In continuation of our endeavor with respect to the development of potent and effective isatin-based anticancer agents, we adopted the molecular hybridization approach to design and synthesize four different sets of isatin-quinazoline (6a–f and 7a–e)/phthalazine (8a–f)/quinoxaline (9a–f) hybrids. The antiproliferative activity of the target hybrids was assessed towards HT-29 (colon), ZR-75 (breast) and A-549 (lung) human cancer cell lines. Hybrids 8b–d emerged as the most active antiproliferative congener in this study. Compound 8c induced apoptosis via increasing caspase 3/7 activity by about 5-fold in the A-549 human cancer cell line. In addition, it exhibited an increase in the G1 phase and a decrease in the S and G2/M phases in the cell cycle effect assay. Furthermore, it displayed an inhibitory concentration 50% value of 9.5 µM against multidrug-resistant NCI-H69AR lung cancer cell line. The hybrid 8c was also subjected to in vitro metabolic investigations through its incubation with rat liver microsomes and analysis of the resulting metabolites with the aid of liquid chromatography-mass spectrometry.

This study investigates the crystal structure, physicochemical properties, and pharmacokinetic profile of Ethyl 2-amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylate (EAMT) as a potential therapeutic agent. The crystal structure was analyzed using Hirshfeld surface analysis in conjunction with the quantum theory of atoms in molecules (QT-AIM). Non-covalent interactions were evaluated through reduced-density gradient reduction, revealing that the EAMT crystal is stabilized by hydrogen bonds between EAMT molecules in the crystal and between EAMT molecules and water molecules. The molecular electrostatic nature of interactions was examined using MESP, while global and local descriptors were calculated to assess the compound’s reactivity. Molecular docking with the Adenosine A1 receptor was performed and validated through a 50 ns molecular dynamics simulation (MDS). Results suggest that EAMT influences protein structure, potentially stabilizing specific secondary structure elements. The compactness analysis showed a slightly more compact protein conformation and a marginally increased solvent exposure in the presence of the EAMT ligand, as indicated by R_g and SASA values. The total binding free energy (ΔG total) was determined to be −114.56 kcal/mol. ADMET predictions demonstrated EAMT’s compliance with Lipinski’s and Pfizer’s rule of five, indicating good oral availability. The compound may exhibit low-potency endocrine activity. In conclusion, EAMT presents potential as a therapeutic candidate, warranting further exploration of its molecular interactions, pharmacokinetics, and potential safety concerns.

Ribociclib (RBC, Kisqali®) is a highly selective CDK4/6 inhibitor that has been approved for breast cancer therapy. Initially, prediction of susceptible sites of metabolism and reactivity pathways were performed by the StarDrop WhichP450™ module and the Xenosite web predictor tool, respectively. Later, in vitro metabolites and adducts of RBC were characterized from rat liver microsomes using LC-MS/MS. Subsequently, in silico data was used as a guide for the in vitro work. Finally, in silico toxicity assessment of RBC metabolites was carried out using DEREK software and structural modification was proposed to reduce their side effects and to validate the bioactivation pathway theory using the StarDrop DEREK module. In vitro phase I metabolic profiling of RBC was performed utilizing rat liver microsomes (RLMs). Generation of reactive metabolites was investigated using potassium cyanide (KCN) as a trapping nucleophile for the transient and reactive iminium intermediates to form a stable cyano adduct that can be identified and characterized using mass spectrometry. Nine phase I metabolites and one cyano adduct of RBC were characterized. The proposed metabolic pathways involved in generation of these metabolites are hydroxylation, oxidation and reduction. The reactive intermediate generation mechanism of RBC may provide an explanation of its adverse reactions. Aryl piperazine is considered a structural alert for toxicity as proposed by the DEREK report. We propose that the generation of only one reactive metabolite of RBC in a very small concentration is due to the decreased reactivity of the piperazine ring compared to previous reports of similar drugs. Docking analysis was performed for RBC and its proposed derivatives at the active site of the human CDK6 enzyme. Methyl-RBC exhibited the best ADMET and docking analysis and fewer side effects compared to RBC and fluoro-RBC. Further drug discovery studies can be conducted taking into account this concept allowing the development of new drugs with enhanced safety profiles that were confirmed by using StarDrop software. To the best of our knowledge, this is the first literature report of RBC in vitro metabolic profiling and structural characterization and toxicological properties of the generated metabolites.

Background: Talazoparib (BMN673) is a new poly(ADP-ribose) polymerase inhibitor that has been FDA approved for patients suffering from metastatic breast cancer with germline BRCA mutations. Method and Results: In the current study, an accurate and efficient liquid chromatography-tandem mass spectrometry (LC–MS/MS) analytical methodology was developed for TZB estimation in addition to its metabolic stability assessment. TZB and lapatinib (LAP) (which is chosen as an internal standard; IS) were separated using reversed phase elution system (Hypersil C ₁₈ column) with an isocratic mobile phase. The linearity range of the established method was 5– 500 ng/mL (r ² ≥ 0.999) in the human liver microsomes (HLMs) matrix. Different parameters were calculated to confirm the method sensitivity (limit of quantification was 2.0 ng/mL), and reproducibility (intra- and inter-day precision and accuracy were below 3.1%) of our methodology. For evaluation of TZB metabolic stability in HLM matrix, intrinsic clearance (9.59 μL/min/mg) and in vitro half-life (72.7 mins) were calculated. TZB treatment discontinuations were reported due to adverse events and dose accumulation, so in silico metabolic vulnerability (experimental and in silico) and toxicity assessment (in silico) of TZB were performed utilizing P450 Metabolism and DEREK modules of StarDrop software. Conclusion: TZB is slowly metabolized by the liver. TZB was reported to be minimally metabolized by the liver that approved our outcomes. We do recommend that plasma levels be monitored in cases when talazoparib is used for a long period of time, since it is possible for TZB to bioaccumulate after multiple doses to toxic levels. According to our knowledge, the current method is considered the first LC-MS/MS methodology for evaluating TZB metabolic stability. Further drug discovery studies can be done depending on this concept allowing the designing of new series of compounds with more safety profile through reducing side effects and improving metabolic behavior.