Bruton's tyrosine kinase (BTK) is a non-RTK cytoplasmic kinase predominantly expressed by hemopoietic lineages, particularly B-cells. A new oxindole-based focused library was designed to identify potent compounds targeting the BTK protein as anticancer agents. This study used rational approaches like structure-based pharmacophore modeling, docking, and ADME properties to select compounds. Molecular dynamics simulations carried out at 20 ns supported the stability of compound 9g within the binding pocket. All the compounds were synthesized and subjected to biological screening on two BTK-expressing cancer cell lines, RAMOS and K562; six non-BTK cancer cell lines, A549, HCT116 (parental and p53-/-), U2OS, JURKAT, and CCRF-CEM; and two non-malignant fibroblast lines, BJ and MRC-5. This study resulted in the identification of four new compounds, 9b, 9f, 9g, and 9h, possessing free binding energies of -10.8, -11.1, -11.3, and -10.8 kcal/mol, respectively, and displaying selective cytotoxicity against BTK-high RAMOS cells. Further analysis demonstrated the antiproliferative activity of 9h in RAMOS cells through selective inhibition of pBTK (Tyr223) without affecting Lyn and Syk, upstream proteins in the BCR signaling pathway. In conclusion, we identified a promising oxindole derivative (9h) that shows specificity in modulating BTK signaling pathways.

Czech Advanced Technologies and Research Institute Institute of Molecular and Translational Medicine Palacký University Olomouc Olomouc 77900 Czech Republic

Department of Chemistry GITAM School of Science GITAM Deemed to Be University Hyderabad Telangana 502329 India

GITAM School of Pharmacy GITAM Deemed to Be University Hyderabad Telangana 502329 India

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacký University and University Hospital Olomouc Hněvotínská 1333 5 Olomouc 77900 Czech Republic

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Wang X.; Kokabee L.; Kokabee M.; Conklin D. S. Bruton’s Tyrosine Kinase and Its Isoforms in Cancer. Front. Cell Dev. Biol. 2021, 9, 668996.10.3389/fcell.2021.668996. PubMed DOI PMC

Krämer J.; Bar-Or A.; Turner T. J.; Wiendl H. Bruton tyrosine kinase inhibitors for multiple sclerosis. Nat. Rev. Neurol. 2023, 19, 289–304. 10.1038/s41582-023-00800-7. PubMed DOI PMC

Estupiñán H. Y.; Wang Q.; Berglöf A.; Schaafsma G. C. P.; Shi Y.; Zhou L.; Mohammad D. K.; Yu L.; Vihinen M.; Zain R.; et al. BTK gatekeeper residue variation combined with cysteine 481 substitution causes super-resistance to irreversible inhibitors acalabrutinib, ibrutinib and zanubrutinib. Leukemia 2021, 35, 1317–1329. 10.1038/s41375-021-01123-6. PubMed DOI PMC

Krajčovičová S.; Jorda R.; Vanda D.; Soural M.; Kryštof V. 1,4,6-Trisubstituted imidazo[4,5-c]pyridines as inhibitors of Bruton’s tyrosine kinase. Eur. J. Med. Chem. 2021, 211, 113094.10.1016/j.ejmech.2020.113094. PubMed DOI

Zhao Y.; Shu Y.; Lin J.; Chen Z.; Xie Q.; Bao Y.; Lu L.; Sun N.; Wang Y. Discovery of novel BTK PROTACs for B-Cell lymphomas. Eur. J. Med. Chem. 2021, 225, 113820.10.1016/j.ejmech.2021.113820. PubMed DOI

Zhang D.; Xu G.; Zhao J.; Wang Y.; Wu X.; He X.; Li W.; Zhang S.; Yang S.; Ma C.; Jiang Y.; Ding Q. Structure-activity relationship investigation for imidazopyrazole-3-carboxamide derivatives as novel selective inhibitors of Bruton’s tyrosine kinase. Eur. J. Med. Chem. 2021, 225, 113724.10.1016/j.ejmech.2021.113724. PubMed DOI

Fang X.; Liu C.; Zhang K.; Yang W.; Wu Z.; Shen S.; Ma Y.; Lu X.; Chen Y.; Lu T.; Hu Q.; Jiang Y. Discovery of orally active 1,4,5,6,8-pentaazaacenaphthylens as novel, selective, and potent covalent BTK inhibitors for the treatment of rheumatoid arthritis. Eur. J. Med. Chem. 2023, 246, 114940.10.1016/j.ejmech.2022.114940. PubMed DOI

Yang M.; Jiang H.; Yang Z.; Liu X.; Sun H.; Hao M.; Hu J.; Chen X.; Jin J.; Wang X. Design, synthesis, and biological evaluation of pyrrolopyrimidine derivatives as novel Bruton’s tyrosine kinase (BTK) inhibitors. Eur. J. Med. Chem. 2022, 241, 114611.10.1016/j.ejmech.2022.114611. PubMed DOI

Ran F.; Xie X.; Wu Q.; Wu H.; Liu Y.; Tao W.; Sun Y.; Wang R.; Zhang Y.; Ling Y. Development of novel hydrazidoarylaminopyrimidine-based BTK/FLT3 dual inhibitors with potent in vivo anti-hematological malignancies effects. Eur. J. Med. Chem. 2023, 245 (Pt 1), 114913.10.1016/j.ejmech.2022.114913. PubMed DOI

Li Y.-Q.; Lannigan W. G.; Davoodi S.; Daryaee F.; Corrionero A.; Alfonso P.; Rodriguez-Santamaria J. A.; Wang N.; Haley J. D.; Tonge P. J. Discovery of Novel Bruton’s Tyrosine Kinase PROTACs with Enhanced Selectivity and Cellular Efficacy. J. Med. Chem. 2023, 66, 7454–7474. 10.1021/acs.jmedchem.3c00176. PubMed DOI PMC

Guo Y.; Hu N.; Liu Y.; Zhang W.; Yu D.; Shi G.; Zhang B.; Yin L.; Wei M.; Yuan X.; et al. Discovery of BGB-8035, a Highly Selective Covalent Inhibitor of Bruton’s Tyrosine Kinase for B-Cell Malignancies and Autoimmune Diseases. J. Med. Chem. 2023, 66, 4025–4044. 10.1021/acs.jmedchem.2c01938. PubMed DOI

Zhang J.; Che J.; Luo X.; Wu M.; Kan W.; Jin Y.; Wang H.; Pang A.; Li C.; Huang W.; et al. Structural Feature Analyzation Strategies toward Discovery of Orally Bioavailable PROTACs of Bruton’s Tyrosine Kinase for the Treatment of Lymphoma. J. Med. Chem. 2022, 65, 9096–9125. 10.1021/acs.jmedchem.2c00324. PubMed DOI

Dou D.; Diao Y.; Sha W.; Su R.; Tong L.; Li W.; Leng L.; Xie L.; Yu Z.; Song H.; et al. Discovery of Pteridine-7(8H)-one Derivatives as Potent and Selective Inhibitors of Bruton’s Tyrosine Kinase (BTK). J. Med. Chem. 2022, 65, 2694–2709. 10.1021/acs.jmedchem.1c02208. PubMed DOI

Ma C.; Li Q.; Zhao M.; Fan G.; Zhao J.; Zhang D.; Yang S.; Zhang S.; Gao D.; Mao L.; et al. Discovery of 1-Amino-1H-imidazole-5-carboxamide Derivatives as Highly Selective, Covalent Bruton’s Tyrosine Kinase (BTK) Inhibitors. J. Med. Chem. 2021, 64, 16242–16270. 10.1021/acs.jmedchem.1c01559. PubMed DOI

Sabat M.; Dougan D. R.; Knight B.; Lawson J. D.; Scorah N.; Smith C. R.; Taylor E. R.; Vu P.; Wyrick C.; Wang H.; et al. Discovery of the Bruton’s Tyrosine Kinase Inhibitor Clinical Candidate TAK-020 (S)-5-(1-((1-Acryloylpyrrolidin-3-yl)oxy)isoquinolin-3-yl)-2,4-dihydro-3H-1,2,4-triazol-3-one, by Fragment-Based Drug Design. J. Med. Chem. 2021, 64, 12893–12902. 10.1021/acs.jmedchem.1c01026. PubMed DOI

Sharma S.; Monga Y.; Gupta A.; Singh S. 2-Oxindole and related heterocycles: synthetic methodologies for their natural products and related derivatives. RSC Adv. 2023, 13, 14249–14267. 10.1039/D3RA02217J. PubMed DOI PMC

Kaur M.; Singh M.; Chadha N.; Silakari O. Oxindole: A Chemical Prism Carrying Plethora of Therapeutic Benefits. Eur. J. Med. Chem. 2016, 123, 858–894. 10.1016/j.ejmech.2016.08.011. PubMed DOI

Khetmalis Y. M.; Shivani M.; Murugesan S.; Chandra Sekhar K. V. G. Oxindole and its derivatives: A review on recent progress in biological activities. Biomed. Pharmacother. 2021, 141, 111842.10.1016/j.biopha.2021.111842. PubMed DOI

Koraboina C. P.; Maddipati V. C.; Annadurai N.; Gurská S.; Džubák P.; Hajdúch M.; Das V.; Gundla R. Synthesis and biological evaluation of oxindole sulfonamide derivatives as bruton’s tyrosine kinase inhibitors. ChemMedChem 2024, 19, e20230051110.1002/cmdc.202300511. PubMed DOI

Claußen H.; Buning C.; Rarey M.; Lengauer T. FLEXE: Efficient molecular docking considering protein structure variations. J. Mol. Biol. 2001, 308, 377–395. 10.1006/jmbi.2001.4551. PubMed DOI

Andér M.; Luzhkov V. B.; Åqvist J. Ligand binding to the voltage-gated Kv1.5 potassium channel in the open state - Docking and computer simulations of a homology model. Biophys. J. 2008, 94, 820–831. 10.1529/biophysj.107.112045. PubMed DOI PMC

Sestito S.; Nesi G.; Daniele S.; Martelli A.; Digiacomo M.; Borghini A.; Pietra D.; Calderone V.; Lapucci A.; Falasca M.; Parrella P.; et al. Design and synthesis of 2-oxindole based multi-targeted inhibitors of PDK1/Akt signaling pathway for the treatment of glioblastoma multiforme. Eur. J. Med. Chem. 2015, 105, 274–288. 10.1016/j.ejmech.2015.10.020. PubMed DOI

Guan H.; Laird A. D.; Blake R. A.; Tang C.; Liang C. Design and synthesis of aminopropyltetrahydroindole-based indolin-2-ones as selective and potent inhibitors of Src and Yes tyrosine kinase. Bioorg. Med. Chem. Lett. 2004, 14, 187–190. 10.1016/j.bmcl.2003.09.069. PubMed DOI

Kravchenko D. V.; Kuzovkova Y. A.; Kysil V. M.; Tkachenko S. E.; Maliarchouk S.; Okun I. M.; Balakin K. V.; Ivachtchenko A. V. Synthesis, and Structure-Activity Relationship of 4-Substituted 2-(2-Acetyloxyethyl)-8-(morpholine-4-sulfonyl) pyrrolo [3, 4-c] quinoline-1, 3-diones as Potent Caspase-3 Inhibitors. J. Med. Chem. 2005, 48, 3680–3683. 10.1021/jm048987t. PubMed DOI

Kim M. H.; Tsuhako A. L.; Co E. W.; Aftab D. T.; Bentzien F.; Chen J.; Cheng W.; Engst S.; Goon L.; Klein R. R.; Le D. T.; et al. The design, synthesis, and biological evaluation of potent receptor tyrosine kinase inhibitors. Bioorg. Med. Chem. Lett. 2012, 22, 4979–4985. 10.1016/j.bmcl.2012.06.029. PubMed DOI

Islam I.; Bryant J.; Chou Y. L.; Kochanny M. J.; Lee W.; Phillips G. B.; Yu H.; Adler M.; Whitlow M.; Ho E.; Lentz D.; et al. Indolinone based phosphoinositide-dependent kinase-1 (PDK1) inhibitors, Part 1: design, synthesis and biological activity. Bioorg. Med. Chem. Lett. 2007, 17, 3814–3818. 10.1016/j.bmcl.2007.04.071. PubMed DOI

Liu C. W.; Lai C. L.; Lin Y. H.; Teng L. W.; Yang S. C.; Wei W. Y.; Lin S. F.; Yang J. Y.; Huang H. J.; Wang R. W.; Chiang C. C.; et al. Design and synthesis of pyrrole-5-(2, 6-dichloro benzyl) sulfonylindolin-2-ones with C-3′ side chains as potent Met kinase inhibitors. RSC Adv. 2014, 4, 58990–58998. 10.1039/C4RA08720H. DOI

Mamand S.; Allchin R. L.; Ahearne M. J.; Wagner S. D. Comparison of interleukin-2-inducible kinase (ITK) inhibitors and potential for combination therapies for T-cell lymphoma. Sci. Rep. 2018, 8, 14216.10.1038/s41598-018-32634-5. PubMed DOI PMC

Chu Y.; Lee S.; Shah T.; Yin C.; Barth M.; Miles R. R.; Ayello J.; Morris E.; Harrison L.; Van de Ven C.; Galardy P.; Goldman S. C.; Lim M. S.; Hermiston M.; McAllister-Lucas L. M.; Giulino-Roth L.; Perkins S. L.; Cairo M. S. Ibrutinib significantly inhibited Bruton’s tyrosine kinase (BTK) phosphorylation, in-vitro proliferation and enhanced overall survival in a preclinical Burkitt lymphoma (BL) model. OncoImmunology 2019, 8, e151245510.1080/2162402X.2018.1512455. PubMed DOI PMC

Pastwa E.; Somiari R. I.; Malinowski M.; Somiari S. B.; Winters T. A. In vitro non-homologous DNA end joining assays-the 20th anniversary. Int. J. Biochem. Cell Biol. 2009, 41, 1254.10.1016/j.biocel.2008.11.007. PubMed DOI PMC

Ping L.; Ding N.; Shi Y.; Feng L.; Li J.; Liu Y.; Lin Y.; Shi C.; Wang X.; Pan Z.; Song Y.; Zhu J. The Bruton’s tyrosine kinase inhibitor ibrutinib exerts immunomodulatory effects through regulation of tumor-infiltrating macrophages. Oncotarget 2017, 8, 39218–39229. 10.18632/oncotarget.16836. PubMed DOI PMC

Bender A. T.; Gardberg A.; Pereira A.; Johnson T.; Wu Y.; Grenningloh R.; Head J.; Morandi F.; Haselmayer P.; Liu-Bujalski L. Ability of Bruton’s tyrosine kinase inhibitors to sequester Y551 and prevent phosphorylation determines potency for inhibition of Fc receptor but not B-cell receptor signaling. Mol. Pharmacol. 2017, 91, 208–219. 10.1124/mol.116.107037. PubMed DOI

Sterling T.; Irwin J. J. ZINC 15 - Ligand Discovery for Everyone. J. Chem. Inf. Model. 2015, 55, 2324–2337. 10.1021/acs.jcim.5b00559. PubMed DOI PMC

Dallakyan S.; Olson A. J.. Small-Molecule Library Screening by Docking with PyRx. In Chemical Biology. Methods in Molecular Biology; Hempel J., Williams C., Hong C., Eds.; Humana Press: New York, NY; Vol. 1263. PubMed

Daina A.; Michielin O.; Zoete V. SwissADME: A free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci. Rep. 2017, 7, 42717.10.1038/srep42717. PubMed DOI PMC

Veber D. F.; Johnson S. R.; Cheng H. Y.; Smith B. R.; Ward K. W.; Kopple K. D. Molecular properties that influence the oral bioavailability of drug candidates. J. Med. Chem. 2002, 45, 2615–2623. 10.1021/jm020017n. PubMed DOI

Desmond Molecular Dynamics System, Schrödinger Release 2020–1; D. E. Shaw Research: New York, 2020.

Maestro-Desmond Interoperability Tools; Schrödinger: New York, 2020.

Grüner B.; Brynda J.; Das V.; Šícha V.; Štěpánková J.; Nekvinda J.; Holub J.; Pospíšilová K.; Fábry M.; Pachl P.; et al. Metallacarborane Sulfamides: Unconventional, Specific, and Highly Selective Inhibitors of Carbonic Anhydrase IX. J. Med. Chem. 2019, 62, 9560–9575. 10.1021/acs.jmedchem.9b00945. PubMed DOI