Pharmaceutical application of third-generation EGFR inhibitor
A technology of inhibitors and uses, applied in the field of clinical medicine, can solve problems such as insurmountable drug resistance and limited application
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Embodiment 1
[0041] Example 1 EGFR enzymatic experimental study
[0042] 1. EGFR mutant enzymology experiment:
[0043] Preparation: Compounds were dissolved in DMSO, and the highest concentration of compounds detected was 100 nM, 3-fold dilution, a total of ten concentrations.
[0044] Methods: In this experiment, fluorescence resonance energy transfer (TR-FRET) method was used to test the inhibitory effect of compounds on T790M, Del, L858R, T790M / Del19 and T790M / L858R mutant EGFR enzymes, and half of the compounds' activity on the enzyme was obtained. Inhibitory concentration IC 50 .
[0045] 1) Add 1-5 μL of EGFR mutant enzyme solution to the 384-well plate, and the final concentration of the enzyme is 0.1-1 nM.
[0046] 2) Add 1-5 μL of the compound solution diluted in gradient.
[0047] 3) Incubate at room temperature for 10 minutes.
[0048] 4) Add 1-5 μL of the substrate mixture containing the final concentration of substrate polypeptide of 5-50 nM and the final concentration of ...
Embodiment 2
[0070] Example 2 Study on the inhibitory effect of the compound on the proliferation of mutant and wild-type cells
[0071] Preparation: Compounds were dissolved in DMSO, and the highest concentration of compounds detected was 1000 nM, 3-fold dilution, a total of 9 concentrations.
[0072] Methods: In this experiment, the method of CellTiter-Glo was used to test compounds against HCC827 (Del19), PC9 (Del19), NCI-H1975 (T790M / L858R), PC9-GR (T790M / Del19) and wild-type human epidermal cell carcinoma cell line A431 Inhibitory effect of cell proliferation, and obtain the half inhibitory concentration IC of compound inhibiting cell proliferation activity 50 .
[0073] 1) Inoculate 90 μL of cell suspension in a 96-well cell culture plate at a density of 1 to 5×10 3 cells / ml, the culture plate was cultured in an incubator for 16-24 hours (37°C, 5% CO2).
[0074] 2) Add the solution of the compound to be tested at different concentrations diluted by gradient to the cells of the cul...
Embodiment 3
[0082] Example 3 Animal Model Clinical Trials
[0083] In a nude mouse tumor model, the compounds of formula I potently inhibited tumor growth in EGFR T790M-mutated NSCLC H1975 and LU1868 cell lines, and also inhibited tumor growth in EGFR-sensitive mutant NSCLC HCC827 cell lines.
[0084] In the high-dose 20 mg / kg group, the compound of formula I or a pharmaceutically acceptable salt thereof caused almost complete regression of EGFR-mutated tumors. The compound of formula I has weak inhibitory effect on the WT EGFR A431 cell line, suggesting that the compound of formula I has better safety than other EGFR inhibitors (data not shown). The compound of formula I potently and specifically inhibits the resistance mutation of EGFR T790 but has a weak effect on WT EGFR, which fully reflects the characteristics of a new generation of EGFR inhibitors.
[0085] Purpose:
[0086] The in vivo pharmacodynamic experiments confirmed that the compound has a good therapeutic effect on local...
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