Article pubs.acs.org/jmc
Discovery of N‑((4-([1,2,4]Triazolo[1,5‑a]pyridin-6-yl)-5(6-methylpyridin-2-yl)‑1H‑imidazol-2-yl)methyl)-2-fluoroaniline (EW-7197): A Highly Potent, Selective, and Orally Bioavailable Inhibitor of TGF‑β Type I Receptor Kinase as Cancer Immunotherapeutic/Antifibrotic Agent Cheng Hua Jin,† Maddeboina Krishnaiah,† Domalapally Sreenu,†,# Vura B. Subrahmanyam,†,∥ Kota S. Rao,†,⊥ Hwa Jeong Lee,† So-Jung Park,‡ Hyun-Ju Park,‡ Kiho Lee,§,∇ Yhun Yhong Sheen,† and Dae-Kee Kim*,† †
Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, 11-1 Daehyun-dong, Seodaemun-gu, Seoul 120-750, Korea ‡ School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Korea § Bioevaluation Center, Korea Research Institute of Bioscience and Biotechnology, Cheongwon, Chungbuk 363-883, Korea S Supporting Information *
ABSTRACT: A series of 2-substituted-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)imidazoles was synthesized and evaluated to optimize a prototype inhibitor of TGF-β type I receptor kinase (ALK5), 6. Combination of replacement of a quinoxalin-6-yl moiety of 6 with a [1,2,4]triazolo[1,5-a]pyridin6-yl moiety, insertion of a methyleneamino linker, and a o-F substituent in the phenyl ring markedly increased ALK5 inhibitory activity, kinase selectivity, and oral bioavailability. The 12b (EW-7197) inhibited ALK5 with IC50 value of 0.013 μM in a kinase assay and with IC50 values of 0.0165 and 0.0121 μM in HaCaT (3TP-luc) stable cells and 4T1 (3TP-luc) stable cells, respectively, in a luciferase assay. Selectivity profiling of 12b using a panel of 320 protein kinases revealed that it is a highly selective ALK5/ALK4 inhibitor. Pharmacokinetic study with 12b·HCl in rats showed an oral bioavailability of 51% with high systemic exposure (AUC) of 1426 ng × h/mL and maximum plasma concentration (Cmax) of 1620 ng/mL. Rational optimization of 6 has led to the identification of a highly potent, selective, and orally bioavailable ALK5 inhibitor 12b.
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INTRODUCTION Transforming growth factor-β (TGF-β) belongs to the TGF-β superfamily, which consists of TGF-β1, TGF-β2, TGF-β3, activins, inhibins, and bone morphogenetic proteins. TGF-β is involved in many cellular processes, including cell proliferation, cell migration, invasion, epithelial−mesenchymal transition (EMT), extracellular matrix production, and immune suppression. TGF-β and its receptors are often chronically overexpressed in various human diseases, including cancer, tissue fibrosis, inflammation, and autoimmunity. Therefore, blockade of TGF-β signaling pathway is an attractive target for drug development. TGF-β signals via two related transmembrane type I and type II serine/threonine kinase receptors. Following TGF-β binding to the constitutively active type II receptor, the type I receptor (activin receptor-like kinase 5 (ALK5)) is phosphorylated and creates a binding site for Smad2/Smad3 proteins, which are further phosphorylated. Phosphorylated Smad2/Smad3 proteins form a heteromeric complex with Smad4, which translocates into the nucleus, assembles with specific DNA-binding cofactors and comodulators, and binds to the promoters of TGF-β target genes involved in cell © 2014 American Chemical Society
differentiation, proliferation, apoptosis, migration, and extracellular matrix production.1−3 Small molecule ATP-competitive ALK5 inhibitors such as 1 (SB-505124),4 2 (SB-525334),5 3 (GW6604),6 4 (SD-208),7,8 and 5 (LY-2157299)9 (Figure 1) specifically inhibited the Smad pathway by occupying the ATP binding site of the ALK5 kinase domain, which is essential for the phosphorylation of its substrates, Smad2/Smad3 proteins. The ALK5 inhibitors 4 and 5 showed promise in blocking TGF-β-mediated tumor progression, metastasis, and suppression of antitumor immunity, and 2, 3, and 4 effectively retarded progressive tissue fibrosis in animal models. Among them, 5 has progressed to phase II trials for glioma, glioblastoma, hepatocellular carcinoma, and pancreatic cancer.3 We have previously reported an imidazole-based ALK5 inhibitor, IN-1130 (6) (Figure 1).10 The inhibitor 6 effectively prevented tumor progression and enhanced immune response in mice bearing prostate cancer.11 It showed its remarkable activity as a suppressor of fibrogenic Received: January 22, 2014 Published: May 2, 2014 4213
dx.doi.org/10.1021/jm500115w | J. Med. Chem. 2014, 57, 4213−4238
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Figure 1. ALK5 inhibitors under development.
inhibitory activity.19 To further optimize the linker, we compared the potential of methyleneoxy or methylenethio group with that of methyleneamino group. It was one of our concerns whether an incorporated heteroatom such as O, S, or N into those three linkers might form another binding interaction with ALK5.
process in models of unilateral ureteral obstruction (UUO) in adult mice12 and rats,13 ameliorated experimental autoimmune encephalomyelitis in mice,14 and significantly reduced tunical fibrosis and corrected penile curvature in rats.15 Although 6 demonstrated its pronounced efficacy in various animal models of cancer and fibrosis, there were two drawbacks to develop it as a clinical candidate: (1) pharmacokinetic studies with 6 showed good oral bioavailability in dog (85%) and monkey (34%) but much lower in mouse (9%) and rat (11%), and (2) the kinase selectivity index of 6 against p38α having one of the most homologous kinase domains to that of ALK5 is 176. Because 9a, 9b, and 12a showed the similar level of potency in ALK5 inhibition in a kinase assay, a cell-based luciferase assay was performed using HaCaT cells permanently transfected with p3TP-luciferase reporter construct25 at a lower concentration, 0.03 μM, to discriminate their ALK5 inhibitory activity (Table 2). Contrary to the kinase assay, 9a (11% inhibition) and 9b (8% inhibition) were much less inhibitory than 12a (52% inhibition) in a cellbased luciferase assay, suggesting that a methyleneoxy or methylenethio linker contributes to the lower cellular permeability compared to a methyleneamino linker. We next determined the solubility of 9a, 9b, and 12a in pH 1.2 and 6.8 aqueous buffers (Table 2). Indeed, although 9a, 9b, and 12a were almost insoluble in pH 6.8 aqueous buffer (10.00 1.98 1.94 1.53 1.10 3.68 2.56 1.30 2.05 3.64 3.94 1.63 2.83 0.82 0.73 0.87 1.31 1.18 1.21 2.43 1.86 0.70 1.46 1.50 1.54
376 297 176 246 >1111 283 139 139 79 263 213 130 146 182 131 109 71 91 122 174 146 148 76 174 81 100 122 300 171
a
compd
X
R
ALK5a
p38αb
selectivity indexc
12ab 12ac 12ad 12ae 12af 12ag 12ah 12ai 12aj 12ak 12al 12am 12an 12ao 12ap 12aq 12ar 12as 12at 12au 12av 12aw 12ax 12ay 12az 12ba 12bb 12bc 12bd 12be 12bf 12bg 12bh 12bi 1 6
NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH
3-CH2CN 3-COMe 3-CO2Me 3-NHSO2Me 3-NHCOMe 4-F 4-Cl 4-Br 4-Me 4-i-Pr 4-OMe 4-OCF3 4-SMe 4-CHCH2 4-CN 4-CONH2 4-CH2CN 4-COMe 4-CO2Me 4-NHSO2Me 4-NHCOMe 2,3-F2 2,3-Cl2 2,3-Me2 2,3-(OMe)2 2,3-(CN)2 3,4-F2 3,4-Cl2 3,4-Me2 3,4-(OMe)2 3,5-F2 3,5-Cl2 3,5-Me2 3,5-(OMe)2
0.011 0.013 0.017 0.015 0.015 0.017 0.011 0.017 0.016 0.046 0.048 0.060 0.040 0.030 0.032 0.055 0.025 0.059 0.078 0.047 0.039 0.009 0.012 0.018 0.058 0.021 0.010 0.008 0.017 0.044 0.006 0.015 0.014 0.014 0.054 0.017
1.41 1.28 1.39 2.27 3.98 3.93 9.30 >10.00 >10.00 >10.00 >10.00 >10.00 >10.00 >10.00 7.81 >10.00 >10.00 >10.00 >10.00 >10.00 >10.00 2.54 8.64 3.49 >10.00 >10.00 1.25 3.89 >10.00 >10.00 0.50 1.07 3.00 2.85 0.59 0.48
128 98 82 151 265 231 845 >588 >625 >217 >208 >166 >250 >333 244 >181 >400 >169 >128 >212 >256 282 720 194 >172 >476 125 486 >588 >227 83 71 214 204 11 28
ALK5 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) using casein as a substrate. bp38α MAP kinase was expressed as untagged human recombinant protein in E. coli. The enzyme was purified by Ni-NTH-agarose (Qiagen). A proprietary radioisotopic protein kinase assay (33PanQinase activity assay) was performed at ProQinase GmbH (Freiburg, Germany) using ATF2 as a substrate. cIC50 of p38α/IC50 of ALK5.
be not accommodated favorably into the ATP binding pocket of ALK5. In the 13 disubstituted compounds, di-F substituted compounds 12aw (R = 2,3-F2, IC50 = 0.009 μM), 12bb (R = 3,4-F2, IC50 = 0.010 μM), and 12bf (R = 3,5-F2, IC50 = 0.006 μM) and di-Cl substituted compounds 12ax (R = 2,3-Cl2, IC50 = 0.012 μM) and 12bc (R = 3,4-Cl2, IC50 = 0.008 μM) were more inhibitory than 12a. Considering the IC50 of ALK5 inhibition and selectivity index against p38α, the 12b−d, 12p−s, 12x, 12z, 12aa, 12ai, 12aj, 12aw, and 12bf were chosen and their ALK5 inhibitory activity was compared at a concentration of 0.03 μM in a luciferase assay (Table 3). All the tested compounds except 12aa (2% inhibition) showed higher inhibitory activity (37−71% inhibition) than the competitor 1 (26% inhibition) in this luciferase assay. In the
impact on ALK5 inhibitory activity, thus, 12b (R = 2-F, IC50 = 0.007 μM), 12c (R = 2-Cl, IC50 = 0.009 μM), and 12d (R = 2-Br, IC50 = 0.007 μM) displayed higher ALK5 inhibitory activity than the unsubstituted 12a (IC50 = 0.013 μM) in a kinase assay (Table 1). At meta-position, halogen atoms, small alkyl groups such as Me and Et, and small functional groups, SMe and CN, were beneficial to the ALK5 inhibitory activity. Hence, 12p (R = 3-F, IC50 = 0.009 μM), 12q (R = 3-Cl, IC50 = 0.006 μM), 12r (R = 3-Br, IC50 = 0.005 μM), 12s (R = 3-Me, IC50 = 0.009 μM), 12t (R = 3-Et, IC50 = 0.008 μM), 12x (R = 3-SMe, IC50 = 0.007 μM), and 12z (R = 3-CN, IC50 = 0.005 μM) were more inhibitory than 12a. In contrast with the ortho- and meta-substituted compounds, 15 out of 16 para-substituted compounds displayed lower ALK5 inhibitory activity than 12a, suggesting that a para-substituent in the phenyl ring seemed to 4216
dx.doi.org/10.1021/jm500115w | J. Med. Chem. 2014, 57, 4213−4238
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less soluble in pH 6.8 aqueous buffer (10 >10
% inhibition at 10 μMa 23.3 30.1 30.7 14.6 44.8
± ± ± ± ±
3.9 3.3 4.0 1.6 5.3
Mean ± SD (n = 3).
The safety pharmacology studies of 12b were conducted to assess its effect on the cardiovascular system, central nervous system, and respiratory system. Inhibition of hERG channels is associated with potential risk of QT prolongation. The whole cell patch-clamp technique was used to record inward rectifying potassium currents (IKr) from single HEK293 cells stably expressing the potassium hERG channel (MPI Research, Inc. Mattawan, MI).50 Superfusion of 12b at concentrations of 1, 10, 30, and 100 μM produced mean concentration-dependent inhibition of 1.79%, 22.13%, 50.84%, and 76.60%, respectively, with an IC50 value of 31.04 μM, indicating low cardiac toxicity risk (Table 14). Table 14. Effect of 12b on Cloned hERG Channels Expressed in Human Embryonic Kidney (HEK293) Cellsa compd
concentration (μM)
vehicleb 12b 12b 12b 12b cisaprided
0 1 10 30 100 0.1
hERG current inhibition (%) 1.18 1.79 22.13 50.84 76.60 83.60
± ± ± ± ± ±
0.343c 0.457 3.579 0.219 2.135 0.977
IC50 (μM) 31.04
a
Assay was performed at MPI Research, Inc. (Mattawan, MI). Physiological salt solution (PSS) with 0.1% DMSO. cMean ± SE (n = 3). dPositive control compound.
b
The potential cardiovascular effect of 12b was evaluated in conscious freely moving beagle dogs instrumented with radiotelemetry transmitters measuring electrocardiogram (ECG), heart rate, and blood pressure (MPI Research, Inc. Mattawan, MI). Oral administration of 12b at dose levels of 10, 30, and 100 mg/kg was without effect on clinical observations, body weight, body temperature, ECG, heart rate, and blood pressure, establishing a no-observed-effect level (NOEL) of at least 100 mg/kg. The potential acute neurobehavioral toxicity of 12b and the potential effect of 12b on respiratory function were evaluated in SD rats (MPI Research, Inc. Mattawan, MI). Acute oral
Figure 5. Docking pose of candidate compounds in the active site of ALK5 (PDB: 1RW8). (A) Docking pose of 12b (magenta capped sticks). (B) 12a (cyan capped sticks). (C) 9a (yellow capped sticks). (D) 9b (green capped sticks). Gray capped sticks represent key amino acid residues within the binding site and ribbon is backbone of ALK5. Ball and stick is water molecule. Red dashed lines are hydrogen bonding interactions (
