To study the in vitro and in vivo pharmacodynamics of dirozalkib.

Prepare dirozalkib solutions with concentrations ranging from 3.81×10^-3 to 1 000.00 nmol·L^-1. The activities of dirozalkib against kinases anaplastic lymphoma kinase (ALK), ALK-L1196M, and ALK-G1202R were evaluated using the Mobility Shift Assay and Radioisotope Filter Binding methods. The inhibitory effect of dirozalkib on cell proliferation was evaluated using the CellTiter-Glo assay in multiple Ba/F3 cell lines transfected with EML4-ALK fusion genes and drug-resistant mutants, at concentrations ranging from 1.53×10^-2 to 1 000.00 nmol·L^-1. Ba/F3-EML4-ALK-L1196M (crizotinib-resistant) cells were subcutaneously inoculated in NOD SCID mice. The tumor-bearing mice were randomly divided into blank group (solvent control), experimental group (50 mg·kg^-1 dirozalkib), control group (50 mg·kg^-1 crizotinib), each group consisted of 9 mice. The mice were gavage once a day, continuous administration over a 14-day period. BALB/c nude mice were used to establish a human lung cancer tumor tissue-derived LU-01-0319R (crizotinib-resistant) patient-derived tumor xenograft (PDX) model. The tumor-bearing mice were randomly divided into blank group (solvent control), experimental group (24 mg·kg^-1 dirozalkib), control group (24 mg·kg^-1 crizotinib), each group consisted of 9 mice. The mice were gavage once a day, continuous administration over a 21-day period. Human lung cancer H228-luc cells were intracranially inoculated in BALB/c nude mice. The tumor-bearing mice were randomly divided into blank group (solvent control), experimental group (100 mg·kg^-1 dirozalkib), control group (100 mg·kg^-1 crizotinib), each group consisted of 9 mice. The mice were gavage once a day, continuous administration over a 14-day period. Assessed the anti-tumor efficacy of dirozalkib.

Dirozalkib significantly inhibited the kinase activities of ALK, ALK-L1196M, and ALK-G1202R [50% inhibiting concentration (IC₅₀) <1.0 nmol·L^-1]. In cells, dirozalkib exhibited significant proliferation inhibitory activity against NCI-H3122 cells harboring EML4-ALK fusion (IC₅₀ = 3.00 nmol·L^-1). Additionally, it demonstrated potent inhibitory activity against multiple cell lines with EML4-ALK fusion and drug-resistant mutants of crizotinib and second-generation ALK inhibitors (IC₅₀: 1.92-62.85 nmol·L^-1). In the crizotinib-resistant Ba/F3-EML4-ALK-L1196M xenograft model, dirozalkib at a dose of 50 mg·kg^-1 had a significant anti-tumor effect, with a relative tumor proliferation rate of 6.14%. In the LU-01-0319R human lung cancer xenograft model (crizotinib-resistant PDX model), the 24 mg·kg^-1 dirozalkib also had a significant tumor inhibitory effect, with a relative tumor proliferation rate of 13.19%. For the H228-luc human lung cancer intracranial inoculation model, dirozalkib at 100 mg·kg^-1 could significantly inhibit tumor growth [BLI value was (569.34±153.65) × 10⁵ photons·s^-1]and could significantly prolong the survival time of the animals.

Dirozalkib effectively inhibits ALK and various resistance mutations associated with first- and second-generation ALK-TKIs, and shows significant antitumor activity in multiple tumor models, including intracranial implantation models.