Identification of 1H-pyrazolo[3,4-b]pyridine derivatives as novel and potent TBK1 inhibitors: design, synthesis, biological evaluation, and molecular docking study

sABSTRACT TANK-binding kinase 1 (TBK1), a noncanonical member of the inhibitor-kappaB kinases (IKKs) family, plays a vital role in coordinating the signalling pathways of innate immunity, involving in the process of neuroinflammation, autophagy, and oncogenesis. In current study, based on rational drug design strategy, we discovered a series of 1H-pyrazolo[3,4-b]pyridine derivatives as potent TBK1 inhibitors and dissected the structure–activity relationships (SARs). Through the several rounds of optimisation, compound 15y stood out as a potent inhibitor on TBK1 with an IC50 value of 0.2 nM and also displayed good selectivity. The mRNA detection of TBK1 downstream genes showed that compound 15y effectively inhibited TBK1 downstream IFN signalling in stimulated THP-1 and RAW264.7 cells. Meanwhile, compound 15y exhibited a micromolar antiproliferation effect on A172, U87MG, A375, A2058, and Panc0504 cell lines. Together, current results provided a promising TBK1 inhibitor 15y as lead compound for immune- and cancer-related drug discovery.


5-bromo-N-isopropylbenzene-1,2-diamine(17)
A well stirred mixture of 16 (4.64 g, 18 mol), reduced iron powder (6.05 g, 108 mmol), and ammonium chloride (2.91 g, 54 mmol) in ethanol and water (30 mL, v/v=3/l) was heated to 80 ℃ for 2 h. After cooling to room temperature, it was filtered on Celite, the filtrate was concentrated under reduced pressure to give a brown oil in 93% yield, which was used in the next step without further purification.

6-bromo-1-isopropyl-1H-benzo[d]imidazole (18)
Compound 17 (1.50 g, 6.57 mmol) was added into formic acid (50 mL), and the reaction was refluxed for 6 h. After cooling to room temperature, formic acid was removed under reduce pressure, the pH of the residue was adjusted to about 7 with saturated solution of sodium bicarbonate, extracted with dichloromethane (50×3). The organic layer was combined, washed with brine, dried over anhydrous sodium sulfate. The filtrate was concentrated under reduced pressure, and the filtrate was separated on column chromatography (dichloromethane/methanol (v/v) = 50 : l) to afford a brown-yellow solid in 92% yield.

N-cyclopropylacetamide(23)
In an ice bath, acetic anhydride (16 mL) was slowly added into the solution of triethylamine (23 mL) and compund 22 (9 g) in dichloromethane (100 mL). After the addition was completed, the reaction was stirred overnight at r.t.. The dichloromethane was removed under reduced pressure. Diethyl ether was added, followed by a large amount of potassium carbonate, and the mixture was stirred 10 h. The solid was filtered, and the filtrate was concentrated under reduced pressure to give a colorless clear oil in 95% yield.

6-bromo-4-fluoro-lH-indazole(32)
At room temperature, hydrazine hydrate (10 mL) was added into the compound 31 (5.00 g, 22.60 mmol) in 1,4-dioxane (10 mL), and the reaction was reacted at 90 ℃ for 5 h. After cooling to room temperature, it was added into the mixture of ice and water, extracted with ethyl acetate (50 mL×3). The organic layer was combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to afford a yellow solid in 62% yield.

5-bromo-7-fluoro-2-methylbenzo[d]oxazole(37)
Compound 36 (700 mg, 3.41mmol) was added into triethyl orthoacetate (20 mL), and the reaction was refluxed at 150 ℃ for 6 h. After cooling to room temperature, the reaction was quenched by adding saturated solution of sodium bicarbonate, extracted with ethyl acetate (50 mL×3), the combined organic layer was washed with water and brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was separated by column chromatography (petroleum ether/ethyl acetate (v/v) = 1 : 1), to afford a pale yellow solid in 68% yield.

6-bromo-4-fluoro-2-(trifluoromethyl)-1H-benzo[d]imidazole(41)
Compound 40 (920mg, 4.01 mmol) was added into trifluoroacetic acid (20 mL), and the reaction was reacted at 70 ℃ for 4 h. After cooling to room temperature, S14 trifluoroacetic acid was removed under reduce pressure, the pH of the residue was adjusted to about 7 with saturated solution of sodium bicarbonate, extracted with dichloromethane (50×3). The organic layer was combined, washed with brine, dried over anhydrous sodium sulfate. The filtrate was concentrated under reduced pressure, and the filtrate was separated on column chromatography (dichloromethane/methanol (v/v) = 20 : l) to afford a pale yellow solid in 70% yield.