The Discovery of Pyrazolopyridones as a Novel Class of Gyrase B Inhibitors Using Structure Guided Design

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1 Supporting Information for: The Discovery of Pyrazolopyridones as a Novel Class of Gyrase B Inhibitors Using Structure Guided Design Jason B. Cross,* Jing Zhang, Qingyi Yang, Michael F. Mesleh, Jan Antoinette C. Romero, Bin Wang, Doug Bevan, Katherine M. Poutsiaka, Felix Epie, Terence Moy, Anu Daniel, Joseph Shotwell, Brian Chamberlain, Nicole Carter, Ole Andersen, John Barker, M. Dominic Ryan, Chester A. Metcalf III, Jared Silverman, Kien Nguyen, Blaise Lippa, Roland E. Dolle Cubist Pharmaceuticals, Inc., 65 Hayden Avenue, Lexington, Massachusetts 02421, United States Evotec UK, Ltd., 114 Innovation Drive, Milton Park, Abingdon, Oxfordshire OX14 4RZ, United Kingdom Bacterial GyrB and TopoIV IC50 Determination. Proteins were obtained from Inspiralis Ltd. (Norwich, United Kingdom). Sa gyrase was used at final concentration of 7.5 nm in a solution of 40 mm HEPES-KOH ph 7.6, 10 mm magnesium acetate, 10 mm dithreitol, 50 g/l BSA, 500 mm potassium glutamate, 1% DMSO, 100 mm ATP, and 10 nm linear pbr322 DNA. Sa topo IV was used at final concentration of 8.5 nm in a solution of 100 mm Tris ph 7.5, 2 mm magnesium chloride, 1 mm dithreitol, 50 g/l BSA, 200 mm potassium glutamate, 1% DMSO, 300 mm ATP, and 10 nm linear pbr322 DNA. Reactions were carried out in triplicate in a volume of 10 microliters per well and were initiated with the addition of ATP, then incubated at 20 C for 30 minutes. To quantify ADP concentration, reactions were stopped by addition of 10 microliters of Transcreener ADP2 FP assay reagent and fluorescence polarization measurements were made according to the manufacturer s protocol (Bellbrook Labs, Madison, WI).

2 MIC Determination. Antibacterial activity of all the compounds was demonstrated by the minimum inhibitory concentrations (MIC) of the compounds, performed in triplicate, against various bacteria measured by the broth microdilution assay performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines with modifications described below. Individual colonies were isolated by streaking frozen glycerol stock of the bacterial species being tested onto rich, nonselective, tryptic soy agar containing 5% sheep s blood (TSAB), and incubated at 37 C for hrs. Streptococcus pneumoniae strain was streaked on TSAB plates and incubated at 37 C with 5% CO2 for hrs. On the day of the assay, primary cultures were started by inoculating 5-10 colonies from the TSAB plates into ~5 ml of Mueller Hinton Broth (MHB) in 14 ml culture tubes and incubated at 37 C with aeration (200 rpm) for ~2 hrs until the OD600 was 0.1. Inoculum cultures were prepared by standardizing the primary cultures in MHB so that the final inoculum density was ~105 colony forming units per milliliter. 50 µl of the diluted inoculum cultures was added to 96 well broth microdilution assay plates along with 50 µl of MHB containing compound (concentrations ranging from µg/ml in two-fold dilutions) for a final volume 100 µl per well with a final culture OD600 of approximately For S. pneumoniae, 5-10 colonies from TSAB plates were re-suspended into MHB to an OD600 of 0.1. This material was used to prepare inoculum culture as mentioned above. The final DMSO concentration in the assay plates was 2%. Plates were incubated for hours at 37 C with aeration (200 rpm). Assay plates containing S. pneumoniae were incubated at 37 C with 5% CO2 for hrs. Following incubation, growth was defined as turbidity that could be detected with the naked eye or achieving minimum OD600 of 0.1. MIC values were defined as the lowest concentration producing no visible turbidity.

3 X-ray Crystal Structure Determination. A loop-deleted 24 kda construct of S. aureus GyrB was used for X-ray crystallography. Protein crystals were grown using the hanging drop method at ph 7.6, followed by soaking of fragments into the crystallization buffer. Data were collected at the Diamond Light Source. Structures were solved by molecular replacement using PDB entry 1KZN as a search model. Refinement and model building were completed using REFMAC5 and Coot, respectively. EXPERIMENTAL SECTION General Procedures. All common solvents and chemicals were used as purchased without further purification. The progress of all reactions was monitored on Aldrich precoated silica gel plates (with fluorescence indicator UV254) using ethyl acetate/n-hexane or methanol/dichloromethane as solvent system, and by a Waters UPLC-MS with model number C10UPB090A. Column chromatography was performed with Aldrich silica gel 60 ( mesh ASTM) with the solvent mixtures specified in the corresponding experiment. Purity of all final compounds was 95% or higher as checked by Waters UPLC-MS and Bruker Avance 500 MHz NMR.. Spots were visualized by irradiation with ultraviolet light (254 nm). Proton (1H) NMR spectra were recorded on Bruker Avance 500 or 300 MHz using solvents as indicated in the experimental section. Chemical shifts are given in parts per million (ppm) (δ relative to residual solvent peak for 1 H). Ethyl 2-(pyridin-3-yl)thiazole-5-carboxylate (5) A mixture of ethyl 2-bromothiazole-5- carboxylate (4.7 g, 0.02 mol), pyridin-3-ylboronic acid (2.9 g, mol), Pd(dppf)Cl 2 (1.4 g,

4 0.002 mol) and Na 2 CO 3 (4.2 g, 0.04 mol) in 1,4-dioxane (50 ml) and water (10 ml) was heated to 100 o C for 4 h with stirring. The mixture was cooled to room temperature and concentrated in vacuo. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 1/8) to afford ethyl 2-(pyridin-3-yl)thiazole-5-carboxylate 5 (2.5 g, 0.01 mol, 55%) as a brown solid. 1 H-NMR (500 MHz, DMSO-d 6 ): δ 9.22 (dd, J = 0.5 Hz, J = 2.0 Hz, 1H), 8.75 (dd, J = 1.5 Hz, J = 4.5 Hz, 1H), 8.57 (s, 1H), (m, 1H), (m, 1H), 4.37 (q, J = 7.5 Hz, 2H), 1.33 (t, J = 7.5 Hz, 3H). MS (EI +, m/z): [M+H] +. 3-Oxo-3-(2-(pyridin-3-yl)thiazol-5-yl)propanenitrile (6) To a stirred solution of n-buli (1.25 M in hexane, 6.4 ml, mol) in THF (20 ml) was added dropwise a solution of CH 3 CN (368 mg, mol) in THF (20 ml) at -78 o C for 1 h. Then a solution of ethyl 2-(pyridin-3- yl)thiazole-5-carboxylate 5 (9.4 g, mol) in THF (10 ml) was added at -78 o C. The mixture was stirred for 2 h at this temperature, quenched with water (50 ml) and warmed up to room temperature. The resulting precipitates were collected, washed with water (20 ml), dried in vacuo to afford 3-oxo-3-(2-(pyridin-3-yl)thiazol-5-yl)propanenitrile 6 (2.3 g, 0.01 mol, 35%) as a pale yellow solid. 1 H-NMR (500 MHz, CDCl 3 ): δ 9.25 (d, J = 2.0 Hz, 1H), 8.77 (dd, J = 2.0 Hz, J = 4.5 Hz, 1H), 8.49 (s, 1H), (m, 1H), (m, 1H), 4.05 (s, 2H). MS (EI +, m/z): [M+Na] +. 1-Tert-butyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazol-5-amine (7) A mixture of 3-oxo-3- (2-(pyridin-3-yl)thiazol-5-yl)propanenitrile 6 (1.55 g, 6.77 mmol), tert-butylhydrazine hydrochloride (1.69 g, mmol) and triethylamine (1.37 g, mmol) in isopropanol (100 ml) was stirred at 80 o C overnight and then cooled to room temperature. The mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (methanol

5 / dichloromethane = 1/40) to afford 1-tert-butyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazol-5- amine 7 (1.0 g, 3.34 mmol, 50%) as a pale yellow solid. 1 H-NMR (500 MHz, DMSO-d 6 ): δ 9.13 (t, J = 1.5 Hz, 1H), 8.64 (dd, J = 1.0 Hz, J = 4.5 Hz, 1H), (m, 1H), 8.07 (s, 1H), (m, 1H), 5.84 (s, 1H), 5.15 (s, 2H), 1.57 (s, 9H). MS (EI +, m/z): [M+H] +. (E)-N'-(1-tert-butyl-4-formyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazol-5-yl)-N,Ndimethylformimidamide (8) To a solution of 1-tert-butyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1Hpyrazol-5-amine 7 (24.5 g, 82.0 mmol) in DMF (140 ml) was added POCl 3 (38.0 g, mmol) dropwise over 15 minutes. The mixture was heated at 70 o C for 2 hours, poured into icewater (400 ml), adjusted ph to 8 with 40% of aq. NaOH. The resulting precipitates were collected, washed with cold water, and dried over high vacuum to afford (E)-N'-(1-tert-butyl-4- formyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1h-pyrazol-5-yl)-n,n-dimethylformimidamide 8 (33.0 g, 100%) as a brown solid. MS (EI +, m/z): [M+H] +. 5-Amino-1-tert-butyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazole-4-carbaldehyde (9) A mixture of (E)-N'-(1-tert-butyl-4-formyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazol-5-yl)-N,Ndimethylformimidamide 8 (33.0 g, 82.0 mmol) in 40% aq. NaOH (150 ml) and ethanol (200 ml) was heated at 90 o C overnight. The mixture was cooled to room temperature, diluted with water (500 ml), and extracted with ethyl acetate (500 ml x 3). The combined organic phase was washed with brine (100 ml), dried over sodium sulfate and concentrated. The crude product was purified by silica gel column chromatography eluting with 50% ethyl acetate in petroleum ether to afford 5-amino-1-tert-butyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazole-4-carbaldehyde 9 (14.0 g, 52%) as a pale yellow solid. 1 H-NMR (500 MHz, DMSO-d 6 ): δ 9.50 (s, 1H), 9.13 (t, J =

6 1.5 Hz, 1H), 8.64 (dd, J = 1.0 Hz, J = 4.5 Hz, 1H), (m, 1H), 8.07 (s, 1H), (m, 1H), 5.25 (s, 2H), 1.57 (s, 9H). MS (EI +, m/z): [M+H] +. 1-Tert-butyl-5-(ethylamino)-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazole-4-carbaldehyde (10c) To a mixture of 5-amino-1-tert-butyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazole-4- carbaldehyde 9 (3.27 g, 0.01 mol) and Cs 2 CO 3 (6.52 g, 0.02 mol) in 40 ml of DMF was added bromoethane (3.27 g, 0.03 mol) at 5 o C. After stirring at 5 o C for 3 days, the mixture was diluted with water (100 ml), extracted with ethyl acetate (100 ml x 2). The organic phase was separated, washed with brine (100 ml), dried over sodium sulfate and concentrated to give crude 1-tert-butyl-5-(ethylamino)-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazole-4-carbaldehyde 10c (1.3 g, 37%) as a pale yellow solid. The crude product was taken to the next step without further purification. ESI-MS (EI +, m/z): [M+H] +. 5-(Ethylamino)-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazole-4-carbaldehyde (11c) A solution of 1-tert-butyl-5-(ethylamino)-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazole-4- carbaldehyde 10c (1.3 g, 3.66 mmol) in trifluoroacetic acid (13 ml) was heated at 70 o C overnight and concentrated. Ethyl acetate (50 ml) and diethyl ether (50 ml) were added. The resulting solid was filtered, dried to afford 5-(ethylamino)-3-(2-(pyridin-3-yl)thiazol-5-yl)-1Hpyrazole-4-carbaldehyde 11c (1.25 g, 70%) as a pale yellow solid. MS (EI +, m/z): [M+H] +. Ethyl 7-ethyl-6-oxo-3-(2-(pyridin-3-yl)thiazol-5-yl)-6,7-dihydro-1H-pyrazolo[3,4- b]pyridine-5-carboxylate (12c) A mixture of 5-(ethylamino)-3-(2-(pyridin-3-yl)thiazol-5-yl)- 1H-pyrazole-4-carbaldehyde 11c (1.25 g, 3.15 mmol), piperidine (2.7 g, 31.5 mmol) and diethyl malonate (2.5 g, mmol) in ethanol (80 ml) was heated at 80 o C for 16 hours. The mixture was concentrated, and treated with ethyl acetate (50 ml) and diethyl ether (50 ml). The

7 resulting solid was filtered to afford ethyl 7-ethyl-6-oxo-3-(2-(pyridin-3-yl)thiazol-5-yl)-6,7- dihydro-1h-pyrazolo[3,4-b]pyridine-5-carboxylate 12c (1.25 g, 86%) as a brown solid. MS (EI +, m/z): [M+H] +. 7-ethyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (2c) A mixture of ethyl 7-ethyl-6-oxo-3-(2-(pyridin-3-yl)thiazol-5-yl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine- 5-carboxylate 12c (300 mg, 0.75 mmol) in aq. HBr (40%, 8.0 ml) was heated at 140 o C for 2 hours in the microwave. The mixture was purified by prep-hplc to afford 7-ethyl-3-(2-(pyridin- 3-yl)thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)-one 2c (130 mg, 53%) as a pale yellow solid. 1 H-NMR (500 MHz, DMSO-d 6 ): δ 9.20 (d, J = 1.5 Hz, 1H), 8.70 (d, J = 4.5 Hz, 1H), 8.51 (s, 1H), 8.36 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.58 (dd, J = 5.0 Hz, J = 7.5 Hz, 1H), 6.28 (d, J = 9.5 Hz, 1H), 4.11 (q, J = 7.0 Hz, 2H), 1.25 (t, J = 7.0 Hz, 3H). MS (EI +, m/z): [M+H] +. 7-Methyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (2b) 2b was prepared according to the general procedure of 2c starting from 9 and iodomethane. 1 H-NMR (500 MHz, DMSO-d 6 ): δ 9.19 (d, J = 1.5 Hz, 1H), 8.70 (dd, J = 4.5 Hz, J = 1.0 Hz, 1H), 8.52(s, 1H), (m, 1H), 7.58 (dd, J = 4.5 Hz, J = 8.0 Hz, 1H), 6.29 (d, J = 4.5 Hz, 1H), 3.52 (s, 3H). MS (EI +, m/z): [M+H] +. 7-Propyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (2d) 2d was prepared according to the general procedure of 2c starting from 9 and 1-iodopropane. 1 H-NMR (500 MHz, DMSO-d 6 ): δ 9.22 (s, 1H), 8.73 (s, 1H), 8.55(s, 1H), 8.41 (d, J = 6.0 Hz, 1H), 8.10 (s, 1H), 7.62 (t, J = 4..8 Hz, 1H), 6.31 (d, J = 8.8 Hz, 1H), 4.03 (t, J = 8.4 Hz, 2H), (m, 2H), 0.92 (t, J = 7.2 Hz, 3H). MS (EI +, m/z): [M+H] +.

8 7-Allyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (2e) 2e was prepared according to the general procedure of 2c starting from 9 and 3-bromoprop-1-ene. 1 H- NMR (500 MHz, DMSO-d 6 ): δ 9.21 (d, J = 1.5 Hz, 1H), 8.72 (d, J = 4.5 Hz, 1H), 8.55 (s, 1H), 8.39 (d, J = 8.0 Hz, 1H), 8.14 (s, 1H), 7.61 (dd, J = 4.5 Hz, J = 8.0 Hz, 1H), (m, 1H), (m, 1H), 5.15 (d, J = 10.0 Hz, 1H), 5.08 (d, J = 17.0 Hz, 1H), 4.70 (d, J = 4.5 Hz, 2H). MS (EI +, m/z): [M+H] +. 7-(2,3-Dihydroxypropyl)-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)- one To a mixture of 7-allyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)- one 2e (380 mg, 1.13 mmol) in acetone (40 ml) and water (20 ml) was added OsO 4 (58 mg, 0.23 mmol) and N-methylmorphline oxide (530 mg, 4.54 mmol) at 10 o C and the reaction mixture was stirred overnight. The reaction mixture was diluted with water (50 ml), extracted with ethyl acetate (50 ml x 2). The combined organic phase was washed with brine (50 ml), dried over sodium sulfate and concentrated to afford 7-(2,3-dihydroxypropyl)-3-(2-(pyridin-3- yl)thiazol-5-yl)-1h-pyrazolo[3,4-b]pyridin-6(7h)-one (230 mg, 53%) as a pale yellow solid. MS (EI +, m/z): [M+H] +. 2-(6-Oxo-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin-7(6H)-yl)acetaldehyde To a solution of 7-(2,3-dihydroxypropyl)-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazolo[3,4- b]pyridin-6(7h)-one (200 mg, 0.54 mmol) in methanol (25 ml) and water (25 ml) was added NaIO 4 (350 mg, 1.63 mmol) at 10 o C and was stirred overnight. The reaction mixture was concentrated, and treated with water (15 ml). The resulting precipitates were collected, washed with water (25 ml) and dried to afford 2-(6-oxo-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-

9 pyrazolo[3,4-b]pyridin-7(6h)-yl)acetaldehyde (120 mg, 66%) as a pale yellow solid. MS (EI +, m/z): [M+H] +. 7-(2-Hydroxyethyl)-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (2f) To a solution of 2-(6-oxo-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin-7(6H)- yl)acetaldehyde (120 mg, 0.35 mmol) in methanol (15 ml) was added NaBH 4 (40 mg, 1.07 mmol) at 10 o C. After stirring for 1 hour, the solvent was removed under reduced pressure. The crude was purified by prep-hplc to afford 7-(2-hydroxyethyl)-3-(2-(pyridin-3-yl)thiazol-5-yl)- 1H-pyrazolo[3,4-b]pyridin-6(7H)-one 2f (40 mg, 33%) as off-white solid. 1 H-NMR (500 MHz, DMSO-d 6 ): δ 9.20 (d, J = 1.5 Hz, 1H), 8.71 (t, J = 6.5 Hz, 1H), 8.54 (s, 1H), 8.37 (d, J = 8.0 Hz, 1H), 8.11 (d, J = 9.5 Hz, 1H), 7.59 (dd, J = 2.5 Hz, J = 7.5 Hz, 1H), 6.31 (d, J = 9.5 Hz, 1H), 4.18 (t, J = 6.5 Hz, 1H), 3.70 (d, J = 6.0 Hz, 2H), 1.07 (s, 1H). MS (EI +, m/z): [M+H] +. 5-Bromo-7-ethyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (14) To a solution of ethyl 7-ethyl-6-oxo-3-(2-(pyridin-3-yl)thiazol-5-yl)-6,7-dihydro-1Hpyrazolo[3,4-b]pyridine-5-carboxylate 12c (158 mg, 0.40 mmol) in tetrahydrafuran (10 ml) and methanol (5.0 ml) was treated with a solution of LiOH (50 mg, 1.2 mmol) in water (5.0 ml) and heated to 80 o C for 12 h. The reaction mixture was cooled to room temperature to give the crude solution of 7-ethyl-6-oxo-3-(2-(pyridin-3-yl)thiazol-5-yl)-6,7-dihydro-1H-pyrazolo[3,4- b]pyridine-5-carboxylate 13. NBS (143 mg, 0.8 mmol) was added and the reaction mixture was stirred at room temperature for 3 hours. The mixture was diluted with water (50 ml), extracted with ethyl acetate (100 ml x 2). The organic phases were separated, washed with brine (50 ml), dried over sodium sulfate and concentrated to afford 5-bromo-7-ethyl-3-(2-(pyridin-3-yl)thiazol-

10 5-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)-one 14 (140 mg, 87%) as a pale yellow solid. MS (EI +, m/z): [M+H] +. 7-Ethyl-5-methyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (2g) A mixture of 5-bromo-7-ethyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)- one 14 (140 mg, 0.35 mmol), Pd(dppf)Cl 2 (20 mg, mmol), 2,4,6-trimethyl-1,3,5,2,4,6- trioxatriborinane (89 mg, 0.7 mmol) and aq. Na 2 CO 3 (10 ml, 2.0 M) in 1,4-dioxane (20 ml) was heated at 100 o C for 2 days and concentrated. The residue was purified by prep-hplc to afford 7- ethyl-5-methyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1h-pyrazolo[3,4-b]pyridin-6(7h)-one 2g (15 mg, 12%) as a pale yellow solid. 1 H-NMR (500 MHz, DMSO-d 6 ): δ (br, 1H), δ 9.20 (d, J = 1.5 Hz, 1H), 8.70 (d, J = 4.5 Hz, 1H), 8.51 (s, 1H), 8.36 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.58 (dd, J = 5.0 Hz, J = 7.5 Hz, 1H), 4.12 (q, J = 7.0 Hz, 2H), 2.20 (s, 3H), 1.25 (t, J = 7.0 Hz, 3H). MS (EI +, m/z): [M+H] +. 7-Ethyl-5-(hydroxymethyl)-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin- 6(7H)-one (2h) A mixture of 5-bromo-7-ethyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazolo[3,4- b]pyridin-6(7h)-one 14 (100 mg, 0.25 mmol), Pd(PPh 3 ) 2 Cl 2 (35 mg, 0.05 mmol) and tributylstannyl)methanol (400 mg, 1.25 mmol) in THF (5.0 ml) was heated at 120 o C for 30 minutes in the microwave. The reaction mixture was concentrated and purified by prep-hplc to afford 7-ethyl-5-(hydroxymethyl)-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin- 6(7H)-one 2h (14 mg, 10%) as a pale yellow solid. 1 H-NMR (500 MHz, DMSO-d 6 ): δ (br, 1H), 9.30 (d, J = 1.5 Hz, 1H), 8.70 (d, J = 3.5 Hz, 1H), 8.57 (s, 1H), (m, 2H), 7.59 (dd, J = 4.5 Hz, J = 7.5 Hz, 1H), 5.19 (s, 1H), 4.40 (d, J = 4.5 Hz, 2H), (m, 2H), (m, 3H). MS (EI +, m/z): [M+H] +.

11 7-Ethyl-N-methyl-6-oxo-3-(2-(pyridin-3-yl)thiazol-5-yl)-6,7-dihydro-1H-pyrazolo[3,4- b]pyridine-5-carboxamide (2i) To a solution of ethyl 7-ethyl-6-oxo-3-(2-(pyridin-3-yl)thiazol- 5-yl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxylate 12c (600 mg, 1.52 mmol) in tetrahydrafuran (20 ml) and methanol (10.0 ml) was treated with a solution of LiOH (190 mg, 4.56 mmol) in water (18.0 ml) and heated to 80 o C for 12 h. The reaction mixture was cooled to room temperature and acidified with 1M HCl. The resulting precipitates were collected, dried to afford 7-ethyl-6-oxo-3-(2-(pyridin-3-yl)thiazol-5-yl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5- carboxylic acid 13 (580 mg, 94%) as a pale yellow solid. MS (EI +, m/z): [M+H] +. To a mixture of 13 (206 mg, 0.50 mmol) in DMF (5.0 ml) was added methylamine hydrochloride (203 mg, 3.0 mmol), DIPEA (505 mg, 5.0 mmol) and HATU (380 mg, 1.0 mmol). The mixture was stirred at room temperature overnight and purified by prep-hplc to afford 7-ethyl-N- methyl-6-oxo-3-(2-(pyridin-3-yl)thiazol-5-yl)-6,7-dihydro-1h-pyrazolo[3,4-b]pyridine-5- carboxamide 2i (40 mg, 20%) as a pale yellow solid. 1 H-NMR (500 MHz, DMSO-d 6 ): δ 9.58 (s, 1H), 9.22 (s, 1H), 8.90 (s, 1H), 8.69 (d, J = 2.5 Hz, 1H), 8.49 (s, 1H), 8.38 (d, J = 8.0 Hz, 1H), 8.22 (s, 1H), 7.56 (d, J = 5.5 Hz, 1H), 4.26 (d, J = 6.5 Hz, 2H), 2.90 (d, J = 3.5 Hz, 3H), 1.32 (t, J = 6.5 Hz, 3H). MS (EI +, m/z): [M+H] +. 4-Methyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (2j) A mixture of 1-tert-butyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazol-5-amine 7 (0.30 g, 1.00 mmol) and ethyl 3-oxobutanoate (0.24 g, 2.00 mmol) in acetic acid (2.0 ml) was stirred at 110 o C overnight and cooled to room temperature. The mixture was concentrated, purified by silica gel column chromatography eluting with 1% methanol in dichloromethane to afford 1-tert-butyl-4- methyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1h-pyrazolo[3,4-b]pyridin-6(7h)-one (0.11 g, 25%) as a pale yellow solid. MS (EI +, m/z): [M+H] +.

12 A mixture of 1-tert-butyl-4-methyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin- 6(7H)-one (0.12 g, 0.33 mmol) in trifluoroacetic acid (5.0 ml) was stirred at 75 o C overnight. The reaction mixture was then concentrated and purified by prep-hplc (Boston C18 21*250mm 10µm, Mobile phase: A: 0.1% aqueous formic acid; B: acetonitrile) to afford 4- methyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1h-pyrazolo[3,4-b]pyridin-6(7h)-one 2j (23.0 mg, 20%) as a pale yellow solid. 1 H-NMR (500 MHz, DMSO-d 6 ): δ (s, 1H), 9.18 (s, 2H), 8.69 (d, J = 4.5 Hz, 1H), 8.35 (d, J = 3.0 Hz, 1H), 7.57 (dd, J = 4.5 Hz, J = 8.0 Hz, 1H), 6.10 (s, 1H), 2.37 (s, 3H). MS (EI +, m/z): [M+H] +. 3-(2-(Pyridin-3-yl)thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (2a) 2a was prepared according to the similar procedure to 2j via ethyl 3-oxopropanoate and 7. 1 H-NMR (500 MHz, DMSO-d 6 ): δ (s, 1H), 9.20 (d, J = 1.5 Hz, 1H), 8.70 (d, J = 3.5 Hz, 1H), 8.57 (s, 1H), (m, 2H), 7.58 (dd, J = 4.5 Hz, J = 7.5 Hz, 1H), 6.44(s, 1H). MS (EI +, m/z): [M+H] +. 4-Phenyl-3-(2-(pyridin-3-yl)thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (2k) 2k was prepared according to the similar procedure to 2j via ethyl 3-oxo-3-phenylpropanoate and 7. 1 H- NMR (500 MHz, DMSO-d 6 ): δ (s, 1H), 9.16 (d, J = 2.0 Hz, 1H), 8.67 (dd, J = 1.5 Hz, J = 5.0 Hz, 1H), 8.54 (s, 1H), 8.34 (dd, J = 2.0 Hz, J = 6.0 Hz, 1H), 7.90 (t, J = 6.5 Hz, 2H), (m, 4H), 6.54 (s, 1H), 6.13 (s, 1H). MS (EI +, m/z): [M+H] +. 1-Propylpiperidine-2,6-dione (16) To a solution of dihydro-2h-pyran-2,6(3h)-dione 15 (70 g, 614 mmol) in acetic acid (150 ml) was added n-propan-1-amine (44 g, 737 mmol, 1.2 eq) at 0oC. The reaction mixture was then stirred at 130oC for 17 hours. After cooling to room temperature, the mixture was concentrated. The residue was purified by distillation under

13 reduced pressure to afford compound 16 as colorless oil (50 g, 50%). MS (EI +, m/z):156.0 [M+H] +. 2,6-Dioxo-1-propylpiperidine-3-carbaldehyde (17) To a solution of 16 (50 g, 323 mmol) in THF (150 ml) at -78oC was added n-buli (190 ml, 484 mmol, 2.5 M in hexane). After stirring for 2 hours, ethyl formate (35.9 g, mmol, 1.5 eq) was added slowly at -78oC. The mixture was stirred at -78oC for another 2 hours. The reaction was then quenched with saturated aq. NH 4 Cl solution (100 ml). The mixture was slowly warmed up to room temperature and adjusted to ph=6-7 with aq. citric acid solution. The mixture was then extracted with ethyl acetate (200 ml 2). The combined organic phase was dried over sodium sulfate and concentrated to give 17 as a colorless oil, which was used in the next step without further purification. MS (EI +, m/z): [M+H] +. 7-Propyl-4,5-dihydro-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (18) Absolute EtOH (precooled to 0 o C, 200 ml) was added with precaution to a cooled stirred mixture of compound 17 (40.00 g, 0.22 mol) and excess P 2 O 5 (350 g, 2.2 mol). Then hydrazine hydrate (112 g, 2.2 mol) was added drop wise while keeping the reaction mixture at around 0 o C. The mixture was then refluxed for 20 hours. After cooling to room temperature, saturated aq. Na 2 CO 3 solution (500 ml) was added slowly at 0 o C. The reaction mixture was concentrated to remove the organic contents. The resulting aqueous solution was then extracted with petroleum ether (200 ml 3). The aqueous phase was adjusted to ph = 7-8 with saturated aq. Na 2 CO 3 solution and then extracted with ethyl acetate (200 ml 3). The combined ethyl acetate extract was washed with saturated NaHCO 3 (200 ml 3) and brine (200 ml 2), and dried over sodium sulfate. The solution was concentrated and purified via silica gel column chromatography eluting with 20-35% ethyl

14 acetate in petroleum ether to give 18 as a brown solid (12 g, 25% over two steps). MS (EI +, m/z): [M+H] +. 3-Iodo-7-propyl-4,5-dihydro-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (19) A mixture of compound 18 (21.7 g 121 mmol) and N-iodosuccinimide (40.7 g, mmol) in 1, 2- dichloroethane (400 ml) was stirred at 85 C for 8 hours. After cooling to room temperature, the reaction mixture was washed sequentially with saturated aq. Na 2 S 2 O 3 (300 ml 4), H 2 O (300 ml), and brine (300 ml), dried over Na 2 SO 4 and concentrated. The residue was purified via silica gel column chromatography eluting with 50% ethyl acetate in petroleum ether to afford the product 19 as a pale-yellow solid (25 g, 68%). 1 H-NMR (500 MHz, DMSO-d 6 ): δ (s, 1H), 7.44 (s, 1H), (t, J=7.5 Hz, 2H), 2.65 (t, J=7.5 Hz, 2H), 2.54 (t, J=7.0 Hz, 2H), (m, 2H), 0.85 (t, 3H, J=7.5 Hz). MS (EI +, m/z): 306.0[M+H] +. 3-Iodo-7-propyl-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (20) To a solution of compound 19 (7.4 g, 24.3 mmol) in 1,4-dioxane (100 ml) at 15 o C was added DDQ (8.3 g, 36.4 mmol). The reaction mixture was stirred at 110 o C for 6 hours, cooled to room temperature, filtered, and washed with 1,4-dioxane (30 ml). The filtrate was concentrated and treated with ethyl acetate (100 ml). After stirring at room temperature for 20 minutes, the precipitates were collected to give pure 20 as a first batch (4.8 g). The filtrate was washed with saturated aq. NaHCO 3 (100 ml), brine (50 ml), dried over Na 2 SO 4 and concentrated to give crude 20 as a second batch (2.5 g). The combined product (7.3 g, 99%) were used for the next step without further purification. MS (EI +, m/z): [M+H] +.

15 3-Iodo-7-propyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (21) To a suspension of compound 20 (7.3 g, 24 mmol) in 3,4-dihydro-2H-pyran (70 ml), was added a few drops of TFA. The mixture was stirred at 95 C for 4 hours. After cooling to room temperature, the mixture was concentrated. EtOAc (80 ml) and H 2 O (50 ml) was added to the residue and the mixture was stirred at room temperature for 30minutes. The resulting solid precipitates was filtered, washed with H 2 O (10 ml), EtOAc (10 ml), and dried over high vacuum to give 21 (7.4 g, 80%) as a pale yellow oil. MS (EI +, m/z): [M+H] +. 7-Propyl-1-(tetrahydro-2H-pyran-2-yl)-3-(trimethylstannyl)-1H-pyrazolo[3,4-b]pyridin- 6(7H)-one (22) A mixture of compound 21 (3.8 g, 9.8 mmol), hexamethylditin (3.86 g, 11.8 mmol) and Pd(PPh 3 ) 2 Cl 2 (700 mg, 1.0 mmol) in 1,4-dioxane (50 ml) was stirred at 110 C for 16 hours. After cooling to 0 C, a solution of KF in water (1 M, 20 ml) was added. The reaction mixture was filtered and washed with ethyl acetate. The filtrate was dried over sodium sulfate and concentrated. The residue was purified via basic Al 2 O 3 column chromatography eluting with 10% ethyl acetate in petroleum ether to afford the product 22 as an off-white solid (1.55 g, 37%). MS (EI +, m/z): [M+H] +. 3-(2-(6-(dimethylamino)pyridin-3-yl)thiazol-5-yl)-7-propyl-1H-pyrazolo[3,4-b]pyridin- 6(7H)-one (3a)

16 5-Bromo-N,N-dimethylpyridin-2-amine A mixture of 2,5-dibromopyridine (1.7 g, 7.17 mmol) and dimethylamine in THF (15 ml, 2.0 M) was stirred at 120 o C for 20 hours in a sealed tube, the reaction mixture was cooled to room temperature and treated with water (50 ml). The resulting solid was filtered, washed with cold water (20 ml x 3), and dried over sodium sulfate to afford 5-bromo-N,N-dimethylpyridin-2-amine (1.2 g, 83%) as an off-white solid. MS (EI +, m/z): [M+H] +. N,N-dimethyl-5-(thiazol-2-yl)pyridin-2-amine A mixture of 5-bromo-N,N-dimethylpyridin-2- amine (1.92 g, 9.6 mmol), 2-(tributylstannyl)thiazole (9.95 g, 28.7 mmol), trifuran-2-yl phosphine (0.89 g, 3.84 mmol) and Pd(PPh 3 ) 2 Cl 2 (1.35 g, 1.92 mmol) in 1, 4-dioxane (60 ml) was stirred at 100 C overnight under nitrogen atmosphere. The mixture was concentrated and purified by silica gel column chromatography eluting with 30% ethyl acetate in petroleum ether to afford N,N-dimethyl-5-(thiazol-2-yl)pyridin-2-amine (1.0 g, 51%) as a pale yellow solid. MS (EI +, m/z): [M+H] +. 5-(5-Bromothiazol-2-yl)-N,N-dimethylpyridin-2-amine To a solution of N,N-dimethyl-5- (thiazol-2-yl)pyridin-2-amine (805 mg, 3.93 mmol) in DMF (10 ml) was added N-

17 bromosuccinimide (700 mg, 3.93 mmol) at room temperature. The mixture was stirred for 1 hour and diluted with cold water (50 ml). The ph was adjusted to 8-9 with saturated aq. Na 2 CO 3 solution. The reaction mixture was then extracted with ethyl acetate (50 ml x 3). The combined organic phases were washed with brine (50 ml), dried over sodium sulfate and concentrated. The residue was purified via silica gel column chromatography eluting with 30% ethyl acetate in petroleum ether to afford 5-(5-bromothiazol-2-yl)-N,N-dimethylpyridin-2-amine (500 mg, 45%) as a pale yellow solid. MS (EI +, m/z): [M+H] +. 3-(2-(6-(Dimethylamino)pyridin-3-yl)thiazol-5-yl)-7-propyl-1-(tetrahydro-2H-pyran-2-yl)- 1H-pyrazolo[3,4-b]pyridin-6(7H)-one A mixture of 5-(5-bromothiazol-2-yl)-N,Ndimethylpyridin-2-amine (500 mg, 1.76 mmol), 7-propyl-1-(tetrahydro-2H-pyran-2-yl)-3- (trimethylstannyl)-1h-pyrazolo[3,4-b]pyridin-6(7h)-one 22 (897 mg, 2.11 mmol), trifuran-2-yl phosphine (162 mg, 0.70 mmol) and Pd(PPh 3 ) 2 Cl 2 (247 mg, 0.35 mmol) in 1, 4-dioxane (20 ml) was stirred at 100 C overnight under nitrogen atmosphere. The mixture was concentrated and purified via silica gel column chromatography eluting with 20% ethyl acetate in petroleum ether to afford 3-(2-(6-(dimethylamino)pyridin-3-yl)thiazol-5-yl)-7-propyl-1-(tetrahydro-2H-pyran-2- yl)-1h-pyrazolo[3,4-b]pyridin-6(7h)-one (300 mg, 37%) as a pale yellow solid. MS (EI +, m/z): [M+H] +. 3-(2-(6-(Dimethylamino)pyridin-3-yl)thiazol-5-yl)-7-propyl-1H-pyrazolo[3,4-b]pyridin- 6(7H)-one (3a) A solution of 3-(2-(6-(dimethylamino)pyridin-3-yl)thiazol-5-yl)-7-propyl-1- (tetrahydro-2h-pyran-2-yl)-1h-pyrazolo[3,4-b]pyridin-6(7h)-one (300 mg, 0.65 mmol) in TFA (20 ml) was stirred at room temperature overnight and concentrated. The residue was purified via prep-hplc to afford 3-(2-(6-(dimethylamino)pyridin-3-yl)thiazol-5-yl)-7-propyl-1H-

18 pyrazolo[3,4-b]pyridin-6(7h)-one 3a (60 mg, 25%) as a pale yellow solid. 1 H-NMR (500 MHz, DMSO-d 6 ): δ (br, 1H), 8.71 (s, 1H), 8.34 (s, 1H), 8.03 (s, 1H), 8.05 (s, 1H), 6.77 (d, J = 9.0 Hz, 1H), 6.28 (d, J = 9.0 Hz, 1H), 4.03 (t, J = 7.5 Hz, 2H), 3.33 (s, 6H), 1.70 (q, J = 7.0 Hz, 2H), 0.91 (t, J = 8.0 Hz, 3H). MS (EI +, m/z): [M+H] +. 3-(2-(6-(4-Methylpiperazin-1-yl)pyridin-3-yl)thiazol-5-yl)-7-propyl-1H-pyrazolo[3,4- b]pyridin-6(7h)-one (3b) Prepared according to the similar procedure of 3a starting from 2,5- dibromopyridine and 4-N-methylpiperazine. 1 H-NMR (500 MHz, DMSO-d 6 ): δ (br, 1H), 8.72 (d, J = 2.0 Hz, 1H), 8.34 (s, 1H), (m, 2H), 6.96 (d, J = 9.0 Hz, 1H), 6.27 (d, J = 9.5 Hz, 1H), 4.02 (t, J = 7.5 Hz, 2H), (m, 4H), (m, 4H), 1.70 (q, J = 7.0 Hz, 2H), 0.91 (t, J = 8.0 Hz, 3H). MS (EI +, m/z): [M+H] +. 3-(2-(6-Methylpyridin-3-yl)thiazol-5-yl)-7-propyl-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (3c) Prepared according to the similar procedure of 3a starting from 5-bromo-2-methylpyridine and 2-(tributylstannyl)thiazole. 1 H-NMR (500 MHz, DMSO-d 6 ): δ (br, 1H), 9.06 (d, J = 2.5 Hz, 1H), 8.50 (s, 1H), 8.26 (dd, J = 2.5 Hz, J = 8.0 Hz, 1H), 8.15 (br, 1H), 7.44 (d, J = 8.0 Hz, 1H), (m, 1H), 4.02 (t, J = 8.0 Hz, 2H), 2.56 (s, 3H), 1.70 (t, J = 7.5 Hz, 2H), 0.92 (t, J = 7.5 Hz, 3H). MS (EI +, m/z): [M+H] +. 3-(2-(6-(Hydroxymethyl)pyridin-3-yl)thiazol-5-yl)-7-propyl-1H-pyrazolo[3,4-b]pyridin- 6(7H)-one (3d) Prepared according to the similar procedure of 3a starting from (5- bromopyridin-2-yl)methanol and 2-(tributylstannyl)thiazole. 1 H-NMR (500 MHz, DMSO-d 6 ): δ (br, 1H), 9.10 (d, J = 2.0 Hz, 1H), 8.52 (s, 1H), 8.38 (dd, J = 2.0 Hz, J = 7.5 Hz, 1H), 8.10 (br, 1H), 7.64 (d, J = 8.5 Hz, 1H), 6.31(d, J = 9.0 Hz, 1H), 5.58 (t, J = 5.5 Hz, 2H),4.64 (d, J =

19 5.5 Hz, 2H), 4.04 (t, J = 5.5 Hz, 2H), (m, 2H), 0.92 (t, J = 7.5 Hz, 3H). MS (EI +, m/z): [M+H] +. 3-(2-(6-Methylpyridin-3-yl)thiazol-5-yl)-7-propyl-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (3e) Prepared according to the similar procedure of 3a starting from 5-bromo-2-isopropylpyridine and 2-(tributylstannyl)thiazole. 1 H-NMR (500 MHz, DMSO-d 6 ): δ 9.10 (d, J = 2.0 Hz, 1H), 8.48 (s, 1H), 8.28 (dd, J = 2.0 Hz, J = 8.0 Hz, 1H), 8.07 (d, J = 9.5 Hz, 1H), 7.46 (d, J = 8.5 Hz, 1H), 6.28(d, J = 9.5 Hz, 1H), (m, 2H), (m, 1H), (m, 2H), 1.28 (d, J = 6.5 Hz, 6H), 0.92 (t, J = 7.5 Hz, 3H). MS (EI +, m/z): [M+H] +. 3-(2-(6-(morpholinomethyl)pyridin-3-yl)thiazol-5-yl)-7-propyl-1H-pyrazolo[3,4-b]pyridin- 6(7H)-one (3f) Prepared according to the similar procedure of 3a starting from 4-((5- bromopyridin-2-yl)methyl)morpholine and 2-(tributylstannyl)thiazole. 1 H-NMR (500 MHz, DMSO-d 6 ): δ (br, 1H), 9.11 (d, J = 2.0 Hz, 1H), 8.52 (s, 1H), 8.35 (dd, J = 2.0 Hz, J = 8.0 Hz, 1H), 8.09 (s, 1H), 7.63 (d, J = 8.0 Hz, 1H), 6.31(d, J = 8.5 Hz, 1H), 4.04 (t, J = 7.5 Hz, 2H), 3.68 (s, 2H), 3.61 (t, J = 4.5 Hz, 4H), 2.50 (t, J = 4.5 Hz, 4H), (m, 2H), 0.92 (t, J = 7.0 Hz, 3H). MS (EI +, m/z):437.2 [M+H] +. 3-(2-(6-((4-Methylpiperazin-1-yl)methyl)pyridin-3-yl)thiazol-5-yl)-7-propyl-1Hpyrazolo[3,4-b]pyridin-6(7H)-one (3g) Prepared according to the similar procedure of 3a starting from 1-((5-bromopyridin-2-yl)methyl)-4-methylpiperazine and 2- (tributylstannyl)thiazole. 1 H-NMR (500 MHz, DMSO-d 6 ): δ (br, 1H), 9.10 (d, J = 2.0 Hz, 1H), 8.52 (s, 1H), 8.35 (dd, J = 1.5 Hz, J = 7.5 Hz, 1H), 8.10 (d, J = 9.5 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 6.31(d, J = 9.5 Hz, 1H), 4.04 (t, J = 7.5 Hz, 2H), 3.66 (s, 2H), (m, 8H), 2.17 (s, 3H), (m, 2H), 0.92 (t, J = 7.0 Hz, 3H). MS (EI +, m/z):450.2 [M+H] +.

20 3-(2-(6-((Diethylamino)methyl)pyridin-3-yl)thiazol-5-yl)-7-propyl-1H-pyrazolo[3,4- b]pyridin-6(7h)-one (3h) Prepared according to the similar procedure of 3a starting from N-((5- bromopyridin-2-yl)methyl)-n-ethylethanamine and 2-(tributylstannyl)thiazole. 1 H-NMR (500 MHz, DMSO-d 6 ): δ 9.11 (d, J=2.0 Hz,1H), 8.52 (s, 1H), 8.36 (dd, J = 2.5 Hz, J = 8.0 Hz, 1H), 8.14 (s, 1H), 7.65 (d, J=8.0 Hz, 1H), 6.32 (d, J=9.0 Hz, 1H), 4.05 (d, J=8.0 Hz, 2H), 3.80 (s, 2H), (m, 4H), (m, 2H), 1.02 (t, J=7.0 Hz, 6H), 0.92 (t, J=7.0 Hz, 3H). MS (EI +, m/z): [M+H] +. Methyl yl)picolinate (3i) Prepared according to the similar procedure of 3a starting from methyl 5- bromopicolinate and 2-(tributylstannyl)thiazole. 1 H-NMR (500 MHz, DMSO-d 6 ): δ 9.32 (d, J=2.0 Hz,1H), 8.60 (s, 1H), 8.56 (dd, J = 2.0 Hz, J = 8.0 Hz, 1H), 8.20 (d, J=8.0 Hz, 1H), 8.12 (d, J=10.0 Hz, 1H), 6.29 (d, J=12.0 Hz, 1H), 4.05 (d, J=7.5 Hz, 2H), 3.93 (s, 3H), (m, 2H), 0.92 (t, J=7.0 Hz, 3H). MS (EI +, m/z): [M+H] +. 5-(5-(6-oxo-7-propyl-6,7-dihydro-1H-pyrazolo[3,4-b]pyridin-3-yl)thiazol-2- N,N-dimethyl-5-(5-(6-oxo-7-propyl-6,7-dihydro-1H-pyrazolo[3,4-b]pyridin-3-yl)thiazol-2- yl)picolinamide (3j) Prepared according to the similar procedure of 3a starting from 5-bromo- N,N-dimethylpicolinamide and 2-(tributylstannyl)thiazole. 1 H-NMR (500 MHz, DMSO-d 6 ): δ 9.18 (d, J=1.5 Hz,1H), 8.55 (s, 1H), 8.47(dd, J = 2.0 Hz, J = 8.0 Hz, 1H), 8.09 (d, J=9.5 Hz, 1H), 7.72 (d, J=8.0 Hz, 1H), 6.29 (d, J=9.5 Hz, 1H), 4.05 (d, J=7.5 Hz, 2H), 3.04 (s, 3H), 2.99 (s, 3H), (m, 2H), 0.92 (t, J=7.0 Hz, 3H). MS (EI +, m/z): [M+H] +. N-(2-(diethylamino)ethyl)-5-(5-(6-oxo-7-propyl-6,7-dihydro-1H-pyrazolo[3,4-b]pyridin-3- yl)thiazol-2-yl)picolinamide (3k) Prepared according to the similar procedure of 3a starting from 5-bromo-N-(2-(diethylamino)ethyl)picolinamide and 2-(tributylstannyl)thiazole. 1 H-NMR

21 (500 MHz, DMSO-d 6 ): δ 9.24 (d, J=2.5 Hz,1H), 8.81 (s, 1H), 8.59 (s, 1H), 8.55(dd, J = 1.5 Hz, J = 8.0 Hz, 1H), 8.24 (s, 1H), 8.18 (d, J=8.5 Hz, 1H), 8.11 (d, J=9.0 Hz, 1H), 6.32 (d, J=10.0 Hz, 1H), 4.05 (d, J=7.0 Hz, 2H), (m, 2H), 2.64 (t, J=7.5 Hz, 2H), (m, 4H), (m, 2H), 1.60 (t, J=7.5 Hz, 6H), 0.92 (t, J=7.5 Hz, 3H). MS (EI +, m/z): [M+H] +. N-(2-morpholinoethyl)-5-(5-(6-oxo-7-propyl-6,7-dihydro-1H-pyrazolo[3,4-b]pyridin-3- yl)thiazol-2-yl)picolinamide (3l) Prepared according to the similar procedure of 3a starting from 5-bromo-N-(2-morpholinoethyl)picolinamide and 2-(tributylstannyl)thiazole. 1 H-NMR (500 MHz, DMSO-d 6 ): δ 9.24 (d, J=2.0 Hz,1H), 8.82 (s, 1H), 8.59 (s, 1H), 8.55(dd, J = 2.0 Hz, J = 8.0 Hz, 1H), 8.21 (s, 1H), 8.18 (d, J=8.0 Hz, 1H), 8.12 (d, J=9.0 Hz, 1H), 6.32 (d, J=9.0 Hz, 1H), 4.04 (t, J=7.5 Hz, 2H), (m, 4H), (m, 4H), (m, 4H), (m, 2H), 0.92 (t, J=7.0 Hz, 3H). MS (EI +, m/z): [M+H] +. 7-Propyl-3-(thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (4a) A mixture of 5- bromothiazole (252 mg, 1.53 mmol), 7-propyl-1-(tetrahydro-2H-pyran-2-yl)-3- (trimethylstannyl)-1h-pyrazolo[3,4-b]pyridin-6(7h)-one 22 (993 mg, 2.34 mmol), trifuran-2-yl phosphine (36 mg, mmol) and Pd(PPh 3 ) 2 Cl 2 (180 mg, mmol) in 1, 4-dioxane (30 ml) was stirred at 100 C overnight under nitrogen atmosphere. The mixture was concentrated and purified via silica gel column chromatography eluting with 50% ethyl acetate in petroleum ether to afford 7-propyl-1-(tetrahydro-2H-pyran-2-yl)-3-(thiazol-5-yl)-1H-pyrazolo[3,4- b]pyridin-6(7h)-one (360 mg, 45%) as a pale yellow solid. MS (EI +, m/z): [M+H] +. A solution of 7-propyl-1-(tetrahydro-2H-pyran-2-yl)-3-(thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin- 6(7H)-one (400 mg, 1.16 mmol) in dichloromethane (10 ml) and TFA (5.0 ml) was stirred at room temperature for 0.5 hour and concentrated. The residue was purified via prep-hplc to

22 afford 7-propyl-3-(thiazol-5-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)-one 4a (50 mg, 16%) as a pale yellow solid 1 H-NMR (500 MHz, DMSO-d 6 ): δ (br, 1H), 9.21 (s, 1H), 8.47 (s, 1H), 8.01(d, J = 9.5 Hz, 1H), 6.27 (d, J=9.0 Hz, 1H), 4.03 (t, J=7.5 Hz, 2H), (m, 2H), 0.91(t, J=7.5 Hz, 3H). MS (EI +, m/z): [M+H] +. 7-Propyl-3-(5-(pyridin-3-yl)thiazol-2-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (4b) Prepared according to the similar procedure of 3a starting from 3-bromopyridine and 5- (tributylstannyl)thiazole. 1 H-NMR (500 MHz, DMSO-d 6 ): δ (br, 1H), 9.01 (t, J = 2.5 Hz, 1H), 8.59(d, J = 5.0 Hz, 1H), 8.54 (s, 1H), 8.18(d, J = 8.0 Hz, 1H), 8.15(d, J = 9.5 Hz, 1H), 6.34 (d, J=9.0 Hz, 1H), 4.05 (t, J=7.0 Hz, 2H), (m, 2H), 0.92 (t, J=7.5 Hz, 3H). MS (EI +, m/z): [M+H] +. Methyl 2-(6-oxo-7-propyl-1-(tetrahydro-2H-pyran-2-yl)-6,7-dihydro-1H-pyrazolo[3,4- b]pyridin-3-yl)-5-(pyridin-3-yl)thiazole-4-carboxylate (4c) Prepared according to the similar procedure of 3a starting from 3-bromopyridine and methyl 5-(tributylstannyl)thiazole-4- carboxylate. 1 H-NMR (500 MHz, DMSO-d 6 ): δ (br, 1H), 9.01 (t, J = 2.5 Hz, 1H), 8.59(d, J = 5.0 Hz, 1H), 8.54 (s, 1H), 8.18(d, J = 8.0 Hz, 1H), 8.15(d, J = 9.5 Hz, 1H), 6.34 (d, J=9.0 Hz, 1H), 4.05 (t, J=7.0 Hz, 2H), 3.78 (s, 3H), (m, 2H), 0.92 (t, J=7.5 Hz, 3H). MS (EI +, m/z): [M+H] +. N,N-dimethyl-2-(6-oxo-7-propyl-6,7-dihydro-1H-pyrazolo[3,4-b]pyridin-3-yl)-5-(pyridin-3- yl)thiazole-4-carboxamide (4d) A mixture of methyl 2-(6-oxo-7-propyl-6,7-dihydro-1Hpyrazolo[3,4-b]pyridin-3-yl)-5-(pyridin-3-yl)thiazole-4-carboxylate 4c (395 mg, 1 mmol) in THF (10 ml) and MeOH (2 ml) solution was treated with LiOH (72 mg, 3 mmol) in 0.5 ml water solution. The mixture was stirred at room temperature for 12 h. After acidifying with 1M HCl,

23 the resulting precipitates were collected to afford 2-(6-oxo-7-propyl-6,7-dihydro-1Hpyrazolo[3,4-b]pyridin-3-yl)-5-(pyridin-3-yl)thiazole-4-carboxylic acid (343 mg, 90%). 2-(6- oxo-7-propyl-6,7-dihydro-1h-pyrazolo[3,4-b]pyridin-3-yl)-5-(pyridin-3-yl)thiazole-4-carboxylic acid (150 mg, 0.39 mmol), dimethylamine hydrochloride (253 mg, 3.1 mmol), diisopropylethylamine (761 mg, 5.9 mmol) and HATU (760 mg, 2.0 mmol) in DMF (5 ml) was stirred at 15 o C for 16 hours. The reaction mixture was purified directly by prep-hplc to give 4d (60 mg, 37%) as an off white solid. 1 H-NMR (500 MHz, DMSO-d 6 ): δ 8.76 (d, J=2.0 Hz, 1H), 8.62 (t, J=4.0 Hz, 1H), 8.08 (d, J=9.0 Hz, 1H), (m, 1H), 7.53(dd, J=5.0 Hz, J=8.0 Hz, 1H), 6.31 (d, J=10.0 Hz, 1H), 4.07 (t, J=7.5Hz, 2H), 3.04(s, 3H), 2.89(s, 3H), (m, 2H), 0.92(t, J=7.0Hz, 3H). MS (EI +, m/z): [M+H] +. Synthesis of 5-methyl-2-(4-(6-oxo-7-propyl-6,7-dihydro-1H-pyrazolo[3,4-b]pyridin-3- yl)piperidin-1-yl)thiazole-4-carboxylic acid (4e) Step 1: tert-butyl 4-(6-oxo-7-propyl-1-(tetrahydro-2H-pyran-2-yl)-6,7-dihydro-1H-pyrazolo[3,4- b]pyridin-3-yl)-5,6-dihydropyridine-1(2h)-carboxylate

24 A mixture of 3-iodo-7-propyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)- one 21 (387 mg, 1.0 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6- dihydropyridine-1(2h)-carboxylate (340 mg, 1.1 mmol), Pd(dppf) 2 Cl 2 (82 mg, 0.1 mmol), Na 2 CO 3 (212 mg, 2.0 mmol), 1,4-dioxane (20 ml) and H 2 O (10 ml) was heated at 100 o C for 5 hours. The mixture was cooled and concentrated. The residue was purified via silica gel column chromatography eluting with 10% ethyl acetate in petroleum ether to afford tert-butyl 4-(6-oxo- 7-propyl-1-(tetrahydro-2H-pyran-2-yl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridin-3-yl)-5,6- dihydropyridine-1(2h)-carboxylate (344 mg, 78%) as an off-white solid. MS (EI +, m/z): [M+H] +. Step 2: tert-butyl 4-(6-oxo-7-propyl-1-(tetrahydro-2H-pyran-2-yl)-6,7-dihydro-1H-pyrazolo[3,4- b]pyridin-3-yl)piperidine-1-carboxylate To a solution of tert-butyl 4-(6-oxo-7-propyl-1-(tetrahydro-2H-pyran-2-yl)-6,7-dihydro-1Hpyrazolo[3,4-b]pyridin-3-yl)-5,6-dihydropyridine-1(2H)-carboxylate (221mg, 0.5 mmol) in MeOH (10 ml) was added Pd/C (40 mg). The mixture was stirred under 1 atm H 2 for 17 hours at 40 o C. The resulting solution was filtered, and concentrated to afford tert-butyl 4-(6-oxo-7- propyl-1-(tetrahydro-2h-pyran-2-yl)-6,7-dihydro-1h-pyrazolo[3,4-b]pyridin-3-yl)piperidine-1- carboxylate (195 mg, 88%) as an off-white solid, which was used for the next step directly. MS (EI +, m/z): [M+H] +. Step 3: 3-(piperidin-4-yl)-7-propyl-1H-pyrazolo[3,4-b]pyridin-6(7H)-one To a solution of tert-butyl 4-(6-oxo-7-propyl-1-(tetrahydro-2H-pyran-2-yl)-6,7-dihydro-1Hpyrazolo[3,4-b]pyridin-3-yl)piperidine-1-carboxylate (890 mg, 2.0 mmol) in dichloromethane

25 (20 ml) was added TFA (5.0 ml) at room temperature. The reaction mixture was stirred for 2 hours at room temperature and concentrated. The residue was diluted with ethyl acetate (60 ml) and washed with saturated aq. NaHCO 3 solution (30 ml). The organic layer was separated, dried over sodium sulfate and concentrated to afford 3-(piperidin-4-yl)-7-propyl-1H-pyrazolo[3,4- b]pyridin-6(7h)-one (420 mg, 80%) as an off-white solid, which was used for the next step directly. MS (EI +, m/z): [M+H] +. Step 4: ethyl 5-methyl-2-(4-(6-oxo-7-propyl-6,7-dihydro-1H-pyrazolo[3,4-b]pyridin-3- yl)piperidin-1-yl)thiazole-4-carboxylate To a solution of 3-(piperidin-4-yl)-7-propyl-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (260 mg, 1.0 mmol) and ethyl 2-chloro-5-methylthiazole-4-carboxylate (308 mg, 1.5 mmol) in DMSO (5.0 ml) was added CsF (760 mg, 5.0 mmol). The reaction mixture was heated at 50 o C for 27 hours. After cooling to room temperature, the solution was diluted with ethyl acetate (80 ml) and washed with water (20 ml). The organic layers was dried over sodium sulfate and concentrated to afford ethyl 5-methyl-2-(4-(6-oxo-7-propyl-6,7-dihydro-1H-pyrazolo[3,4-b]pyridin-3- yl)piperidin-1-yl)thiazole-4-carboxylate (220 mg, 51%) as a pale yellow solid, which was used for the next step directly. MS (EI +, m/z): [M+H] +. Step 5: 5-methyl-2-(4-(6-oxo-7-propyl-6,7-dihydro-1H-pyrazolo[3,4-b]pyridin-3-yl)piperidin-1- yl)thiazole-4-carboxylic acid 4f A mixture of ethyl 5-methyl-2-(4-(6-oxo-7-propyl-6,7-dihydro-1H-pyrazolo[3,4-b]pyridin-3- yl)piperidin-1-yl)thiazole-4-carboxylate (220 mg, 0.51 mmol), LiOH (105 mg, 2.5 mmol), H 2 O (3.0 ml) and THF (5.0 ml) was stirred at 20 o C for 17 hours. The reaction mixture was ph-

26 adjusted to 3, and concentrated. The residue was purified by prep-hplc to afford 5-methyl-2-(4- (6-oxo-7-propyl-6,7-dihydro-1H-pyrazolo[3,4-b]pyridin-3-yl)piperidin-1-yl)thiazole-4- carboxylic acid 4f (56 mg, 29%) as an off-white solid. 1 H NMR (500 MHz, DMSO-d 6 ): (br, 1H), (br, 1H), 7.81 (d, J =9.0 Hz, 1H), 6.12 (d, J =9.5 Hz, 1H), (m, 4H), (m, 1H), (m, 2H), 2.51 (s, 3H), (m, 2H), (m, 2H), (m, 2H), 0.88 (t, J =7.5 Hz, 3H). MS (EI +, m/z): [M+H] +.