LIU HAIBO (US)
POSY SHOSHANA L (US)
MU YUCHENG (US)
BRONSON JOANNE JEWETT (US)
D’AGOSTINO LAURA AKULLIAN (US)
WO2019246513A1 | 2019-12-26 | |||
WO2022056281A1 | 2022-03-17 | |||
WO2022192598A1 | 2022-09-15 | |||
WO2023150150A1 | 2023-08-10 |
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CLAIMS What is claimed is: 1. A compound having the following structure: wherein, independently for each occurrence: R1 is selected from the group consisting of: −H, alkyl, −OCH3, substituted alkyl, alkoxyl, amine, secondary amine, tertiary amine, halogen, aryl, −CH2CH3, −CN, −OCH3, cyclopropyl, cyclopropoxy, cyclohexyl, −CF3, -OH, -Ph, -CH2CH3, - N(CH3)2, -NHCH3, and cycloalkyl; R2 is selected from the group consisting of: −H, alkyl, −CN, −OCH3, cycloalkyl, −CF3, −C(CH3)2R7, aryl, substituted alkyl, alkoxyl, -CH(CH3)2, -C(CH3)3, - OCF3, -OH, and benzyloxy; R3 is selected from the group consisting of: −H, alkyl, −OCH3, substituted alkyl, amine, secondary amine, tertiary amine, −CHF2, halogen, −CN, −OCH3, −N(CH3)2, −OCHF2, alkoxyl, -NHCH3, -OH, -CH2CH3, and morpholin-4-yl; R4 is selected from the group consisting of: −H , alkyl, −CH2CH3, −OCH3, -OH, and −CF3; R5 is selected from the group consisting of : -H, cycloalkyl, alkyl, and substituted alkyl. 2. The compound according to claim 1, wherein: R1 is selected from the group consisting of: −H, −CH3, −OCH3, and cyclopropyl; R2 is selected from the group consisting of: −H, −CH3, −CN, and −OCH3; R3 is selected from the group consisting of: −H, −CH3, and −OCH3; R4 is selected from the group consisting of: −H and −CH3. 3. The compound according to claim 1, wherein: R1 is cyclopropyl; R2 is −H; R3 is −H; R4 is −H; R5 is -H. 4. The compound according to claim 1, wherein: R1 is −OCH3; R2 is alkyl; R3 is −H; R4 is −H; R5 is -H. 5. The compound according to claim 1, wherein: R1 is −OCH3; R2 is −H; R3 is −H; R4 is −H; R5 is -H. 6. The compound according to claim 1, wherein: R1 is −OCH3; R2 is −H; R3 is alkyl; R4 is −H; R5 is -H. 7. The compound according to claim 1, wherein: R1 is −H; R2 is −OCH3; R3 is −H; R4 is −H; R5 is -H. 8. The compound according to claim 1, wherein: R1 is alkyl; R2 is −CN; R3 is −H; R4 is −H; R5 is -H. 9. The compound according to claim 1, wherein: R1 is −H; R2 is −H; R3 is −H; R4 is alkyl; R5 is -H. 10. The compound according to claim 1, wherein: R1 is −H; R2 is −H; R3 is −OCH3; R4 is −H; R5 is -H. 11. A compound selected from the group consisting of: 5-[6-fluoro-4-[[(6-methoxy-2-pyridyl)amino]methyl]-1H-indazol-7-yl]-1,1-dioxo- 1,2,5-thiadiazolidin-3-one; 5-(6-fluoro-4-(((5-methoxypyridin-2-yl)amino)methyl)-1H-indazol-7-yl)-1,2,5- thiadiazolidin-3-one 1,1-dioxide; 5-[6-fluoro-4-[[(4-methoxy-2-pyridyl)amino]methyl]-1H-indazol-7-yl]-1,1-dioxo- 1,2,5-thiadiazolidin-3-one; 5-[4-[[(4-cyclopropyl-2-pyridyl)amino]methyl]-6-fluoro-1H-indazol-7-yl]-1,1- dioxo-1,2,5-thiadiazolidin-3-one; 5-[6-fluoro-4-[[(4-methyl-2-pyridyl)amino]methyl]-1H-indazol-7-yl]-1,1-dioxo- 1,2,5-thiadiazolidin-3-one; 5-(4-(((4,6-dimethylpyridin-2-yl)amino)methyl)-6-fluoro-1H-indazol-7-yl)-1,2,5- thiadiazolidin-3-one 1,1-dioxide; 5-(6-fluoro-4-(((4-methoxy-5-methylpyridin-2-yl)amino)methyl)-1H-indazol-7- yl)-1,2,5-thiadiazolidin-3-one 1,1-dioxide; 6-[[6-fluoro-7-(1,1,4-trioxo-1,2,5-thiadiazolidin-2-yl)-1H-indazol-4- yl]methylamino]-4-methyl-pyridine-3-carbonitrile; 5-[6-fluoro-4-[[(4-methoxy-6-methyl-2-pyridyl)amino]methyl]-1H-indazol-7-yl]- 1,1-dioxo-1,2,5-thiadiazolidin-3-one; 5-[6-fluoro-4-[[(3-methyl-2-pyridyl) amino] methyl]-1H-indazol-7-yl]-1,1-dioxo- 1,2,5-thiadiazolidin-3-one; or pharmaceutically acceptable salts thereof. 12. A pharmaceutical composition comprising a compound of Formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier. 13. A method for treating cancer comprising administering to said patient a therapeutically effective amount of a compound of Formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof wherein the cancer/disease is selected from: human cancers, carcinomas, sarcomas, adenocarcinomas, papillary adenocarcinomas, lymphomas, leukemias, melanomas, solid lymphoid cancers, kidney cancer, breast cancer, lung cancer, bladder cancer, colon cancer, ovarian cancer, prostate cancer, pancreatic cancer, stomach cancer, brain cancer, head and neck cancer, skin cancer, uterine, testicular, glioma, esophagus, liver cancer, including hepatocarcinoma, lymphoma, including B-acute lymphoblastic lymphoma, non-Hodgkin's lymphomas, Burkitt's lymphoma, Small lymphomas, Hodgkin's lymphoma, leukemia, and multiple myeloma. 14. A method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of a compound of claim 1 in combination with an additional therapeutic agent. 15. The method of claim 14 wherein the additional therapeutic agent is an immunotherapeutic agent. 16. The method of claim 15 wherein the immunotherapeutic agent is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, and an anti-CTLA-4 antibody. 17. A method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of a pharmaceutically acceptable composition of claim 1. 18. The method of claim 14 wherein the method of treating cancer is selected from radiation, surgery, chemotherapy, or administration of a biologic drug. 19. The method of claim 18 wherein the method of treating cancer is the administration of a biologic drug, wherein the biologic drug is a drug that stimulates the immune system. 20. The method of claim 19 wherein the method comprises administering to the subject an inhibitor of DGKα and/or DGKζ, an antagonist of the PD1/PD-L1 axis, and an antagonist of CTLA4. |
The first aspect of the present invention provides at least one compound of Formula (I): A compound having the following structure: wherein, independently for each occurrence: R 1 is selected from the group consisting of: −H, alkyl, −OCH 3 , substituted alkyl, alkoxyl, amine, secondary amine, tertiary amine, halogen, aryl, −CH 2 CH 3 , −CN, −OCH 3 , cyclopropyl, cyclopropoxy, cyclohexyl, −CF 3 , -OH, -Ph, -CH2CH3, -N(CH3)2, - NHCH3, and cycloalkyl; R 2 is selected from the group consisting of: −H, alkyl, −CN, −OCH 3, cycloalkyl, −CF 3 , −C(CH 3 ) 2 R 7 , aryl, substituted alkyl, alkoxyl, -CH(CH3)2, -C(CH3)3, -OCF3, -OH, and benzyloxy; R 3 is selected from the group consisting of: −H, alkyl, −OCH 3 , substituted alkyl, amine, secondary amine, tertiary amine, −CHF 2 , halogen, −CN, −OCH 3 , −N(CH 3 ) 2 , −OCHF 2 , alkoxyl, -NHCH3, -OH, -CH2CH3, and morpholin-4-yl; R 4 is selected from the group consisting of: −H , alkyl, −CH 2 CH 3 , −OCH 3 , -OH, and −CF 3 ; R 5 is selected from the group consisting of : -H, cycloalkyl, alkyl, and substituted alkyl.In one embodiment of the compound of formula I: In one embodiment of the compound of formula I: R 1 is selected from the group consisting of: −H, −CH 3 , −OCH 3 , and cyclopropyl; R 2 is selected from the group consisting of: −H, −CH 3 , −CN, and −OCH 3 ; R 3 is selected from the group consisting of: −H, −CH 3 , and −OCH 3 ; R 4 is selected from the group consisting of: −H and −CH 3 . In another embodiment of the compound of formula I: R 1 is cyclopropyl; R 2 is −H; R 3 is −H; R 4 is −H; R 5 is -H. In one embodiment of the compound of formula I: R 1 is −OCH 3 ; R 2 is alkyl; R 3 is −H; R 4 is −H; R 5 is -H. In another embodiment of the compound of formula I: R 1 is −OCH 3 ; R 2 is −H; R 3 is −H; R 4 is −H; R 5 is -H. In one embodiment of the compound of formula I: R 1 is −OCH 3 ; R 2 is −H; R 3 is alkyl; R 4 is −H; R 5 is -H. In one embodiment of the compound of formula I: R 1 is −H; R 2 is −OCH 3 ; R 3 is −H; R 4 is −H; R 5 is -H. In another embodiment of the compound of formula I: R 1 is alkyl; R 2 is −CN; R 3 is −H; R 4 is −H; R 5 is -H. In one embodiment of the compound of formula I: R 1 is −H; R 2 is −H; R 3 is −H; R 4 is alkyl; R 5 is -H. In another embodiment of the compound of formula I: R 1 is −H; R 2 is −H; R 3 is −OCH 3 ; R 4 is −H; R 5 is -H. In one embodiment of the compound of formula I, the compound is selected from a group consisting of: 5-[6-fluoro-4-[[(6-methoxy-2-pyridyl)amino]methyl]-1H-indazo l-7-yl]-1,1-dioxo- 1,2,5-thiadiazolidin-3-one; 5-(6-fluoro-4-(((5-methoxypyridin-2-yl)amino)methyl)-1H-inda zol-7-yl)-1,2,5- thiadiazolidin-3-one 1,1-dioxide; 5-[6-fluoro-4-[[(4-methoxy-2-pyridyl)amino]methyl]-1H-indazo l-7-yl]-1,1-dioxo- 1,2,5-thiadiazolidin-3-one; 5-[4-[[(4-cyclopropyl-2-pyridyl)amino]methyl]-6-fluoro-1H-in dazol-7-yl]-1,1- dioxo-1,2,5-thiadiazolidin-3-one; 5-[6-fluoro-4-[[(4-methyl-2-pyridyl)amino]methyl]-1H-indazol -7-yl]-1,1-dioxo- 1,2,5-thiadiazolidin-3-one; 5-(4-(((4,6-dimethylpyridin-2-yl)amino)methyl)-6-fluoro-1H-i ndazol-7-yl)-1,2,5- thiadiazolidin-3-one 1,1-dioxide; 5-(6-fluoro-4-(((4-methoxy-5-methylpyridin-2-yl)amino)methyl )-1H-indazol-7- yl)-1,2,5-thiadiazolidin-3-one 1,1-dioxide; 6-[[6-fluoro-7-(1,1,4-trioxo-1,2,5-thiadiazolidin-2-yl)-1H-i ndazol-4- yl]methylamino]-4-methyl-pyridine-3-carbonitrile; 5-[6-fluoro-4-[[(4-methoxy-6-methyl-2-pyridyl)amino]methyl]- 1H-indazol-7-yl]- 1,1-dioxo-1,2,5-thiadiazolidin-3-one; 5-[6-fluoro-4-[[(3-methyl-2-pyridyl) amino] methyl]-1H-indazol-7-yl]-1,1-dioxo- 1,2,5-thiadiazolidin-3-one; or pharmaceutically acceptable salts thereof. In another embodiment the invention comprises a pharmaceutical composition comprising a compound of Formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier. In an embodiment the invention comprises a method for treating cancer comprising administering to said patient a therapeutically effective amount of a compound of Formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof wherein the cancer/disease is selected from: human cancers, carcinomas, sarcomas, adenocarcinomas, papillary adenocarcinomas, lymphomas, leukemias, melanomas, solid lymphoid cancers, kidney cancer, breast cancer, lung cancer, bladder cancer, colon cancer, ovarian cancer, prostate cancer, pancreatic cancer, stomach cancer, brain cancer, head and neck cancer, skin cancer, uterine, testicular, glioma, esophagus, liver cancer, including hepatocarcinoma, lymphoma, including B-acute lymphoblastic lymphoma, non-Hodgkin's lymphomas, Burkitt's lymphoma, Small lymphomas, Hodgkin's lymphoma, leukemia, and multiple myeloma. In another embodiment, the invention comprises a method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of a compound of formula I in combination with an additional therapeutic agent. In one embodiment, the additional therapeutic agent is an immunotherapeutic agent. In another embodiment, the immunotherapeutic agent is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, and an anti-CTLA-4 antibody. In one embodiment, the method of treating cancer in a patient in need thereof, comprises administering to the patient an effective amount of a pharmaceutically acceptable composition of the compound of formula I. In another embodiment, the method of treating cancer is selected from radiation, surgery, chemotherapy, or administration of a biologic drug. In one embodiment, the method of treating cancer is the administration of a biologic drug and the biologic drug is a drug that stimulates the immune system. In another embodiment, the method of treating cancer comprises administering to the subject an inhibitor of DGKα and/or DGKζ, an antagonist of the PD1/PD-L1 axis and an antagonist of CTLA4. These embodiments are not intended to limit the scope of the invention. SYNTHETIC METHODS The compounds of the invention may be prepared by the methods and examples presented below and by methods known to those of ordinary skill in the art. In each of the examples below, the R groups are as defined above for each formula unless noted. Optimum reaction conditions and reaction times may vary according to the reactants used. Unless otherwise specified, solvents, temperatures, pressures, and other reaction conditions may be readily selected by one of ordinary skill in the art. The intermediates used in the syntheses below are either commercially available or easily prepared by methods known to those skilled in the art. Reaction progress may be monitored by conventional methods such as thin-layer chromatography (TLC) or high- pressure liquid chromatography-mass spec (HPLC-MS). Intermediates and products may be purified by methods known in the art, including column chromatography, HPLC, preparative TLC, or Preparatory HPLC. Preparation of Examples Preparation of synthetic intermediates (Int-2) Preparation of 7-(1,1-dioxido-4-oxo-1,2,5-thiadiazolidin-2-yl)-6-fluoro-2-( 4- methoxybenzyl)-2H-indazole-4-carbaldehyde (Int-2) as shown in Scheme 1. Scheme 1:
B Step 1: Synthesis of 4-bromo-6-fluoro-7-nitro-1H-indazole (1-2) To a round-bottom-flask with 4-bromo-6-fluoro-1H-indazole (10.00 g, 46.95 mmol) and sulfuric acid (100 mL) was added potassium nitrate (4.98 g, 49.30 mmol) dissolved in concentrated sulfuric acid (100 mL) dropwise at 0 o C. The resulting mixture was stirred at room temperature overnight. After completion of the reaction monitored by LCMS, the mixture was poured into 1.0 L of ice water, resulting in the formation of a light-yellow precipitate. This mixture was filtrated. The solid was washed with water and dried to obtain the desired product. The crude product was purified by a silica column chromatography (5%~10% ethyl acetate in petroleum ether) to afford 4-bromo-6-fluoro- 7-nitro-1H-indazole (9.33 g, 35.88 mmol, 76.42% yield) as a yellow solid. 1 H NMR (400 MHz, DMSO-d 6 ) δ 14.23 (s, 1H), 8.33 (d, J = 1.7 Hz, 1H), 7.74 (dd, J = 11.5, 1.9 Hz, 1H). Note: The desired isomer was identified by 2D NMR because there is no correlation between the aromatic proton and the NH group of the pyrazole moiety. Step 2: Synthesis of 4-bromo-6-fluoro-2-(4-methoxybenzyl)-7-nitro-2H-indazole (1- 3) To a stirred mixture of 4-bromo-6-fluoro-7-nitro-1H-indazole (9.33 g, 35.88 mmol) in DCM (400 mL) were added 4-methoxybenzyl 2,2,2-trichloroacetimidate (12.68 g, 44.98 mmol) and TsOH (1.21 g, 7.02 mmol) at room temperature. The resulting mixture was stirred at room temperature overnight. LCMS showed the starting material was consumed completely. The solution was diluted with DCM and washed with saturated sodium bicarbonate. The organic phase was dried over anhydrous sodium sulfate, filtrated, and concentrated. The resulting residue was purified by a silica gel column chromatography eluting with DCM to afford 4-bromo-6-fluoro-2-[(4-methoxyphenyl)methyl]-7-nitro- indazole (12.22 g, 32.15 mmol, 89.60% yield) as a yellow solid. MS: m/z: Calc’d for C 15 H 11 BrFN 3 O 3 [M+H] + 380, found 380. Step 3: Synthesis of 4-bromo-6-fluoro-2-(4-methoxybenzyl)-2H-indazol-7-amine (1- 4) To a stirred mixture of 4-bromo-6-fluoro-2-[(4-methoxyphenyl)methyl]-7-nitro-indazol e (12.22 g, 32.15 mmol) in ethanol (200 mL) and water (20 mL) were added Fe (17.77 g, 318.26 mmol) and NH 4 Cl (17.22 g, 321.91 mmol) at room temperature. The resulting mixture was stirred at 80 o C for 2 h under a nitrogen atmosphere. LCMS showed the reaction was completed. The reaction mixture was filtrated. The filtrate was concentrated to remove ethanol under reduced pressure, and diluted with water (200 mL). The solution was extracted with ethyl acetate (3 x 200 mL). The combined organic layers were washed with brine (200 mL), dried over anhydrous sodium sulfate, filtrated, and concentrated. The resulting residue was purified by a silica gel column chromatography (6% ethyl acetate in dichloromethane) to afford 4-bromo-6-fluoro-2-[(4- methoxyphenyl)methyl]indazol-7-amine (10 g, 28.56 mmol, 88.82% yield) as a pink solid. MS: m/z: Calc’d for C 15 H 13 BrFN 3 O [M+H] + 350, found 350. Step 4: Synthesis of ethyl (4-bromo-6-fluoro-2-(4-methoxybenzyl)-2H-indazol-7-yl) glycinate (1-5) To a stirred solution of 4-bromo-6-fluoro-2-[(4-methoxyphenyl)methyl]indazol-7-amine (7.5 g, 21.42 mmol) and 50% ethyl 2-oxoacetate in toluene (6.58 g, 32.13 mmol) in DMF (100 mL) was added TMSCl (6.8 mL, 53.54 mmol) at 0 o C. The mixture was stirred at ambient temperature for 40 mins. A solution of NaBH 3 CN (3.37 g, 53.54 mmol) in DMF (20 mL) was added slowly to the above mixture at 0 o C. The resulting mixture was stirred at ambient temperature for 3 h until the starting material has been fully consumed. The mixture was quenched by the addition of a saturated NH 4 Cl (200 mL) at 0 o C. The solution was extracted with ethyl acetate 2 times. The combined organic phase was washed with brine 3 times. The organic layers were dried over anhydrous sodium sulfate, filtrated, and concentrated. The resulting residue was purified by a silica gel column chromatography (20%~30% ethyl acetate in petroleum ether) to obtain ethyl 2-[[4-bromo-6-fluoro-2-[(4-methoxyphenyl)methyl]indazol-7- yl]amino]acetate (5.5 g, 12.60 mmol, 58.86% yield) as a light-yellow oil. MS: m/z: Calc’d for C 19 H 19 BrFN 3 O 3 [M+H] + 436, found 436. Step 5: Synthesis of ethyl N-(4-bromo-6-fluoro-2-(4-methoxybenzyl)-2H-indazol-7- yl)-N-sulfamoylglycinate (1-6) To a stirred solution of ethyl 2-[[4-bromo-6-fluoro-2-[(4- methoxyphenyl)methyl]indazol-7-yl]amino]acetate (5.5 g, 12.61 mmol) in DMA (50 mL) was added a solution of sulfamoyl chloride (9.47 g, 81.94 mmol) in DMA(15 mL) at 0 o C. The reaction mixture was stirred at ambient temperature overnight. LCMS showed the starting material was consumed completely. The mixture was diluted with ethyl acetate and washed with brine 7 times until the DMA was washed out completely. The organic phase was dried over anhydrous sodium sulfate, filtrated, and concentrated to obtain 6.7 g of the products as a light-brown semi-solid. The crude product was used in the next step directly. MS: m/z: Calc’d for C 19 H 20 BrFN 4 O 5 S [M+H] + 515, found 515. Step 6: Synthesis of 5-(4-bromo-6-fluoro-2-(4-methoxybenzyl)-2H-indazol-7-yl)- 1,2,5-thiadiazolidin-3-one 1,1-dioxide (Int-1) To a stirred solution of ethyl 2-[[4-bromo-6-fluoro-2-[(4-methoxyphenyl)methyl]indazol- 7-yl]-sulfamoyl-amino]acetate (6.7 g, 13 mmol) in Methanol (60 mL) was added 30% NaOMe in MeOH (14.03 g, 78.01 mmol) at 0 o C. The reaction mixture was stirred at ambient temperature for 2 h. After completion of the reaction monitored by LCMS, the mixture was diluted with ethyl acetate and concentrated. The resulting suspension was dissolved with water (500 mL), diluted with ethyl acetate, acidified by 1 N HCl solution to pH = 3, and extracted with ethyl acetate 3 times. The combined organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by reversed-phase column to obtain 5-[4- bromo-6-fluoro-2-[(4-methoxyphenyl)methyl]indazol-7-yl]-1,1- dioxo-1,2,5- thiadiazolidin-3-one (4 g, 8.5237 mmol, 65.56% yield) as a light yellow solid. MS: m/z: Calc’d for C 17 H 14 BrFN 4 O 4 S [M+H] + 469, found 469. Step 7: Synthesis of 5-(6-fluoro-2-(4-methoxybenzyl)-4-vinyl-2H-indazol-7-yl)-1,2 ,5- thiadiazolidin-3-one 1,1-dioxide (1-7) To a stirred solution of 5-[4-bromo-6-fluoro-2-[(4-methoxyphenyl)methyl]indazol-7-yl] - 1,1-dioxo-1,2,5-thiadiazolidin-3-one (2 g, 4.26 mmol) and tributyl(vinyl)stannane (4.05 g, 12.79 mmol) in DMA (20 mL) were added Pd 2 (dba) 3 (0.39 g, 0.43 mmol) and P(t- Bu) 3 HBF 4 (0.41 g, 0.85 mmol). The resulting mixture was evacuated and backfilled with N 2, 3 times. Then, the mixture was stirred at 100 °C overnight. LCMS showed the starting material was consumed completely. The reaction mixture was purified by reversed-phase column (0.05% NH 4 HCO 3 in water and MeCN) to obtain 5-[6-fluoro-2- [(4-methoxyphenyl)methyl]-4-vinyl-indazol-7-yl]-1,1-dioxo-1, 2,5-thiadiazolidin-3-one (1.1 g, 2.64 mmol, 61.98% yield) as a yellow solid. MS: m/z: Calc’d for C 19 H 17 FN 4 O 4 S [M+H] + 417, found 417. Step 8: Synthesis of 7-(1,1-dioxido-4-oxo-1,2,5-thiadiazolidin-2-yl)-6-fluoro-2-( 4- methoxybenzyl)-2H-indazole-4-carbaldehyde (Int-2) To a stirred solution of 5-[6-fluoro-2-[(4-methoxyphenyl)methyl]-4-vinyl-indazol-7-yl ]- 1,1-dioxo-1,2,5-thiadiazolidin-3-one (1.1 g, 2.64 mmol), NMO (0.62 g, 5.28 mmol) and Citric Acid (1.11 g, 5.28 mmol) in a mixed solvent of tert-butanol (10 mL) and water (10 mL) was added K 2 OsO 4 (0.09 g, 0.26 mmol) at room temperature. The resulting mixture was stirred at room temperature for 2 h. LCMS showed the starting material was converted to the intermediate completely. Then, NaIO 4 (1.69 g, 7.92 mmol) was added to the mixture at 0 o C in batches. The resulting mixture was stirred at room temperature for 2 h. LCMS showed the reaction was completed. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate, 7 times. The combined organic phase was dried over anhydrous sodium sulfate, filtrated, and concentrated. The resulting residue was purified by reversed-phase column chromatography (0.05% NH 4 HCO 3 in water and MeCN) to afford 6-fluoro-2-[(4-methoxyphenyl)methyl]-7-(1,1,4-trioxo-1,2,5- thiadiazolidin-2-yl)indazole-4-carbaldehyde (600 mg, 1.43 mmol, 54.28% yield) as a yellow solid. MS: m/z: Calc’d for C 18 H 15 FN 4 O 5 S [M+H] + 419, found 419. EXAMPLE 1: 5-[6-fluoro-4-[[(6-methoxy-2-pyridyl)amino]methyl]-1H-indazo l-7-yl]- 1,1-dioxo-1,2,5-thiadiazolidin-3-one S Step 1: Synthesis of 5-(6-fluoro-2-(4-methoxybenzyl)-4-(((6-methoxypyridin-2- yl)amino)methyl)-2H-indazol-7-yl)-1,2,5-thiadiazolidin-3-one 1,1-dioxide (2-1) To a stirred solution of 6-fluoro-2-[(4-methoxyphenyl)methyl]-7-(1,1,4-trioxo-1,2,5- thiadiazolidin-2-yl)indazole-4-carbaldehyde (Int-2, 60 mg, 0.14 mmol) and 6- methoxypyridin-2-amine (21.36 mg, 0.17 mmol) in DCM (5 mL) was added Trimethylsilyl trifluoromethanesulfonate (63.89 mg, 0.28 mmol) at 0 o C. The mixture was stirred at room temperature for 1 h. NaBH(OAc) 3 (27.53 mg, 0.29 mmol) was added to above mixture at 0 o C. The reaction mixture was stirred at room temperature for an additional 2hrs. After completion of the reaction monitored by LCMS, the mixture was diluted with DCM (10 mL) and concentrated in vacuo. The resulting residue was purified by reversed-phase column chromatography (0.05% NH 4 HCO 3 in water and MeCN) to obtain 5-[6-fluoro-1-[(4-methoxyphenyl)methyl]-4-[[(6-methoxy-2- pyridyl)amino]methyl]indazol-7-yl]-1,1-dioxo-1,2,5-thiadiazo lidin-3-one (50 mg, 0.095 mmol, 66.21% yield) as a light yellow solid. MS: m/z: Calc’d for C 24 H 23 FN 6 O 5 S [M+H] + 527; found 527. Step 2: Synthesis of 5-[6-fluoro-4-[[(6-methoxy-2-pyridyl)amino]methyl]-1H- indazol-7-yl]-1,1-dioxo-1,2,5-thiadiazolidin-3-one (2-2) To a solution of 5-[6-fluoro-1-[(4-methoxyphenyl)methyl]-4-[[(6-methoxy-2- pyridyl)amino]methyl]indazol-7-yl]-1,1-dioxo-1,2,5-thiadiazo lidin-3-one (50 mg, 0.09 mmol) in DCE (2 mL) was added TFA (2 mL) at room temperature, the mixture was stirred at 60 °C for 4 h. After completion of the reaction monitored by LCMS, the mixture was concentrated. The resulting residue was purified by reversed-phase column chromatography (0.05% NH 4 HCO 3 in water and MeCN) and further purified by Prep- HPLC to obtain 5-[6-fluoro-4-[[(6-methoxy-2-pyridyl)amino]methyl]-1H-indazo l-7-yl]- 1,1-dioxo-1,2,5-thiadiazolidin-3-one (14.5 mg, 0.03 mmol, 37.08% yield) as a white solid. MS: m/z: Calc’d for C 16 H 15 FN 6 O 4 S [M+H] + 407; found 407. 1 H NMR (400 MHz, DMSO-d 6 ) δ 13.28 (s, 1H), 8.33 (d, J = 3.8 Hz, 1H), 7.58-7.13 (m, 2H), 7.01 (d, J = 11.0 Hz, 1H), 6.09 (dd, J = 7.9, 3.9 Hz, 1H), 5.90 (dd, J = 7.8, 3.9 Hz, 1H), 4.77 (s, 2H), 4.45 (d, J = 3.8 Hz, 2H), 3.66 (d, J = 3.0 Hz, 3H). Prep-HPLC purification conditions: Column: SunFire Prep C18 OBD Column, 19*150 mm, 5μm; Mobile Phase A: Water(0.1%FA), Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 40% B to 60% B in 6.5 min, 60% B; Wave Length: 254/210 nm. EXAMPLE 2: 5-(6-fluoro-4-(((5-methoxypyridin-2-yl)amino)methyl)-1H-inda zol-7-yl)- 1,2,5-thiadiazolidin-3-one 1,1-dioxide The title compound was prepared in 11.53% overall yield as a white solid according to the preparation of EXAMPLE 1 using 5-methoxypyridin-2-amine in STEP 1. MS: m/z: Calc’d for C 16 H 15 FN 6 O 4 S [M+H] + 407; Found 407. 1 H NMR (400 MHz, DMSO-d 6 ) δ 13.26 (s, 1H), 8.59 (s, 1H), 8.26 (s, 1H), 7.70 (dd, J = 9.7, 2.8 Hz, 1H), 7.54 (d, J = 2.9 Hz, 1H), 7.13 – 6.98 (m, 2H), 4.85 (s, 2H), 4.17 (s, 2H), 3.77 (s, 3H). Prep-HPLC purification conditions: Column: HALO C18, Column 3.0*30 mm, 2.0 um; Mobile phaseA: water/0.05%TFA, Mobile phaseB: ACN/0.05%TFA; Flow rate: 1.5000 L/min; Gradient: 5% B to 40% B in 1.69 min; 40% B to 95% B in 0.60 min; 95% B to 95 % B hold in 0.5 min; Wave Length: 254 nm. EXAMPLE 3: 5-[6-fluoro-4-[[(4-methoxy-2-pyridyl)amino]methyl]-1H-indazo l-7-yl]- 1,1-dioxo-1,2,5-thiadiazolidin-3-one The title compound was prepared in 21.62% overall yield as a white solid according to the preparation of EXAMPLE 1 using 4-methoxypyridin-2-amine; hydrochloride in STEP 1. MS: m/z: Calc’d for C 16 H 15 FN 6 O 4 S [M+H] + 407; Found 407. 1 H NMR (400 MHz, DMSO-d 6 ) δ 13.25 (s, 1H), 12.79 (s, 1H), 8.75 (s, 1H), 8.26 (s, 1H), 7.87 (d, J = 7.2 Hz, 1H), 7.06 (d, J = 11.5 Hz, 1H), 6.58 (dd, J = 7.2, 2.4 Hz, 1H), 6.48 (d, J = 2.4 Hz, 1H), 4.89 (d, J = 5.6 Hz, 2H), 4.12 (s, 2H), 3.90 (s, 3H). Prep-HPLC purification conditions: SunFire Prep C18 OBD Column, 19*150 mm, 5μm; Mobile Phase A: Water (0.05% TFA), Mobile Phase B: ACN; Flow rate: 25 mL/min; Gradient: 20% B to 40% B in 5.3 min, 40% B; Wave Length: 254/210 nm. EXAMPLE 4: 5-[4-[[(4-cyclopropyl-2-pyridyl)amino]methyl]-6-fluoro-1H-in dazol-7- yl]-1,1-dioxo-1,2,5-thiadiazolidin-3-one The title compound was prepared in 7.78% overall yield as a white solid according to the preparation of EXAMPLE 1 using 4-cyclopropylpyridin-2-amine in STEP 1. MS: m/z: Calc’d for C 18 H 17 FN 6 O 3 S [M+H] + 417; Found 417. 1 H NMR (400 MHz, DMSO-d 6 +D 2 O) δ 8.28 (d, J = 5.0 Hz, 1H), 7.77 – 7.67 (m, 1H), 7.07 (dd, J = 11.5, 4.2 Hz, 1H), 6.77 (d, J = 11.4 Hz, 1H), 6.54 (t, J = 6.8 Hz, 1H), 4.88 (d, J = 5.7 Hz, 2H), 4.15 (s, 2H), 2.10 – 1.90 (m, 1H), 1.30 – 1.10 (m, 2H), 1.00 – 0.70 (m, 2H). Prep-HPLC purification conditions: SunFire Prep C18 OBD Column, 19*150 mm, 5μm; Mobile Phase A: Water (0.1% FA), Mobile Phase B: ACN; Flow rate: 20 mL/min; Gradient: 10% B to 25% B in 6 min, 25% B; Wave Length: 210/254 nm. EXAMPLE 5: 5-[6-fluoro-4-[[(4-methyl-2-pyridyl)amino]methyl]-1H-indazol -7-yl]-1,1- dioxo-1,2,5-thiadiazolidin-3-one The title compound was prepared in 10.71% overall yield as a white solid according to the preparation of EXAMPLE 1 using 4-methylpyridin-2-amine in STEP 1. MS: m/z: Calc’d for C 16 H 15 FN 6 O 3 S, [M+H] + 391; Found 391. 1 H NMR (400 MHz, DMSO-d 6 ) δ 13.28 (s, 1H), 8.99 (s, 1H), 8.26 (s, 1H), 7.87 (d, J = 6.5 Hz, 1H), 7.20-6.89 (m, 2H), 6.80 (d, J = 6.5 Hz, 1H), 4.88 (d, J = 5.5 Hz, 2H), 4.14 (s, 2H), 2.35 (s, 3H). Prep-HPLC purification conditions: SunFire Prep C18 OBD Column, 19*150 mm, 5μm; Mobile Phase A: Water (0.1% FA), Mobile Phase B: ACN; Flow rate: 25 mL/min; Gradient: 50% B to 70% B in 5.3 min, 70% B; Wave Length: 210/254 nm. EXAMPLE 6: 5-(4-(((4,6-dimethylpyridin-2-yl)amino)methyl)-6-fluoro-1H-i ndazol-7- yl)-1,2,5-thiadiazolidin-3-one 1,1-dioxide The title compound was prepared in 13.75% overall yield as a white solid according to the preparation of EXAMPLE 1 using 4,6-dimethylpyridin-2-amine in STEP 1. MS: m/z: Calc’d for C 17 H 18 FN 6 O 3 S, [M+H] + 405; Found 405. 1 H NMR (500 MHz, DMSO-d 6 ) δ 13.28 (s, 1H), 8.99 (s, 1H), 8.26 (s, 1H), 7.21 (s, 1H), 7.11 (s, 1H), 7.07 - 6.98 (m, 2H), 6.74 (br s, 1H), 6.67 - 6.55 (m, 1H), 4.89 (br d, J=5.2 Hz, 2H), 4.11 (br s, 2H), 2.40 (s, 3H), 2.27 (s, 3H). Prep-HPLC purification conditions: XBridge C18 Column, 19*200 mm, 5 μm; Mobile Phase A: ACN/H 2 O (5:95) with 10 mM AA; Mobile Phase B: ACN/H2O (95:5) with 10 mM AA; Flow Rate: 20 mL/min; Gradient: 0% B to 40% B in 20 min, 40% B; Wave Length: 220 nm. EXAMPLE 7: 5-(6-fluoro-4-(((4-methoxy-5-methylpyridin-2-yl)amino)methyl )-1H- indazol-7-yl)-1,2,5-thiadiazolidin-3-one 1,1-dioxide The title compound was prepared in 23.80% overall yield as a white solid according to the preparation of EXAMPLE 1 using 4-methoxy-5-methylpyridin-2-amine in STEP 1. MS: m/z: Calc’d for C 17 H 17 FN 6 O 4 S, [M+H] + 421; Found 421. 1 H NMR (500 MHz, DMSO-d 6 ) δ 13.28 (s, 1H), 8.99 (s, 1H), 8.23 (s, 1H), 7.70 (s, 1H), 7.22-6.94 (m, 2H), 4.89 – 4.82 (m, 2H), 4.09 (s, 2H), 3.88 (s, 3H), 1.99 (s, 3H). Prep-HPLC purification conditions: XBridge C18 Column, 19*200 mm, 5 μm; Mobile Phase A: ACN/H 2 O (5:95) with 10 mM AA; Mobile Phase B: ACN/H2O (95:5) with 10 mM AA; Flow Rate: 20 mL/min; Gradient: 0% B to 40% B in 20 min, 40% B; Wave Length: 220 nm. EXAMPLE 8: 6-[[6-fluoro-7-(1,1,4-trioxo-1,2,5-thiadiazolidin-2-yl)-1H-i ndazol-4- yl]methylamino]-4-methyl-pyridine-3-carbonitrile The title compound was prepared in 17.62% overall yield as a white solid according to the preparation of EXAMPLE 1 using 6-amino-4-methyl-pyridine-3-carbonitrile in STEP 1. MS: m/z: Calc’d for C 17 H 14 FN 7 O 3 S, [M+H] + 416; Found 416. 1 H NMR (400 MHz, DMSO-d 6 ) δ 13.29 (s, 1H), 8.95 – 7.77 (m, 3H), 6.93 (d, J = 11.3 Hz, 1H), 6.56 (s, 1H), 4.86 (s, 2H), 4.41 (s, 2H), 2.28 (d, J = 4.4 Hz, 3H). Prep-HPLC purification conditions: Aeris PEPTIDE 5um XB-C18 Axia, 21.2 mm X 250 mm, 5 μm; Mobile Phase A: Water (0.05%TFA), Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 50% B to 70% B in 6.6 min, 70% B; Wave Length: 254/210 nm. EXAMPLE 9: 5-[6-fluoro-4-[[(4-methoxy-6-methyl-2-pyridyl)amino]methyl]- 1H- indazol-7-yl]-1,1-dioxo-1,2,5-thiadiazolidin-3-one S Step 1: To a stirred mixture of 6-fluoro-2-[(4-methoxyphenyl)methyl]-7-(1,1,4-trioxo- 1,2,5-thiadiazolidin-2-yl)indazole-4-carbaldehyde (Int-2, 50 mg, 0.12 mmol) and 4- methoxy-6-methyl-pyridin-2-amine (25 mg, 0.18 mmol) in DCE (4 mL) was added Ti(i- PrO) 4 (68 mg, 0.24 mmol). The resulting mixture was stirred at room temperature for 1 h. NaBH3CN (31 mg, 0.48 mmol) was added to the above mixture at 0 °C. The resulting mixture was stirred at room temperature for 1 h. After completion of the reaction monitored by LCMS, the mixture was concentrated. The resulting residue was purified by a reversed-phase column to afford 5-[6-fluoro-4-[[(4-methoxy-6-methyl-2- pyridyl)amino]methyl]-2-[(4-methoxyphenyl)methyl]indazol-7-y l]-1,1-dioxo-1,2,5- thiadiazolidin-3-one (44 mg, 0.08 mmol, 68% yield) as a light yellow solid. MS: m/z: Calc’d for C 25 H 25 FN 6 O 5 S, [M+H] + 541; Found 541. Step 2: The title compound was prepared in 10.00% yield as a white solid according to the preparation of EXAMPLE 1 in STEP 2. MS: m/z: Calc’d for C 17 H 17 FN 6 O 4 S, [M+H] + 421; Found 421. 1 H NMR (400 MHz, DMSO-d 6 ) δ 13.24 (s, 1H), 12.68 (s, 1H), 8.29 (s, 2H), 7.08 (d, J = 11.5 Hz, 1H), 6.50 (s, 1H), 6.35 (s, 1H), 4.92 (d, J = 5.9 Hz, 2H), 4.12 (d, J = 4.9 Hz, 2H), 3.86 (s, 3H), 2.40 (s, 3H). Prep-HPLC purification conditions: SunFire Prep C18 OBD Column, 19*150 mm, 5μm; Mobile Phase A: Water (0.05%TFA), Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 40% B to 60% B in 6.5 min, 60% B; Wave Length: 254/210 nm. EXAMPLE 10: 5-[6-fluoro-4-[[(3-methyl-2-pyridyl) amino] methyl]-1H-indazol-7-yl]- 1,1-dioxo-1,2,5-thiadiazolidin-3-one The title compound was prepared in a 6.21% overall yield as a white solid according to the preparation of EXAMPLE 9 using 3-methylpyridin-2-amine in STEP 1. MS: m/z: Calc’d for C 16 H 15 FN 6 O 3 S, [M+H] + 391; Found 391. 1 H NMR (400 MHz, DMSO-d 6 ) δ 13.26 (s, 1H), 8.59 (s, 1H), 8.26 (s, 1H), 8.84 – 8.65 (m, 2H), 6.92 – 6.71 (m, 2H), 4.85 (s, 2H), 4.06 (s, 2H), 2.23 (s, 3H). Prep-HPLC purification conditions: SunFire Prep C18 OBD Column, 19*150 mm, 5μm; Mobile Phase A: Water (0.05% TFA), Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 5% B to 20% B in 6.5 min, 20% B; Wave Length: 254/210 nm.
Example Compounds Prepared by the Aforementioned Procedures are listed in Table 1 Table 1 BIOLOGICAL ASSAYS The pharmacological properties of the compounds of this invention may be confirmed by a number of biological assays known in the art. The exemplified biological assays which follow have been carried out with compounds of the invention. Data related to the preferred embodiments can be found in Table 2. PhosphoSens Assays A PhosphoSens® kinase assay was performed as described by the vendor (AssayQuant Technologies, Marlborough, MA). Briefly, 1000X solutions of compounds were prepared in DMSO via serial dilution of the 10 mM DMSO stocks using 3-fold intervals in a 384-well reagent plate.50 nL of the compound dilution series was then added to the corresponding wells of a 384-well assay plate. 40 mL of 1.25X substrate (AQT0264) in 1X assay buffer (50 mM HEPES pH 7.5, 500 µM EGTA, 10 nM MgCl2, 0.01% Brij-35, 1% Glycerol, 1 mM DTT, and 0.2 mg/mL BSA) was transferred to each well of the assay plate to achieve a final substrate concentration of 20 µM. Finally, 10 mL of 5X PTPN2 enzyme stock was added to each well of the assay plate for a final enzyme concentration of 150 pM. Reaction progress curves were collected by sampling fluorescence intensity at the excitation wavelength 360 nm (λex360) and emission wavelength 480 nm (λem480) every 71 seconds for one hour using a Synergy H4 plate reader (BioTek Instruments/Agilent Technologies, Winooski, VT) at room temperature. Phosphotase activity assay using DIFMUP as substrate: The PTPN2 biochemical assay was performed as follows, a 5X stock solution of human PTPN2 (SRP5075, MilliporeSigma, Burlington, MA) and a 1.25X stock solution of DiFMUP (D6567, ThermoFisher Scientific, Waltham, MA), were prepared in 1X reaction buffer consisting of 50 mM HEPES, pH 7.4, 1 mM EDTA, 150 mM NaCl, 0.2 mg/mL BSA, 100 U/mL catalase and 10 mM DTT.40 mL of the DiFMUP substrate solution, for a final concentration of 25 mM DiFMUP substrate, was added to a Corning 3574384-well, white, non-binding surface microtiter plate containing 0.05 mL of serially diluted test compounds prepared in DMSO. The reactions were started with the addition of 10 mL of the enzyme solution, for a final PTPN2 concentration of 0.15 nM, and monitored every 105 seconds for 60 minutes at λ EX 360/λ EM 460 in a BioTek Synergy HTX plate reader (Agilent Technologies, Santa Clara, CA) at room temperature. The initial linear portions of the progress curves were fit according to a linear equation to yield the slopes and converted to % inhibition based on a value of 100% activity for the no inhibitor treated control. IC 50 values of each compound were obtained by fitting the % inhibition-compound concentration curves using Dotmatics software (Dotmatics, Bishops Stortford, Hertfordshire, England). Cell proliferation assay protocol B16-F10 cells (ATCC, Manassas, VA, #CRL-6475) were cultured in DMEM growth medium (ThermoFisher Scientific, Waltham, MA, #11995-040) supplemented with 10% heat-inactivated FBS (ThermoFisher Scientific, #16140-071) and 1% pen/strep (ThermoFisher Scientific, #15140-122). The cells were seeded into two white opaque 384-well tissue culture-treated microplates (PerkinElmer, Waltham, MA, #6007688) at a density of 100 cells/well in 20uL total volume and incubated overnight at 37C and 5% CO2.30nL of compounds dissolved in DMSO were then transferred from a source plate into target wells with the Echo650 acoustic liquid handler (Beckman Coulter, Indianapolis, IN). Negative control wells received 30nL of DMSO only (0.15% final concentration). Plates were returned to the incubator for 1 hour and then cells were treated with either 5uL of growth medium or 5uL of growth medium containing 50 ng/mL of recombinant mouse IFN-gamma protein (R&D Systems, Minneapolis, MN, #485-MI/CF, 10 ng/mL final concentration) using the Assist automated pipetting platform (INTEGRA Biosciences, Hudson, NH). Plates were incubated at 37C for 4 days and cell proliferation was assayed with the CellTiter-Glo reagent (Promega, Madison, WI, #G7573, 25uL per well). Luminescence signal intensity was collected with the EnVision 2105 plate reader (PerkinElmer) 15 minutes after CellTiter-Glo reagent addition and analyzed with the Dotmatics software platform to calculate compound IC50 values. Off-target compound-mediated cytotoxicity was identified by checking for growth inhibition in the absence of IFNg. Phospho-STAT1 assay protocol B16-F10 cells (ATCC, Manassas, VA, #CRL-6475) were cultured in DMEM growth medium (ThermoFisher Scientific, Waltham, MA, #11995-040) supplemented with 10% heat-inactivated FBS (ThermoFisher Scientific, #16140-071) and 1% pen/strep (ThermoFisher Scientific, #15140-122). The cells were seeded into a white opaque 384- well tissue culture treated microplate (PerkinElmer, Waltham, MA, #6007688) at a density of 10,000 cells/well in 20uL total volume and incubated overnight at 37C and 5% CO2.30nL of compounds dissolved in DMSO were then transferred from a source plate into target wells with the Echo650 acoustic liquid handler (Beckman Coulter, Indianapolis, IN). Negative control wells received 30nL of DMSO only (0.15% final concentration). Plates were returned to the incubator for 1 hour and then cells were treated with either 5uL of growth medium or 5uL of growth medium containing 500 ng/mL of recombinant mouse IFN-gamma protein (R&D Systems, Minneapolis, MN, #485-MI/CF, 100 ng/mL final concentration) using the Assist automated pipetting platform (INTEGRA Biosciences, Hudson, NH). Plates were incubated at 37C for 1 hour and assayed for phosphorylated STAT1 protein levels with the phospho-STAT1 (Tyr701) HTRF kit (Cisbio, Bedford, MA, #63ADK026PEH) according to the manufacturer’s instructions. HTRF signal intensity was collected with the EnVision 2105 plate reader (PerkinElmer) 24 hours later and analyzed with the Dotmatics software platform to calculate compound IC50 values.
Biological Assay Data Table 2 is a summary of Biological Assay data for Examples/Embodiments Prepared. For IC50 data, High DDT concentration and/or DiFMUP substrate assays were used; a skilled artisan may use either assay. A row or column with a double asterisk indicates that one IC50 value or embodiment has been provided. Table 2