Protocol full text hidden due to copyright restrictions
Open the protocol to access the free full text link
Protocol full text hidden due to copyright restrictions
Open the protocol to access the free full text link
ClusterBlast and SubclusterBlast outputs for the balhimycin (23 (link)) biosynthesis gene cluster. The top six hits of each analysis module are shown. The ClusterBlast module shows the homology between the balhimycin gene cluster and the vancomycin, VEG, A40926 and teicoplanin biosynthesis gene clusters. Homologous genes are shown in identical colors, whereas white-colored genes have no BLAST hits between the gene clusters. The novel SubclusterBlast module can identify homologous sub-clusters encoding the biosynthesis of specific chemical moieties. In this case, SubclusterBlast is able to identify the dihydroxyphenylglycine (dHpg), hydroxyphenylglycine (Hpg) and hydroxytyrosine (Bht) precursor biosynthesis sub-clusters, as well as the vancosamine-like sugar biosynthesis sub-cluster.
Example 18
Step 1: Methyl 2-methoxy-5-(4,7,8-trichloroquinolin-2-yl)benzoate. To a suspension of 2,4,7,8-tetrachloroquinoline (200 mg, 0.75 mmol) with (4-methoxy-3-(methoxycarbonyl)phenyl) boronic acid (205.3 mg, 0.98 mmol) and Na2CO3 (178 mg, 1.68 mmol) in dioxane (8.0 mL) and water (2.0 mL) was added Pd(PPh3)4 (79.2 mg). The resultant mixture was vacuumed and purged with N2 for three cycles, then stirred and heated at 80° C. over two hours. After cooling to room temperature, the reaction mixture was dissolved in DCM (50 mL) and washed with water and brine. The resultant organic layer was separated and dried over anhydrous Na2SO4. A silica gel flash column chromatography eluting with DCM/Hexane afforded the desired colorless product (MS: [M+1]+ 396).
Step 2: 2-Hydroxy-5-(4,7,8-trichloroquinolin-2-yl)benzoic acid (I-75). Methyl 2-methoxy-5-(4,7,8-trichloroquinolin-2-yl) benzoate (17 mg) in DCM (1.5 mL) was treated with 1M BBr3 in DCM (0.1 mL) at room temperature over 8 hours. The resulting mixture was diluted with EtOAc (25 mL), washed with water (10 mL) and dried over Na2SO4. Concentration under vacuum afforded the desired light brown solid (11 mg), 2-hydroxy-5-(4,7,8-trichloroquinolin-2-yl) benzoic acid (MS: [M+1]+ 368).
Step 3: 5-(7,8-dichloro-4-(1H-imidazol-1-yl)quinolin-2-yl)-2-hydroxybenzoic acid. To a solution of 2-hydroxy-5-(4,7,8-trichloroquinolin-2-yl) benzoic acid (11 mg) in DMF (0.5 mL) was added imidazole (55 mg) and K2CO3 (50 mg). The resultant solution was stirred and heated at 120° C. over 5 hours until the starting material was completely consumed. The reaction mixture was diluted with water (3 mL) and treated with Dowex resin until the pH to 3. The resultant colorless solid was isolated by filtration and washed with water (3 mL). After drying under vacuum, the desired product was obtained (MS: [M+1]+ 400).
Example 12
Step 1: Methyl 5-(7-chloro-4-(1H-imidazol-1-yl)quinolin-2-yl)-2-methoxybenzoate was prepared according to the procedure above to prepare compound I-2.
Step 2: 5-(7-Chloro-4-(1H-imidazol-1-yl)quinolin-2-yl)-2-hydroxybenzoic acid (I-16) was prepared according to the procedure above to prepare compound I-26. (MS: [M+1]+ 366).
Step 3: Ethyl 5-(7-chloro-4-(1H-imidazol-1-yl)quinolin-2-yl)-2-ethoxybenzoate. To a vial were added 5-(7-chloro-4-(1H-imidazol-1-yl)quinolin-2-yl)-2-hydroxybenzoic acid (14 mg, 0.0383 mmol), ethyl iodide (22 μl, 0.274 mmol), Cs2CO3 (62 mg, 0.191 mmol) and DMF (1.0 mL). The resulting reaction mixture was stirred at room temperature overnight. The mixture was diluted with ethyl acetate (40 mL), washed by H2O (4×20 mL) and brine (15 mL). After concentration, the crude was used in next step.
Step 4: 5-(7-Chloro-4-(1H-imidazol-1-yl)quinolin-2-yl)-2-ethoxybenzoic acid. To the crude product above were added MeOH (0.6 mL), THF (0.4 mL) and a solution of NaOH in H2O (7.66 mg/0.2 mL). The resulting reaction mixture was stirred at room temperature overnight. Then the mixture was diluted by H2O (3 mL) and acidified by HOAc to pH 4. The cloudy mixture was centrifuged and the residue was washed by H2O (2×1.5 mL), dried over high vacuum to afford the title product as off-white solid (6 mg) (MS: [M+1]+ 394).
Example 11
Step 1: 3-(4,7-dichloroquinolin-2-yl)-5-methoxybenzoic acid was prepared essentially by the same methods described above to prepare I-2.
Step 2: 3-(7-chloro-4-(1H-imidazol-1-yl)quinolin-2-yl)-5-methoxybenzoic acid. To a vial were added 3-(4,7-dichloroquinolin-2-yl)-5-methoxybenzoic acid (50 mg, 0.144 mmol), imidazole (49 mg, 0.718 mmol), Cs2CO3 (70 mg, 0.216 mmol) and DMF (1.0 mL). The resulting reaction mixture was stirred at 110° C. overnight. At room temperature the reaction mixture was diluted by H2O (4 mL) and acidified by HCl (1 N) to pH about 3. A lot of white solid precipitated, centrifuged and washed by H2O (2×4 mL). The residue was dried in vacuo to afford the titled compound as white solid (30 mg). MS: [M+1]+ 380.
Step 3: 3-(7-chloro-4-(1H-imidazol-1-yl)quinolin-2-yl)-5-hydroxybenzoic acid. To a vial were added 3-(7-chloro-4-(1H-imidazol-1-yl)quinolin-2-yl)-5-methoxybenzoic acid (7.9 mg, 0.0208 mmol), NaI (20 mg, 0.133 mmol) and HBr in HOAc (33%, 1.0 mL). The resulting mixture was stirred at 50° C. overnight. The mixture was cooled to room temperature and diluted by H2O (10 mL), centrifuged, and washed with H2O (4 mL). The residue was dried under high vacuum to afford the title product as off-white solid (18 mg) (MS: [M+1]+ 366).