tert-butyl (1R,4R)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate

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

Procedure: A mixture of 5-chloro-7-(2-chloro-4-pyridyl)-8-(3-(trifluoromethyl)-phenyl)-1,2,4-triazolo[4,3-c]pyrimidine (820 mg, 2.0 mmole), potassium carbonate (ca. 1.0 g) and (−)-2-(tert-butoxycarbonyl)-(1R, 4R)-2,5-diaza-bicyclo[2.2.1]heptane (436 mg, 2.2 mmole) in 10 ml of DMF was stirred at room temperature for 16 hours. The reaction solution was poured into water (50 mL), and precipitate was filtered and washed with water and dried under vacuum to give product as off white solid; MS m/z (M)+ 572.3; C27H25ClF3N7O2 requir. 572. was filtered washed with water dried under vacuum to give product as off white solid

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

Procedure: A mixture of N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}-N′-[(3′-formyl-6-methyl-3-biphenylyl)methyl]-2,6-pyridinedicarboxamide (610 mg, 0.925 mmol), 1,1-dimethylethyl (1R,4R)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (183 mg, 0.925 mmol), sodium triacetoxyborohydride (392 mg, 1.849 mmol) and acetic acid (0.064 mL, 1.109 mmol) in DCM (10 mL) was stirred at room temperature overnight. The reaction mixture was quenched with saturated NaHCO3 and extracted with DCM twice. The combined organic layers were concentrated under vacuum to give a crude residue. It was purified with Companion, eluting with 0 to 100% ethyl acetate in hexane to get rid of impurities and then 10% methanol in DCM to elute the product. The product fractions were combined and concentrated under vacuum to give 1,1-dimethylethyl (1S,4S)-5-[(5′-{[({6-[({[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}amino)carbonyl]-2-pyridinyl}carbonyl)amino]methyl}-2′-methyl-3-biphenylyl)methyl]-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate as a solid. It was re-dissolved in 25% TFA in DCM (2 mL) and stirred at room temperature for 2 h. Solvent was evaporated under a stream of nitrogen and then the residue was purified with a Gilson HPLC (with 0.1% TFA in the solvents), eluting with 10 to 70% CH3CN in water at a flow rate of 20 mL/min. The product fractions were combined and converted to the free base with 1 N NaOH, and the basified solution was extracted with ethyl acetate twice. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and then concentrated under vacuum to give N-({3′-[(1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-ylmethyl]-6-methyl-3-biphenylyl}methyl)-N′-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}-2,6-pyridinedicarboxamide as a solid (431 mg, 62.8%). LC-MS m/z 743 (M+H)+, 1.18 min (ret time). The reaction mixture was quenched with saturated NaHCO3 extracted with DCM twice The combined organic layers were concentrated under vacuum to give a crude residue It was purified with Companion eluting with 0 to 100% ethyl acetate in hexane to get rid of impurities 10% methanol in DCM to elute the product concentrated under vacuum

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

Procedure: Using amide formation method 1, (R,R)-2,5-Diaza-bicyclo[2.2.1]heptane-2-c arboxylic acid tert-butyl ester (1.0 g, 4.26 mmol, 1.0 eq) was combined with 6-Amino-nicotinic acid (588 mg, 4.26 mmol, 1.0 eq) to give 5-(6-Amino-pyridine-3-carbonyl)-(R,R)-2,5-diaza-bicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester (420 mg, 31% yield). 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.35-1.40 (m, 3H) 1.40-1.51 (m, 9H) 1.89 (br. s., 2H) 3.31-3.50 (m, 2H) 3.55 (d, J=9.09 Hz, 1H) 3.67 (dd, J=13.14, 6.57 Hz, 1H) 4.50 (br. s., 1H) 4.64 (br. s., 1H) 4.91 (br. s., 1H) 6.51 (d, J=6.57 Hz, 1H) 7.64 (br. s., 1H) 8.24 (br. s., 1H); MS m/z 637.0 (M+H)+.

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

Procedure: Using amide formation method 1, (R,R)-2,5-Diaza-bicyclo[2.2.1]heptane-2-c arboxylic acid tert-butyl ester (1.0 g, 4.26 mmol, 1.0 eq) was combined with 6-Amino-nicotinic acid (588 mg, 4.26 mmol, 1.0 eq) to give 5-(6-Amino-pyridine-3-carbonyl)-(R,R)-2,5-diaza-bicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester (420 mg, 31% yield). 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.35-1.40 (m, 3H) 1.40-1.51 (m, 9H) 1.89 (br. s., 2H) 3.31-3.50 (m, 2H) 3.55 (d, J=9.09 Hz, 1H) 3.67 (dd, J=13.14, 6.57 Hz, 1H) 4.50 (br. s., 1H) 4.64 (br. s., 1H) 4.91 (br. s., 1H) 6.51 (d, J=6.57 Hz, 1H) 7.64 (br. s., 1H) 8.24 (br. s., 1H); MS m/z 637.0 (M+H)+.

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

Procedure: 5-{(1R)-2-[(1R,4R)-2,5-Diazabicyclo[2.2.1]hept-2-yl]-1-hydroxyethyl}-4-methyl-2-benzofuran-1 (3H)-one was prepared in an analogous fashion as described for the synthesis of INTERMEDIATE 3 starting from 4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1 (3H)-one (INTERMEDIATE 2) and commercially available tert-butyl (1R,4R)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate.

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

Procedure: 5-{(1R)-2-[(1R,4R)-2,5-Diazabicyclo[2.2.1]hept-2-yl]-1-hydroxyethyl}-4-methyl-2-benzofuran-1 (3H)-one was prepared in an analogous fashion as described for the synthesis of INTERMEDIATE 3 starting from 4-methyl-5-[(2R)-oxiran-2-yl]-2-benzofuran-1 (3H)-one (INTERMEDIATE 2) and commercially available tert-butyl (1R,4R)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate.

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

Procedure: A solution of 1.06 g of 3-methyl-5-trichloromethyl-1,2,4-oxadiazole prepared by the procedure of F. Eloy, Bull. Soc. Chim. Belg., 1964, 73, 793, in 2 ml of toluene is added slowly to a solution of 1.05 g of product from Example 1 and 0.82 g of diazabicyclo[3.6.0]unodecene-5 in 1 ml of toluene. The resulting mixture is stirred at room temperature for 3 days and then concentrated in vacuo to dryness. The residue is purified by chromatography (aluminum oxide, Activity Grade 2.5; methylene chloride) to give 0.74 g of the desired product as an off-white solid: concentrated in vacuo to dryness The residue is purified by chromatography (aluminum oxide, Activity Grade 2.5; methylene chloride)

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

Procedure: Using amide formation method 1, (R,R)-2,5-Diaza-bicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester (1.0 g, 4.26 mmol, 1.0 eq) was combined with 6-Amino-nicotinic acid (588 mg, 4.26 mmol, 1.0 eq) to give 5-(6-Amino-pyridine-3-carbonyl)-(R,R)-2,5-diaza-bicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester (420 mg, 31% yield). 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.35-1.40 (m, 3H) 1.40-1.51 (m, 9H) 1.89 (br. s., 2H) 3.31-3.50 (m, 2H) 3.55 (d, J=9.09 Hz, 1H) 3.67 (dd, J=13.14, 6.57 Hz, 1H) 4.50 (br. s., 1H) 4.64 (br. s., 1H) 4.91 (br. s., 1H) 6.51 (d, J=6.57 Hz, 1H) 7.64 (br. s., 1H) 8.24 (br. s., 1H); MS m/z 637.0 (M+H)+.

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

Procedure: A mixture of 5-chloro-7-(2-chloro-4-pyridyl)-8-(3-(trifluoromethyl)-phenyl)-1,2,4-triazolo[4,3-c]pyrimidine (820 mg, 2.0 mmole), potassium carbonate (ca. 1.0 g) and (−)-2-(tert-butoxycarbonyl)-(1R, 4R)-2,5-diaza-bicyclo[2.2.1]heptane (436 mg, 2.2 mmole) in 10 ml of DMF was stirred at room temperature for 16 hours. The reaction solution was poured into water (50 mL), and precipitate was filtered and washed with water and dried under vacuum to give product as off white solid; MS m/z (M)+ 572.3; C27H25ClF3N7O2 requir. 572. was filtered washed with water dried under vacuum to give product as off white solid

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

Procedure: A mixture of N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}-N′-[(3′-formyl-6-methyl-3-biphenylyl)methyl]-2,6-pyridinedicarboxamide (610 mg, 0.925 mmol), 1,1-dimethylethyl (1R,4R)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (183 mg, 0.925 mmol), sodium triacetoxyborohydride (392 mg, 1.849 mmol) and acetic acid (0.064 mL, 1.109 mmol) in DCM (10 mL) was stirred at room temperature overnight. The reaction mixture was quenched with saturated NaHCO3 and extracted with DCM twice. The combined organic layers were concentrated under vacuum to give a crude residue. It was purified with Companion, eluting with 0 to 100% ethyl acetate in hexane to get rid of impurities and then 10% methanol in DCM to elute the product. The product fractions were combined and concentrated under vacuum to give 1,1-dimethylethyl (1S,4S)-5-[(5′-{[({6-[({[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}amino)carbonyl]-2-pyridinyl}carbonyl)amino]methyl}-2′-methyl-3-biphenylyl)methyl]-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate as a solid. It was re-dissolved in 25% TFA in DCM (2 mL) and stirred at room temperature for 2 h. Solvent was evaporated under a stream of nitrogen and then the residue was purified with a Gilson HPLC (with 0.1% TFA in the solvents), eluting with 10 to 70% CH3CN in water at a flow rate of 20 mL/min. The product fractions were combined and converted to the free base with 1 N NaOH, and the basified solution was extracted with ethyl acetate twice. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and then concentrated under vacuum to give N-({3′-[(1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-ylmethyl]-6-methyl-3-biphenylyl}methyl)-N′-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}-2,6-pyridinedicarboxamide as a solid (431 mg, 62.8%). LC-MS m/z 743 (M+H)+, 1.18 min (ret time). The reaction mixture was quenched with saturated NaHCO3 extracted with DCM twice The combined organic layers were concentrated under vacuum to give a crude residue It was purified with Companion eluting with 0 to 100% ethyl acetate in hexane to get rid of impurities 10% methanol in DCM to elute the product concentrated under vacuum

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

Procedure: Using amide formation method 1, (R,R)-2,5-Diaza-bicyclo[2.2.1]heptane-2-c arboxylic acid tert-butyl ester (1.0 g, 4.26 mmol, 1.0 eq) was combined with 6-Amino-nicotinic acid (588 mg, 4.26 mmol, 1.0 eq) to give 5-(6-Amino-pyridine-3-carbonyl)-(R,R)-2,5-diaza-bicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester (420 mg, 31% yield). 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.35-1.40 (m, 3H) 1.40-1.51 (m, 9H) 1.89 (br. s., 2H) 3.31-3.50 (m, 2H) 3.55 (d, J=9.09 Hz, 1H) 3.67 (dd, J=13.14, 6.57 Hz, 1H) 4.50 (br. s., 1H) 4.64 (br. s., 1H) 4.91 (br. s., 1H) 6.51 (d, J=6.57 Hz, 1H) 7.64 (br. s., 1H) 8.24 (br. s., 1H); MS m/z 637.0 (M+H)+.