This work presents a multifaceted mechanism of the anticancer action of a 2-styrylquinazoline derivative. Extensive analysis of various aspects related to tyrosine kinase inhibition and effects on cellular targets at both the gene and protein levels revealed the potential of this IS20 compound for future research. This study presents a detailed analysis of the relationship between ABL and SRC kinase affecting the inhibition of the EGFR/mTOR signaling pathway in a non-obvious manner. The study was supported by experiments using various molecular biology techniques to confirm the induction of oxidative stress, inhibition of the cell cycle in the G2/M phase and the triggering of cell death via both the apoptosis and autophagy pathways. The cell models included those with different p53 protein status, which affected the cellular response in the form of altered Ndrg1 expression. Finally, the appropriate physicochemical properties of IS20 for adequate bioavailability and toxicity to the body were observed in an in vivo model.

Department of Chemical Biology Palacky University Olomouc Slechtitelu 27 Olomouc 779 00 Czech Republic

Department of Medical Chemistry Medical University of Lublin Chodźki 4a Lublin 20 093 Poland

Department of Systems Biology and Engineering Silesian University of Technology Akademicka 16 Gliwice 44 100 Poland

Institute of Chemistry University of Silesia in Katowice 75 Pułku Piechoty 1a Chorzów 41 500 Poland

Institute of Physics University of Silesia in Katowice 75 Pułku Piechoty 1a Chorzów 41 500 Poland

Zobrazit více v PubMed

Mrozek-Wilczkiewicz, A. et al. Investigating the anti-proliferative activity of styrylazanaphthalenes and azanaphthalenediones. Bioorg. Med. Chem.18, 2664–2671 (2010). PubMed

Das, D. & Hong, J. Recent advancements of 4-aminoquinazoline derivatives as kinase inhibitors and their applications in medicinal chemistry. Eur. J. Med. Chem.170, 55–72 (2019). PubMed

Musiol, R. et al. Investigating the Activity Spectrum for Ring-Substituted 8-Hydroxyquinolines.Molecules15, 288–304. (2010). PubMed PMC

Bedi, P. M., Kumar, V. & Mahajan, M. P. Synthesis and biological activity of novel antibacterial Quinazolines. Bioorg. Med. Chem. Lett.14, 5211–5213 (2004). PubMed

Alagarsamy, V. et al. Synthesis of novel 3-butyl-2-substituted amino-3H-quinazolin-4-ones as analgesic and anti-inflammatory agents. Chem. Biol. Drug Des.70, 254–260 (2007). PubMed

Held, F. E. et al. Facile access to potent antiviral Quinazoline heterocycles with fluorescence properties via merging metal-free domino reactions. Nat. Commun.8, 15071 (2017). PubMed PMC

Lee, J. Y., Park, J. H., Lee, S. J., Park, H. & Lee, Y. S. Styrylquinazoline derivatives as HIV-1 integrase inhibitors. Arch. Pharm., 335 (2002). PubMed

Hricoviniova, J., Hricoviniova, Z. & Kozics, K. Antioxidant, cytotoxic,genotoxic, and DNA-Protective potential of 2,3-Substituted Quinazolinones: Structure-Activity relationship study. Int. J. Mol. Sci., 22 (2021). PubMed PMC

Jampilek, J. et al. Investigating biological activity spectrum for novel styrylquinazoline analogues. Molecules14, 4246–4265 (2009). PubMed PMC

Shagufta, S. & Ahmad, I. An insight into the therapeutic potential of Quinazoline derivatives as anticancer agents. MedChemComm8, 871–885 (2017). PubMed PMC

Wischhusen, J., Naumann, U., Ohgaki, H., Rastinejad, F. & Weller, M. CP-31398, a novel p53-stabilizing agent, induces p53-dependent and p53-independent glioma cell death. Oncogene22, 8233–8245 (2003). PubMed

Rippin, T. M. et al. Characterization of the p53-rescue drug CP-31398 in vitro and in living cells. Oncogene21, 2119–2129 (2002). PubMed

Mularski, J., Malarz, K., Pacholczyk, M. & Musiol, R. The p53 stabilizing agent CP-31398 and multi-kinase inhibitors. Designing, synthesizing and screening of styrylquinazoline series. Eur. J. Med. Chem.163, 610–625 (2019). PubMed

Zhou, D. et al. Small molecules inhibit STAT3 activation, autophagy, and cancer cell anchorage-independent growth. Bioorg. Med. Chem.25, 2995–3005 (2017). PubMed PMC

Xu, D. et al. Structure-Based Target-Specific screening leads to Small-Molecule camkii inhibitors. ChemMedChem12, 660–677 (2017). PubMed PMC

Wei, X. W. et al. Su, 2-Styryl-4-aminoquinazoline derivatives as potent DNA-cleavage, p53-activation and in vivo effective anticancer agents. Eur. J. Med. Chem.186, 111851 (2020). PubMed

Dvorakova, M. et al. Synthesis, inhibitory activity, and in Silico modeling of selective COX-1 inhibitors with a Quinazoline core. ACS Med. Chem. Lett.12, 610–616 (2021). PubMed PMC

Malarz, K., Mularski, J., Pacholczyk, M. & Musiol, R. Styrylquinazoline derivatives as ABL inhibitors selective for different DFG orientations. J. Enzyme Inhib. Med. Chem.38, 2201410 (2023). PubMed PMC

Malarz, K., Mularski, J., Kuczak, M., Mrozek-Wilczkiewicz, A. & Musiol, R. Novel benzenesulfonate scaffolds with a high anticancer activity and G2/M cell cycle arrest. Cancers. 13, 1790 (2021). PubMed PMC

Simabuco, F. M. et al. p53 and metabolism: from mechanism to therapeutics. Oncotarget9, 23780–23823 (2018). PubMed PMC

Rivlin, N., Brosh, R., Oren, M. & Rotter, V. Mutations in the p53 tumor suppressor gene: important milestones at the various steps of tumorigenesis. Genes Cancer. 2, 466–474 (2011). PubMed PMC

Liu, D., Xu, Y. & Aging p53, Oxidative Stress, and Antioxid. Redox. Signal., 15 1669–1678. (2011). PubMed PMC

Niedbala, M., Malarz, K., Sharma, G., Kramer-Marek, G. & Kaspera, W. Glioblastoma: pitfalls and opportunities of immunotherapeutic combinations. Onco Targets Ther.15, 437–468 (2022). PubMed PMC

Cancer Genome Atlas Research. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature455, 1061–1068 (2008). PubMed PMC

Pang, Y. et al. Report of canonical BCR-ABL1 fusion in glioblastoma. JCO Precis. Oncol.5, 1348–1353 (2021). PubMed PMC

Palande, V. et al. Detection of gene mutations and gene–gene fusions in Circulating cell‐free DNA of glioblastoma patients: an avenue for clinically relevant diagnostic analysis. Mol. Oncol.16, 2098–2114 (2022). PubMed PMC

Goethe, E., Carter, B. Z., Rao, G. & Pemmaraju, N. Glioblastoma and acute myeloid leukemia: malignancies with striking similarities. J. Neurooncol. 136, 223–231 (2018). PubMed

Nie, Y., Li, H. H., Bula, C. M. & Liu, X. Stimulation of p53 DNA binding by c-Abl requires the p53 C terminus and tetramerization. Mol. Cell. Biol.20 741–748. (2000). PubMed PMC

Weller, M. et al. Predicting chemoresistance in human malignant glioma cells: the role of molecular genetic analyses. Int. J. Cancer. 79, 640–644 (1998). PubMed

Law, J. C., Ritke, M. K., Yalowich, J. C., Leder, G. H. & Ferrell, R. E. Mutational inactivation of the p53 gene in the human erythroid leukemic K562 cell line. Leuk. Res.17, 1045–1050 (1993). PubMed

Freed-Pastor, W. A. & Prives, C. Mutant p53: one name, many proteins. Genes Dev.26, 1268–1286 (2012). PubMed PMC

Malarz, K., Mularski, J., Pacholczyk, M. & Musiol, R. The landscape of the Anti-Kinase activity of the IDH1 inhibitors. Cancers. 12, 536 (2020). PubMed PMC

Trachootham, D., Alexandre, J. & Huang, P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat. Rev. Drug Discov. 8, 579–591 (2009). PubMed

Zou, Z., Chang, H., Li, H. & Wang, S. Induction of reactive oxygen species: an emerging approach for cancer therapy. Apoptosis22, 1321–1335 (2017). PubMed

Perillo, B. et al. ROS in cancer therapy: the bright side of the Moon. Exp. Mol. Med.52, 192–203 (2020). PubMed PMC

Malarz, K. et al. The role of oxidative stress in activity of anticancer thiosemicarbazones. Oncotarget9, 17689–17710 (2018). PubMed PMC

Malarz, K., Zych, D., Gawecki, R., Kuczak, M. & Musioł, R. A. Mrozek-Wilczkiewicz, New derivatives of 4’-phenyl-2,2’:6’,2″-terpyridine as promising anticancer agents. Eur. J. Med. Chem., 113032. (2020). PubMed

Malarz, K., Zych, D., Kuczak, M., Musioł, R. & Mrozek-Wilczkiewicz, A. Anticancer activity of 4’-phenyl-2,2’:6’,2″-terpyridines - behind the metal complexation. Eur. J. Med. Chem.189, 112039 (2020). PubMed

Mrozek-Wilczkiewicz, A., Malarz, K., Rejmund, M., Polanski, J. & Musiol, R. Anticancer activity of the thiosemicarbazones that are based on di-2-pyridine ketone and Quinoline moiety. Eur. J. Med. Chem.171, 180–194 (2019). PubMed

Arihara, Y. et al. Small molecule CP-31398 induces reactive oxygen species-dependent apoptosis in human multiple myeloma. Oncotarget8, 65889–65899 (2017). PubMed PMC

Zhong, B. et al. A p53-stabilizing agent, CP-31398, induces p21 expression with increased G2/M phase through the YY1 transcription factor in esophageal carcinoma defective of the p53 pathway. Am. J. Cancer Res.9, 79–93 (2019). PubMed PMC

Sivaraman, G. et al. Chemically diverse small molecule fluorescent chemosensors for copper ion. Coord. Chem. Rev.357, 50–104 (2018).

Chai, L. Q., Chai, Y. M., Li, C. G. & Zhou, L. Two mono- and dinuclear Cu (II) complexes derived from 3-ethoxy salicylaldehyde: X-ray structures, spectroscopic, electrochemical, antibacterial activities, Hirshfeld surfaces analyses, and time-dependent density functional theory studies. Appl. Organomet. Chem.36, e6475 (2022).

Sarkar, S. et al. A multi-responsive thiosemicarbazone-based probe for detection and discrimination of group 12 metal ions and its application in logic gates. New. J. Chem.42, 15157–15169 (2018).

Franco, R. & Cidlowski, J. A. Apoptosis and glutathione: beyond an antioxidant.Cell. Death Differ.16, 1303–1314 (2009). PubMed

Ighodaro, O. M. & Akinloye, O. A. First line defence antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): their fundamental role in the entire antioxidant defence grid. Alex. J. Med.54, 287–293 (2018).

Ryter, S. W. & Tyrrell, R. M. The Heme synthesis and degradation pathways: role in oxidant sensitivity. Heme Oxygenase has both pro- and antioxidant properties. Free Radic. Biol. Med.28, 289–309 (2000). PubMed

Chau, L. Y. Heme oxygenase-1: emerging target of cancer therapy. J. Biomed. Sci., 22 (2015). PubMed PMC

Chiang, S. K., Chen, S. E. & Chang, L. C. The role of HO-1 and its crosstalk with oxidative stress in Cancer cell survival. Cells10, 2401 (2021). PubMed PMC

Mrozek-Wilczkiewicz, A. et al. The synthesis and anticancer activity of 2-styrylquinoline derivatives. A p53 independent mechanism of action. Eur. J. Med. Chem.177, 338–349 (2019). PubMed

Fang, B. A. et al. Molecular functions of the iron-regulated metastasis suppressor, NDRG1, and its potential as a molecular target for cancer therapy. Biochim. Biophys. Acta. 1845, 1–19 (2014). PubMed

Nakahara, Y. et al. A tumor suppressor gene, N-myc Downstream-Regulated gene 1 (NDRG1), in gliomas and glioblastomas. Brain Sci.12, 473 (2022). PubMed PMC

Sun, B. et al. Decreased expression of NDRG1 in glioma is related to tumor progression and survival of patients. J. Neurooncol.94, 213–219 (2009). PubMed

Menezes, S. V., Sahni, S., Kovacevic, Z. & Richardson, D. R. Interplay of the iron-regulated metastasis suppressor NDRG1 with epidermal growth factor receptor (EGFR) and oncogenic signaling. J. Biol. Chem.292, 12772–12782 (2017). PubMed PMC

Chen, Z. et al. The Iron chelators Dp44mT and DFO inhibit TGF-β-induced Epithelial-Mesenchymal transition via Up-Regulation of N-Myc Downstream-regulated gene 1 (NDRG1). J. Biol. Chem.287, 17016–17028 (2012). PubMed PMC

Le, N. T. V. & Richardson, D. R. Iron chelators with high antiproliferative activity up-regulate the expression of a growth inhibitory and metastasis suppressor gene: a link between iron metabolism and proliferation. Blood104, 2967–2975 (2004). PubMed

Cao, S. S. & Kaufman, R. J. Endoplasmic reticulum stress and oxidative stress in cell fate decision and human disease. Antioxid. Redox Signal.21, 396–413 (2014). PubMed PMC

Merlot, A. M. et al. The metastasis suppressor, NDRG1, differentially modulates the Endoplasmic reticulum stress response. Biochim. Biophys. Acta Mol. Basis Dis.1865, 2094–2110 (2019). PubMed

Waser, M., Mesaeli, N., Spencer, C. & Michalak, M. Regulation of calreticulin gene expression by calcium. J. Cell Biol.138, 547–557 (1997). PubMed PMC

Yang, Y. et al. The ER-localized Ca2+-binding protein calreticulin couples ER stress to autophagy by associating with microtubule-associated protein 1A/1B light chain 3. J. Biol. Chem.294, 772–782 (2019). PubMed PMC

Sahni, S. et al. The metastasis suppressor, N-myc Downstream-regulated gene 1 (NDRG1), inhibits Stress-induced autophagy in Cancer cells. J. Biol. Chem.289, 9692–9709 (2014). PubMed PMC

Pollak, N., Dolle, C. & Ziegler, M. The power to reduce: pyridine nucleotides–small molecules with a multitude of functions. Biochem. J.402, 205–218 (2007). PubMed PMC

Parker, S. J. & Metallo, C. M. Metabolic consequences of oncogenic IDH mutations. Pharmacol. Ther.152, 54–62 (2015). PubMed PMC

Shi, J. et al. An IDH1 mutation inhibits growth of glioma cells via GSH depletion and ROS generation. Neurol. Sci.35, 839–845 (2014). PubMed

Kovacevic, Z. et al. The metastasis suppressor, N-MYC downstream-regulated Gene-1 (NDRG1), Down-regulates the erbb family of receptors to inhibit downstream oncogenic signaling pathways. J. Biol. Chem.291, 1029–1052 (2016). PubMed PMC

Liu, W. et al. The proto-oncogene c-Src and its Downstream Signaling Pathways Are Inhibited by the Metastasis Suppressor, NDRG1 Oncotarget, 68851–8874 (2015). PubMed PMC

Hwang, B., Engel, L., Goueli, S. A. & Zegzouti, H. A homogeneous bioluminescent immunoassay to probe cellular signaling pathway regulation. Commun. Biol.3 (2020). PubMed PMC

Ito, H. et al. Bidirectional regulation between NDRG1 and GSK3β controls tumor growth and is targeted by differentiation inducing Factor-1 in glioblastoma. Cancer Res.80, 234–248 (2020). PubMed

Ma, W., Na, M., Tang, C., Wang, H. & Lin, Z. Overexpression of N-myc downstream-regulated gene 1 inhibits human glioma proliferation and invasion via phosphoinositide 3-kinase/AKT pathways. Mol. Med. Rep.12, 1050–1058 (2015). PubMed PMC

Takekawa, M. & Saito, H. A family of stress-inducible GADD45-like proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK, Cell. 95, 521–530. (1998). PubMed

Kovacevic, Z. et al. The metastasis suppressor, N-myc downstream regulated gene 1 (NDRG1), upregulates p21 via p53-independent mechanisms. Carcinogenesis32, 732–740 (2011). PubMed

Schlafli, A. M. et al. Prognostic value of the autophagy markers LC3 and p62/SQSTM1 in early-stage non-small cell lung cancer. Oncotarget7, 39544–39555 (2016). PubMed PMC

Gomez-Sanchez, R. et al. Gonzalez-Polo, mRNA and protein dataset of autophagy markers (LC3 and p62) in several cell lines. Data Brief.7, 641–647 (2016). PubMed PMC

Li, L., Tan, J., Miao, Y., Lei, P. & Zhang, Q. ROS and autophagy: interactions and molecular regulatory mechanisms. Cell. Mol. Neurobiol.35, 615–621 (2015). PubMed PMC

Kim, Y. C. & Guan, K. L. mTOR: a Pharmacologic target for autophagy regulation. J. Clin. Invest.125, 25–32 (2015). PubMed PMC

Cande, C. et al. Apoptosis-inducing factor (AIF): a novel caspase-independent death effector released from mitochondria. Biochimie84, 215–222 (2002). PubMed

Usuda, J. et al. Restoration of p53 gene function in 12-O-tetradecanoylphorbor 13-acetate-resistant human leukemia K562/TPA cells. Int. J. Oncol.22, 81–86 (2003). PubMed

Ni, J., Lan, F., Xu, Y., Nakanishi, H. & Li, X. Extralysosomal cathepsin B in central nervous system: mechanisms and therapeutic implications. Brain Pathol.32, e13071 (2022). PubMed PMC

Xie, Z. et al. Cathepsin B in programmed cell death machinery: mechanisms of execution and regulatory pathways. Cell. Death Dis.14, 255 (2023). PubMed PMC

Sax, J. K., Fei, P., Murphy, M. E., Bernhard, E. & Korsmeyer, S. J. El-Deiry, BID regulation by p53 contributes to chemosensitivity. Nat. Cell. Biol.4, 842–849 (2002). PubMed

Kerns, E. H., Di, L. & Properties, D. L. Concepts. Structure Design and Methods: from ADME To Toxicity Optimization (Academic, 2008).

Krüger, A., Gonçalves Maltarollo, V., Wrenger, C. & Kronenberger, T. ADME Profiling in Drug Discovery and a New Path Paved on Silica (In: Drug Discovery and Development - New Advances, 2020).

Lipinski, C. A., Lombardo, F., Dominy, B. W. & Feeney, P. J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings1PII of original article: S0169-409X(96)00423-1. Adv. Drug Deliv. Rev. 46 (2001) 3–26. (1997). PubMed

Jimenez, D. G. et al. Molecular properties, including chameleonicity, as essential tools for designing the next generation of oral beyond rule of five drugs. ADMET DMPK. 12, 721–736 (2024). PubMed PMC

Avdeef, A. Often neglected steps in transforming drug solubility from single measurement in pure water to physiologically-appropriate solubility-pH. ADMET DMPK, 13 (2025). PubMed PMC

Jampilek, J. & Kralova, K. Insights into Lipid-Based Delivery Nanosystems of Protein-Tyrosine Kinase Inhibitors for Cancer Therapy. Pharmaceutics 14, 2706. (2022). PubMed PMC

Lipinski, C. A. Compound properties and drug quality. In: (ed Wermuth, C. G.) The Practice of Medicinal Chemistry. Academic, New York, 481–490. (2008).

Veber, D. F., Johnson, S. R., Cheng, H. Y., Smith, B. R. & Ward, K. W. Kopple, molecular properties that influence the oral bioavailability of drug candidates. J. Med. Chem.45, 2615–2623 (2002). PubMed

Muehlbacher, M., Spitzer, G. M., Liedl, K. R. & Kornhuber, J. Qualitative prediction of blood–brain barrier permeability on a large and refined dataset. J. Comput. Aided Mol. Des.25, 1095–1106 (2011). PubMed PMC

Carpenter, S. et al. Felice, A method to predict Blood-Brain barrier permeability of Drug-Like compounds using molecular dynamics simulations. Biophys. J.107, 630–641 (2014). PubMed PMC

Chunduri, V. & Maddi, S. Role of in vitro two-dimensional (2D) and three-dimensional (3D) cell culture systems for ADME-Tox screening in drug discovery and development: a comprehensive review. ADMET DMPK. 11, 1–32 (2023). PubMed PMC

Shen, C. & Zuo, Z. Zebrafish (Danio rerio) as an excellent vertebrate model for the development, reproductive, cardiovascular, and neural and ocular development toxicity study of hazardous chemicals. Environ. Sci. Pollut. Res. Int.27, 43599–43614 (2020). PubMed

Zhao, W., Chen, Y., Hu, N., Long, D. & Cao, Y. The uses of zebrafish (Danio rerio) as an in vivo model for toxicological studies: A review based on bibliometrics. Ecotoxicol. Environ. Saf.272, 116023 (2024). PubMed

Caballero, M. V. & Candiracci, M. Zebrafish as toxicological model for screening and recapitulate human diseases. J. Unexplored Med. Data. 3, 4 (2018).

Rahman, I., Kode, A. & Biswas, S. K. Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat. Protoc.1, 3159–3165 (2006). PubMed