Identification of 4-(6-((2-methoxyphenyl)amino)pyrazin-2-yl)benzoic acids as CSNK2A inhibitors with antiviral activity and improved selectivity over PIM3

We report the synthesis of 2,6-disubstituted pyrazines as potent cell active CSNK2A inhibitors. 4’-Carboxyphenyl was found to be the optimal 2-pyrazine substituent for CSNK2A activity, with little tolerance for additional modification. At the 6-position, modifications of the 6-isopropylaminoindazole substituent were explored to improve selectivity over PIM3 while maintaining potent CSNK2A inhibition. The 6-isopropoxyindole analogue 6c was identified as a nanomolar CSNK2A inhibitor with 30-fold selectivity over PIM3 in cells. Replacement of the 6-isopropoxyindole by isosteric ortho-methoxy anilines, such as 7c, generated analogues with selectivity for CSNK2A over PIM3 and improved the kinome-wide selectivity. The optimized 2,6-disubstituted pyrazines showed inhibition of viral replication consistent with their CSNK2A activity.


Chemistry Methods
All reagents and solvents used were purchased from commercial sources and were used without further purification.NMR spectra were obtained using a Bruker 850 MHz or INOVA 400 MHz spectrometers at room temperature; chemical shifts are expressed in parts per million (ppm, δ units) and are referenced to the residual protons in the deuterated solvent used.
Coupling constants are given in units of hertz (Hz).Splitting patterns describe apparent multiplicities and are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br s (broad singlet), dd (doublet of doublets), ddd (double double doublet), tt (triplet of triplets).The purity of compounds submitted for biological screening was determined to be ≥95% as measured by NMR.Analytical thin layer chromatography (TLC) was performed on silica gel plates, 200 μm with an F254 indicator.Column chromatography was performed using RediSep Rf ® preloaded silica gel cartridges on Isolera one Biotage automated purification systems.Samples for high-resolution mass spectrometry were analyzed with a ThermoFisher Q Exactive HF-X (ThermoFisher, Bremen, Germany) mass spectrometer coupled with a Waters Acquity H-class liquid chromatograph system.Samples were introduced via a heated electrospray source (HESI) at a flow rate of 0.3 mL/min.Electrospray source conditions were set as: spray voltage 3.0 kV, sheath gas (nitrogen) 60 arb, auxillary gas (nitrogen) 20 arb, sweep gas (nitrogen) 0 arb, nebulizer temperature 375 degrees C, capillary temperature 380 °C, RF funnel 45 V.The mass range was set to 150-2000 m/z.All measurements were recorded at a resolution setting of 120,000.Separations were conducted on a Waters Acquity UPLC BEH C18 column (2.1 x 50 mm, 1.7 m particle size).LC conditions were set at 95 % water with 0.1% formic acid (A) ramped linearly over 5.0 mins to 100% acetonitrile with 0.1% formic acid (B) and held until 6.0 mins.At 7.0 mins the gradient was switched back to 95% (A) and allowed to re-equilibrate until 9.0 mins.Injection volume for all samples was 3 L.Analytical LC/MS data was obtained using a Waters Acquity Ultrahigh-performance liquid chromatography (UPLC) system equipped with a photodiode array (PDA) detector using the following method: solvent A = Water + 0.2% FA, solvent B = ACN + 0.1% FA, flow rate = 1mL/min.The gradient started at 95% A for 0.05 min.Afterwards, it was ramped up to 100% B over 2 min and held for an additional minute at this concentration, before returning to the initial gradient.Compounds were purified on prep HPLC using an Agilent 1100 equipped with a Phenomenex column (Phenyl-Hexyl, 75 x 30 mm, 5 μm) using the following method: Solvent A: water + 0.05 % TFA; Solvent B: MeOH; flow rate: 70.00 mL/min.LC conditions were set at 90 % (A) ramped linearly over 8.0 mins to 100% (B) and held until 10.0 mins at 100% B. At 10.0 mins the gradient was switched back to 90% (A).

1-(6-Chloropyrazin-2-yl)-6-nitro-1H-indazole (I)
To a stirring solution of 6-nitro-1H-indazole (2.00 g, 1.00 Eq, 12.3 mmol) in DMF (25 mL), was added NaH (883 mg, 3 Eq, 36.8 mmol) at 0 °C.The reaction was stirred for 30 minutes at this temperature, and then 2,6-dichloropyrazine (2.74 g, 1.50 Eq, 18.4 mmol) was added.The reaction was allowed to warm to room temperature and stirred for 16 hours.The majority of DMF was removed by air drying.The resulting crude slurry was poured onto ice water and stirred for 15 minutes.Afterwards, the resulting suspension was filtered.The filter cake was washed with water several times and then left to dry in vacuo.The crude residue was collected and purified by silica gel chromatography using a system of hexanes and DCM starting to afford the title material as a yellowish-white solid (1.08 g, 32%).

1-(6-Chloropyrazin-2-yl)-1H-indazol-6-amine (II)
To a stirring suspension of I (600 mg, 1 Eq, 2.18 mmol) in EtOH/water (28 mL, 3:1), was added Fe (365 mg, 3 Eq, 6.53 mmol) and NH4Cl (582 mg, 5 Eq, 10.9 mmol), and the reaction was heated to reflux and stirred for 4 hours.The reaction mixture was filtered on celite, and the celite pad was washed several times with DCM.The solvent was removed in vacuo.The crude residue was dissolved in DCM, and the organic layer was washed with water several times to remove excess NH4Cl.The layers were separated, and the combined organic fractions were washed one more time with brine, and then dried with Na2SO4.The solvent was removed in vacuo to afford the title material as a yellow solid (470 mg, 88%).
Upon completion of the reaction, water and ethyl acetate were added.The layers were separated, and the aqueous layer was extracted with ethyl acetate.The combined organic extracts were washed with brine, filtered, and the solvent was removed in vacuo.The crude product was purified by silica gel chromatography using a gradient of hexanes and ethyl acetate to afford the title material as a brown solid (30 mg, 43%).To a stirring solution of III (30 mg, 1 Eq, 0.087 mmol) and NaBH3CN (16 mg, 3 Eq, 0.26 mmol) in DMF (0.60 mL), were added benzaldehyde (0.018 mL, 2 Eq, 0.17 mmol) and AcOH (0.005 mL, 1 Eq, 0.087 mmol), and the resulting mixture was heated to 70 °C and stirred for 18 hours.The reaction was stopped by adding water.The aqueous layer was extracted with ethyl acetate.The combined organic extracts were washed with water, brine, and dried with Na2SO4.
The solvent was removed in vacuo, and the crude product was purified by silica gel chromatography using a gradient of hexanes and ethyl acetate to afford the desired product as a yellow solid (20 mg, 42%).
Afterwards, water was added, and the aqueous layer was extracted with ethyl acetate.The combined organic extracts were washed with brine, and then directly transferred to a flask.The solvent was removed in vacuo, and the crude product was purified by silica gel chromatography using a gradient of DCM and ethyl acetate to afford the title material as a brown solid (10 mg, 12%).

Methyl-4-(6-(6-acetamido-1H-indazol-1-yl)pyrazin-2-yl)benzoate (3h)
To a stirring solution of III (50 mg, 1 Eq, 0.14 mmol) and DIPEA (37 mg, 0.050 mL, 2 Eq, 0.29 mmol) in THF (1.00 mL) at 0 °C, was added acetyl chloride (17 mg, 0.015 mL, 1.50 Eq, 0.22 mmol), dropwise.The reaction was stirred at 0 °C for 5 minutes, and then allowed to warm to room temperature and stirred for an additional 6 hours.Water and DCM were added.The layers were separated, and the organic layer was washed with water, brine, and then dried with Na2SO4.The solvent was removed in vacuo, and the crude product was purified by silica gel chromatography using a system of DCM/ethyl acetate to afford the title material as white solid (23 mg, 41%).

4-(6-(6-Amino-1H-indazol-1-yl)pyrazin-2-yl)benzoic acid (4a)
To a stirring solution of III (30 mg, 1 Eq, 0.087 mmol) in THF (0.50 mL), was added 2 M LiOH solution (0.22 mL).The reaction was heated to 50 °C and stirred overnight.Afterwards, the solvents were removed in vacuo.The residue was then dissolved in water, and the pH was adjusted to ~ 2 using 1 M HCl solution.The resulting precipitate was filtered and washed with water, and then collected.The collected residue was purified using preparative HPLC to afford the desired product as a yellow solid (15 mg, 52%).

4-(6-(6-(Ethylamino)-1H-indazol-1-yl)pyrazin-2-yl)benzoic acid (4c)
To a stirring solution of 3b (30 mg, 0.080 mmol) in THF (1.00 mL), was added 2 M LiOH solution (1.0 mL), and the reaction was stirred at room temperature overnight.The solvents were removed in vacuo, and the solid residue was then dissolved in water.A 1 M HCl solution was used to adjust the pH of the aqueous solution to 4-5 (or until appearance of precipitate).The suspension was filtered, and the residue was washed with water and dried to afford the title material as a yellow solid (11 mg, 38%).

4-(6-(6-((2-(Methylamino)ethyl)amino)-1H-indazol-1-yl)pyrazin-2-yl)benzoic acid (4g)
To a stirring solution of 3g in THF (1.00 mL) was added 2 M LiOH solution (0.40 mL), and the reaction was stirred at rt overnight .The solvents were evaporated.The residue was dissolved in water, and the pH was adjusted to 3 using 1 M HCl solution.The resulting suspension was filtered, and the residue was washed with water, and then collected.The residue was taken up in 1.20 mL of DCM/MeOH (1:4) followed by addition of TFA (0.20 mL), and the reaction was stirred at room temperature for 3 days.The solvents were removed in vacuo, and the crude product was purified by reverse phase chromatography using a gradient of water/0.5%TFA and ACN to afford the title material as a yellow solid (16 mg, 62%).

4-(6-(6-((2-Morpholinoethyl)amino)-1H-indazol-1-yl)pyrazin-2-yl)benzoic acid (4h)
To a stirring solution of 3h (9.7 mg, 1 Eq, 0.021 mmol) in THF (0.80 mL) was added 2 M LiOH solution (0.80 mL), and the reaction was stirred at room temperature for 3 days .The solvents were evaporated.The residue was dissolved in water, and TFA was added until reaching a pH of 4 (or until a precipitate is formed).The resulting suspension was filtered, and the residue was washed with water, and then left to dry before being collected.The crude product was purified using preparative HPLC to afford the TFA salt of the title material as a yellow solid (3 mg, 25%).

2-Fluoro-4-(6-(6-(isopropylamino)-1H-indazol-1-yl)pyrazin-2-yl)benzoic acid (5c)
The ester precursor was synthesized using the same Suzuki conditions for making compound III starting with V (200 mg, 1.00 Eq, 0.527 mmol) and (3-fluoro-4-(methoxycarbonyl)phenyl)boronic acid (115 mg, 1.10 Eq, 0.580 mmol) to afford desired material as a yellow solid.The isolated product (50 mg, 0.12 mmol) was taken up in THF (1.00 mL), and then excess amount of 2 M LiOH solution was added (1 mL).The reaction was stirred at room temperature overnight.The solvents were evaporated, and the residue was triturated with DCM (x3).The residual DCM was evaporated, and the residue was then dissolved in water and the pH was adjusted 4-5 using a 1 M HCl solution.The resulting precipitate was filtered, and the residue was washed with water and dried to afford the title material as a yellow solid (30 mg, 62%).
Then, Pd(dppf)Cl2 (21.2 mg, 0.10 Eq, 0.029 mmol) was added to the reaction mixture, and the reaction flask was heated to 70 °C and stirred overnight.The reaction was cooled to room temperature and then poured onto water.The aqueous layer was extracted with ethyl acetate.
The combined organic extracts were washed with brine, dried with Na2SO4, and the solvent was removed in vacuo.The crude product was purified by silica gel chromatography using a gradient of DCM and ethyl acetate to afford the ester precursor as a yellow solid.The isolated product (60 mg, 0.14 mmol) was subjected to the same hydrolysis conditions as 5c to afford the title material as a yellow solid (13 mg, 22%).

6-Isopropoxy-1H-indazole (VIII)
A mixture of 1H-indazol-6-ol (1.00 g, 1 Eq, 7.46 mmol), 2-iodopropane (0.97 mL, 1.30 Eq, 9.69 mmol) and Cs2CO3 (3.643 g, 1.50 Eq, 11.18 mmol) in DMF (10 mL), was stirred at room temperature for 24 hours.Water and ethyl acetate were added.The layers were separated, and the aqueous layer was extracted with ethyl acetate.The combined organic extracts were washed with brine and then dried with Na2SO4.The solvent was removed in vacuo, and the crude product was purified by silica gel chromatography using a gradient of hexanes and ethyl acetate to afford the desired product as a white solid (830 mg, 63%).

4-(6-(6-(Isopropylamino)-1H-indol-1-yl)pyrazin-2-yl)benzoic acid (6d)
The ester precursor was synthesized using similar procedure for V starting with XI (320 mg, 1 Eq, 1.29 mmol) and N-isopropyl-1H-indol-6-amine (247 mg, 1.10 Eq, 1.42 mmol) to afford the desired product as a yellow solid.The isolated product (60 mg, 1 Eq, 0.16 mmol) was subjected to the same hydrolysis conditions as 5c to afford the title material a white solid (10 mg, 17%).(380 mg, 2 Eq, 1.17 mmol), BINAP (36.3 mg, 0.10 Eq, 0.058 mmol), and Pd2(dba)3 (26.7 mg, 0.05 Eq, 0.029 mmol) were mixed in dioxane (3.50 mL).The solvent was purged with argon gas, and then the flask was sealed.The reaction mixture was heated to 90 °C and stirred for 16 hours.Upon completion of the reaction, it was allowed to cool to room temperature, diluted with DCM, and then filtered over a celite pad.The residue on the celite pad was washed several times with DCM.The filtrate was collected and concentrated in vacuo.The crude compound was purified using silica gel chromatography using a gradient of hexanes and ethyl acetate to afford the desired product as a yellow solid (95 mg, 44%).To a stirring suspension of XIIa (90 mg, 1 Eq, 0.24 mmol) in 3:1 EtOH/Water (3.50 mL), were added Fe (68 mg, 5 Eq, 1.2 mmol) and NH4Cl (39 mg, 3 Eq, 0.73 mmol), and the reaction was heated to reflux and stirred for 3 hours.The reaction was stopped and filtered through celite.
The filtering agent was washed several times with ethyl acetate.The filtrate was concentrated in vacuo.The crude product was dissolved in Ethyl acetate.Water was added, and the organic phase was washed with water and brine and then dried with Na2SO4.The solvent was removed in vacuo to afford the desired amine as a green solid which was carried to the next step without further purification.
The solvent was removed in vacuo.The crude product was purified by a silica gel chromatography using a gradient of hexanes and ethyl acetate to afford the desired product as brown solid.The isolated product (10 mg, 0.026 mmol) was subjected to the same hydrolysis conditions as 5c to afford the title material as a yellow solid (9 mg, 93%).The ester precursor was synthesized using similar procedure to 7a starting with XIIIb (180 mg, 1 Eq, 0.526 mmol) to afford the desired product as brown solid.The isolated product (93 mg, 0.24 mmol) was subjected to the same hydrolysis conditions as 5c.After ester hydrolysis, the product was purified by reverse phase HPLC chromatography to afford the trifluoroacetate salt of the title material as a yellow solid (17 mg, 19%).

4-Isopropoxy-1-methoxy-2-nitrobenzene (XIV)
A mixture of 4-methoxy-3-nitrophenol (500 mg, 1 Eq, 2.96 mmol), 2-iodopropane (653 mg, 0.384 mL, 1.30 Eq, 3.84 mmol) and Cs2CO3 (1.44 g, 1.50 Eq, 4.43 mmol) in DMF (5.00 mL) was stirred at room temperature for 24 hours, and at 60 °C for 4 hours .Water was added and the aqueous layer was extracted with ethyl acetate.The combined organic extracts were washed with brine and then dried with Na2SO4.The solvent was removed in vacuo.The crude product was purified by silica gel chromatography using a gradient of hexanes and ethyl acetate to afford the desired product as a yellow liquid (454 mg, 73%).

5-Isopropoxy-2-methoxyaniline (XVI)
Synthesized using same procedure for XIIIa starting with XIV (400 mg, 1.89 mmol) to afford the title material in quantitative yield and taken to the next step without purification.

4-(6-((5-Isopropoxy-2-methoxyphenyl)amino)pyrazin-2-yl)benzoic acid (7c)
The ester precursor was synthesized using same procedure for XIIa starting with XI (250 mg, 1 Eq, 1.01 mmol) and XVI (219 mg, 1.20 Eq, 1.21 mmol) to afford the desired product as a yellow solid.The isolated product (100 mg, 1 Eq, 0.254 mmol) was subjected to the same hydrolysis conditions as 5c to afford the title material as a yellow solid (90 mg, 93%).FuGENE HD (Promega) was added at 30 µl/mL to form a lipid:DNA complex.The solution was then mixed and incubated at room temperature for 20 min.The transition complex was mixed with a 20x volume of HEK293 cells at 20,000 cells per mL in DMEM/FBS and 100 µL per well was added to a 96-well plate that was incubated overnight at 37°C, 5% CO2.The following day, the media was removed via aspiration and replaced with 85 μL of Opti-MEM without phenol red.
A total of 5 μL per well of 20x Sigma) supplemented with 10% fetal bovine serum (Gibco) and penicillin and streptomycin (Sigma).DBT cells were plated in 96 well plates to be 80% confluent at the start of the assay.
Test compounds were diluted to 15 µM in DMEM.Serial 4-fold dilutions were made in DMEM, providing a concentration range of 15 µM to 0.22 µM.Media was aspirated from the DBT cells and 100 μL of the diluted test compounds were added to the cells for 1 h at 37°C.After 1 h, MHV-nLuc 5 was added at an MOI of 0.1 in 50 μL DMEM so that the final concentration of the first dilution of compound was 10 μM (T=0).After 10 h, the media was aspirated, and the cells were washed with PBS and lysed with passive lysis buffer (Promega) for 20 min at room temperature.Relative light units (RLUs) were measured using a luminometer (Promega; GloMax).Triplicate data was analyzed in Prism Graphpad to generate IC50 values.
Complex structures were achieved by soaking the apo crystals for at least 24h with the desired inhibitor dissolved in reservoir solution.Final concentration of the inhibitor was 0.5 mM.
Data collection, structure solution and refinement.Diffraction data were collected at beamline X06SA (Villigen, CH) at a wavelength of 1.0 Å at 100 K.The reservoir solution supplemented with 20% ethylene glycol was used as cryoprotectant.Data were processed using XDS 4 and scaled with aimless 5 .The PDB structure with the accession code 6Z83

5 ,
500 mM NaCl, 0.5 mM TCEP) at 4 C. Additionally, TEV protease was added (protein:TEV 1:20 molar ratio) to remove the tag.The next day the protein solution was loaded onto Nickel-Sepharose column beads again to remove the TEV protease and cleaved Tag.The combined flow through fraction and the wash fraction (25 mM imidazole) containing the protein were concentrated to approximately 4-5 mL and loaded onto Superdex 75 16/60 Hi-Load gel filtration column equilibrated with final buffer.The protein was concentrated to approximately 9 mg/mL.

Table S1 .
Kinase selectivity of 2, 6c, and 7c by thermal stability assay.Compounds were tested at a concentration of 10 µM.Tm shifts are shown in ºC.Kinases with a Tm shift >5 ºC are highlighted in red.
Raw milliBRET (mBRET) values were obtained by dividing the acceptor emission values (600 nm) by the donor emission values (450 nm) and multiplying by 1000.Averaged control values were used to represent complete inhibition (no tracer control: Opti-MEM + DMSO only) and no inhibition (tracer only control: no compound, Opti-MEM + DMSO + Tracer K10 only) and were plotted alongside the raw mBRET values.The data was first normalized and then fit using Sigmoidal, 4PL binding curve in Prism Software to determine IC50 values.MHV Assay.DBT cells were cultured at 37°C in Dulbecco's modified Eagle medium (DMEM; solution.A total of 50 μL of the 3X substrate/extracellular NL inhibitor were added to each well.The plates were read within 30 min on a GloMax Discover luminometer (Promega) equipped with 450 nm BP filter (donor) and 600 nm LP filter (acceptor) using 0.3 s integration time.
6was used as an initial search MR model using the program MOLREP6. Te final model was built manually using Coot 7 and refined with REFMAC5 8 .Data collection and refinement statistics: *Values for the highest resolution shell are shown in parentheses.