Figures & data
Scheme 1. Reagents and conditions: (i) a) NaNO2, 15% HCl, H2O, 0 °C, 30 min; b) Ethyl acetylacetate, C2H5COONa, EtOH/H2O, 0–25 °C, 2 h; (ii) Ph3P = CHCOOC2H5, PhMe, 110 °C, 12 h; (iii) 10% NaOH, EtOH/THF, 50 °C, 4 h; (iv) 2-fluoro-4-nitrophenol, Pyridine, PhCl, 135 °C, 72 h; (v) Phenyl chloroformate, Pyridine, CH2Cl2, 0 °C, 30 min; (vi) 3-((tert-butyldiphenylsilyl)oxy)azetidine Et3N, THF, 70 °C, 4 h; (vii) Pd/C, H2, EtOH, r.t, 5 h; (viii) 10, HATU, DIPEA, DMF, r.t, 12 h; (ix) TBAF, THF, 0 °C to r.t, 30 min.
![Scheme 1. Reagents and conditions: (i) a) NaNO2, 15% HCl, H2O, 0 °C, 30 min; b) Ethyl acetylacetate, C2H5COONa, EtOH/H2O, 0–25 °C, 2 h; (ii) Ph3P = CHCOOC2H5, PhMe, 110 °C, 12 h; (iii) 10% NaOH, EtOH/THF, 50 °C, 4 h; (iv) 2-fluoro-4-nitrophenol, Pyridine, PhCl, 135 °C, 72 h; (v) Phenyl chloroformate, Pyridine, CH2Cl2, 0 °C, 30 min; (vi) 3-((tert-butyldiphenylsilyl)oxy)azetidine Et3N, THF, 70 °C, 4 h; (vii) Pd/C, H2, EtOH, r.t, 5 h; (viii) 10, HATU, DIPEA, DMF, r.t, 12 h; (ix) TBAF, THF, 0 °C to r.t, 30 min.](/cms/asset/aaf27b58-6c9c-4a6a-a5a1-0b8b46c3abab/ienz_a_2286435_sch0001_c.jpg)
Figure 3. (A). The chemical structure of LAH-1; (B). The IC50 determination of LAH-1 against c-Met enzyme in vitro. (C). Kinase profile of LAH-1 against a panel of 18 kinases at 300 nM.
![Figure 3. (A). The chemical structure of LAH-1; (B). The IC50 determination of LAH-1 against c-Met enzyme in vitro. (C). Kinase profile of LAH-1 against a panel of 18 kinases at 300 nM.](/cms/asset/f641a40b-5f64-433a-9c7c-46c1a79d3cbc/ienz_a_2286435_f0003_c.jpg)
Figure 4. IC50 determination of LAH-1 against 5 c-Met addicted cancer cell lines and 1 non-addicted cell line. IC50 values were determined after exposure of cells to derivatives for 72 h, and data are expressed as the mean ± SD of two independent experiments.
![Figure 4. IC50 determination of LAH-1 against 5 c-Met addicted cancer cell lines and 1 non-addicted cell line. IC50 values were determined after exposure of cells to derivatives for 72 h, and data are expressed as the mean ± SD of two independent experiments.](/cms/asset/7cd1e787-f604-4185-80f4-35bebab03e00/ienz_a_2286435_f0004_c.jpg)
Figure 5. Western blot analyses of EBC-1 cells. Cells were treated with LAH-1 at the indicated concentrations for 2 h, and GADPH was used as a loading control. Each experiment was done in double, and representative images are shown, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as compared with control.
![Figure 5. Western blot analyses of EBC-1 cells. Cells were treated with LAH-1 at the indicated concentrations for 2 h, and GADPH was used as a loading control. Each experiment was done in double, and representative images are shown, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as compared with control.](/cms/asset/e5c11abb-4f07-4db9-b9d0-3ad3edb5808f/ienz_a_2286435_f0005_c.jpg)
Figure 6. Quantification of apoptotic EBC-1 cells by Annexin V-FITC/PI dual staining, the data shown are the mean ± SD from two independent experiments. Q1: Early apoptotic cells, Q2: Late apoptotic cells. Q3: Necrotic cells, Q4: Living cells. *p < 0.05, **p < 0.01 as compared with control.
![Figure 6. Quantification of apoptotic EBC-1 cells by Annexin V-FITC/PI dual staining, the data shown are the mean ± SD from two independent experiments. Q1: Early apoptotic cells, Q2: Late apoptotic cells. Q3: Necrotic cells, Q4: Living cells. *p < 0.05, **p < 0.01 as compared with control.](/cms/asset/10744184-25fa-475c-bc53-e8fec2a51959/ienz_a_2286435_f0006_c.jpg)
Figure 7. (A). EBC-1 cells were treated with different concentration of LAH-1. Cell plates were stained with crystal violet and imaged. Two independent experiments were carried out, and a representative plate is shown. (B). LAH-1 suppressed HGF-induced NCI-H441 cell invasion and migration. Representative images are shown. The relative migration (C) and invasion (D) fold change were plotted. The data shown are the mean from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as compared with control.
![Figure 7. (A). EBC-1 cells were treated with different concentration of LAH-1. Cell plates were stained with crystal violet and imaged. Two independent experiments were carried out, and a representative plate is shown. (B). LAH-1 suppressed HGF-induced NCI-H441 cell invasion and migration. Representative images are shown. The relative migration (C) and invasion (D) fold change were plotted. The data shown are the mean from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as compared with control.](/cms/asset/c9199916-5587-4020-b2da-0e87e57235b3/ienz_a_2286435_f0007_c.jpg)
Table 1. In Vitro ADME and in vivo PK parameters for LAH-1.