IN Cell Investigator Developer Toolbox (GE Healthcare) was used to analyze the assay data

IN Cell Investigator Developer Toolbox (GE Healthcare) was used to analyze the assay data. the construction to the slower eluting enantiomer, whereas the faster eluting compound was associated to the construction (Table 4). Table 4 Alkene seriesa construction (compound (= 8.5 Hz, 1H), 7.20 (d, = 1.9 Hz, 1H), 7.04 (dd, = 8.5, 1.9 Hz, 1H), 3.97 (s, 3H). Methyl 4-bromo-2-((4-methoxybenzyl)oxy)benzoate 12a A solution of phenol 6 (1.70 g, 7.36 mmol), 4-methoxybenzyl alcohol (1.29 g, 9.30 mmol) and triphenylphosphine (2.81 g, 10.71 mmol) in DCM (35 mL) was cooled at 0 C and treated with di-373 (M+Na). 1H NMR (500 MHz, CDCl3) 7.70 (d, = 8.3 Hz, 1H), 7.43 C 7.40 (m, 2H), 7.20 (d, = 1.8 Hz, 1H), 7.15 (dd, = 8.3, 1.8 Hz, 1H), 6.96 C 6.93 (m, 2H), 5.11 (s, 2H), 3.89 (s, 3H), 3.83 (s, 3H). Methyl 4-(2-amino-5-bromopyridin-3-yl)-2-((4-methoxybenzyl)oxy)benzoate 14b A solution of bromide 12a (1.10 g, 3.13 mmol), bis(pinacolato)diboron (1.20 g, 4.72 mmol), potassium acetate (925 mg, 9.44 mmol) and 1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II)DCM (130 mg, 0.16 mmol) in DMF (15 mL) was stirred at 100 C less than microwave irradiation for 1 h 30 min. The reaction was quenched with brine and extracted with AcOEt. The combined organics were washed with brine, dried (Na2SO4), and concentrated c-di-AMP to afford the crude boronic ester 13a. A solution of crude boronic ester 13a (ca. 3.13 mmol), sodium bicarbonate (480 mg, 5.71 mmol), 1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II)DCM (125 mg, 0.15 mmol) and 5-bromo-3-iodopyridin-2-amine (850 mg, 2.84 mmol) in DMF/water (8/1, 15 mL) was stirred at 100 C less than microwave irradiation for 1 h 30 min. The reaction was quenched with brine and extracted with EtOAc. The combined organics were washed with brine, Rabbit polyclonal to ABCG5 dried (Na2SO4), concentrated and purified by Biotage column chromatography (0C30% EtOAc/cyclohexane) to give bromopyridine 14b (1.02 g, 81%). HRMS (ESI) calcd for C21H20BrN2O4 (M+H) 443.0601, found 443.0617. 1H NMR (500 MHz, CDCl3) 8.13 (d, = 2.4 Hz, 1H), 7.90 (d, = 7.8 Hz, 1H), 7.45 (d, = 2.4 Hz, 1H), 7.43 C 7.40 (m, 2H), 7.07 C 7.04 (m, 2H), 6.95 C 6.92 (m, 2H), 5.17 (s, 2H), 4.57 (br. s, 2H), 3.94 (s, 3H), 3.83 (s, 3H). Methyl 4-(2-amino-5-(4-((dimethylamino)methyl)thiophen-2-yl)pyridin-3-yl)-2-((4-methoxybenzyl)oxy)benzoate 16lA remedy of bromide 14b (1.01 g, 2.28 mmol), bis(pinacolato)diboron (870 mg, 3.43 mmol), potassium acetate (680 mg, 6.94 mmol) and 1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II)DCM (200 mg, 0.25 mmol) in DMF (11 mL) was stirred at 100 C under microwave irradiation for 1 h 30 min. The reaction was quenched with brine and extracted with AcOEt. The combined organics were washed with brine, dried (Na2SO4), and concentrated to afford the crude boronic acid 15b. A solution of crude boronic acid 15b (ca. 2.28 mmol), 1-(5-bromothiophen-3-yl)-504 (M+H). 1H NMR (500 MHz, CDCl3) 8.36 (s, 1H), 7.92 (d, = 7.9 Hz, 1H), 7.56 (d, = 2.3 Hz, 1H), 7.44 C 7.41 (m, 2H), 7.24 (s, 1H), 7.14 C 7.10 (m, 3H), 6.97 C 6.91 (m, 2H), 5.19 (s, 2H), 4.59 (br. s, 2H), 3.94 (s, 3H), 3.82 (s, 3H), 3.54 (s, 2H), 2.36 (s, 6H). Methyl 4-(2-amino-5-(4-((dimethylamino)methyl)thiophen-2-yl)pyridin-3-yl)-2-hydroxybenzoate 18A remedy of phenol ether 16l (560 mg, 1.11 mmol) in DCM (7 mL) was treated with trifluoroacetic acid (800 L, 10.81 mmol) at 0 C. After 1 h 30 min. the reaction was brought to pH ca. 5C6 with 1M NaOH and 1M HCl, the aqueous coating separated and extracted with DCM. The combined organic layers were concentrated and purified by Biotage column chromatography (0C15% MeOH/DCM) to give phenol 18 (394 mg, 92%). HRMS (ESI) calcd for C20H22N3O3S (M+H) 384.1376, found 384.1391. 1H NMR (500 MHz, MeOD) 8.28 (d, = 2.4 Hz, 1H), 7.99 (d, = 8.2 Hz, 1H), 7.68 (d, = 2.4 Hz, 1H), 7.58 (d, = 1.4 Hz, 1H), 7.39 (d, = 1.4 Hz, 1H), 7.10 (d, = 1.7 Hz, 1H), 7.07 (dd, = 8.2, 1.7 Hz, 1H), 4.30 (s, 2H), 4.00 (s, 3H), 2.88 (s, 6H). ()-(calcd for C25H27F3N3O3S (M+H) 506.1720, found.The medium was aspirated and the cells were washed with PBS (Invitrogen, Paisley, United Kingdom), trypsinized (Internal supply, 0.25% versene trypsin with EDTA), neutralized, and counted. Ile14 and Gly92. The dimethylamino group is clearly resolved and superimposes well with the basic center of the piperidine ring of construction. Pleasingly, aminopyridine configured enantiomer is definitely significantly more potent than the enantiomer.6 We therefore separated the two enantiomers of aminopyridine configuration is preferred for Nek2 inhibition, we assigned the configuration to the slower eluting enantiomer, whereas the faster eluting compound was associated to the configuration (Table 4). Table 4 Alkene seriesa construction (compound (= 8.5 Hz, 1H), 7.20 (d, = 1.9 Hz, 1H), 7.04 (dd, = 8.5, 1.9 Hz, 1H), 3.97 (s, 3H). Methyl 4-bromo-2-((4-methoxybenzyl)oxy)benzoate 12a A solution of phenol 6 (1.70 g, 7.36 mmol), 4-methoxybenzyl alcohol (1.29 g, 9.30 mmol) and triphenylphosphine (2.81 g, 10.71 mmol) in DCM (35 mL) was cooled at 0 C and treated with di-373 (M+Na). 1H NMR (500 MHz, CDCl3) 7.70 (d, = 8.3 Hz, 1H), 7.43 C 7.40 (m, 2H), 7.20 (d, = 1.8 Hz, 1H), 7.15 (dd, = 8.3, 1.8 Hz, 1H), 6.96 C 6.93 (m, 2H), 5.11 (s, 2H), 3.89 (s, 3H), 3.83 (s, 3H). Methyl 4-(2-amino-5-bromopyridin-3-yl)-2-((4-methoxybenzyl)oxy)benzoate 14b A solution of bromide 12a (1.10 g, 3.13 mmol), bis(pinacolato)diboron (1.20 g, 4.72 mmol), potassium acetate (925 mg, 9.44 mmol) and 1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II)DCM (130 mg, 0.16 mmol) in DMF (15 mL) was stirred at 100 C less than microwave irradiation for 1 h 30 min. The reaction was quenched with brine and extracted with AcOEt. The combined organics were washed with brine, dried (Na2SO4), and concentrated to afford the crude boronic ester 13a. A solution of crude boronic ester 13a (ca. 3.13 mmol), sodium bicarbonate (480 mg, 5.71 mmol), 1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II)DCM (125 mg, 0.15 mmol) and 5-bromo-3-iodopyridin-2-amine (850 mg, 2.84 mmol) in DMF/water (8/1, 15 mL) was stirred at 100 C less than microwave irradiation for 1 h 30 min. The reaction was quenched with brine and extracted with EtOAc. The combined organics were washed with brine, dried (Na2SO4), concentrated and purified by Biotage column chromatography (0C30% EtOAc/cyclohexane) to give bromopyridine 14b (1.02 g, 81%). HRMS (ESI) calcd for C21H20BrN2O4 (M+H) 443.0601, found 443.0617. 1H NMR (500 MHz, CDCl3) 8.13 (d, = 2.4 Hz, 1H), 7.90 (d, = 7.8 Hz, 1H), 7.45 (d, = 2.4 Hz, 1H), 7.43 C 7.40 (m, 2H), 7.07 C 7.04 (m, 2H), 6.95 C 6.92 (m, 2H), 5.17 (s, 2H), 4.57 (br. s, 2H), 3.94 (s, 3H), 3.83 (s, 3H). Methyl 4-(2-amino-5-(4-((dimethylamino)methyl)thiophen-2-yl)pyridin-3-yl)-2-((4-methoxybenzyl)oxy)benzoate 16lA remedy of bromide 14b (1.01 g, 2.28 mmol), bis(pinacolato)diboron (870 mg, 3.43 mmol), potassium acetate (680 mg, 6.94 mmol) and 1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II)DCM (200 mg, 0.25 mmol) in DMF (11 mL) was stirred at 100 C under microwave irradiation for 1 h 30 min. The reaction was quenched with brine and extracted with AcOEt. The combined organics were washed with brine, dried (Na2SO4), and concentrated to afford the crude boronic acid 15b. A solution of crude boronic acid 15b (ca. 2.28 mmol), 1-(5-bromothiophen-3-yl)-504 (M+H). 1H NMR (500 MHz, CDCl3) 8.36 (s, 1H), 7.92 (d, = 7.9 Hz, 1H), 7.56 (d, = 2.3 Hz, 1H), 7.44 C 7.41 (m, 2H), 7.24 (s, 1H), 7.14 C 7.10 (m, 3H), 6.97 C 6.91 (m, 2H), 5.19 (s, 2H), 4.59 (br. s, 2H), 3.94 (s, 3H), 3.82 (s, 3H), 3.54 (s, 2H), 2.36 (s, 6H). Methyl 4-(2-amino-5-(4-((dimethylamino)methyl)thiophen-2-yl)pyridin-3-yl)-2-hydroxybenzoate 18A remedy of phenol ether 16l (560 mg, 1.11 mmol) in DCM (7 mL) was treated with trifluoroacetic acid (800 L, 10.81 mmol) at 0 C. After 1 h 30 min. the reaction was brought to pH ca. 5C6 with 1M NaOH and 1M HCl, the aqueous coating separated and extracted with DCM. The combined organic layers were concentrated and purified by Biotage column chromatography (0C15% MeOH/DCM) to give phenol 18 (394 mg, 92%). HRMS (ESI) calcd for C20H22N3O3S (M+H) 384.1376, found 384.1391. 1H NMR (500 MHz, MeOD) 8.28 (d, = 2.4 Hz, 1H), 7.99 (d, = 8.2 Hz, 1H), 7.68 (d, = 2.4 Hz, 1H), 7.58 (d, = 1.4 Hz, 1H), 7.39 (d, = 1.4 Hz, 1H), 7.10 (d, = 1.7 Hz, 1H), 7.07 (dd, = 8.2, 1.7 Hz, 1H), 4.30 (s, 2H), 4.00 (s, 3H), 2.88 (s, 6H). ()-(calcd for C25H27F3N3O3S (M+H) 506.1720, found 506.1701. 1H NMR (500 MHz, MeOD) .Cells were maintained inside a humidified atmosphere of 5% CO2 at 37C. and superimposes well with the basic center of the piperidine ring of construction. Pleasingly, aminopyridine configured enantiomer is definitely significantly more potent than the enantiomer.6 We therefore separated the two enantiomers of aminopyridine configuration is preferred for Nek2 inhibition, we assigned the configuration to the slower eluting enantiomer, whereas the faster eluting compound was associated to the configuration (Table 4). Table 4 Alkene seriesa construction (compound (= 8.5 Hz, 1H), 7.20 (d, = 1.9 Hz, 1H), 7.04 (dd, = 8.5, 1.9 Hz, 1H), 3.97 (s, 3H). Methyl 4-bromo-2-((4-methoxybenzyl)oxy)benzoate 12a A solution of phenol 6 (1.70 g, 7.36 mmol), 4-methoxybenzyl alcohol (1.29 g, 9.30 mmol) and triphenylphosphine (2.81 g, 10.71 mmol) in DCM (35 mL) was cooled at 0 c-di-AMP C and treated with di-373 (M+Na). 1H NMR (500 MHz, CDCl3) 7.70 (d, = 8.3 Hz, 1H), 7.43 C 7.40 (m, 2H), 7.20 (d, = 1.8 Hz, 1H), 7.15 (dd, = 8.3, 1.8 Hz, 1H), 6.96 C 6.93 (m, 2H), 5.11 (s, 2H), 3.89 (s, 3H), 3.83 (s, 3H). Methyl 4-(2-amino-5-bromopyridin-3-yl)-2-((4-methoxybenzyl)oxy)benzoate 14b A solution of bromide 12a (1.10 g, 3.13 mmol), bis(pinacolato)diboron (1.20 g, 4.72 mmol), potassium acetate (925 mg, 9.44 mmol) and 1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II)DCM (130 mg, 0.16 mmol) in DMF (15 mL) was stirred at 100 C less than microwave irradiation for 1 h 30 min. The reaction was quenched with brine and extracted with AcOEt. The combined organics were washed with brine, dried (Na2SO4), and concentrated to afford the crude boronic ester 13a. A solution of crude boronic ester 13a (ca. 3.13 mmol), sodium bicarbonate (480 mg, 5.71 mmol), 1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II)DCM (125 mg, 0.15 mmol) and 5-bromo-3-iodopyridin-2-amine (850 mg, 2.84 mmol) in DMF/water (8/1, 15 mL) was stirred at 100 C less than microwave irradiation for 1 h 30 min. The reaction was quenched with brine and extracted with EtOAc. The combined organics were washed with brine, dried (Na2SO4), concentrated and purified by Biotage column chromatography (0C30% EtOAc/cyclohexane) to give bromopyridine 14b (1.02 g, 81%). HRMS (ESI) calcd for C21H20BrN2O4 (M+H) 443.0601, found 443.0617. 1H NMR (500 MHz, CDCl3) 8.13 (d, = 2.4 Hz, 1H), 7.90 (d, = 7.8 Hz, 1H), 7.45 (d, = 2.4 Hz, 1H), 7.43 C 7.40 (m, 2H), 7.07 C 7.04 (m, 2H), 6.95 C 6.92 (m, 2H), 5.17 (s, 2H), 4.57 (br. s, 2H), 3.94 (s, 3H), 3.83 (s, 3H). Methyl 4-(2-amino-5-(4-((dimethylamino)methyl)thiophen-2-yl)pyridin-3-yl)-2-((4-methoxybenzyl)oxy)benzoate 16lA remedy of bromide 14b (1.01 g, 2.28 mmol), bis(pinacolato)diboron (870 mg, 3.43 mmol), potassium acetate (680 mg, 6.94 mmol) and 1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II)DCM (200 mg, 0.25 mmol) in DMF (11 mL) was stirred at 100 C under microwave irradiation for 1 h 30 min. The reaction was quenched with brine and extracted with AcOEt. The combined organics were washed with brine, dried (Na2SO4), and concentrated to afford the crude boronic acid 15b. A solution of crude boronic acid 15b (ca. 2.28 mmol), 1-(5-bromothiophen-3-yl)-504 (M+H). 1H NMR (500 MHz, CDCl3) 8.36 (s, 1H), 7.92 (d, = 7.9 Hz, 1H), 7.56 (d, = 2.3 Hz, 1H), 7.44 C 7.41 (m, 2H), 7.24 (s, 1H), 7.14 C 7.10 (m, 3H), 6.97 C 6.91 (m, 2H), 5.19 (s, 2H), 4.59 (br. s, 2H), 3.94 (s, 3H), 3.82 (s, 3H), 3.54 (s, 2H), 2.36 (s, 6H). Methyl 4-(2-amino-5-(4-((dimethylamino)methyl)thiophen-2-yl)pyridin-3-yl)-2-hydroxybenzoate 18A remedy of phenol ether 16l (560 mg, 1.11 mmol) in DCM (7 mL) was treated with trifluoroacetic acid (800 L, 10.81 mmol) at 0 C. After 1 h 30 min. the reaction was brought to pH ca. 5C6 with 1M NaOH and 1M HCl, the aqueous coating separated and extracted with DCM. The combined organic layers were concentrated and purified by Biotage column chromatography (0C15% MeOH/DCM) to give phenol 18 (394 mg, 92%). HRMS (ESI) calcd for C20H22N3O3S (M+H) 384.1376, found 384.1391..s, 1H), 7.53 (d, = 2.4 Hz, 1H), 7.26 (d, = 1.5 Hz, 1H), 7.22 (dd, = 8.0, 1.5 Hz, 1H), 7.18 (s, 1H), 7.09 (d, = 1.5 Hz, 1H), 6.41 (dd, = 12.1, 8.5 Hz, 1H), 6.09 C 6.04 (m, 1H), 5.98 (s, 2H), 5.58 C 5.54 (m, 1H), 3.36 (s, 2H), 2.15 (s, 6H), 1.53 (d, = 6.3 Hz, 3H). Aminopyridine calcd for C24H26F3N4O2S (M+H) 491.1723, found 491.1722. in hydrophobic contacts with Ile14 and Gly92. The dimethylamino group is clearly resolved and superimposes well with the basic center of the piperidine ring of construction. Pleasingly, aminopyridine configured enantiomer is definitely significantly more potent than the enantiomer.6 We therefore separated the two enantiomers of aminopyridine configuration is preferred for Nek2 inhibition, we assigned the configuration to the slower eluting enantiomer, whereas the faster eluting compound was associated to the configuration (Table 4). Table 4 Alkene seriesa construction (compound (= 8.5 Hz, 1H), 7.20 (d, = 1.9 Hz, 1H), 7.04 (dd, = 8.5, 1.9 Hz, 1H), 3.97 (s, 3H). Methyl 4-bromo-2-((4-methoxybenzyl)oxy)benzoate 12a A solution of phenol 6 (1.70 g, 7.36 mmol), 4-methoxybenzyl alcohol (1.29 g, 9.30 mmol) and triphenylphosphine (2.81 g, 10.71 mmol) in DCM (35 mL) was cooled at 0 C and treated with di-373 (M+Na). 1H NMR (500 MHz, CDCl3) 7.70 (d, = 8.3 Hz, 1H), 7.43 C 7.40 (m, 2H), 7.20 (d, = 1.8 Hz, 1H), 7.15 (dd, = 8.3, 1.8 Hz, 1H), 6.96 C 6.93 (m, 2H), 5.11 (s, 2H), 3.89 (s, 3H), 3.83 (s, 3H). Methyl 4-(2-amino-5-bromopyridin-3-yl)-2-((4-methoxybenzyl)oxy)benzoate 14b A solution of bromide 12a (1.10 g, 3.13 mmol), bis(pinacolato)diboron (1.20 g, 4.72 mmol), potassium acetate (925 mg, 9.44 mmol) and 1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II)DCM (130 mg, 0.16 mmol) in DMF (15 mL) was stirred at 100 C less than microwave irradiation for 1 h 30 min. The reaction was quenched with brine and extracted with AcOEt. The combined organics were washed with brine, dried (Na2SO4), and concentrated to afford the crude boronic ester 13a. A solution of crude boronic ester 13a (ca. 3.13 mmol), sodium bicarbonate (480 mg, 5.71 mmol), 1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II)DCM (125 mg, 0.15 mmol) and 5-bromo-3-iodopyridin-2-amine (850 mg, 2.84 mmol) in DMF/water (8/1, 15 mL) was stirred at 100 C less than microwave irradiation for 1 h 30 min. The reaction was quenched with brine and extracted with EtOAc. The combined organics were washed with brine, dried (Na2SO4), concentrated and purified by Biotage column chromatography (0C30% EtOAc/cyclohexane) to give bromopyridine 14b (1.02 g, 81%). HRMS (ESI) calcd for C21H20BrN2O4 (M+H) 443.0601, found 443.0617. 1H NMR (500 MHz, CDCl3) 8.13 (d, = 2.4 Hz, 1H), 7.90 (d, = 7.8 Hz, 1H), 7.45 (d, = 2.4 Hz, 1H), 7.43 C 7.40 (m, 2H), 7.07 C 7.04 (m, 2H), 6.95 C 6.92 (m, 2H), 5.17 (s, 2H), 4.57 (br. s, 2H), 3.94 (s, 3H), 3.83 (s, 3H). Methyl 4-(2-amino-5-(4-((dimethylamino)methyl)thiophen-2-yl)pyridin-3-yl)-2-((4-methoxybenzyl)oxy)benzoate 16lA remedy of bromide 14b (1.01 g, 2.28 mmol), bis(pinacolato)diboron (870 mg, 3.43 mmol), potassium acetate (680 mg, 6.94 mmol) and 1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II)DCM (200 mg, 0.25 mmol) in DMF (11 mL) was stirred at 100 C under microwave irradiation for 1 h 30 min. The reaction was quenched with brine and extracted with AcOEt. The combined organics were washed with brine, dried (Na2SO4), and concentrated to afford the crude boronic acid 15b. A solution of crude boronic acid 15b (ca. 2.28 mmol), 1-(5-bromothiophen-3-yl)-504 (M+H). 1H NMR (500 MHz, CDCl3) 8.36 (s, 1H), 7.92 (d, = 7.9 Hz, 1H), 7.56 (d, = 2.3 Hz, 1H), 7.44 C 7.41 (m, 2H), 7.24 (s, 1H), 7.14 C 7.10 (m, 3H), 6.97 C 6.91 (m, 2H), 5.19 (s, 2H), 4.59 (br. s, 2H), 3.94 (s, 3H), 3.82 (s, 3H), 3.54 (s, 2H), 2.36 (s, 6H). Methyl 4-(2-amino-5-(4-((dimethylamino)methyl)thiophen-2-yl)pyridin-3-yl)-2-hydroxybenzoate 18A remedy of phenol ether 16l (560 mg, 1.11 mmol) in DCM (7 mL) was treated with trifluoroacetic acid (800 L, 10.81 mmol) at 0 C. After 1 h 30 min. the reaction was brought to pH ca. 5C6 with 1M NaOH and 1M HCl, the aqueous coating separated and extracted with DCM. The combined organic layers were concentrated and purified by Biotage column chromatography (0C15% MeOH/DCM) to give phenol 18 (394 mg, 92%). HRMS (ESI) calcd for C20H22N3O3S (M+H) 384.1376, found 384.1391. 1H NMR (500 MHz, MeOD) 8.28 (d, = 2.4 Hz, 1H), 7.99 (d, = 8.2 Hz, 1H), 7.68 (d, = 2.4 Hz, 1H), 7.58 (d, = 1.4 Hz, 1H), 7.39 (d, = 1.4 Hz, 1H), 7.10 (d, = 1.7 Hz, 1H), 7.07 (dd, = 8.2, 1.7 Hz, 1H), 4.30 (s, 2H), 4.00 (s, 3H), 2.88 (s, 6H). ()-(calcd for C25H27F3N3O3S (M+H) 506.1720, found 506.1701. 1H NMR (500 MHz, MeOD) 8.29 (d, = 2.4 Hz, 1H), 7.88 (d, = 8.0 Hz, 1H), 7.65 (d, = 2.4 Hz, 1H), 7.56 (s, 1H), 7.37 (d, = 1.5 Hz, 1H), 7.22 (dd, = 8.0, 1.5 Hz, 1H), 7.17 (s, 1H), 6.27 (dd, = 12.1,.Simultaneously, the cells were treated in triplicate wells with test compound in DMSO at 1% v/v final concentration (Fisher Scientific, Loughborough, Leicestershire, United Kingdom) and incubated for 3 hours. to the slower eluting enantiomer, whereas the faster eluting compound was associated to the configuration (Table 4). Table 4 Alkene seriesa configuration (compound (= 8.5 Hz, 1H), 7.20 (d, = 1.9 Hz, 1H), 7.04 (dd, = 8.5, 1.9 Hz, 1H), 3.97 (s, 3H). Methyl 4-bromo-2-((4-methoxybenzyl)oxy)benzoate 12a A solution of phenol 6 (1.70 g, 7.36 mmol), 4-methoxybenzyl alcohol (1.29 g, 9.30 mmol) and triphenylphosphine (2.81 g, 10.71 mmol) in DCM (35 mL) was cooled at 0 C and treated with di-373 (M+Na). 1H NMR (500 MHz, CDCl3) 7.70 (d, = 8.3 Hz, 1H), 7.43 C 7.40 (m, 2H), 7.20 (d, = 1.8 Hz, 1H), 7.15 (dd, = 8.3, 1.8 Hz, 1H), 6.96 C 6.93 (m, 2H), 5.11 (s, 2H), 3.89 (s, 3H), 3.83 (s, 3H). Methyl 4-(2-amino-5-bromopyridin-3-yl)-2-((4-methoxybenzyl)oxy)benzoate 14b A solution of bromide 12a (1.10 g, 3.13 mmol), bis(pinacolato)diboron (1.20 g, 4.72 mmol), potassium acetate (925 mg, c-di-AMP 9.44 mmol) and 1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II)DCM (130 mg, 0.16 mmol) in DMF (15 mL) was stirred at 100 C under microwave irradiation for 1 h 30 min. The reaction was quenched with brine and extracted with AcOEt. The combined organics were washed with brine, dried (Na2SO4), and concentrated to afford the crude boronic ester 13a. A solution of crude boronic ester 13a (ca. 3.13 mmol), sodium bicarbonate (480 mg, 5.71 mmol), 1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II)DCM (125 mg, 0.15 mmol) and 5-bromo-3-iodopyridin-2-amine (850 mg, 2.84 mmol) in DMF/water (8/1, 15 mL) was stirred at 100 C under microwave irradiation for 1 h 30 min. The reaction was quenched with brine and extracted with EtOAc. The combined organics were washed with brine, dried (Na2SO4), concentrated and purified by Biotage column chromatography (0C30% EtOAc/cyclohexane) to give bromopyridine 14b (1.02 g, 81%). HRMS (ESI) calcd for C21H20BrN2O4 (M+H) 443.0601, found 443.0617. 1H NMR (500 MHz, CDCl3) 8.13 (d, = 2.4 Hz, 1H), 7.90 (d, = 7.8 Hz, 1H), 7.45 (d, = 2.4 Hz, 1H), 7.43 C 7.40 (m, 2H), 7.07 C 7.04 (m, 2H), 6.95 C 6.92 (m, 2H), 5.17 (s, 2H), 4.57 (br. s, 2H), 3.94 (s, 3H), 3.83 (s, 3H). Methyl 4-(2-amino-5-(4-((dimethylamino)methyl)thiophen-2-yl)pyridin-3-yl)-2-((4-methoxybenzyl)oxy)benzoate 16lA answer of bromide 14b (1.01 g, 2.28 mmol), bis(pinacolato)diboron (870 mg, 3.43 mmol), potassium acetate (680 mg, 6.94 mmol) and 1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II)DCM (200 mg, 0.25 mmol) in DMF (11 mL) was stirred at 100 C under microwave irradiation for 1 h 30 min. The reaction was quenched c-di-AMP with brine and extracted with AcOEt. The combined organics were washed with brine, dried (Na2SO4), and concentrated to afford the crude boronic acid 15b. A solution of crude boronic acid 15b (ca. 2.28 mmol), 1-(5-bromothiophen-3-yl)-504 (M+H). 1H NMR (500 MHz, CDCl3) 8.36 (s, 1H), 7.92 (d, = 7.9 Hz, 1H), 7.56 (d, = 2.3 Hz, 1H), 7.44 C 7.41 (m, 2H), 7.24 (s, 1H), 7.14 C 7.10 (m, 3H), 6.97 C 6.91 (m, 2H), 5.19 (s, 2H), 4.59 (br. s, 2H), 3.94 (s, 3H), 3.82 (s, 3H), 3.54 (s, 2H), 2.36 (s, 6H). Methyl 4-(2-amino-5-(4-((dimethylamino)methyl)thiophen-2-yl)pyridin-3-yl)-2-hydroxybenzoate 18A answer of phenol ether 16l (560 mg, 1.11 mmol) in DCM (7 mL) was treated with trifluoroacetic acid (800 L, 10.81 mmol) at 0 C. After 1 h 30 min. the reaction was brought to pH ca. 5C6 with 1M NaOH and 1M HCl, the aqueous layer separated and extracted with DCM. The combined organic layers were concentrated and purified by Biotage column chromatography (0C15% MeOH/DCM) to give phenol 18 (394 mg, 92%). HRMS (ESI) calcd for C20H22N3O3S (M+H) 384.1376, found 384.1391. 1H NMR (500 MHz, MeOD) 8.28 (d, = 2.4 Hz, 1H), 7.99 (d, = 8.2 Hz, 1H), 7.68 (d, = 2.4 Hz, 1H), 7.58 (d, = 1.4 Hz, 1H), 7.39 (d, = 1.4 Hz, 1H), 7.10 (d, = 1.7 Hz, 1H), 7.07 (dd, = 8.2, 1.7 Hz, 1H), 4.30 (s, 2H), 4.00 (s, 3H), 2.88 (s, 6H). ()-(calcd for C25H27F3N3O3S (M+H) 506.1720, found 506.1701. 1H NMR (500 MHz, MeOD) 8.29 (d, = 2.4 Hz, 1H), 7.88 (d, = 8.0 Hz, 1H), 7.65 (d, = 2.4 Hz, 1H), 7.56 (s, 1H), 7.37 (d, = 1.5 Hz, 1H), 7.22 (dd, = 8.0, 1.5 Hz, 1H), 7.17 (s, 1H), 6.27 (dd, = 12.1, 8.7 Hz, 1H), 5.94 C 5.85 (m, 1H), 5.49 C 5.45 (m, 1H), 4.24 (s, 2H), 3.92 (s,.