Structure-activity relationship of 3,5-diaryl-2-aminopyridine ALK2 inhibitors reveals unaltered binding affinity for fibrodysplasia ossificans progressiva causing mutants

Abstract

There are currently no effective therapies for fibrodysplasia ossificans progressiva (FOP), a debilitating and progressive heterotopic ossification disease caused by activating mutations of ACVR1 encoding the BMP type I receptor kinase ALK2. Recently, a subset of these same mutations of ACVR1 have been identified in diffuse intrinsic pontine glioma (DIPG) tumors. Here we describe the structure-activity relationship for a series of novel ALK2 inhibitors based on the 2-aminopyridine compound K02288. Several modifications increased potency in kinase, thermal shift, or cell-based assays of BMP signaling and transcription, as well as selectivity for ALK2 versus closely related BMP and TGF-β type I receptor kinases. Compounds in this series exhibited a wide range of in vitro cytotoxicity that was not correlated with potency or selectivity, suggesting mechanisms independent of BMP or TGF-β inhibition. The study also highlights a potent 2-methylpyridine derivative 10 (LDN-214117) with a high degree of selectivity for ALK2 and low cytotoxicity that could provide a template for preclinical development. Contrary to the notion that activating mutations of ALK2 might alter inhibitor efficacy due to potential conformational changes in the ATP-binding site, the compounds demonstrated consistent binding to a panel of mutant and wild-type ALK2 proteins. Thus, BMP inhibitors identified via activity against wild-type ALK2 signaling are likely to be of clinical relevance for the diverse ALK2 mutant proteins associated with FOP and DIPG.

Figure 1

Previously described BMP inhibitors.

Scheme 1. General Procedure for the Synthesis of 2-Amino-3-(3,4,5-trimethoxyphenyl)pyridine Derivatives

Reagents and conditions: (a) 3,4,5-trimethoxyphenylboronic acid, MeCN/DMF, Na₂CO₃ (aqueous, 1 M), 10 mol % Pd(PPh₃)₄, 90 °C, 8 h, 80%; (b) arylboronic acid, DME, Na₂CO₃ (aqueous, 1 M), 10 mol %, Pd(PPh₃)₄, 90 °C, 8 h, 40–85%.

Scheme 2. General Procedure for the Synthesis of Various 2-Substituted 3-Aryl-5-(piperazinylphenyl)pyridine Derivatives

Reagents and conditions: (a) arylboronic acid, MeCN/DMF, Na₂CO₃ (aqueous, 1 M), 10 mol % Pd(PPh₃)₄, 90 °C, 8 h, 65–85%; (b) [(N-Boc)piperazin-1-yl]phenylboronic acid pinacol ester, DME, Na₂CO₃ (aqueous, 1 M), 10 mol % Pd(PPh₃)₄, 90 °C, 8 h, 70–75%; (c) TFA, DCM, rt, 12 h, 100%.

Scheme 3. Synthesis of a 2-Methyl 3-Aryl-5-(4-piperazinylphenyl)pyridine Derivatives

Reagents and conditions: (a) trimethylboroxine, 1,4-dioxane, K₂CO₃ (2 equiv), 20 mol % Pd(PPh₃)₄, 110 °C, 8 h, 90%; (b) TFA, DCM, rt, 12 h, 100%.

Figure 2

Potency and selectivity of K02288 derivatives based on thermal shift, biochemical kinase activity, and ligand induced transcriptional activity assays. (a) The 2-aminopyridine scaffold of K02288. (b) Modifications to the solvent exposed domain (R₁) of K02288. (c) Thermal shift (ΔT_m), biochemical enzymatic inhibition (IC₅₀) for ALK2 and ALK5 kinase proteins, and inhibition of cell-based BMP6 and TGF-β1-induced transcriptional activity (IC₅₀) by K02288 derivative compounds (nd = not determined). (d) Correlation of thermal shift and cell-based BMP/TGF-β inhibition assays.

Figure 3

Potency and selectivity of compound 15 derivatives based on thermal shift, biochemical kinase activity, and ligand induced transcriptional activity assays. (a) The 2-aminopyridine scaffold of 15. (b) Modifications to the ATP-binding pocket hydrophobic domain (R₂) of compound 15. (c) Thermal shift (ΔT_m), biochemical enzymatic inhibition (IC₅₀) for ALK2 and ALK5 kinase proteins, and inhibition of cell-based BMP6 and TGF-β1-induced transcriptional activity (IC₅₀) by compound 15 derivatives (nd = not determined). (d) Correlation of thermal shift and cell-based BMP/TGF-β inhibition assays.

Figure 4

Potency and selectivity of K02288 derivatives based on thermal shift, biochemical kinase activity, and ligand induced transcriptional activity assays. (a) The 2-aminopyridine scaffold of 15. (b) Modifications to the primary amine kinase hinge binding domain (R₃) of compound 15. (c) Thermal shift (ΔT_m), biochemical enzymatic inhibition (IC₅₀) for ALK2 and ALK5 kinase proteins, and inhibition of cell-based BMP6 and TGF-β1-induced transcriptional activity (IC₅₀) by compound 15 derivatives (nd = not determined). (d) Correlation of thermal shift and cell-based BMP/TGF-β inhibition assays.

Figure 5

Potency and selectivity of K02288 derivatives based on thermal shift, biochemical kinase activity, and ligand induced transcriptional activity assays. (a) Structure of hybrid derivative molecules. (b) Thermal shift (ΔT_m), biochemical enzymatic inhibition (IC₅₀) for ALK2 and ALK5 kinase proteins, and inhibition of cell-based BMP6 and TGF-β1-induced transcriptional activity (IC₅₀) by hybrid molecules (nd = not determined). (c) Correlation of thermal shift and cell-based BMP/TGF-β inhibition assays.

Figure 6

LDN-214117 exhibits selectivity for ALK2-mediated BMP signaling. (a) 10 (LDN-214117) demonstrates selective inhibition of ALK2 and ALK1 in preference to ALK3 kinase activity. (b) In a cell-based assay measuring BMP-mediated transcription (BRE-Luciferase), K02288 (b) and 15 (c) exhibit relatively limited selectivity for diverse BMP ligands, whereas 26 (d) and 10 (e) exhibit relatively selective inhibition of BMP6 versus BMP2 or BMP4, consistent with selective inhibition of ALK2- versus ALK3-mediated signaling, respectively.

Figure 7

Compound 10 exhibits increased kinome selectivity. Kinome dendrogram plot for compound 15 (LDN-212838) (a) and compound 10 (LDN-214117) (b) showing an improved selectivity profile for 10 for BMP type I receptor kinases.

Figure 8

FOP-causing ALK2 mutations do not affect inhibitor binding. (a) Strong correlation of thermal shift data for ATP competitive kinase inhibitors binding to wild-type ALK2 versus known FOP causing GS-domain mutations of ALK2 and (b) known FOP causing kinase domain mutations suggests the potency of ATP competitive inhibitors are not affected by these disease causing mutations. m = slope, R² = correlation coefficient.

Figure 9

Cytotoxicity of FDA-approved kinase inhibitor compounds as compared with BMP inhibitor compounds. Cultured HepG2 cells were exposed to 1, 10, and 100 μM concentrations of compounds for 4 and 24 h. The average cell viability of three experiments is shown (green indicating >75%, orange indicating 25–75%, and red <25% viability).

Figure 10

Binding mode of 26. (a) The inhibitor (yellow) forms a single hydrogen bond to the hinge amide of H286 as well as a water-mediated bond to the catalytic lysine K235. (b) Plot of the interactions of the inhibitor (purple) in the binding pocket of ALK2. The plot was generated by LigPlot+.