Inflammatory bowel disease (IBD) is a chronic immuno-inflammation in gastrointestinal system. particular quality. These three substances demonstrated >75% inhibitory effect against TNF–induced cell adhesion between monocyte and colon epithelial cells at 1?M concentration. Considering that 5-aminosalicylic acid (5-ASA, mesalazine), an active metabolite of sulfasalazine (SSZ) which is usually widely used to treat IBD in the clinical field, has only 3.5% inhibitory activity at the same drug concentration (1?M), the activity of our three compounds can be quite ONO 4817 marvellous. Moreover, efficacy studies using rats with severe colon inflammation induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) have confirmed that our compounds are certainly effective against IBD. When orally administered at the dose of 1 1?mg/kg, those compounds showed very good efficacy demonstrated by ameliorating disease parameters such as % of the recovery in colon- and body-weights (up to 79% and 59%, respectively) and myeloperoxidase (MPO) level. Open in a separate window Physique 1. 2,4,5-Trimethylpyridin-3-ol as anti-IBD scaffold. In this study, we made some changes in the functional group at C(6)-position of the pyridin-3-ol ring and examined how these structural changes affect on efficacy against IBD. We considered five types of functional groups (alkoxy-, ureido-, thioureido-, carbamato-, and sulfonamido-group) to replace 6-amino groups, and synthesised several to a dozen new derivatives for each family (Physique 1). 2.?Materials and methods 2.1. Chemistry Unless noted otherwise, materials were purchased from commercial suppliers and used without further purification. Air- or moisture-sensitive reactions were carried out under an inert gas atmosphere. Reaction progress was monitored by thin-layer-chromatography (TLC) using silica gel F254 plates. The products were purified by flash column chromatography using silica gel 60 (70C230 mesh) or by Biotage Isolera One system with indicated solvents. Melting points were determined using a Fischer-Jones melting point apparatus and were not corrected. NMR spectra were obtained using Bruker spectrometers 250?MHz or 400?MHz for 1H-NMR and 62.5?MHz or 100?MHz for 13?C-NMR. Chemical shifts () were expressed in ppm using solvent as an internal standard and coupling constant (244 [M?+?H]+; 1H-NMR (CDCl3) 13.04 (s, 1H), 7.48C7.32 (m, 5H), 4.70 (s, 2H), 2.28 (s, 3H), 2.17 (s, 3H), 2.10 (s, 3H); 13?C-NMR (CDCl3) 163.20, 145.35, 139.06, 136.95, 133.43, 128.75 (2?C), 128.42, 128.20 (2?C), 123.53, 75.83, 13.98, 13.85, 12.49. 2.1.2. 2,5-Bis(benzyloxy)-3,4,6-trimethylpyridine (7a) To a solution of 5-(benzyloxy)-3,4,6-trimethylpyridin-2-ol (6, 50?mg, 0.21?mmol) in a mixed solvent of DMF (1?ml) and THF (1?ml) was added Ag2CO3 (69?mg, 0.25?mmol) followed by addition of 3-bromo-1-propylbenzeze (38?L, 0.32?mmol). The mixture was stirred at room heat for 24?h. After filtration of the reaction mixture through a Celite pad, the filtrate was diluted with CH2Cl2 and water. The aqueous layer separated was extracted with CH2Cl2, and the combined CH2Cl2 answer was dried over MgSO4, filtered and concentrated. The residue was purified by silica gel column chromatography (Hexanes/EtOAc = 20/1) to give 7a (60?mg, 88%). White solid; PGC1A TLC Rf 0.61 (Hexanes/EtOAc = 10/1); m.p. 64?C; MS (ESI) 334 [M?+?H]+; 1H-NMR (CDCl3) 7.54C7.29 (m, 10H), 5.39 (s, 2H), 4.76 (s, 2H), 2.44 (s, 3H), 2.22 (s, 3H), 2.16 (s, 3H); 13?C-NMR (CDCl3) 156.88, 146.70, 144.54, 141.09, 138.43, 137.33, 128.57 (2?C), 128.28 (2?C), 128.10, 127.92 (2?C), 127.68 (2?C), 127.39, 117.07, 74.97, ONO 4817 67.26, 18.97, 12.78, 11.83. 2.1.3. 3,4,6-Trimethylpyridine-2,5-diol (8a) To a suspension of 10% palladium on activated carbon (5?mg) in MeOH (2?ml) was added 7a (20?mg, 0.06?mmol). The mixture was stirred with hydrogen balloon at room heat for 1?h. After filtration of the reaction mixture through a Celite pad, the filtrate was concentrated. The residue was purified by silica gel column chromatography (CH2Cl2/MeOH = 20/1) to give 8a (9?mg, 98%). ONO 4817 White solid; TLC Rf 0.27 (CH2Cl2/MeOH = 9/1); m.p. 176?C; MS (ESI) 154 [M?+?H]+; 1H-NMR (DMSO-10.96 (s, 1H), 7.51 (s, 1H), 2.07 (s, 3H), 2.03 (s, 3H), 1.90 (s, 3H); 13?C-NMR (DMSO-160.04, 141.97, 134.45, 126.76, 121.30, 13.70, 13.55, 12.27. 2.1.4. 3-(Benzyloxy)-6-butoxy-2,4,5-trimethylpyridine (7?b) To a solution of 5-(benzyloxy)-3,4,6-trimethylpyridin-2-ol (6, 100?mg, 0.41?mmol) in DMF (4?ml) was added Ag2CO3 (136?mg, 0.49?mmol) followed by addition of 1-iodobutane (70?L, 0.62?mmol). The mixture was stirred at 40?C for 2?h. After filtration of the reaction mixture through a Celite pad, the filtrate was concentrated then your residue was diluted with EtOAc and washed with brine and water. The EtOAc option was dried out over MgSO4, filtered and focused. The residue was additional purified by silica gel column chromatography (Hexanes/EtOAc = 30/1) to provide 7?b (87?mg, 71%). Yellow solid Pale; TLC Rf 0.29 (Hexanes/EtOAc = 20/1); m.p. 33?C; MS (ESI) 300 [M?+?H]+; 1H-NMR (CDCl3) 7.55C7.31 (m, 5H), 4.74 (s, 2H), 4.28 (t, 157.57,.