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Synthesis, Biological Evaluation of SPF-32629A-Based 2- and 4-Pyridone Analogs as Chymase Inhibitors

  • Received : 2014.03.24
  • Accepted : 2014.04.08
  • Published : 2014.08.20
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Abstract

Keywords

Experimental

General Procedures for Synthesis of 3a-e. To a solution of 6-methoxy-2-pyridinecarboxaldehyde (1.0 eq) in THF (0.05 M) was added dropwise ArMgBr (1.0 M in Et2O, 1.2 eq) at –78 °C. After stirring at –78 °C for 1 h, the reaction mixture was quenched by 1 N HCl and extracted with EtOAc. A combined organic layer was washed with brine, dried over MgSO4 and concentrated in vacuo. The residue was purified by flash column chromatography (EtOAc/hexanes).

(6-Methoxypyridin-2-yl)(phenyl)methanol (3a): 1HNMR (600 MHz, CDCl3) δ 7.48 (m, 1H), 7.44 (d, 2H, J = 7.2 Hz), 7.35 (m, 2H), 7.29 (m, 1H), 6.73 (d, 1H, J = 7.2 Hz), 6.63 (d, 1H, J = 7.8 Hz), 5.72 (s, 1H), 5.09 (s, 1H), 3.98 (s, 3H); 13C-NMR (150 MHz, CDCl3) δ 163.2, 158.9, 143.2, 139.5, 128.5, 127.8, 127.0, 113.7, 109.2, 74.8, 53.4. LRMS (ESI+) m/z 216.0 (M+H+).

(4-Fluorophenyl)(6-methoxypyridin-2-yl)methanol (3b): Yield 73%. 1H-NMR (600 MHz, CDCl3) δ 7.42 (t, 1H, J = 7.8 Hz), 7.36-7.34 (m, 2H), 6.98-6.96 (m, 2H), 6.7 (d, 1H, J = 7.2 Hz), 6.58 (d, 1H, J =8.4 Hz), 5.64 (d, 1H, J = 4.8 Hz), 5.08 (d, 1H, J = 4.8 Hz), 4.00 (s, 3H); 13C-NMR (150 MHz, CDCl3) δ 163.2, 161.4, 158.9, 139.5, 139.1, 128.6, 115.2, 113.4, 109.2, 74.3, 53.3; LRMS (ESI+) m/z 233.2 (M+H+).

(4-Chlorophenyl)(6-methoxypyridin-2-yl)methanol (3c): Yield 80%. 1H-NMR (600 MHz, CDCl3) δ 7.45 (d, 1H, J = 4.8 Hz), 7.30 (d, 2H, J = 8.4 Hz), 7.06 (d, 2H, J =8.4 Hz), 6.67 (d, 1H, J = 7.2 Hz), 6.60 (s, 1H), 5.62 (s, 1H), 5.03 (s, 1H), 3.92 (s, 3H); 13C-NMR (150 MHz, CDCl3) δ 163.2, 158.4, 141.7, 139.6, 133.4, 128.5, 128.3, 116.7, 113.4, 119.4, 74.2, 60.5, 53.4, 21.0, 14.1; LRMS (ESI+) m/z 250.0 (M+H+).

(6-Methoxypyridin-2-yl)(p-tolyl)methanol (3d): Yield 80%. 1H-NMR (600 MHz, CDCl3) δ 7.48 (t, 1H, J = 7.8 Hz), 7.32 (d, 2H, J = 7.8 Hz), 7.17 (d, 2H, J = 7.8 Hz), 6.73 (d, 1H, J = 7.2 Hz), 6.63 (d, 1H, J =7.8 Hz), 5.68 (d, 1H, J = 3.6 Hz), 4.98 (d, 1H, J = 4.2 Hz), 4.00 (s, 3H), 2.35 (s, 3H); 13CNMR (150 MHz, CDCl3) δ 163.2, 159.2, 140.4, 139.5, 137.3, 129.2, 126.9, 113.6, 109.1, 74.7, 53.4, 21.1. LRMS (ESI+) m/z 230.1 (M+H+).

General Procedures for Synthesis of 4a-e. To a CHCl3 solution of the 3 (1.0 eq) iodotrimethylsilane (3.0 eq) was added at 60 °C. After stirring for 1 h, the reaction mixture was quenched with aq NaHSO3, and extracted with CH2Cl2. The organic phase was washed with brine, dried over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography (CH2Cl2 /MeOH).

6-(Hydroxy(phenyl)methyl)pyridin-2(1H)-one (4a): Yield 76%. 1H-NMR (600 MHz, CD3OD) δ 7.52 (dd, 1H, J = 7.2 9.0 Hz), 7.41-7.40 (m, 2H), 7.36-7.33 (m, 2H), 7.30-7.29 (m, 1H), 6.39 (dd, 1H, J = 9.0, 0.6 Hz), 6.32 (d, 1H, J = 7.2 Hz), 5.62 (s, 1H); 13C-NMR (150 MHz, CD3OD) δ 164.5, 151.0, 142.2, 141.2, 128.2, 127.9, 126.4, 117.3, 104.3, 71.7; LRMS (ESI+) m/z 202.1 (M+H+).

6-((4-Fluorophenyl)(hydroxy)methyl)pyridin-2(1H)-one (4b): Yield 74%. 1H-NMR (600 MHz, CD3OD) δ 7.50 (dd, 1H, J = 9.0, 7.2 Hz), 7.44-7.41 (m, 2H), 7.05 (t, 2H, J = 8.7 Hz), 6.41 (d, 1H, J = 9.0 Hz), 6.32 (d, 1H, J = 7.2 Hz), 5.68 (s, 1H); 13C-NMR (150 MHz, CD3OD) δ 164.4, 150.9, 142.3, 137.3, 128.6, 128.5, 117.4, 104.6, 71.0. LRMS (ESI+) m/z 220.1 (M+H+).

6-((4-Chlorophenyl)(hydroxy)methyl)pyridin-2(1H)-one (4c): Yield 70%. 1H-NMR (600 MHz, CD3OD) δ 7.52 (dd, 1H, J = 7.2, 9.0 Hz), 7.41-7.39 (m, 2H), 7.36-7.34 (m, 2H), 6.40 (d, 1H, J = 9.0 Hz), 6.33 (d, 1H, J = 7.2 Hz), 5.62 (s, 1H); 13C-NMR (150 MHz, CD3OD) δ 164.5, 150.6, 142.2, 140.0, 133.6, 128.3, 128.0, 117.5, 104.4, 70.9; LRMS (ESI+) m/z 216.2 (M+H+).

6-(Hydroxy(p-tolyl)methyl)pyridin-2(1H)-one (4d):Yield 77%. 1H-NMR (600 MHz, CD3OD) δ 7.52 (t, 1H, J = 7.2, 9.0 Hz), 7.27 (d, 2H, J = 7.8 Hz), 7.17 (d, 2H, J = 7.8 Hz), 6.38 (d, 1H, J = 9.0 Hz), 6.31 (d, 1H, J = 7.2 Hz), 5.58 (s, 1H), 2.31 (s, 3H); 13C-NMR (150 MHz, CD3OD) δ 164.4, 151.1, 142.2, 138.2, 137.8, 128.8, 126.4, 117.1, 104.2, 71.5, 19.75. LRMS (ESI+) m/z 216.2 (M+H+).

General Procedures for Synthesis of 5a-e and 6a-e. To a solution of the 2-pyridone 4 (1.0 eq) in CH2Cl2 was added EDCI (1.2 eq), DMAP (0.3 eq) and isovaleric acid (1.2 eq) at 0 °C. After stirring at ambient temperature for 1 h, the reaction mixture was washed with saturated aq. NaHCO3 and extracted with EtOAc. A combined organic layer was washed with brine, dried over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography (CH2Cl2/MeOH) to afford the monoester 5 and diester 6.

(6-Oxo-1,6-dihydropyridin-2-yl)(phenyl)methyl isovaleroate (5a): 1H-NMR (600 MHz, CDCl3) δ 11.85 (br s, 1H), 7.43-7.45 (m, 2H), 7.31-7.37 (m, 4H), 6.64 (s, 1H), 6.45 (dd, 1H, J = 0.6, 9 Hz), 6.09 (dt, 1H, J = 1.2, 6 Hz), 2.33 (d, 2H, J = 7.2 Hz), 2.13 (m, 1H), 0.91 (d, 6H, 6.6 Hz); 13CNMR (150 MHz, CDCl3) δ 171.6, 164.4, 146.4, 140.9, 136.7, 128.9, 128.8, 127.2, 119.8, 104.4, 73.1, 43.2, 25.6, 22.4, 22.3; LRMS (ESI+) m/z 286.0 (M+H+).

(4-Fluorophenyl)(6-oxo-1,6-dihydropyridin-2-yl)methyl isovaleroate (5b): Yield 22%. 1H-NMR (600 MHz, CDCl3) δ 12.96 (s, 1H), 7.47-7.45 (m, 2H), 7.35 (dd, 1H, J = 7.2, 9 Hz), 7.01 (t, 2H, J = 8.4 Hz), 6.62 (s, 1H), 6.11 (d, 2H, J = 6.6 Hz), 6.44 (d, 1H, J = 8.4 Hz), 6.11 (d, 1H, J = 6.6 Hz), 2.32 (dd, 2H, J = 1.8, 7.8 Hz), 2.13-2.09 (m, 1H), 0.89 (d, 1H, J = 6.6); 13C-NMR (150 MHz, CDCl3) 171.6, 165.0, 163.6, 162.0, 146.9, 141.0, 132.9, 132.9, 129.4, 129.4, 119.6, 115.8, 115.6, 104.0, 77.3, 77.1, 76.9, 72.6, 43.2, 25.6, 22.4, 22.4; LRMS (ESI+) m/z 304.1 (M+H+).

(6-Oxo-1,6-dihydropyridin-2-yl)(p-tolyl)methyl isovaleroate (5d): 1H-NMR (600 MHz, CDCl3) δ 11.52 (br s, 1H), 7.30-7.34 (m, 3H), 7.16 (d, 2H, J = 7.8 Hz), 6.61 (s, 1H), 6.43 (d, 1H, 9 Hz), 6.08 (d, 1H, J = 7.2 Hz), 2.31 (dd, 5H, J = 1.2, 1.8 Hz), 2.09-2.16 (m, 1H), 0.91 (d, 6H, J = 6.6 Hz); 13C-NMR (150 MHz, CDCl3) δ 171.6, 164.4, 146.4, 140.9, 138.8, 133.6, 129.5, 127.2, 119.8, 104.2, 73.0, 43.2, 30.3, 25.6, 22.4, 22.3, 21.2; LRMS (ESI+) m/z 300.1 (M+H+).

6-((4-Fluorophenyl)(isovaleryloxy)methyl)pyridin-2-yl isovaleroate (6b): Yield 17%. 1H-NMR (600 MHz, CDCl3) δ 7.75 (t, 1H, J = 7.8 Hz), 7.38-7.36 (m, 2H), 7.31 (d, 1H J = 7.8), 7.0-6.97 (m, 3H), 5.44 (d, 2H, J = 7.2 Hz), 2.31 (d, 2H, J = 7.2 Hz), 2.25-2.18 (m, 1H), 2.17-2.10 (m, 1H), 1.03 (d, 6H, J = 7.2 Hz), 0.93 (d, 6H, J = 7.2 Hz); LRMS (ESI+) m/z 388.1 (M+H+).

General Procedures for Synthesis of 9a-d. To a THF solution of 4-(benzyloxy)picolinonitrile 8 (1.0 eq) was added ArMgBr (1.0 M in Et2O, 1.0 eq) at –78 °C. After stirring for 1 h, the reaction mixture was quenched with 6 N HCl, and stirred for 12 h. The organic phase was washed with brine, dried over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography (EtOAc/hexanes) to afford the ketone 9.

(4-(Benzyloxy)pyridin-2-yl)(phenyl)methanone (9a): Yield 80%. 1H-NMR (600 MHz, CDCl3) δ 8.53 (d, 1H, J = 5.4Hz), 8.07 (dd, 2H J = 1.2, 8.4 Hz), 7.65 (d, 1H, J = 2.4 Hz), 7.59-7.56 (m, 1H), 7.49-7.45 (m, 2H), 7.45-7.40 (m, 4H), 7.34-7.35 (m, 1H), 7.05 (dd, 1H, J = 2.4, 6.0 Hz), 5.17 (s, 2H); 13C-NMR (150 MHz, CDCl3) δ 193.7, 165.6, 156.8, 149.9, 136.3, 135.3, 132.9, 131.0, 128.8, 128.5, 128.1, 127.6, 113.3, 110.8, 70.2; LRMS (ESI+) m/z 290.1 (M+H+).

General Procedures for Synthesis of 10a-d. To a solution of 9 in MeOH was added NaBH4 (1.0 eq) at –78 °C. After stirring for 30 min, the reaction mixture was quenched with MeOH and concentrated in vacuo. The residue was purified by column chromatography (EtOAc/hexanes) to afford 10.

2-(Hydroxyphenylmethyl)-4(1H)-pyridone (10a): Yield 71%. 1H-NMR (600 MHz, CD3OD) δ 7.74 (d, 1H J = 9.0 Hz), 7.45 (t, 2H, J = 1.2, 9.0 Hz), 7.39 (t, 2H, J = 9.6 Hz), 7.35-7.31 (m, 1H), 6.42-6.38 (m, 2H), 5.70 (s, 1H); 13CNMR (125 MHz, CD3OD) δ 180.3, 155.0, 141.3, 138.2, 128.4, 128.0, 126.4, 115.4, 113.4, 72.0; LRMS (ESI+) m/z 202.1 (M+H+).

General Procedures for Synthesis of 11a-d. A solution of the alcohol 10 in MeOH was treated with 10% Pd/C and hydrogenated for 1 h. The reaction mixture was filtered through Celite® and the solvent was removed in vacuo. The residue was purified by flash column chromatography (CH2Cl2/MeOH) to afford the 4-pyridone 11.

(4-(Benzyloxy)pyridin-2-yl)(phenyl)methanone (11a): Yield 80%. 1H-NMR (600 MHz, CDCl3) δ 8.53 (d, 1H, J = 5.4 Hz), 8.08 (dd, 2H, J = 1.2, 8.4 Hz), 7.65 (d, 1H, J = 2.4 Hz), 7.59-7.56 (m, 1H), 7.49-7.45 (m, 2H), 7.45-7.40 (m, 4H), 7.34-7.35 (m, 1H), 7.05 (dd, 1H, J = 2.4, 6.0 Hz), 5.18 (s, 2H); 13C-NMR (150 MHz, CDCl3) δ 193.7, 165.6, 156.8, 149.9, 136.3, 135.3, 132.9, 131.0, 128.8, 128.5, 128.1, 127.6, 113.3, 110.8, 70.2; LRMS (ESI+) m/z 290.1 (M+H+).

2-((4-Fluorophenyl)(hydroxy)methyl)pyridin-4(1H)-one (11b): Yield 95%. 1H-NMR (600 MHz, CD3OD) δ 7.74 (d, 1H, J = 8.4 Hz), 7.49-7.46 (m, 2H), 7.14-7.11 (m, 2H), 6.40 (t, 2H, J = 3.0, 8.4 Hz), 5.71 (s, 1H); 13C-NMR (150 MHz, CD3OD) δ 163.6, 161.7, 137.4, 128.4, 115.4, 113.4, 71.2; LRMS (ESI+) m/z 220.0 (M+H+).

2-(Hydroxy(p-tolyl)methyl)pyridin-4(1H)-one (11d): Yield 93%. 1H-NMR (600 MHz, CD3OD) δ 7.73 (d, 1H, J = 9.0 Hz), 7.32 (d, 2H, J = 9.6 Hz), 7.21 (d, 1H, J = 9.6 Hz), 6.39 (t, 2H, J = 7.2, 3 Hz), 5.65 (s, 1H), 2.34 (s, 3H); 13CNMR (150 MHz, CD3OD) δ 138.3, 138.0, 128.9, 126.4, 115.3, 113.3, 71.8, 19.8; LRMS (ESI+) m/z 216.1 (M+H+).

General Procedures for Synthesis of 12a-e and 13a-e. To a solution of 11 in CH2Cl2 was added EDCI (1.1 eq), DMAP (0.5 eq) and isovaleric acid (1.0 eq) at 0 °C. The reaction mixture was stirred at ambient temperature for 1 h. After the completion of the reaction, saturated aq. NaHCO3 was added for quenching. The mixture was extracted with EtOAc. A combined organic layer was washed with brine, dried over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography (CH2Cl2/MeOH) to afford 12 and 13.

(4-Oxo-1,4-dihydropyridin-2-yl)(phenyl)methyl isovaleroate (12a): Yield 70%. 1H-NMR (600 MHz, CDCl3) δ 7.55 (d, 1H, J = 7.8 Hz), 7.54-7.36 (m, 2H), 7.31-7.29 (m, 3H), 6.71 (s, 1H), 6.60 (s, 1H), 6.37 (dd, 1H, J = 2.4, 8.4 Hz), 5.31 (s, 1H), 3.47 (s, 1H), 2.28 (d, 2H, J = 8.4 Hz), 0.90 (dd, 6H, J = 3.6, 7.8 Hz); 13C-NMR (150 MHz, CDCl3) δ 177.5, 171.7, 152.5, 140.4, 137.1, 128.9, 128.8, 127.4, 115.39, 113.8, 73.9, 53.4, 50.5, 43.1, 29.7, 25.6, 22.3; LRMS (ESI+) m/z 286.1 (M+H+).

(4-Fluorophenyl)(4-oxo-1,4-dihydropyridin-2-yl)methyl isovaleroate (12b): Yield 28%. 1H-NMR (600 MHz, CDCl3) δ 7.61 (d, 1H, J = 7.8 Hz), 7.37 (dd, 2H, J = 6.6, 10.2 Hz), 6.98 (t, 2H, J = 10.2 Hz), 6.72 (s, 1H), 6.61 (d, 1H, J = 3 Hz), 6.41-6.40 (m, 1H), 5.29 (s, 1H), 2.27 (d, 2H, J = 9 Hz), 0.97 (d, 1H, J = 7.8), 0.89 (dd, 6H, J = 3.6, 8.4 Hz); 13CNMR (125 MHz, CDCl3) δ 177.8, 177.1, 171..5, 163.8, 161.8, 152.5, 140.4, 133, 132.9, 129.4, 129.3, 115.9, 115.7, 115.4, 113.5, 72.9, 53.4, 44.2, 43.1, 25.7, 25.6, 22.5, 22.3; LRMS (ESI+) m/z 301.2 (M+H+).

(4-Oxo-1,4-dihydropyridin-2-yl)(p-tolyl)methyl isovaleroate (12d): Yield 21%. 1H-NMR (600 MHz, CDCl3) δ 7.56 (d, 1H, J = 8.4 Hz), 7.28 (d, 1H, J = 12 Hz), 7.11 (d, 1H, J = 9.6 Hz), 6.69 (s, 1H), 6.61 (d, 1H, J = 3 Hz), 6.39 (dd, 1H, J = 3, 8.4 Hz), 5.31 (s, 1H), 2.31 (s, 3H), 2.27 (d, 2H, J = 8.4 Hz), 0.99 (d, 1H, J = 7.8 Hz), 0.90 (dd, 6H, J = 3, 8.4 Hz); 13C-NMR (125 MHz, CDCl3) δ 177.9, 171.6, 152.5, 140.1, 138.8 134.0, 129.5, 127.4, 115.4, 113.7, 73.6, 43.1, 25.7, 25.6, 22.5, 22.4, 22.3, 21.1; LRMS (ESI+) m/z 300.2 (M+H+).

2-((4-Fluorophenyl)(isovaleryloxy)methyl)pyridin-4-yl isovaleroate (13b): 1H-NMR (600 MHz, CDCl3) δ 8.60 (d, 1H, J = 6.6 Hz), 7.42 (dd, 2H, J = 6.6, 10.2 Hz), 7.28 (d, 1H, J = 2.4 Hz), 7.07-7.01 (m, 3H), 6.90 (s, 1H), 2.47 (d, 1H, J = 8.4 Hz), 2.47 (d, 1H, J = 8.4 Hz), 2.36 (d, 2H, J = 8.4 Hz), 1.07 (d, 6H, J = 7.8 Hz), 1.00 (d, 4H, J = 7.8 Hz), 0.96 (d, 6H, J = 7.8 Hz); LRMS (ESI+) m/z 388.1 (M+H+).

2-((Isovaleryloxy)(p-tolyl)methyl)pyridin-4-yl isovaleroate (13d): Yield 90%. 1H-NMR (600 MHz, CDCl3) δ 8.56 (d, 1H, J = 6.6 Hz), 7.35 (d, 2H, J = 9.6 Hz), 7.28 (d, 1H, J = 3 Hz), 7.17 (d, 2H, J = 9.6 Hz), 7.04 (dd, 1H, J = 2.4, 6.6 Hz), 6.91 (s, 1H), 2.48 (d, 2H, J = 8.4 Hz), 2.36 (t, 2H, J = 8.4, 14.4 Hz), 1.09 (d, 6H, J = 7.8 Hz), 0.98 (dd, 6H, J = 1.2, 7.8 Hz); LRMS (ESI+) m/z 384.2 (M+H+).

Chymase Inhibition Assay. Chymase assay was performed with Chymase Activity Assay kit (Sigma-Aldrich) according to the manufacturer’s protocol. Briefly, test compounds dissolved in DMSO were incubated with 0.3 mg of chymase (Cat. Number C8118) in assay buffer (Cat. number A9606). The reaction was initiated by addition of substrate A (Nsuccinyl- Ala-Ala-Pro-Phe p-nitroanilide, Cat. Number S0448), and enzymatic activity was monitored by measuring absorbance at 405 nm for 20 min using Victor X3 (Perkin- Elmer) microplate reader. Percent inhibition and IC50 of each sample were calculated by Prism (GraphPad). Chymostatin was used as positive control.

α-Chymotrypsin Inhibition Assay. α-Chymotrypsin assay was performed in 38 mM Tris–HCl buffer (pH 7.8) with 53 mM CaCl2. α-Chymotrypsin (2-5 units/mL prepared in buffer mentioned above) with various concentrations (1, 10, 100 μM) of test compounds prepared in DW was incubated at 30 °C or 10 min. The reaction was started by the addition of the substrate, p-NA. All the reactions were performed in triplicate in a final volume of 180 μL, by using a plate reader. The plate reader absorbance wavelength was settled at 405 nm. Aprotinin was used as positive control.

Supporting Information. The 1H- and 13C-NMR spectral data of synthesized analogs are available as Supporting Information.

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