Glioblastoma multiforme (GBM) remains a lethal brain cancer with poor prognosis, largely due to the limitations of current therapies. Focal adhesion kinase (FAK) is a promising therapeutic target, given its central role in driving GBM progression, invasion, and microenvironment adaptation. While several FAK inhibitors, such as TAE-226, have entered clinical trials, none have achieved approval, underscoring the need for novel, more effective agents. Through a structure-based molecular hybridization strategy that integrated key pharmacophores from TAE-226 and the clinical candidate GSK2256098, a novel series of N-methoxy-2-(pyrimidin-4-ylamino)benzamide derivatives were designed and synthesized. Among the eighteen synthesized compounds, 7h emerged as the most potent compound. It demonstrated superior enzymatic and cellular activity, with FAK inhibitory activity (IC50 = 3.62 nM) and potent anti-proliferative activity against U87-MG glioblastoma cell line (IC50 = 71 nM), outperforming the positive control TAE-226 (IC50 = 4.81 nM and 0.49 µM, respectively). Furthermore, in the PAMPA-BBB assay, the passive permeability (Pe) of 7h reached 25.97 × 10−6 cm∙s⁻1, which was comparable to and slightly higher than TAE-226 (24.80 × 10−6 cm∙s⁻1), suggesting favorable passive permeation potential that warrants further in vivo brain distribution studies. Mechanistic studies in U87-MG cells revealed that compound 7h induced apoptosis and pronounced G2/M cell cycle arrest compared to TAE-226 at equivalent concentrations, suggesting a potent anti-proliferative effect associated with FAK inhibition. In a U87-MG xenograft mouse model, 7h exhibited favorable in vivo dose-dependent antitumor efficacy with this specific model. At 10 mg/kg/day, its tumor growth inhibition (TGI = 54.1%) exceeded that of TAE-226 (TGI = 50.1%), while a dose of 20 mg/kg/day achieved enhanced TGI of 83.44%. Molecular docking simulations suggested that 7h combined within the ATP pocket of FAK, facilitated by synergistic hydrophobic interactions and key hydrogen bonds with residues ILE-428 and CYS-502. Overall, as a preliminary proof-of-concept, compound 7h represents a novel and promising FAK inhibitor, demonstrating potential for overcoming current therapeutic limitations in GBM.

Design, synthesis and anti-glioblastoma multiforme activity of N-methoxy-2-(pyrimidin-4-ylamino)benzamide derivatives as focal adhesion kinase inhibitors

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发表日期：

2026年7月23日

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Yang Chen,
Ting Wu,
Haolin Yang,
Aoxiang Fan

摘要

Glioblastoma multiforme (GBM) remains a lethal brain cancer with poor prognosis, largely due to the limitations of current therapies. Focal adhesion kinase (FAK) is a promising therapeutic target, given its central role in driving GBM progression, invasion, and microenvironment adaptation. While several FAK inhibitors, such as TAE-226, have entered clinical trials, none have achieved approval, underscoring the need for novel, more effective agents. Through a structure-based molecular hybridization strategy that integrated key pharmacophores from TAE-226 and the clinical candidate GSK2256098, a novel series of N-methoxy-2-(pyrimidin-4-ylamino)benzamide derivatives were designed and synthesized. Among the eighteen synthesized compounds, 7h emerged as the most potent compound. It demonstrated superior enzymatic and cellular activity, with FAK inhibitory activity (IC₅₀ = 3.62 nM) and potent anti-proliferative activity against U87-MG glioblastoma cell line (IC₅₀ = 71 nM), outperforming the positive control TAE-226 (IC₅₀ = 4.81 nM and 0.49 µM, respectively). Furthermore, in the PAMPA-BBB assay, the passive permeability (Pe) of 7h reached 25.97 × 10⁻⁶ cm∙s⁻¹, which was comparable to and slightly higher than TAE-226 (24.80 × 10⁻⁶ cm∙s⁻¹), suggesting favorable passive permeation potential that warrants further in vivo brain distribution studies. Mechanistic studies in U87-MG cells revealed that compound 7h induced apoptosis and pronounced G2/M cell cycle arrest compared to TAE-226 at equivalent concentrations, suggesting a potent anti-proliferative effect associated with FAK inhibition. In a U87-MG xenograft mouse model, 7h exhibited favorable in vivo dose-dependent antitumor efficacy with this specific model. At 10 mg/kg/day, its tumor growth inhibition (TGI = 54.1%) exceeded that of TAE-226 (TGI = 50.1%), while a dose of 20 mg/kg/day achieved enhanced TGI of 83.44%. Molecular docking simulations suggested that 7h combined within the ATP pocket of FAK, facilitated by synergistic hydrophobic interactions and key hydrogen bonds with residues ILE-428 and CYS-502. Overall, as a preliminary proof-of-concept, compound 7h represents a novel and promising FAK inhibitor, demonstrating potential for overcoming current therapeutic limitations in GBM.