URAT1 inhibitor for promoting uric acid excretion

Abstract

The present invention belongs to the field of medicinal chemistry. Specifically disclosed is a class of URAT1 inhibitors for promoting uric acid excretion, which are compounds as represented by the structure of formula (I) or pharmaceutically acceptable salts thereof. Experiments show that the compounds provided by the present invention have a very good inhibitory effect on the transport of uric acid by hURAT1 in HEK293 transfected cells, and that such compounds have a good application prospect in the treatment of hyperuricemia or gout. ##STR00001##

Claims

1. A compound, a (4-Hydroxyphenyl) (diazole) ketone derivative, as represented by the structure of formula (I-1) or pharmaceutically acceptable salts thereof ##STR00023## wherein, A is a non-aromatic six-membered ring; Y is carbonyl or; R.sup.1 is one or more selected from the group consisting of hydrogen, deuterium, hydroxy, halogen, nitro, amino, cyano, C.sub.1-5 alkyl, substituted C.sub.1-5 alkyl, substituted C.sub.1-3 amino, C.sub.1-3 alkoxy, substituted C.sub.1-3 alkoxy and C.sub.1-5 alkylthio; R.sup.2 is one or more selected from the group consisting of hydrogen, deuterium, hydroxy, halogen, nitro, amino, cyano, C.sub.1-4 alkyl, substituted C.sub.1-3 alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl, substituted C.sub.1-3 amino, C.sub.1-5 alkoxy, substituted C.sub.1-5 alkoxy and C.sub.1-5 alkylthio; R.sup.3 is C.sub.1-4 alkyl, substituted C.sub.1-4 alkyl or C.sub.3-4 cycloalkyl; m is an integer from 0 to 3; n is an integer from 1 to 3.

2. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein ring A is a cyclohexene ring or a non-aromatic six-membered ring; the non-aromatic six-membered ring containing at least one O or/and N atom.

3. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R.sup.1 is selected from one or more of the group consisting of hydrogen, deuterium, fluorine, chlorine, bromine, hydroxy, cyano, C.sub.1-3 alkyl, C.sub.1-3 haloalkyl and C.sub.1-3 alkoxy; m is 0, 1 or 2.

4. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R.sup.2 is selected from one or more of the group consisting of hydrogen, deuterium, halogen, cyano, vinyl, ethynyl, C.sub.1-2 alkyl, substituted C.sub.1-2 alkyl, C.sub.1-2 alkoxy, substituted C.sub.1-2 alkoxy, C.sub.1-2 alkylthio, and substituted C.sub.1-2 alkylthio; n is 1 or 2.

5. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R.sup.3 is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, cyclopropyl and cyclobutyl.

6. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from the following: ##STR00024## ##STR00025##

7. A pharmaceutical composition comprising the compound or pharmaceutically acceptable salt thereof according to claim 1 as an active ingredient and pharmaceutically acceptable excipients.

8. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound or pharmaceutically acceptable salt thereof is prepared as a medicament for promoting uric acid excretion.

9. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R.sup.1, or R.sup.2 is selected from the group consisting of hydroxy, halogen, nitro, amino and cyano.

10. The compound or pharmaceutically acceptable salt thereof according to claim 4, wherein R.sup.2 is selected from the group consisting of deuterium, halogen, C.sub.1-2 alkyl, and C.sub.1-2 alkoxy.

Description

EMBODIMENTS

(1) The present invention will be further described below by examples, but the scope of the present invention is not limited hereinto.

Example 1

Synthesis of (3,5-dibromo-4-hydroxyphenyl)(2-ethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-3-yl)methanone (5)

(2) ##STR00007##

(3) Step A: To a mixture of 2-aminopyridine (2.0 g, 21.3 mmol) and triethylamine (2.58 g, 25.5 mmol) in dichloromethane (20 mL) was added dropwise with propionyl chloride (2.07 g, 22.4 mmol) in an ice-water bath. After the addition was completed, the obtained mixture was stirred overnight at room temperature. The mixture was added with water (40 mL) and extracted with dichloromethane (40 mL×3), and the combined organic phase was washed with brine (30 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by flash chromatography (200-300 meshes of silica gel, eluted with ethyl acetate:petroleum ether=1:15 to 1:10) to obtain N-(pyridine-2-yl)-propionamide (1) (2.74 g). The yield was 85.6%.

(4) Step B: A mixture containing compound 1 (300 mg, 2.0 mmol), 2-bromo-1-(4-methoxyphenyl) ethanone (460 mg, 2.0 mmol) and toluene (10 mL) was stirred under reflux for 48 hours. After cooling to room temperature, water (30 mL) was added and the pH was adjusted to 8-9 with a saturated aqueous solution of potassium carbonate. The mixture was extracted with dichloromethane (40 mL×3) and dried over anhydrous sodium sulfate. The solvent is evaporated under reduced pressure and the product is purified by column chromatography (200-300 mesh silica gel, ethyl acetate:petroleum ether=1:30 to 1:1) to give (2-ethylimidazo[1,2-a]pyridin-3-yl)(4-methoxyphenyl)methanone (2) (254 mg). The yield was 45.3%. .sup.1H NMR (DMSO-d.sub.6, 500 MHz) δ 9.18 (d, J=7.0 Hz, 1H), 7.74-7.69 (m, 3H), 7.58-7.55 (m, 1H), 7.17-7.14 (m, 1H), 7.09 (d, J=8.5 Hz, 2H), 3.87 (s, 3H), 2.45 (q, J=7.5 Hz, 2H), 1.11 (t, J=7.5 Hz, 3H). MS (EI, m/z): 281.1 [M+H].sup.+.

(5) Step C: A mixture containing compound 2 (250 mg, 0.89 mmol), 10% palladium carbon (25 mg) and DMF (7 mL) was stirred overnight at 30° C. under hydrogen. Then ethyl acetate (30 mL) was added and the mixture was filtered through a celite pad. The filtrate was washed with water (30 mL×3) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to give (2-ethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-3-yl) (4-methoxyphenyl)methanone (3) (230 mg). The yield was 90.7%.

(6) Step D: To a solution of compound 3 (220 mg, 0.77 mmol) in anhydrous dichloromethane (10 mL) was added dropwise 1.0 M solution of boron tribromide in toluene (2.3 mL) in the ice water bath. After addition, the resulting mixture was stirred at room temperature overnight. The reaction mixture was poured into ice water (30 mL) and the pH was adjusted to 7-8 with saturated sodium bicarbonate. The mixture was extracted with ethyl acetate (40 mL×2) and the combined organic phases was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to give (2-ethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-3-yl)(4-hydroxyphenyl)methanone (4) (205 mg). The yield was 98.0%.

(7) Step E: A solution of bromine (260 mg, 1.63 mmol) in acetic acid (1 mL) was added dropwise to a mixture of compound 4 (200 mg, 0.74 mmol) and anhydrous sodium acetate (182 mg, 2.2 mmol) in acetic acid (8 mL). After the addition was completed, the resulting mixture was stirred at room temperature for 1 hour. A diluted aqueous solution of sodium bisulfite was added dropwise to the reaction mixture until the color faded. The solvent was evaporated under reduced pressure, water (15 mL) was added and then the pH value was adjusted to 7-8 with saturated sodium bicarbonate solution. The mixture was extracted with ethyl acetate (40 mL×2) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the obtained product was recrystallized from petroleum ether/ethyl acetate to give (3,5-dibromo-4-hydroxyphenyl)(2-ethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-3-yl)methanone (5) (175 mg). The yield was 55.3%. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) δ 7.77 (s, 2H), 4.02-4.00 (m, 2H), 2.86-2.83 (m, 2H), 2.28 (q, J=7.6 Hz, 2H), 1.92-1.86 (m, 4H), 1.08 (t, J=7.6 Hz, 3H). MS (EI, m/z): 426.9 [M−H].sup.−.

Example 2

Synthesis of (3,5-dibromo-4-hydroxyphenyl)(5,6,6,7,8-pentadeuterio-2-ethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-3-yl)methanone (10)

(8) ##STR00008##

(9) Step A: 60% sodium hydride (1.68 g, 42 mmol) was added portionwise to a solution of p-methoxyacetophenone (3.0 g, 20.0 mmol) in DMF (15 mL). After the addition was completed, stirring was continued at this temperature for 40 minutes, and then ethyl propionate (2.04 g, 20 mmol) was added dropwise. After the addition was completed, the resulting mixture was stirred at room temperature overnight. After the addition of water (60 mL), the mixture was extracted with ethyl acetate (30 mL×3), the combined organic phases was washed with saturated brine (20 mL×2) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate:petroleum ether=1:30) to give 1-(4-methoxyphenyl)pentane-1.3-dione (6) (3.16 g). The yield was 76.6%.

(10) Step B: 2-amino-5-bromopyridine (2.60 g, 15.0 mmol) and compound 6 (3.72 g, 18.0 mmol) were dissolved in THF (40 mL), and then, in the ice water bath, iodophthalic acid (5.80 g, 18.0 mmol) and boron trifluoride etherate (430 mg, 3.03 mmol)) were added sequentially. After the addition was completed, stirring was continued at room temperature overnight. Water (40 mL) was added, and the pH value was adjusted to 7-8 with a saturated sodium bicarbonate solution and then ethyl acetate (50 mL×3) was added as extraction agent. The combined organic layers was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate:petroleum ether=1:20) to give (6-bromo-2-ethylimidazo[1,2-a]pyridin-3-yl)(4-methoxyphenyl)methanone (7) (1.15 g). The yield was 21.3%.

(11) Step C: Compound 7 (200 mg, 0.557 mmol) was suspended in DMF (10 mL). Heavy water (0.5 mL) and 5% palladium on carbon (20 mg) were added, and the resulting mixture was stirred under atmospheric pressure for 48 hours under deuterium. After filtered through a celite pad, water (40 mL) was added to the filtrate, followed by extraction with ethyl acetate (30 mL×3). The combined organic phases was washed with water (20 mL×3) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to give (4-methoxyphenyl) (5,6,6,7,8-pentadeuterio-2-ethyl-5,6,7, 8-tetrahydroimidazo[1,2-a]-pyridin-3-yl)methanone (8) (164 mg). The yield was 100%. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) δ 7.67 (dd, J=2.0, 6.8 Hz, 2H), 7.06 (dd, J=2.0, 6.8 Hz, 2H), 4.03-4.01 (m, 1H), 3.86 (s, 3H), 2.80-2.78 (m, 1H), 2.18 (q, J=7.6 Hz, 2H), 1.81-1.79 (m, 1H), 0.99 (t, J=7.6 Hz, 3H).

(12) Experimental procedures of Steps D and E were carried out according to the preparation of Steps D and E in Example 1 to give (3,5-dibromo-4-hydroxyphenyl)(5,6,6,7,8-pentadeuterio-2-ethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-3-yl)methanone (10). .sup.1H NMR (DMSO-d.sub.6, 400 MHz) δ 7.80 (s, 2H), 4.05-4.01 (m, 1H), 2.85-2.83 (m, 1H), 2.27 (q, J=7.2 Hz, 2H), 1.83-1.81 (m, 1H), 1.08 (t, J=7.2 Hz, 3H). MS (EI, m/z): 434.0 [M+H].sup.+.

Example 3

Synthesis of 5-(2-ethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-3-carbonyl)-2-hydroxybenzonitrile (16)

(13) ##STR00009##

(14) Step A: 4-methoxyacetophenone (44.0 g, 293 mmol) was added into a mixture of 1-chloromethyl-4-fluoro-1,4-diazabicyclo[2.2.2]octane bis(tetrafluoroborate) (104 g, 294 mmol), iodine (38.6 g, 152 mmol) and acetonitrile (440 mL) in an ice-water bath. After addition was completed, the obtained mixture was stirred overnight at room temperature. The reaction mixture was added with water (1350 mL), and a large amount of solid was separated out. The mixture was filtered and dried to obtain 3-iodo-4-methoxyacetophenone (11) (70.0 g). The yield was 86.5%.

(15) Step B: A mixture of the compound 11 (70.0 g, 254 mmol), cuprous cyanide (34.0 g, 380 mmol) and DMF (400 mL) was stirred overnight at 130° C. The mixture was cooled to room temperature, filtered through a celite pad, added with water (1600 mL) and extracted with ethyl acetate (800 mL×3). The combined organic phase was successively washed with water (400 mL×2) and brine (400 mL), and then dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to obtain a crude compound of 5-acetyl-2-methoxybenzonitrile (12) (50.0 g). The compound was directly used in the next reaction without further treatment.

(16) Step C: A solution of bromine (49.0 g, 307 mmol) in methanol (50 mL) was added dropwise into a solution of the crude compound 12 (45.0 g) in methanol (250 mL). After the addition was completed, the obtained mixture was stirred overnight at room temperature. The mixture was added with water (900 mL), filtered and dried to give 5-(2-bromo-acetyl)-2-hydroxy-3-methylbenzonitrile(13) (41.0 g). The total yield of the reactions in the steps B and C was 70.6%.

(17) Step D: A mixture of the compound 13 (41.0 g, 161 mmol), the compound 1 (24.0 g, 161 mmol) and methylbenzene (600 mL) was refluxed and stirred for 48 hours. The mixture was cooled to room temperature, added with water (400 mL) and adjusted with saturated sodium bicarbonate solution until the pH value was 7-8. The mixture was extracted with ethyl acetate (600 mL×3) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by flash chromatography (200-300 meshes of silica gel, eluted with ethyl acetate:petroleum ether=1:30 to 2:1) to obtain 5-(2-ethylimidazo[1,2-a]pyridine-3-carbonyl)-2-methoxybenzonitrile (14) (25.7 g). The yield was 52.3%.

(18) Step E: A mixture containing compound 14 (1.0 g, 3.28 mmol), 10% palladium carbon (100 mg) and acetic acid (10 mL) was stirred overnight at 30° C. under hydrogen atmosphere. After filtered through a celite pad, the solvent was evaporated under reduced pressure, ethyl acetate (70 mL) was added, and the mixture was washed with water (20 mL) and dried over sodium sulphate. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate:petroleum ether=1:3 to 4:1) to give 5-(2-ethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-3-carbonyl)-2-methoxybenzonitrile (15) (400 mg). The yield was 39.5%.

(19) Step F: 60% sodium hydride (65 mg, 1.63 mmol) was added in portions into a solution of ethanethiol (0.12 mL) in THF (10 mL), the mixture was stirred for about 5 minutes and then filtered, and the filter cake was collected. Subsequently, the filter cake was added to the solution of the compound 15 (100 mg, 0.323 mmol) in DMF (6 mL), and the obtained mixture was stirred for 1 h at 60° C. The mixture was cooled to room temperature, filtered with a celite pad, added with water (40 mL) and adjusted with 2 M citric acid aqueous solution until the pH value was 5-6. The mixture was extracted with ethyl acetate (40 mL×3) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate:petroleum ether=1:2 to 5:1) to give 5-(2-ethyl-5,6,7,8-Tetrahydroimidazo[1,2-a]pyridine-3-carbonyl)-2-hydroxybenzonitrile (16) (52 mg). The yield was 54.5%. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) δ 7.89 (s, 1H), 7.79 (dd, J=2.0, 8.8 Hz, 1H), 7.05 (d, J=8.4 Hz, 1H), 4.04 (t, J=5.6 Hz, 2H), 2.83-2.81 (m, 2H), 2.18 (q, J=7.2 Hz, 2H), 1.89-1.83 (m, 4H), 1.01 (t, J=7.2 Hz, 3H). MS (EI, m/z): 296.2 [M+H].sup.+.

Example 4

Synthesis of 3-bromo-5-(2-ethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-3-carbonyl)-2-hydroxybenzonitrile (17)

(20) ##STR00010##

(21) Step A: A solution of bromine (27 mg, 1.63 mmol) in acetic acid (1 mL) was added dropwise to a solution of compound 16 (50 mg, 0.74 mmol) and anhydrous sodium acetate (28 mg, 2.2 mmol) in acetic acid (8 mL) in. After the addition was completed, the resulting mixture was stirred at room temperature for 1 hour. A dilute sodium bisulfite solution was added dropwise to the reaction mixture until the color faded. The solvent was evaporated under reduced pressure, then water (15 mL) was added, and the pH value was adjusted to 7-8 with saturated sodium bicarbonate. The mixture was extracted with ethyl acetate (40 mL×2) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the obtained product was recrystallized from petroleum ether/ethyl acetate to give 3-bromo-5-(2-ethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-3-carbonyl) 2-hydroxybenzonitrile (17) (30 mg). The yield was 55.3%. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) δ 7.93 (d, J=2.4 Hz, 1H), 7.71 (d, J=2.4 Hz, 1H), 4.00 (t, J=5.6 Hz, 2H), 2.99-2.97 (m, 2H), 2.37-2.35 (m, 2H), 1.95-1.89 (m, 4H), 1.15 (t, J=7.6, 3H). MS (EI, m/z): 376.1 [M+H].sup.+.

Example 5

Synthesis of (3,5-dibromo-4-hydroxyphenyl)(2-ethyl-7-hydroxy-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-3-yl)methanone (23) and (3,5-dibromo-4-hydroxyphenyl)-(2-ethyl-7-methoxy-5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-3-yl)methanone (25)

(22) ##STR00011## ##STR00012##

(23) Step A: 2-amino-4-methoxypyridine (4.9 g, 39.5 mmol) and triethylamine (4.4 g, 43.5 mmol) were dissolved in tetrahydrofuran (30 mL), then propionyl chloride (4.0 g, 43.5 mmol) was added dropwise in an ice water bath, and the resulting mixture was stirred at room temperature overnight. Water (100 mL) was added, and the mixture was extracted with ethyl acetate (60 mL×3). The combined organic layers were washed with saturated brine (30 mL) and the solvent was evaporated under reduced pressure. Potassium carbonate (4.1 g, 29.7 mmol), methanol (50 mL) and water (12 mL) were added to the product, and the resulting mixture was stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure, water (20 mL) was added, the mixture was extracted with ethyl acetate (30 mL×3). The combined organic layers were washed with saturated brine (15 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to give N-(4-methoxypyridin-2-yl)propanamide (18) (4.85 g). The yield was 68.2%.

(24) Step B: A mixture containing compound 18 (4.85 g, 26.9 mmol), 2-bromo-1-(4-methoxyphenyl)ethanone (6.14 g, 26.9 mmol) and toluene (50 mL) was stirred under reflux overnight. After cooling to room temperature, water (50 mL) was added, and the pH value was adjusted to 8-9 with 2 M potassium carbonate solution. The mixture was extracted with dichloromethane (70 mL×3) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate:petroleum ether=1:5 to 2:3) to give (2-ethyl-7-methoxyimidazo[1,2-a]pyridin-3-yl)(4-methoxyphenyl)methanone (19) (900 mg). The yield was 10.8%. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) δ 9.08 (d, J=7.6 Hz, 1H), 7.67 (d, J=8.8 Hz, 2H), 7.17 (d, J=2.4 Hz, 1H), 7.08 (d, J=8.4 Hz, 2H), 6.88-6.86 (m, 1H), 3.91 (s, 3H), 3.87 (s, 3H), 2.38 (q, J=7.2 Hz, 2H), 1.10 (t, J=7.2 Hz, 3H).

(25) Step C: A 1.0 M solution of boron tribromide in toluene (9 mL) was added dropwise to a solution of compound 19 (900 mg, 2.9 mmol) in anhydrous dichloromethane (25 mL). After the addition was completed, the resulting mixture was stirred at room temperature overnight. The reaction mixture was poured into ice water (50 mL) and the pH value was adjusted to 7-8 with saturated sodium bicarbonate. The mixture was extracted with ethyl acetate (40 mL×3) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (200-300 mesh silica gel, methanol:dichloromethane=1:50 to 1:20) to give (2-ethyl-7-hydroxyimidazo[1,2-a]pyridin-3-yl)(4-hydroxyphenyl)methanone (20) (477 mg) and (2-ethyl-7-methoxyimidazo[1,2-a]pyridine-3-yl)(4-hydroxyphenyl)methanone (21) (277 mg). The yields were 58.3% and 32.2% respectively. Compound 20: .sup.1H NMR (DMSO-d.sub.6, 400 MHz) δ 10.83 (s, 1H), 10.22 (s, 1H), 9.06 (d, J=7.6 Hz, 1H), 7.54 (d, J=8.4 Hz, 2H), 6.89-6.84 (m, 3H), 6.77-6.75 (m, 1H), 2.37 (q, J=7.6 Hz, 2H), 1.08 (t, J=7.6 Hz, 3H). Compound 21: .sup.1H NMR (DMSO-d6, 400 MHz) δ 10.25 (s, 1H), 9.03 (d, J=7.6 Hz, 1H), 7.57 (dd, J=2.0, 6.8 Hz, 2H), 7.15 (d, J=2.4 Hz, 1H), 6.91-6.83 (m, 3H), 3.91 (s, 3H), 2.45 (q, J=7.6 Hz, 2H), 1.11 (t, J=7.6 Hz, 3H).

(26) Step D: A mixture containing compound 20 (185 mg, 0.66 mmol), Raney Ni (40 mg) and ethanol (15 mL) was stirred under hydrogen at 60° C. for 6 hours, then Raney Ni was added to the reaction mixture (40 mg) and then stirring was continued for 3 hours at 60° C. under hydrogen. After cooled to room temperature, the mixture was filtered, and the filter cake was subject to a drip washing with a small amount of ethyl acetate. The solvent was evaporated under reduced pressure, and the product was purified by column chromatography (200-300 mesh silica gel, methanol:dichloromethane=1:50-1:30) to give (2-ethyl-7-hydroxy-5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-3-yl)(4-hydroxyphenyl)methanone (22) (106 mg). The yield was 62.7%. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) δ 10.33 (s, 1H), 7.58 (d, J=8.8 Hz, 2H), 6.86 (d, J=8.8 Hz, 2H), 5.14 (d, J=3.2 Hz, 1H), 4.17 (s, 1H), 4.12-4.02 (m, 2H), 3.02-2.96 (m, 1H), 2.74-2.68 (m, 1H), 2.20 (q, J=7.6 Hz, 2H), 1.99-1.88 (m, 2H), 1.00 (t, J=7.6 Hz, 3H).

(27) Step E: Compound 22 (56 mg, 0.22 mmol) was dissolved in DMF (3 mL), NBS (77 mg, 0.44 mmol) was added, and the mixture was stirred in ice water bath for 0.5 hour. After the addition of water (15 mL), the mixture was extracted with ethyl acetate (30 mL×3) and the combined organic layers were washed with water (15 mL×2) and saturated brine (15 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (200˜300 mesh silica gel, methanol:dichloromethane=1:50) to give (3,5-dibromo-4-hydroxyphenyl)(2-ethyl-7-hydroxy-5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-3-yl)methanone (23) (13 mg). The yield was 13.5%. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) δ 7.81 (s, 2H), 5.22 (s, 1H), 4.21-4.19 (m, 1H), 4.12-4.05 (m, 2H), 3.08-3.03 (m, 1H), 2.80-2.76 (m, 1H), 2.26 (q, J=7.6 Hz, 2H), 2.03-1.19 (m, 2H), 1.07 (t, J=7.6 Hz, 3H). MS (EI, m/z): 442.9 [M−H].sup.−.

(28) Using compound 21 as a raw material, the experimental procedures of steps F and G were carried out according to the preparation methods of steps D and E of this example to give (3,5-dibromo-4-hydroxyphenyl)(2-ethyl-7-methoxy-5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-3-yl)methanone (25). .sup.1H NMR (DMSO-d.sub.6, 400 MHz) δ 7.81 (s, 2H), 4.09-4.03 (m, 2H), 3.88-3.87 (m, 1H), 3.33 (s, 3H), 3.07-3.06 (m, 1H), 2.94-2.93 (m, 1H), 2.24 (q, J=7.6 Hz, 2H), 2.17-2.04 (m, 2H), 1.06 (t, J=7.6 Hz, 3H). MS (EI, m/z): 457.0 [M−H].sup.−.

Example 6

Synthesis of 3,5-dibromo-4-hydroxyphenyl)(2-ethyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyridin-3-yl)methanone (32)

(29) ##STR00013##

(30) Step A: A mixture of 1-aminopyridinium iodide (15.5 g, 70.0 mmol), ethyl 2-pentynoate (9.72 g, 77.1 mmol), potassium carbonate (21.26 g, 154 mmol) and DMF (150 mL) was stirred for 4.5 hours at the room temperature. The mixture was added with water (450 mL) and filtered, and the filter cake was washed with water (100 mL) to give a wet compound of ethyl 2-ethylpyrazolo[1,5-a]-pyridine-3-formate (26) (12.25 g). The compound was directly used in the next reaction without drying.

(31) Step B: A mixture of the wet compound 26 (12.25 g), ethanol (30 mL), THF (30 mL) and 2 M sodium hydroxide aqueous solution (70 mL) was stirred overnight at 60° C. About half of the solvent was evaporated under reduced pressure, and the mixture was added with water (150 mL) and adjusted with 2 M hydrochloric acid until the pH value was 5-6. The mixture was filtered to give a wet compound of 2-ethylpyrazolo[1,5-a]pyridine-3-formic acid (27) (10.0 g). The compound was directly used in the next reaction without drying.

(32) Step C: The wet compound 27 (5.60 g) was suspended in water (100 mL) and added with concentrated sulfuric acid (4 mL), and the obtained mixture was stirred for 3 h at 80° C. The mixture was cooled to room temperature and adjusted with 2 M sodium hydroxide aqueous solution until the pH value was 8-9. The mixture was extracted with ethyl acetate (40 mL×3), and the combined organic phase was successively washed with water (30 mL) and brine (20 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to obtain 2-ethylpyrazolo[1,5-a]pyridine (28) (3.18 g). The total yield of the reactions in the steps A, B and C was 47.7%.

(33) Step D: A mixture of the compound 28 (584 mg, 3.99 mmol), 4-methoxybenzoyl chloride (680 mg, 3.99 mmol) and aluminum trichloride (800 mg, 6.0 mmol) was stirred overnight at 100° C. The mixture was cooled slightly, added with ethyl acetate (30 mL) and water (30 mL), and adjusted with 2 M sodium hydroxide aqueous solution until the pH value was 9-10. The mixture was layered, and the organic phase was collected. The water phase was extracted with ethyl acetate (30 mL×2), and the combined organic phase was washed with brine (20 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by flash chromatography (200-300 meshes of silica gel, eluted with ethyl acetate:petroleum ether=1:30 to 1:10) to obtain (2-ethylpyrazolo[1,5-a]pyridine-3-yl)(4-methoxyphenyl)methanone (29) (305 mg). The yield was 27.3%. .sup.1H NMR (DMSO-d.sub.6, 300 MHz) δ 8.79 (d, J=6.9 Hz, 1H), 7.66 (d, J=8.7 Hz, 2H), 7.44-7.39 (m, 1H), 7.33-7.30 (m, 1H), 7.08-7.03 (m, 3H), 3.86 (s, 3H), 2.84 (q, J=7.5 Hz, 2H), 1.20 (t, J=7.5 Hz, 3H).

(34) Step E: 60% sodium hydride (218 mg, 5.45 mmol) was added in portions into a solution of ethanethiol (338 mg, 5.44 mmol) in DMF (3 mL), the reaction mixture was stirred for about 5 minute and then added with a solution of the compound 29 (305 mg, 1.09 mmol) in DMF (3 mL), and the obtained mixture was stirred for 2 hours at 120° C. The mixture was cooled to room temperature, added with water (30 mL) and adjusted with diluted hydrochloric acid until the pH value was 7-8. Subsequently, the mixture was extracted with ethyl acetate (30 mL×3), and the combined organic phase was successively washed with water (20 mL×3) and brine (20 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to obtain (2-ethylpyrazolo[1,5-a]pyridine-3-yl)(4-hydroxyphenyl)methanone (30) (420 mg). The compound was directly used in the next reaction without purification. .sup.1H NMR (DMSO-d.sub.6, 300 MHz) δ 10.27 (s, 1H), 8.76 (d, J=6.6 Hz, 1H), 7.56 (d, J=8.4 Hz, 2H), 7.42-7.31 (m, 2H), 7.05-7.01 (m, 1H), 6.87 (d, J=8.4 Hz, 2H), 2.84 (q, J=7.5 Hz, 2H), 1.20 (t, J=7.5 Hz, 3H).

(35) Step F was carried out according to the preparation method of Step D in Example 4 to give (2-ethyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyridin-3-yl)(4-hydroxyphenyl)methanone (31).

(36) Step G: NBS (86 mg, 0.483 mmol) was added to a solution of compound 31 (65 mg, 0.240 mmol) in DMF (5 mL) and stirred for 1 hour. After the addition of water (20 mL), the mixture was extracted with ethyl acetate (20 mL×3), and the combined organic phases were washed with water (10 mL×3) and saturated brine (10 mL) and dried over sodium sulphate. The solvent was evaporated under reduced pressure, and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate:dichloromethane=1:10) to give 2-ethyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyridin-3-yl)methanone (32). .sup.1H NMR (DMS O-d.sub.6, 400 MHz) δ 7.74 (s, 2H), 4.05 (t, J=6.0 Hz, 2H), 3.36-3.34 (m, 2H), 2.57-2.51 (m, 2H), 1.96-1.95 (m, 2H), 1.72-1.70 (m, 2H), 1.08 (t, J=7.6 Hz, 3H). MS (EI, m/z): 429.0 [M+H].sup.+.

Example 7

Synthesis of (3,5-dibromo-4-hydroxyphenyl)(2-ethyl-4,5,6,7-tetrahydro-2H-indazol-3-yl)methanone (41)

(37) ##STR00014## ##STR00015##

(38) Step A: To a solution of cyclohexanone (9.81 g, 100 mmol) and diethyl oxalate (14.6 g, 100 mmol) in THF (100 mL) was added portionwise 60% sodium hydride (4.8 g, 120 mmol). After the addition was completed, the mixture was heated to 40° C. and stirred for 0.5 hour, and then raised to 50° C. and stirred for 1.5 hours. After cooling to room temperature, the reaction solution was poured into a solution of acetic acid (8 mL) in water (200 mL). The mixture was extracted with methyl tert-butyl ether (100 mL×2), and the combined organic phases were washed with saturated brine (40 mL). The solvent was evaporated under reduced pressure and the product was purified by column chromatography (200-300 mesh silica gel, eluted with petroleum ether) to give ethyl 2-oxy-2-(2-oxocyclohexyl)acetate (33) (10.5 g). The yield was 53.0%.

(39) Step B: A solution of compound 33 (10.1 g, 51.0 mmol) and 85% hydrazine hydrate (1.84 g, 48.8 mmol) in ethanol (40 mL) was stirred at 60° C. for 2 hours. The solvent was evaporated under reduced pressure, water (40 mL) was added, the mixture was extracted with ethyl acetate (40 mL×3) and the combined organic layers was washed with brine (30 mL). The solvent was evaporated under reduced pressure and the product was purified by column chromatography (200-300 mesh silica gel, petroleum ether:ethyl acetate=1:100 to 1:3) to give 4,5,6,7-tetrahydro-2H-indazole-3-carboxylic acid ethyl ester (34) (5.0 g). The yield was 50.5%.

(40) Step C: A mixture containing compound 34 (2.3 g, 11.8 mmol), ethyl iodide (3.69 g, 23.7 mmol), cesium carbonate (5.79 g, 17.8 mmol) and DMF (25 mL) was stirred at room temperature overnight. After the addition of water (75 mL), the mixture was extracted with ethyl acetate (50 mL×3) and the combined organic layers was washed with water (20 mL×2) and saturated brine (15 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (200-300 mesh silica gel, petroleum ether:ethyl acetate=1:20 to 1:10) to give 2-ethyl-4,5,6,7-tetrahydro-2H-indazole-3-carboxylic acid ethyl ester (35) (1.58 g, petroleum ether:ethyl acetate=1:1, R.sub.f=0.8) and 1-ethyl-4,5,6,7-tetrahydro-2H-indazole-3-carboxylic acid ethyl ester (36) (1.01 g, petroleum ether:ethyl acetate=1:1, R.sub.f=0.5). The yields were 60.2% and 38.5% respectively.

(41) Step D: A mixture containing compound 35 (1.58 g, 7.24 mmol), sodium hydroxide (580 mg, 14.5 mmol), methanol (5 mL) and water (15 mL) was stirred at 40° C. for 1 hour. The water was evaporated under reduced pressure, and then water was carried twice with toluene. Thionyl chloride (6 mL) and DMF (1 drop) were added to the residue, and the resulting mixture was stirred under reflux for 1 hour. The solvent was evaporated under reduced pressure, then THF (15 mL) was added, and the above THF solution was added portionwise to a concentrated aqueous ammonia (15 mL) in an ice water bath. After the addition was completed, stirring was continued for 20 minutes. After added water (30 mL), the mixture was extracted with ethyl acetate (30 mL×3) and the combined organic layers was washed with brine (20 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to give 2-ethyl-4,5,6,7-tetrahydro-2H-indazole-3-carboxamide (37) (1.18 g). The yield was 84.3%.

(42) Step E: A solution of trifluoroacetic anhydride (1.92 g, 9.14 mmol) and compound 37 (1.1 g, 5.69 mmol) in THF (20 mL) was stirred at room temperature for 3 hours. The solvent was evaporated under reduced pressure, water (20 mL) was added, and the pH value was adjusted to 8-9 with a 2 M aqueous sodium hydroxide solution. The mixture was extracted with ethyl acetate (30 mL×2) and the combined organic layers was washed with brine (15 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate:petroleum ether=1:20) to give 2-ethyl-4,5,6,7-tetrahydro-2H-indazole-3-carbonitrile (38) (640 mg). The yield was 64.2%. 1H NMR (DMSO-d6, 400 MHz) δ 4.22 (q, J=7.2 Hz, 2H), 2.59-2.50 (m, 4H), 1.76-1.68 (m, 4H), 1.37 (t, J=7.2 Hz, 3H).

(43) Step F: 1.0 M of 4-methoxyphenylmagnesium bromide in THF (5.7 mL) was added dropwise to a solution of compound 38 (500 mg, 2.85 mmol) in THF (10 mL). After the addition was completed, the resulting mixture was stirred at room temperature overnight. 6 M hydrochloric acid solution (5 mL) was added, and the pH value was adjusted to 8-9 with a 2 M aqueous sodium hydroxide solution after stirring for about 1 hour. The mixture was extracted with ethyl acetate (40 mL×2) and the combined organic layers was washed with brine (20 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate:petroleum ether=1:100 to 1:1) to give (2-ethyl-4,5,6,7-tetrahydro-2H-indazol-3-yl)(4-methoxyphenyl)methanone (39) (300 mg). The yield was 37.0%.

(44) Step G: A 1.0 M solution of boron tribromide in toluene (3.2 mL) was added dropwise to a solution of compound 39 (300 mg, 1.05 mmol) in anhydrous dichloromethane (6 mL). After addition, the resulting mixture was stirred at room temperature overnight. The reaction mixture was poured into ice water (30 mL) and the pH value was adjusted to 7-8 with saturated sodium bicarbonate. The mixture was extracted with ethyl acetate (30 mL×3) and the combined organic layers was washed with brine (20 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to give (2-ethyl-4,5,6,7-tetrahydro-2H-indazol-3-yl)(4-hydroxyphenyl)methanone (40) (280 mg). The yield was 98.6%. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) δ 10.22 (s, 1H), 7.38 (d, J=8.4 Hz, 2H), 6.81 (d, J=8.4 Hz, 2H), 3.99-3.96 (m, 2H), 2.58-2.55 (m, 2H), 2.10-2.08 (m, 2H), 1.72-1.70 (m, 2H), 1.58-1.57 (m, 2H), 1.21 (t, J=7.2 Hz, 3H).

(45) Step H: A solution of bromine (124 mg, 0.776 mmol) in acetic acid (3 mL) was added dropwise to a mixture of compound 40 (100 mg, 0.370 mmol) and anhydrous sodium acetate (89 mg, 1.11 mmol) in acetic acid (15 mL). After the addition was completed, the resulting mixture was stirred at room temperature for 1 hour. A diluted aqueous solution of sodium bisulfite was added dropwise to the reaction mixture until the color faded. The solvent was evaporated under reduced pressure, water (15 mL) was added, and the pH value was adjusted to 7-8 with saturated sodium bicarbonate. The mixture was extracted with ethyl acetate (40 mL×2) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate:petroleum ether=1:100 to 1:1) to give (3,5-dibromo-4-hydroxyphenyl) (2-ethyl-4,5,6,7-tetrahydro-2H-indazol-3-yl)methanone (41). .sup.1H NMR (DMSO-d.sub.6, 400 MHz) δ 7.83 (s, 2H), 4.20-4.18 (m, 2H), 2.60-2.58 (m, 2H), 2.14-2.11 (m, 2H), 1.73-1.72 (m, 2H), 1.57-1.56 (m, 2H), 1.31 (t, J=7.2 Hz, 3H). MS (EI, m/z): 426.9 [M−H].sup.−.

Example 8

Synthesis of 2,6-dibromo-4-[(2-ethyl-5,6,7,8-tetrahydroimidazo[1,2-a]-pyridin-3-yl)hydroxymethyl]phenol (42)

(46) ##STR00016##

(47) Lithium aluminum hydride (18 mg, 0.474 mmol) was added to a solution of compound 5 (135 mg, 0.315 mmol) in THF (15 mL), and the mixture was stirred at this temperature for 0.5 hour. After adding water (15 mL), the pH value was adjusted to 5-6 with 2 M citric acid solution, and the mixture was extracted with ethyl acetate/THF mixture (20 mL×3). The combined organic phases was washed with brine (15 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (200-300 mesh silica gel, dichloromethane:methanol=1:100 to 1:30) to give 2,6-dibromo-4-[(2-ethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-3-yl)-hydroxymethyl]phenol (42). .sup.1H NMR (DMSO-d.sub.6, 400 MHz) δ 7.36 (s, 2H), 5.95 (s, 1H), 5.78 (s, 1H), 3.88-3.85 (m, 2H), 2.67-2.65 (m, 2H), 2.32 (q, J=7.6 Hz, 2H), 1.76-1.69 (m, 4H), 1.04 (t, J=7.6 Hz, 3H). MS (EI, m/z): 431.0 [M+H].sup.+.

Example 9

Synthesis of (3,5-dibromo-4-hydroxyphenyl)(2-ethyl-6-methyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-3-yl)methanone (43)

(48) ##STR00017##

(49) The synthesis of Compound 43 was carried out according to the procedures of Example 1, in which 2-aminopyridine in Step A of Example 1 was replaced with 2-amino-5-methylpyridine. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) δ 7.79 (s, 2H), 4.16-4.11 (m, 1H), 3.57-3.51 (m, 1H), 2.96-2.79 (m, 2H), 2.27 (q, J=7.6 Hz, 2H), 2.03-1.91 (m, 2H), 1.56-1.47 (m, 1H), 1.09-1.03 (m, 6H). MS (EI, m/z): 441.0 [M−H].sup.−.

Example 10

Synthesis of (3-bromo-4-hydroxy-5-methylphenyl)(2-ethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-3-yl)methanone (48)

(50) ##STR00018##

(51) Step A: A solution of bromoacetyl bromide (9.9 g, 49.0 mmol) in dichloromethane (10 mL) was added dropwise into a solution of 2-methylanisole (5.0 g, 40.9 mmol) and aluminum trichloride (6.0 g, 45.0 mmol) in dichloromethane (40 mL) for about 20 minute at 0-5° C. After the addition was completed, the obtained mixture was continuously stirred for 2 hours at this temperature. The reaction solution was poured into a proper amount of ice water in batches and extracted with dichloromethane (60 mL×3). The combined organic phase was successively washed with water (30 mL), saturated sodium bicarbonate aqueous solution (30 mL×2), water (30 mL) and brine (30 mL) and then dried over anhydrous sodium sulfate. The organic phase was filtered by a short silica gel column. The solvent was evaporated under reduced pressure, and the product was purified by flash chromatography (200-300 meshes of silica gel, ethyl acetate:petroleum ether=1:100 to 1:30) to obtain 2-bromo-1-(3-methyl-4-methoxyphenyl)ethanone (44) (3.0 g). The yield was 30.2%.

(52) Steps B, C, D and E were carried out according to the preparation of Steps B, C, D and E in Example 1 to give (3-bromo-4-hydroxy-5-methylphenyl)(2-ethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-3-yl)methanone (48). .sup.1H NMR (DMSO-d.sub.6, 400 MHz) δ 7.57 (s, 1H), 7.28 (s, 1H), 3.89-3.87 (m, 2H), 2.76-2.73 (m, 2H), 2.29 (q, J=7.6 Hz, 2H), 1.96 (s, 3H), 1.87-1.80 (m, 4H), 1.05 (q, J=7.6 Hz, 3H). MS (EI, m/z): 363.1 [M+H].sup.+.

Example 11

Synthesis of (3,5-dibromo-4-hydroxyphenyl)(2-ethyl-2,4,6,7-tetrahydropyrano[4,3-c]pyrazol-3-yl methanone (58)

(53) ##STR00019## ##STR00020##

(54) Experimental procedures of Steps A, B, and C were carried out according to the preparation of Steps A, B, and C in Example 7, wherein the cyclohexanone in Step A of Example 1 was replaced with tetrahydropyrone.

(55) Step D: A solution of compound 51 (2.5 g, 11.1 mmol) in THF (10 mL) was added dropwise to a mixture containing lithium aluminum hydride (846 mg, 22.3 mmol) and THF (15 mL). After the addition was completed, the resulting mixture was further stirred at this temperature for 1 hour. Water (1 mL), 10% sodium hydroxide solution (2 mL) and water (3 mL) were added dropwise to the reaction mixture. After filtrated, the filter cake was subject to a drip washing with THF (15 mL) and the filtrate was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to give (2-ethyl-2,4,6,7-tetrahydropyrano[4,3-c]pyrazol-3-yl)methanol (53) (2.1 g). The yield was 100%.

(56) Step E: A mixture containing compound 53 (2.0 g, 11.0 mmol), manganese dioxide (4.78 g, 55.0 mmol) and chloroform (15 mL) was stirred at 45° C. overnight. The insolubles were removed by filtration, and the solvent was evaporated under reduced pressure. The product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate:petroleum ether=1:20 to 1:6) to give (2-ethyl-2,4,6,7-tetrahydropyrano[4,3-c]pyrazol-3-yl)carbaldehyde (54) (928 mg). The yield was 46.8%.

(57) Step F: 1.0 M solution of 4-methoxyphenylmagnesium bromide in THF (5.5 mL) was added dropwise to a solution of compound 54 (900 mg, 4.99 mmol) in THF (15 mL) at −70° C. After the addition was completed, the resulting mixture was stirred at this temperature for further 20 minutes. The reaction mixture was slowly added dropwise to ice water (20 mL), and extracted with ethyl acetate (30 mL×3). The combined organic phases were dried over brine (30 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate:petroleum ether=1:10 to 1:1) to give (2-ethyl-2,4,6,7-tetrahydropyrano[4,3-c]pyrazol-3-yl)(4-methoxyphenyl)methanol (55) (1.4 g). The yield was 97.3%.

(58) Step G: A mixture containing compound 55 (1.38 g, 4.79 mmol), 2-iodobenzoic acid (1.74 g, 55.0 mmol) and DMSO (15 mL) was stirred at room temperature for 1.5 hours. After the addition of water (45 mL), the mixture was extracted with ethyl acetate (30 mL×3) and the combined organic phases were washed successively with water (20 mL×2) and brine (20 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to give (2-ethyl-2,4,6,7-tetrahydropyrano[4,3-c]pyrazol-3-yl)(4-methoxyphenyl)-methanone (56) (1.32 g). The yield was 96.3%.

(59) Step H Experimental procedures were carried out according to the preparation method of Step F in Example 3 to give (2-ethyl-2,4,6,7-tetrahydropyrano[4,3-c]pyrazol-3-yl) (4-hydroxyphenyl)methanone (57).

(60) Step I Experimental procedures were carried out according to the preparation method of Step E in Example 1 to give (3,5-dibromo-4-hydroxyphenyl)(2-ethyl-2,4,6,7-tetrahydropyran)-[4,3-c]pyrazol-3-yl)methanone (58). .sup.1H NMR (DMSO-d.sub.6, 400 MHz) δ 7.84 (s, 2H), 4.26 (q, J=7.2 Hz, 2H), 4.22 (s, 2H), 3.86 (t, J=6.4 Hz, 2H), 2.71 (t, J=6.4 Hz, 2H), 1.35 (t, J=7.2 Hz, 3H). MS (EI, m/z): 431.0 [M+H].sup.+.

Example 12

Synthesis of (3,5-dibromo-4-hydroxyphenyl)(2-ethyl-6-fluoro-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-3-yl)methanone (62)

(61) ##STR00021##

(62) Step A: 2-amino-5-fluoropyridine (750 mg, 6.69 mmol) and compound 6 (1.65 g, 8.00 mmol) were dissolved in THF (15 mL), then, iodophthalic acid (2.59 g, 8.05 mmol) and boron trifluoride etherate (192 mg, 1.35 mmol)) were added sequentially in the ice water bath. After the addition was completed, stirring was continued at room temperature overnight. Water (30 mL) was added, and the pH value was adjusted to 7-8 with a saturated sodium bicarbonate solution and then the mixture was extracted with ethyl acetate (30 mL×3). The combined organic layers was washed with brine (20 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate:petroleum ether:dichloromethane=1:30:1 to 1:6:1) to give (6-fluoro-2-ethylimidazo[1,2-a]pyridin-3-yl)-(4-methoxyphenyl)methanone (59) (390 mg). The yield was 19.5%.

(63) Experimental procedures of Steps B, C and D were carried out according to the preparation methods of Steps C, D and E in Example 1 to give (3,5-dibromo-4-hydroxyphenyl)(2-ethyl-6-fluoro-5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-3-yl) methanone (62). .sup.1H NMR (DMSO-d.sub.6, 400 MHz) δ 7.80 (s, 2H), 4.98-4.96 (m, 1H), 4.56-4.51 (m, 1H), 4.42-4.37 (m, 1H), 3.01 (t, J=6.4 Hz, 2H), 2.33-2.21 (m, 4H), 1.06 (t, J=7.2 Hz, 3H). MS (EI, m/z): 433.0 [M+H].sup.+.

Example 13

Synthesis of 3-bromo-5-((2-ethyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-3-yl)hydroxymethyl)-2-hydroxyl benzonitrile (63)

(64) ##STR00022##

(65) Sodium borohydride (90 mg, 2.4 mmol) was added to a solution of compound 17 (90 mg, 0.24 mmol) in anhydrous THF (7 mL), stirred at room temperature for 1 hour, and the pH value was adjusted to 5-6 with 2 M citric acid solution after adding water (10 mL). The mixture was extracted with ethyl acetate (15 mL×2) and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (200-300 mesh silica gel, ethyl acetate:petroleum ether=1:2 to 5:1) to give 3-bromo-5-((2-ethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-3-yl)hydroxymethyl)-2-hydroxybenzonitrile (63) (8 mg). The yield was 8.89%. MS (EI, m/z): 376.10 [M+H].sup.+.

Example 14

Inhibition Assay of Uric Acid Transport for Compounds in HEK293-hURAT1 Transfection Cell Line

(66) I. Materials

(67) Zurampic was purchased from Chengdu Yichao Pharmaceutical Technology Co., Ltd. The plasmid pCMV6-hURAT1 was purchased from Origene Technologies, Inc. Geneticin (G418) was purchased from Sangon Biotech Co., Ltd. HEK293 cell line was purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences. Poly-lysine was purchased from Sigma-Aldrich Co. LLC. .sup.14C-uric acid was purchased from American Radiolabeled Chemicals, Inc. Sodium gluconate, potassium gluconate, calcium gluconate, KH.sub.2PO.sub.4, MgSO.sub.4, glucose and HEPES were purchased from Sinopharm Chemical Reagent Co., Ltd. DMEM culture medium and fetal bovine serum were purchased from Thermo Fisher Scientific Inc.

(68) II. Experimental Methods and Results

(69) 1. Construction of a HEK293 stable cell line with high expression of hURAT1:

(70) The plasmid pCMV6-hURAT1 was transfected into HEK293 cells, then the stable strain was obtained by the G418 (final concentration of 500 μg/mL) resistance screening, which is the high expression of hURAT1 transporter membrane protein. It can be used for in vitro inhibition assay of uric acid transporter hURAT1 (Weaver Y M, Ehresman D J, Butenhoff J L, et al. Roles of rat renal organic anion transporters in transporting perfluorinated carboxylates with different chain lengths. Toxicological Sciences, 2009, 113(2):305-314).

(71) 2. Coating 24-well plate: to a coated 24-well plate was added 200 μl of 0.1 mg/mL poly-lysine per well and the plate was left overnight. Poly-lysine was removed from wells. The wells were cleaned thoroughly with steriled water and dried for use.

(72) 3. To the above coated 24-well plate was added HEK293-hURAT1 stable cells (2×10.sup.5 cells per well). The cells were cultured at 37° C. under 5% CO.sub.2 for 3 days.

(73) 4. Preparation of HBSS buffer: the following reagents were weighed according to the final concentration of 125 mM sodium gluconate, 4.8 mM potassium gluconate, 1.3 mM calcium gluconate, 1.2 mM KH.sub.2PO.sub.4, 1.2 mM MgSO.sub.4, 5.6 mM glucose and 25 mM HEPES. Deionized water was added to reach the corresponding volume, and the solution was fully mixed to give HBSS (pH value: 7.4). The buffer was stored at −20° C. in a refrigerator.

(74) 5. At the day of experiment, the HBSS buffer was taken out of the refrigerator and warmed to 37° C. in a water bath. Taken out the 24-well plate with HEK293-hURAT1 stable cells, removed the culture medium and washed cells with HBSS, then add 160 μL of HBSS and 20 μL test compound per well. The final concentration of tested compound per well is 500 nM. The blank control well contains only 180 μL of HBSS without tested compound. The plate was placed at room temperature for 10 minutes.

(75) 6. To each well was added 20 μL of 50 μM .sup.14C-uric acid. The 24-well plate was placed at room temperature for 20 minutes.

(76) 7. The solution in each well was removed and the cells in each well were washed with the pre-cooled HBSS buffer. To each well was added 0.2 M NaOH to dissolve the cells. The solution containing cell fragments was collected and the appropriate amount of scintillation liquid was added. The radioisotope intensity of the .sup.14C-Uric acid (CPM value) was then detected by using PerkinElmer MicroBeta Trilux 1450 liquid scintillation analyzer.

(77) 8. In HEK293 transfected cell lines, the formula for calculating the inhabitation rate of uric acid transport for compounds was shown as below (Table 1), the CPM value of the tested compounds was represented by CPM.sub.(tested compound) and the CPM value of the blank control was represented by CPM.sub.(blank control). All tests were repeated three times, and the results were averaged and the standard deviation (SD) was calculated:
Inhibition rate(%).sub.(500 nM compound concentration)=(CPM.sub.(blank control)−CPM.sub.(test compound))/CPM.sub.(blank control)×100%

(78) III. Experimental Results

(79) The results showed that in comparison with the control drug Zurampic at the concentration of 500 nM, the compounds of the invention (in particular, 5, 10, 17, 23, 25, 41, 42, 43 and 48) have very good inhibitory effects of uric acid transport in HEK293-hURAT1 transfection cell line.

(80) TABLE-US-00001 TABLE 1 Inhibition rates of uric acid transport for test compounds and Zurampic in HEK293-hURAT1 transfection cell line Compound number Inhibition rate of uric acid transport, ±SD (%) or drug (compound concentraton: 500 nM) Zurampic 27.59 ± 2.89 5 53.43 ± 4.54 10 47.53 ± 3.12 17 47.46 ± 0.14 23 47.54 ± 1.65 25 47.58 ± 4.12 32 43.39 ± 2.40 41 46.24 ± 6.18 42 47.65 ± 3.08 43 53.17 ± 9.36 48 46.11 ± 4.91 58 38.62 ± 6.30 63 42.32 ± 2.60