Novel ALK inhibitors containing DAAP fragments: Rational drug design and anti-tumor activity research

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase belonging to the insulin receptor subfamily, involved in cellular signaling pathways associated with insulin. While ALK gene remains inactive in normal human tissues, it exhibits high expression specifically in the central nervous system. However, dysregulation of the ALK gene has been implicated in non-small cell lung cancer (NSCLC) development. Although commercially available ALK inhibitors demonstrate favorable clinical efficacy against most Ceritinib-resistant mutants, they exhibit resistance towards G1202R mutants. Therefore, developing novel ALK inhibitors is crucial for addressing drug resistance in patients. We designed and synthesized 48 novel 2,4-diarylpyrimidine-based ALK inhibitors and investigated their antitumor activities. Among them, Ld-10 showed significant inhibitory activity against ALK kinase with an IC₅₀ value of 1135 nM and demonstrated excellent antiproliferative activity against lung cancer cells H2228 with an IC₅₀ value of 1.35 ± 0.13 μM. To further validate the antitumor potential of Ld-10, we conducted a series of in vitro pharmacological experiments. These included a hemolysis assay to confirm its low toxicity profile, an AO assay, a JC-1 staining assay, and a Calcein-AM/PI cell double staining assay for assessing apoptosis induction. Additionally, we performed dose-dependent arrest at G0/G1 phase to evaluate inhibition of cell growth and carried out cell cycle analysis and cloning experiments to provide evidence for significant tumor growth inhibition by compound Ld-10. In vivo pharmacological experiments demonstrated effective tumor growth inhibition without any significant toxic effects on mouse organs caused by Ld-10 administration. Based on these comprehensive findings from our experimental investigations, it can be concluded that Ld-10 holds promising potential as a novel ALK inhibitor.