1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)-3-(2-(dimethylamino)ethyl)imidazolidin-2-one (ZX-42), a novel ALK inhibitor, induces apoptosis and protective autophagy in H2228 cells

J Pharm Pharmacol. 2020 Oct;72(10):1370-1382. doi: 10.1111/jphp.13315. Epub 2020 Jun 28.


Objectives: To examine the antiproliferative effects of 1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)-3-(2-(dimethylamino)ethyl)imidazolidin-2-one (ZX-42) on the echinoderm microtubule-associated protein-4/anaplastic lymphoma kinase fusion gene (EML4-ALK) positive lung cancer cell line H2228 and its underlying mechanism.

Methods: The MTT assay was used to study the effect of ZX-42 on H2228 cell growth. Propidium iodide (PI) staining and Western blotting were used to investigate the cell cycle changes. ZX-42-induced cell apoptosis was determined using the Annexin V-FITC/PI (AV/PI) apoptotic assay kit, acridine orange/ethidium bromide (AO/EB) and Hoechst 33258 staining, Rhodamine 123 (Rh 123) fluorescence assay and Western blotting. ZX-42-induced reactive oxygen species (ROS) production was examined by ROS assay kit. Transmission electron microscope, monodansylcadaverine (MDC) staining and the AV/PI apoptotic assay kit were used to demonstrate the relationship between autophagy and apoptosis.

Key findings: ZX-42 had good cell viability inhibitory effect on H2228 cells. ZX-42 dramatically inhibited ALK and its downstream pathways. ZX-42 also blocked H2228 cell cycle at G1 phase and then induced apoptosis by activating the mitochondrial pathway. Next, ZX-42 induced the production of ROS, and antioxidant N-acetylcysteine (NAC) reduced ROS production and also decreased apoptotic rates. We also found that ZX-42 induced protective autophagy in H2228 cells.

Conclusions: In summary, ZX-42 is a novel ALK inhibitor that significantly inhibits the cell viability of H2228 cells and ultimately induces apoptosis through the mitochondrial pathway, in which autophagy plays a protective role. Therefore, inhibition of autophagy might enhance the anti-cancer effect of ZX-42.

Keywords: EML4-ALK; H2228 cells; ZX-42; autophagy; mitochondrial apoptosis.

MeSH terms

  • A549 Cells
  • Anaplastic Lymphoma Kinase / antagonists & inhibitors*
  • Anaplastic Lymphoma Kinase / metabolism
  • Antineoplastic Agents / chemistry
  • Antineoplastic Agents / pharmacology*
  • Apoptosis / drug effects*
  • Apoptosis / physiology
  • Autophagy / drug effects*
  • Autophagy / physiology
  • Cytoprotection / drug effects*
  • Cytoprotection / physiology
  • Dose-Response Relationship, Drug
  • HEK293 Cells
  • Humans
  • Protein Kinase Inhibitors / chemistry
  • Protein Kinase Inhibitors / pharmacology*
  • Protein Structure, Secondary


  • Antineoplastic Agents
  • Protein Kinase Inhibitors
  • ALK protein, human
  • Anaplastic Lymphoma Kinase