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Tyrosine kinase inhibitors have often been reported to treat early-stage hormone-receptor-positive breast cancers. In particular, neratinib has shown positive responses in stage I and II cases in women with HER2-positive breast cancers with trastuzumab. In order to augment the biopharmaceutical attributes of the drug, the work designed endeavors to explore the therapeutic benefits of neratinib in combination with naringenin, a phytoconstituent with reported uses in breast cancer. A UPLC-MS/MS method was developed for the simultaneous estimation of neratinib and naringenin in rat plasma, while imatinib was selected as the internal standard (IS). Acetonitrile was used as the liquid extractant. The reversed-phase separation was achieved on a C18 column (100 mm × 2.1 mm, 1.7 µm) with the isocratic flow of mobile phase-containing acetonitrile (0.1% formic acid) and 0.002 M ammonium acetate (50:50, % v/v) at flow rate 0.5 mL·min−1. The mass spectra were recorded by multiple reaction monitoring of the precursor-to-product ion transitions for neratinib (m/z 557.138→111.927), naringenin (m/z 273.115→152.954), and the IS (m/z 494.24→394.11). The method was validated for selectivity, trueness, precision, matrix effect, recovery, and stability over a concentration range of 10–1280 ng·mL−1 for both targets and was acceptable. The method was also assessed for greenness profile by an integrative qualitative and quantitative approach; the results corroborated the eco-friendly nature of the method. Therefore, the developed method has implications for its applicability in clinical sample analysis from pharmacokinetic studies in human studies to support the therapeutic drug monitoring (TDM) of combination drugs. [ABSTRACT FROM AUTHOR]

[Display omitted] • Quinazolinone derivatives were designed keeping Ispinesib and Idelalisib as reference structures. • Compounds 3a, 3b, 3e, 3g and 3h revealed nanomolar potency against KSP and PI3Kδ. • Compounds were found to be active against MDA-MB-231 breast cancer cells. • Anticancer activity was found to be associated with their P53 -dependent apoptotic activity. Triple-negative breast cancer (TNBC) comprises 10 % to 20 % of breast cancer, however, it is more dangerous than other types of breast cancer, because it lacks druggable targets, such as the estrogen receptors (ER) and the progesterone receptor (PR), and has under expressed receptor tyrosine kinase, ErbB2. Present targeted therapies are not very effective and other choices include invasive procedures like surgery or less invasive ones like radiotherapy and chemotherapy. This study investigated the potential anticancer activity of some novel quinazolinone derivatives that were designed on the structural framework of two approved anticancer drugs, Ispinesib (KSP inhibitor) and Idelalisib (PI3Kδ inhibitor), to find out solutions for TNBC. All the designed derivatives (3a-l) were subjected to extra precision molecular docking and were synthesized and spectrally characterized. In vitro enzyme inhibition assay of compounds (3a, 3b, 3e, 3 g and 3 h) revealed their nanomolar inhibitory potential against the anticancer targets, KSP and PI3Kδ. Using MTT assay, the cytotoxic potential of compounds 3a, 3b and 3e were found highest against MDA-MB-231 cells with an IC 50 of 14.51 µM, 16.27 µM, and 9.97 µM, respectively. Remarkably, these compounds were recorded safe against the oral epithelial normal cells with an IC 50 values of 293.60 µM, 261.43 µM, and 222 µM, respectively. The anticancer potential of these compounds against MDA-MB-231 cells was revealed to be associated with their apoptotic activity. This was established by examination with the inverted microscope that revealed the appearance of various apoptotic features like cell shrinkage, apoptotic bodies, and membrane blebbing. Using flow cytometry, the Annexin V/PI-stained cancer cells showed an increase in early and late apoptotic cells. In addition, DNA fragmentation was revealed to occur after treatment with the tested compounds by gel electrophoresis. The relative gene expression of pro-apoptotic and anti-apoptotic genes revealed an overexpression of the P53 and BAX genes and a downregulation of the BCL-2 gene by real-time PCR. So, this work proved that compounds 3a, 3b, and 3e could be developed as anticancer candidates, via their P53-dependent apoptotic activity. [ABSTRACT FROM AUTHOR]