Synthesis, biological evaluation and molecular modelling of 2,4-disubstituted-5-(6-alkylpyridin-2-yl)-1H-imidazoles as ALK5 inhibitors

Figures & data

Figure 1. Small-molecule ATP-competitive ALK5 inhibitors in clinical trials.

Scheme 1. Reagents and conditions: (a) NH₄OAc, AcOH, 120 °C, 3 h; (b) 28% H₂O₂, 6 N NaOH, EtOH, 55 °C, 3 h.

Scheme 2. Reagents and conditions: (a) NaNO₂, 5 N HCl, rt, 1 h; (b) 5a or 3-(2-oxoethyl)benzonitrile (5d), NH₄OAc, t-BuOMe/MeOH, rt, overnight, Ar atmosphere; (c) (i) 28% H₂O₂, 6 N NaOH, EtOH, DMSO, 55 °C, overnight; (ii) triethyl phosphite, anhydrous DMF, 110 °C, 72 h.

Scheme 3. Reagents and conditions: (a) 28% H₂O₂, 6 N NaOH, MeOH, 55 °C, 2 h; (b) 1 N HCl, THF, reflux, 1 h.

Scheme 4. Reagents and conditions: (a) 14a or 14 b, NH₄OAc, t-BuOMe/MeOH, 30 °C, overnight, Ar atmosphere.

Table 1. ALK5 inhibitory activity and Caco-2 cell permeability of 2,4-disubstitubed-5–(6-alkylpyridin-2-yl)-1H-imidazoles 7a–c, 11a–h, and 16a–h.

Compound	R	CONH2	n	IC50^a,b (μM)	p3TP-luciferase activity^d (% control)	Caco-2 permeability (%)
Mock					2 ± 0^e
TGF-β					100 ± 5.0
7a			1	0.224	28 ± 7.1	35 ± 0.7^f
7b			2	0.093	26 ± 7.9	23 ± 3.0
7c			3	0.338	60 ± 9.5	32 ± 1.1
11a	Me	p-CONH2	1	0.088	16 ± 4.5	25 ± 1.3
11b	Et	p-CONH2	1	0.112	33 ± 21.3	26 ± 3.5
11c	i-Pr	p-CONH2	1	2.195	39 ± 11.4	30 ± 9.0
11d	n-Bu	p-CONH2	1	2.700	85 ± 32.5	41 ± 5.3
11e	Me	m-CONH2	1	0.036^c	8 ± 5.8	56 ± 26.8
11f	Et	m-CONH2	1	0.075	17 ± 3.7	38 ± 13.8
11g	i-Pr	m-CONH2	1	1.250	69 ± 6.7	34 ± 15.6
11h	n-Bu	m-CONH2	1	1.038	83 ± 21.1	n.d.^g
16a	Me	p-CONH2	2	0.030	15 ± 8.2	45 ± 5.3
16b	Et	p-CONH2	2	0.041	16 ± 8.4	25 ± 3.7
16c	i-Pr	p-CONH2	2	0.653	85 ± 38.9	23 ± 3.1
16d	n-Bu	p-CONH2	2	2.383	76 ± 5.7	24 ± 1.9
16e	Me	m-CONH2	2	0.047	7 ± 0.7	21 ± 9.5
16f	Et	m-CONH2	2	0.066	56 ± 14.6	6 ± 4.1
16g	i-Pr	m-CONH2	2	0.593	48 ± 41.8	0
16h	n-Bu	m-CONH2	2	2.875	92 ± 10.4	0

^aALK5 was expressed in Sf9 insect cells as human recombinant GST-fusion protein by means of the vaculovirus expression system. A Proprietary radioisotopic protein kinase assay (³³PanQinase^® Activity Assay) was performed at ProQinase GmbH (Freiburg, Germany). The assay contained 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl₂, 3 mM MnCl₂, 3 μM Na-orthovanadate, 1.2 mM DTT, 50 μg/mL PEG₂₀₀₀₀, 1 μM [γ-³³P]-ATP (approximately 5 × 10⁵ cpm per well). One hundred ng/50 μL of ALK5 and 1000 ng/50 μL of casein as a substrate were used per well.

^bSinglicate.

^cMean of triplicates.

^dHaCaT (p3TP-luc) stable cells were used. Luciferase activity was determined at a concentration of 0.1 μM of inhibitor.

^eThe mean ± SD of three independent experiments run in triplicates relative to control incubations with DMSO vehicle.

^fThe mean ± SD of quadruplicates.

^gNot determined.

Table 2. ALK5 and p38α inhibitory activity of 11e, 1, and 2.

Compound	IC50 for ALK5 (μM)^a,b	IC50 for p38α (μM)^b,c	Selectivity index^d	IC50 for p3TP-luciferase (μM)^e,f
11e	0.013	0.288	22	0.0196
Galunisertib (1)	0.086	0.320	4	>0.1
Vactosertib (2)	0.013	1.775	137	0.0165

^aA proprietary radioisotopic protein kinase assay (³³PanQinase^® Activity Assay) was performed at ProQinase GmbH (Freiburg, Germany). The assay contained 70 mM HEPES-NaOH, pH 7.5, 3 mM MgCl₂, 3 mM MnCl₂, 3 μM Na-orthovanadate, 1.2 mM DTT, 50 μg/mL PEG₂₀₀₀₀, 1 μM [γ-³³P]-ATP (approximately 6 × 10⁵ cpm per well). Five ng/50 μL of ALK5 and 1000 ng/50 μL of GSK3 (14–27) as a substrate were used per well.

^bMean of duplicates.

^cA proprietary radioisotopic protein kinase assay (³³PanQinase^® Activity Assay) was performed at ProQinase GmbH (Freiburg, Germany) using ATF2 as a substrate.

^dIC₅₀ for p38α/IC₅₀ for ALK5.

^eHaCaT (3TP-luc) stable cells were used.

^fMean of triplicates.

Figure 2. Inhibitory profile of 11e in 28 protein kinase assays.

Figure 3. Docked pose of 11e in the active site of ALK5 (PDBid:1RW8). (A, B) 11e is (magenta carbon atoms) superimposed over the X-ray pose of native ligand (grey carbon atoms). The active site of ALK5 is shown as MOLCAD lipophilic potential surface map (A), and lipophilicity increases from blue (hydrophilic) to brown (liphophilic). Grey capped sticks represent key amino acid residues within the binding site, and the backbone of ALK5 is shown as ribbon. The bound water molecule in the X-ray structure is represented by ball and stick. (C) Intermolecular interaction between 11e and ALK5. Grey capped sticks represent key amino acid residues in the active site. Red dashed lines are hydrogen bonding interactions (<3.0 Å).