O-(cyclopropylmethyl)hydroxylamine;hydrochloride

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A solution comprised of 7-fluoro-6-(4-iodo-2-methyl-phenylamino)-1H-benzoimidazole-5-carboxylic acid, O-cyclopropylmethylhydroxylamine hydrochloride (1.25 equiv.), benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (1.25 equiv.), and diisopropylethylamine (3 equiv.) in 1:1 v/v tetrahydrofuran-dichloromethane is stirred for 30 minutes. The reaction mixture is concentrated in vacuo and the residue is taken up into diethyl ether. The ether phase is washed with dilute aqueous hydrochloric acid, saturated aqueous sodium bicarbonate, and brine, is dried (MgSO4), and is concentrated in vacuo to afford the desired product. The product may be recrystallized with an appropriate solvent like methanol or chloroform if further purification is necessary. The reaction mixture is concentrated in vacuo The ether phase is washed with dilute aqueous hydrochloric acid, saturated aqueous sodium bicarbonate, and brine is dried (MgSO4) is concentrated in vacuo

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: A solution comprised of 7-fluoro-6-(4-iodo-2-methyl-phenylamino)-1H-benzoimidazole-5-carboxylic acid, O-cyclopropylmethylhydroxylamine hydrochloride (1.25 equiv.), benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (1.25 equiv.), and diisopropylethylamine (3 equiv.) in 1:1 v/v tetrahydrofuran-dichloromethane is stirred for 30 minutes. The reaction mixture is concentrated in vacuo and the residue is taken up into diethyl ether. The ether phase is washed with dilute aqueous hydrochloric acid, saturated aqueous sodium bicarbonate, and brine, is dried (MgSO4), and is concentrated in vacuo to afford the desired product. The product may be recrystallized with an appropriate solvent like methanol or chloroform if further purification is necessary. The reaction mixture is concentrated in vacuo The ether phase is washed with dilute aqueous hydrochloric acid, saturated aqueous sodium bicarbonate, and brine is dried (MgSO4) is concentrated in vacuo

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 1,3-Diaminoacetone dihydrochloride monohydrate (2.00 g, 11.2 mmol) was dissolved in refluxing methanol and O-(cyclopropylmethyl)hydroxylamine hydrochloride (2.60 g, 21.1 mmol) was added. After refluxing for 96 hours the solvent was evaporated. The crude product was dissolved in methanol and an excess of trimethylorthoformate was added to the reaction mixture which was heated to reflux. After 48 hours the solvent was removed in vacuo. Crystallization from methanol/ethyl acetate afforded 1.30 g (57%) of light-brown material. Mp. 158.0° C. was added After refluxing for 96 hours the solvent was evaporated The crude product was dissolved in methanol an excess of trimethylorthoformate was added to the reaction mixture which was heated to reflux After 48 hours the solvent was removed in vacuo Crystallization from methanol/ethyl acetate

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: 1,3-Diaminoacetone dihydrochloride monohydrate (2.00 g, 11.2 mmol) was dissolved in refluxing methanol and O-(cyclopropylmethyl)hydroxylamine hydrochloride (2.60 g, 21.1 mmol) was added. After refluxing for 96 hours the solvent was evaporated. The crude product was dissolved in methanol and an excess of trimethylorthoformate was added to the reaction mixture which was heated to reflux. After 48 hours the solvent was removed in vacuo. Crystallization from methanol/ethyl acetate afforded 1.30 g (57%) of light-brown material. Mp. 158.0° C. was added After refluxing for 96 hours the solvent was evaporated The crude product was dissolved in methanol an excess of trimethylorthoformate was added to the reaction mixture which was heated to reflux After 48 hours the solvent was removed in vacuo Crystallization from methanol/ethyl acetate

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: HATU (67 mg, 0.18 mmol) and triethylamine (50 μL, 0.35 mmol) were added to a solution of sodium 1-(2-fluoro-4-iodo-benzyl)-1H-pyrrolo[2,3-c]pyridine-2-carboxylate (49 mg, 0.12 mmol) and O-cyclopropylmethyl-hydroxylamine hydrochloride (21.8 mg, 0.178 mmol) in DMF (500 μL). The reaction mixture was stirred at room temperature for 18 h, then a further 11 mg (0.09 mmol) of O-cyclopropylmethyl-hydroxylamine hydrochloride, 33 mg (0.09 mmol) of HATU, and 50 μL (0.35 mmol) of triethylamine was added. Stirring was continued for a further 18 h, then the reaction mixture was poured into ethyl acetate (15 mL). The organic layer was washed with a 1:1 solution of saturated NaHCO3 and brine (10 mL), dried over Na2SO4, filtered and concentrated to afford a yellow oil. The crude product was purified by reversed phase HPLC to afford the title compound as a white powder (TFA salt, 39 mg, 57%). LCMS (Method C): RT=3.17 min, M+H+=466. 1H NMR (DMSO-D6): δ=0.21 (m, 2H), 0.51 (m, 2H), 1.00 (m, 1H), 3.60 (d, J=7.2 Hz, 2H), 5.96 (s, 2H), 6.61 (t, J=8.0 Hz, 1H), 7.33 (s, 1H), 7.47 (dd, J=8.0, 1.2 Hz, 1H), 7.68 (dd, J=9.6, 1.6 Hz, 1H), 8.24 (d, J=6.4 Hz, 1H), 8.43 (d, J=6.4 Hz, 1H), 9.52 (s, 1H). Stirring was continued for a further 18 h The organic layer was washed with a 1:1 solution of saturated NaHCO3 and brine (10 mL) dried over Na2SO4 filtered concentrated to afford a yellow oil The crude product was purified by reversed phase HPLC

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: HATU (67 mg, 0.18 mmol) and triethylamine (50 μL, 0.35 mmol) were added to a solution of sodium 1-(2-fluoro-4-iodo-benzyl)-1H-pyrrolo[2,3-c]pyridine-2-carboxylate (49 mg, 0.12 mmol) and O-cyclopropylmethyl-hydroxylamine hydrochloride (21.8 mg, 0.178 mmol) in DMF (500 μL). The reaction mixture was stirred at room temperature for 18 h, then a further 11 mg (0.09 mmol) of O-cyclopropylmethyl-hydroxylamine hydrochloride, 33 mg (0.09 mmol) of HATU, and 50 μL (0.35 mmol) of triethylamine was added. Stirring was continued for a further 18 h, then the reaction mixture was poured into ethyl acetate (15 mL). The organic layer was washed with a 1:1 solution of saturated NaHCO3 and brine (10 mL), dried over Na2SO4, filtered and concentrated to afford a yellow oil. The crude product was purified by reversed phase HPLC to afford the title compound as a white powder (TFA salt, 39 mg, 57%). LCMS (Method C): RT=3.17 min, M+H+=466. 1H NMR (DMSO-D6): δ=0.21 (m, 2H), 0.51 (m, 2H), 1.00 (m, 1H), 3.60 (d, J=7.2 Hz, 2H), 5.96 (s, 2H), 6.61 (t, J=8.0 Hz, 1H), 7.33 (s, 1H), 7.47 (dd, J=8.0, 1.2 Hz, 1H), 7.68 (dd, J=9.6, 1.6 Hz, 1H), 8.24 (d, J=6.4 Hz, 1H), 8.43 (d, J=6.4 Hz, 1H), 9.52 (s, 1H). Stirring was continued for a further 18 h The organic layer was washed with a 1:1 solution of saturated NaHCO3 and brine (10 mL) dried over Na2SO4 filtered concentrated to afford a yellow oil The crude product was purified by reversed phase HPLC

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a stirring suspension comprised of O-cyclopropylmethyl-hydroxylamine hydrochloride (5.40 g, 0.0437 mol) in dichloromethane (20 ml) at ambient temperature under a nitrogen atmosphere was added diisopropylethylamine (10.8 ml, 0.062 mol). A solution comprised of 2,3,4-trifluorobenzenesulfonyl chloride (Oakwood Products, Inc., 1.00 g, 4.34×10−3 mol) in dichloromethane (120 ml) was added dropwise to the reaction vessel containing the stirring suspension over a 12 minute period. The reaction mixture was stirred for another 12 minutes and was quenched with 10% aqueous hydrochloric acid (140 ml). The biphasic mixture was stirred vigorously for 16 hours. The layers were separated and the organic phase was dried (MgSO4) and concentrated to 6 ml volume. The concentrated solution was administered to a flash silica column (Biotage, 90 g of silica gel). Elution with dichloromethane afforded 0.8283 g of a white amorphous solid; 68% yield; 1H-NMR (400 MHz; CDCl3 signal offset to δ 7.03; values reported are uncorrected) δ 7.50 (m,1H), 7.10 (s,1H), 6.95 (m,1H), 3.59 (d, 2H, J═7.2 Hz), 0.80 (m, 1H), 0.31 (m, 2H), 0.02 (m, 2H); 19F-NMR (376 MHz; CDCl3) δ−122.65 (m, 1F), −129.37 (m, 1F), −156.20 (m, 1F); MS (APCl−) 280 (M−1, 100), 210 (55), 195 (45). was added dropwise to the reaction vessel containing the stirring suspension over a 12 minute period was quenched with 10% aqueous hydrochloric acid (140 ml) The biphasic mixture was stirred vigorously for 16 hours The layers were separated the organic phase was dried (MgSO4) concentrated to 6 ml volume Elution with dichloromethane

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a stirring suspension comprised of O-cyclopropylmethyl-hydroxylamine hydrochloride (5.40 g, 0.0437 mol) in dichloromethane (20 ml) at ambient temperature under a nitrogen atmosphere was added diisopropylethylamine (10.8 ml, 0.062 mol). A solution comprised of 2,3,4-trifluorobenzenesulfonyl chloride (Oakwood Products, Inc., 1.00 g, 4.34×10−3 mol) in dichloromethane (120 ml) was added dropwise to the reaction vessel containing the stirring suspension over a 12 minute period. The reaction mixture was stirred for another 12 minutes and was quenched with 10% aqueous hydrochloric acid (140 ml). The biphasic mixture was stirred vigorously for 16 hours. The layers were separated and the organic phase was dried (MgSO4) and concentrated to 6 ml volume. The concentrated solution was administered to a flash silica column (Biotage, 90 g of silica gel). Elution with dichloromethane afforded 0.8283 g of a white amorphous solid; 68% yield; 1H-NMR (400 MHz; CDCl3 signal offset to δ 7.03; values reported are uncorrected) δ 7.50 (m,1H), 7.10 (s,1H), 6.95 (m,1H), 3.59 (d, 2H, J═7.2 Hz), 0.80 (m, 1H), 0.31 (m, 2H), 0.02 (m, 2H); 19F-NMR (376 MHz; CDCl3) δ−122.65 (m, 1F), −129.37 (m, 1F), −156.20 (m, 1F); MS (APCl−) 280 (M−1, 100), 210 (55), 195 (45). was added dropwise to the reaction vessel containing the stirring suspension over a 12 minute period was quenched with 10% aqueous hydrochloric acid (140 ml) The biphasic mixture was stirred vigorously for 16 hours The layers were separated the organic phase was dried (MgSO4) concentrated to 6 ml volume Elution with dichloromethane

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The title compound was prepared in an analogous fashion to N-ethoxy-5-(2-fluoro-4-iodophenylamino)imidazo[1,5-a]pyrazine-6-carboxamide, using O-(cyclopropylmethyl)-hydroxylaminehydrochloride as the starting material. 1H NMR (DMSO-d6, 400 MHz) δ ppm 11.82 (s, 1H), 10.36 (s, 1H), 8.82 (s, 1H), 7.95 (s, 1H), 7.91 (s, 1H), 7.73 (dd, J=10.4 Hz, 1.8 Hz, 1H), 7.44 (d, 8.4 Hz, 1H), 6.58 (t, J=8.4 Hz, 1H), 3.67 (d, J=7.2 Hz, 2H), 1.12 to 1.01 (m, 1H), 0.54-0.48 (m, 2H), 0.28-0.23 (m, 2H); LCMS (method D2): RT=1.33 min, [M+H]+=468.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The title compound was prepared in an analogous fashion to N-ethoxy-5-(2-fluoro-4-iodophenylamino)imidazo[1,5-a]pyrazine-6-carboxamide, using O-(cyclopropylmethyl)-hydroxylamine hydrochloride as the starting material. 1H NMR (DMSO-d6, 400 MHz) δ ppm 11.82 (s, 1H), 10.36 (s, 1H), 8.82 (s, 1H), 7.95 (s, 1H), 7.91 (s, 1H), 7.73 (dd, J=10.4 Hz, 1.8 Hz, 1H), 7.44 (d, 8.4 Hz, 1H), 6.58 (t, J=8.4 Hz, 1H), 3.67 (d, J=7.2 Hz, 2H), 1.12 to 1.01 (m, 1H), 0.54-0.48 (m, 2H), 0.28-0.23 (m, 2H); LCMS (method D2): RT=1.33 min, [M+H]+=468.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of (3R,4S)-3-ethyl-4-(6H-pyrrolo[2,3-e][1,2,4]triazolo[4,3-a]pyrazin-1-yl)cyclopentanone (0.05 g, 0.19 mmol, Example #AA.1.59) in EtOH (1 mL) was added TEA (0.04 mL, 0.28 mmol) and O-(cyclopropylmethyl)hydroxylamine hydrochloride (0.02 g, 0.19 mmol, Huhu Technologies). The reaction mixture was stirred for about 18 h at ambient temperature. Additional O-(cyclopropylmethyl)hydroxylamine hydrochloride (0.02 g, 0.19 mmol, Huhu Technologies) was added. The reaction was stirred at ambient temperature for about 5 h. The solvent was removed under reduced pressure. The crude material was purified by silica gel chromatography eluting with a gradient of 1-10% MeOH in DCM to afford (3R,4S)-3-ethyl-4-(6H-pyrrolo[2,3-e][1,2,4]triazolo[4,3-a]pyrazin-1-yl)cyclopentanone O-cyclopropylmethyl oxime (0.051 g, 80%): LC/MS (Table 1, Method a) Rt=1.94 min; MS m/z: 339 (M+H)+. The reaction was stirred at ambient temperature for about 5 h The solvent was removed under reduced pressure The crude material was purified by silica gel chromatography eluting with a gradient of 1-10% MeOH in DCM

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To an 8 mL scintillation vial equipped for stirring was added (R)-2-(6-fluoro-3-oxopyrrolo[4,3,2-de][2,6]naphthyridin-4(1H,3H,5H)-yl)-3-methylbutanoic acid (15 mg, 0.051 mmol). DMF (0.5 mL), O-(cyclopropylmethyl)hydroxylamine hydrochloride (6.4 mg, 0.051 mmol), HOBt (11.83 mg, 0.077 mmol), EDC (14.81 mg, 0.077 mmol) and N,N-dimethylpyridin-4-amine (9.44 mg, 0.077 mmol) were added and the solution was stirred at 25° C. for 4 h. The reaction mixture was purified via preparative mass trigger LC-MS (AcCN/H2O, 20-50%). The fractions were collected and lyophilized to afford the title compound as a white solid (5 mg, 23%). 1H NMR (400 MHz, CD3OD) δ 0.16-0.24 (m, 2 H) 0.39-0.55 (m, 2 H) 0.93 (d, J=6.57 Hz, 3 H) 1.05 (d, J=6.32 Hz, 3 H) 1.06-1.15 (m, 1 H) 2.53 (dt, J=11.24, 6.51 Hz, 1 H) 3.55-3.73 (m, 2 H) 4.94 (d, J=11.37 Hz, 1 H) 5.07 (d, J=19.20 Hz, 1 H) 5.46 (d, J=18.44 Hz, 1 H) 7.86 (s, 1 H) 8.17 (d, J=3.28 Hz, 1 H). [M+H] calc'd for C18H21FN4O3, 361; found, 361.4. To an 8 mL scintillation vial equipped the solution was stirred at 25° C. for 4 h The reaction mixture was purified via preparative mass trigger LC-MS (AcCN/H2O, 20-50%) The fractions were collected