism primarily featured defluorinated-hydroxylation, depentylation, and ester hydrolysis coupled with hydroxylation. Glucuronidation metabolites were exclusive to zebrafish. Based on abundance and stability, H-M4 (ester hydrolysis), D-M1 (ester hydrolysis/depentylation), and Z-M15 (ester hydrolysis/condensation) were identified as key biomarkers for HLM, D. magna, and zebrafish, respectively. Toxicity assessments indicated reduced toxicity for most metabolites versus 5F-ADB. However, H-M7, D-M7, D-M11, and Z-M15 (products of ester hydrolysis/condensation or defluorinated-hydroxylation/oxidation) exhibited comparable toxicity to the parent compound. Critically, D-M7 (defluorinated-hydroxylation/oxidation) demonstrated heightened hydrophilicity and potentially elevated ecotoxicity in D. magna, warranting further ecological risk investigation. This study provides the first multi-trophic metabolic characterization of 5F-ADB, delivering critical data for tracing illicit synthesis, monitoring drug-use distribution, and evaluating environmental hazards of synthetic cannabinoids.