Synthesis and Structure-Activity Relationships of 3,5-Disubstituted-pyrrolo[2,3- b]pyridines as Inhibitors of Adaptor-Associated Kinase 1 with Antiviral Activity

Abstract

There are currently no approved drugs for the treatment of emerging viral infections, such as dengue and Ebola. Adaptor-associated kinase 1 (AAK1) is a cellular serine-threonine protein kinase that functions as a key regulator of the clathrin-associated host adaptor proteins and regulates the intracellular trafficking of multiple unrelated RNA viruses. Moreover, AAK1 is overexpressed specifically in dengue virus-infected but not bystander cells. Because AAK1 is a promising antiviral drug target, we have embarked on an optimization campaign of a previously identified 7-azaindole analogue, yielding novel pyrrolo[2,3- b]pyridines with high AAK1 affinity. The optimized compounds demonstrate improved activity against dengue virus both in vitro and in human primary dendritic cells and the unrelated Ebola virus. These findings demonstrate that targeting cellular AAK1 may represent a promising broad-spectrum antiviral strategy.

Figure 1.

Known AAK1 inhibitors

Figure 2.

AAK1 inhibitors with documented antiviral activity

Figure 3.

A pyrrolo[2,3-b]pyridine (7-aza-indole) based AAK1 inhibitor

Figure 4.

Binding displacement assay of compound 1 against the NAK family members.

Figure 5.

Crystal structure of AAK1 (grey) in complex with compound 1 (purple), PDB ID 5L4Q. A: Overview of the crystal structure of AAK1. Highlighted are areas that are important for kinase function. Compound 1 bound at the hinge is shown. ASCH = Activation Segment C-terminal Helix, a feature unique to kinases of the NAK family. B: 2F_o-F_c Electron density map contoured at 1σ around compound 1, showing the fit of the model to the map. C and D: Detailed views of the interactions of compound 1 in the ATP-binding site from two orientations. Black dotted lines indicate polar interactions, red spheres indicate water molecules. Note that residues 48–63 from β1 and β2 are removed from C for clarity.

Figure 6.. Compounds 8g and 21b suppress DENV infection more effectively than compound 1.

Dose response of DENV infection (blue) and cell viability (black) to compounds 1, 8g, and 21b measured by luciferase and alamarBlue assays, respectively, 48 hours after infection. Data are plotted relative to vehicle control. Shown are representative experiments from at least two conducted, each with 5 biological replicates; shown are means ± SD.

Figure 7.. Compounds 1, 8g and 21b suppress EBOV infection.

Dose response of EBOV infection (blue) and cell viability (black) to compounds 1, 8g and 21b measured by plaque assay (for compound 1) or immunofluorescence assays (for compounds 8g and 21b) and CellTiter-Glo luminescent cell viability assay in Huh7 cells 48 hours after infection. Data are plotted relative to vehicle control. Shown are representative experiments from at least two conducted, each with 3 biological replicates; shown are means ± SD.

Figure 8.. Antiviral effect of compounds 1, 8g and 21b correlate with functional inhibition of AAK1.

Dose response of AP2M1 phosphorylation to treatment with 1 (A), 8g (B), and 21b (C) by Western analysis in lysates derived from Huh7 cells. Representative membranes (from two independent experiments) blotted with anti-phospho-AP2M1 (pAP2M1), anti-AP2M1 (AP2M1), and anti-Actin (Actin) antibodies and quantified data of pAP2M1/actin protein ratio normalized to DMSO controls are shown. ** p < 0.01, *** p < 0.001 by 2-tailed unpaired t test.

Figure 9.. Ex vivo antiviral activity of 21b and 8g in human primary dendritic cells.

Dose response of DENV infection (blue) and cell viability (black) to compounds 21b (A) and 8g (B) measured by plaque assays and alamarBlue assays, respectively, 72 hours after infection of primary human monocyte-derived dendritic cells (MDDCs). Shown is a representative experiment with cells from a single donor, out of 2 independent experiments conducted with cells derived from 2 donors, each with 6 biological replicates; shown are means ± SD.

Figure 10.

Kinome tree of compound 21b. Kinases that bind compound 21b are marked with red circles. The larger the circle, the stronger the binding affinity.

Figure 11.

AAK1 snapshot structures with 3 ligands. Compound 1 (green, lightgreen), compound 8g (magenta, rose) and compound 21b (cyan; lightblue), taken extracted from the last 4 ns of the MD trajectory as representative structure from the biggest cluster using the average linkage clustering method available in cpptraj. The AAK1 and lkb X-ray structures are also shown (orange, yellow). Image made by the Chimera software.

Figure 12.

AAK1 enzyme with compound 21b (same snapshot as in Figure 11). Amino acids making contact with the two methoxy groups (heavy atom distances < 4 Å) are coloured in yellow and magenta. The magenta colour represents also the residues having Van der Waals contacts with the nitrile group of compound 1. Image made by the Chimera software.

Scheme 1.

Synthesis of 3-substituted-5-aryl pyrrolo[2,3-b]pyridines 8a-p Reagents and conditions. a) TMSA, Pd(PPh₃)₂Cl₂, CuI, Et₃N, THF, rt; b) KOtBu, NMP, 80°C; c) HNO₃, 0°C to rt; d) Pd(PPh₃)₄, K₂CO₃, ArB(OH)₂, H₂O, dioxane, 105°C; e)H₂, Pd/C, THF, rt; f)RCOCl, pyridine, THF, 1M NaOH, rt; g)RCOOH, BOP, Et₃N, DMF, rt; h)PhSO₂Cl, pyridine, rt.

Scheme 2.

Synthesis of 3-benzoyl-5-(3,4-dimethoxyphenyl)-pyrrolo[2,3-b]pyridine 14 Reagents and conditions. a) hexamine, H₂O, CH₃COOH, 120°C ; b) NaH, TsCl, 0°C to rt ; c) 3,4-dimethoxyphenylboronic acid, Pd(PPh₃)₄, 2M K₂CO₃, toluene, EtOH, 105°C ; d) 3M PhMgBr, THF, rt; e) MnO₂, THF, rt ; f) KOH, EtOH, 80°C.

Scheme 3.

Synthesis of 3-phenyl-5-(3,4-dimethoxyphenyl)-pyrrolo[2,3-b]pyridine 19 Reagents and conditions. a) NIS, acetone, rt ; b) NaH, TsCl, THF, 0°C to rt ; c) PhB(OH)₂, Pd(PPh₃)₄, K₂CO₃, toluene, EtOH, H₂O, 90°C ; d) 3,4-dimethoxyphenylboronic acid, Pd(PPh₃)₄, K₂CO₃, toluene, EtOH, H₂O, 105°C ; e) KOH, EtOH, 80°C.

Scheme 4.

Synthesis of 3-alkynyl-5-(3,4-dimethoxyphenyl)-pyrrolo[2,3-b]pyridines 21a-f Reagents and conditions. a) RC≡CH, Pd(PPh₃)₂Cl₂, CuI, THF, Et₃N, rt ; b) 3,4-dimethoxyphenylboronic acid, Pd(PPh₃)₄, K₂CO₃, H₂O, dioxane, 105 °C.

Scheme 5.

Synthesis of 1-methyl-1H-pyrrolo[2,3-b]pyridine 26. Reagents and conditions. a) NaH, MeI, THF, 0°C to rt ; b) HNO₃, 0°C to rt ; c) 3,4-dimethoxyphenylboronic acid, Pd(PPh₃)₄, K₂CO₃, H₂O, dioxane, 105°C ; d) H₂, THF, rt ; e) nicotinoyl chloride, pyridine, THF, 1M NaOH, rt.

Scheme 6.

Synthesis of pyrazolo[3,4-b]pyridine 30 Reagents and conditions. a) 3,4-dimethoxyphenylboronic acid, Pd(PPh₃)₄, K₂CO₃, H₂O, dioxane, 105°C ; b) 35% hydrazine hydrate, EtOH, 80°C ; c) nicotinoyl chloride, pyridine, rt.

Scheme 7.

Synthesis of pyrrolo[2,3-b]pyrazine 37 Reagents and conditions. a) NBS, DMSO, rt ; b) Me₃SiC≡CH, Pd(PPh₃)₂Cl₂, CuI, THF, Et₃N, rt ; c) KOtBu, NMP, 100°C ; d) NIS, acetone, rt ; e) 3-ethynylpyridine, Pd(PPh₃)₂Cl₂, CuI, THF, Et₃N, rt ; f) 3,4-dimethoxyphenylboronic acid, Pd(PPh₃)₄, K₂CO₃, H₂O, dioxane, 105 °C.