Discovery of LAH-1 as potent c-Met inhibitor for the treatment of non-small cell lung cancer

Figures & data

Figure 1. Representative structures of reported c-Met inhibitors.

Figure 2. The design strategy of LAH-1 based on QBH-196.

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 = CHCOOC₂H_5, 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.

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 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 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 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 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.

Table 1. In Vitro ADME and in vivo PK parameters for LAH-1.

Metabolic stability^a
Species		T1/2(min)		CL int(μL/min/mg)	Remaining (T = 60min)
RLM		> 125		< 8.6	81.2%
HLM		> 125		< 8.6	88.7%
Permeability assay^b
Egg-PAMPA		Mean Pe (nm/s)		Incubation Time (h)	Permeability
–		3.4		4	Moderate
CYP450 inhibition^c
isozyme		CYP1A2	CYP2D6	CYP2C9	CYP2C19	CYP3A4
% inhibition		12.8	21.5	50.7	15.6	32.2
Pharmacokinetics^d
	Tmax (h)	Cmax(μg/mL)	AUC 0-∞ (ng•h/mL)	T1/2 (h)	CL (mL/min/kg)	F%
iv	–	14.9	16767	1.6	19.2	–
po	0.33	4.46	8210	2.2	–	12.2

^a Testosterone and Diclofenac were used at positive control.

^b The permeability assay was Performed at 10 μM concentration.

^c Performed at 10 μM concentration. α-Naphthoflavone (CYP1A2), Squinidine (CYP2D6), (+)-N-3-benzylnirvanol (CYP2C19), Quinidine (CYP2D6), and ketoconazole (CYP3A4) were used as the positive controls.

^d Determined from intravenous or PO dosing to male SD rats at a dose of 5 mg/kg and 20 mg/kg, respectively (n = 3).

Figure 8. (A) Predicted binding mode of LAH-1 to c-Met (3LQ8). Hydrogen bonds are indicated by yellow dashed lines. Images are generated using PyMol. (B) The overlay of compound LAH-1 with Foretinib in c-Met. (C) Dynamics of LAH-1 bound to c-Met (3LQ8) during 100 ns simulation time.