THIAZOLE-5-CARBOXYLIC ACID DERIVATIVE AND PREPARATION METHOD AND USE THEREOF

Abstract

The present disclosure relates to a thiazole-5-carboxylic acid derivative represented by Formula (I), a stereoisomer and/or pharmaceutically acceptable salt thereof. The compound of Formula (I), the stereoisomer and/or pharmaceutically acceptable salt thereof of the present disclosure can be used to prepare a medicament for preventing or treating hyperuricemia and/or gout, and can be prepared into dosage forms for various administration routes. The compounds provided in the present disclosure have good tolerance, safety and excellent uric acid-lowering activity.

##STR00001##

Claims

1. A thiazole-5-carboxylic acid derivative represented by Formula (I): ##STR00040## wherein: X is selected from oxygen or nitrogen; both Y and Z are carbon; each of R.sub.1, R.sub.2, R.sub.3, R.sub.4 is independently selected from a hydrogen atom, halogen, oxo, and substituted or unsubstituted amino, alkylamino, aldehyde, alkyl, aminoalkyl, hydroxylalkyl, hydroxyl, alkoxyl, alkylcarbonyloxy, carboxyl, alkylcarbonyl or alkyloxycarbonyl; provided that not all R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are hydrogen atoms; or one or more of R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are optionally, together with one or more atoms of X, Y and Z, form a saturated five-membered ring structure, a saturated six-membered ring structure or a derivative structure thereof; provided that a saturated six-membered ring structure composed of R.sub.3 or R.sub.4, together with X, Y, and Z, is excluded, and, a stereoisomer and/or pharmaceutically acceptable salt thereof.

2. The thiazole-5-carboxylic acid derivative according to claim 1, wherein each of one or more hydrogen atoms of the above R.sub.1, R.sub.2, R.sub.3 and R.sub.4 groups is independently substituted by halogen, hydroxyl, alkoxyl, alkylcarbonyloxy, aldehyde, carboxyl, alkylcarbonyl, alkyloxycarbonyl, alkylaminoalkyloxycarbonyl, amino, alkylamino, alkyl, hydroxyalkyl, carboxyalkyl, aminoalkyl or alkylaminoalkyl.

3. The thiazole-5-carboxylic acid derivative according to claim 2, wherein each of the R.sub.1, R.sub.2, R.sub.3 and R.sub.4 is independently selected from a hydrogen atom, halogen, hydroxyl, oxo, aldehyde, carboxyl, amino, alkyl, halogenated alkyl, aminoalkyl, aminoalkylamino, alkoxyl, alkyloxyalkyl, alkyloxyalkyloxy, alkylcarbonyl, alkylcarbonyloxy, alkyloxycarbonyl, alkylamino, alkylaminoalkyl, hydroxylalkylamino, hydroxylalkylamino alkyl, alkylaminoalkyloxycarbonyl, hydroxylalkyl, hydroxylalkyloxy, carboxylalkyl or aminoalkyl.

4. The thiazole-5-carboxylic acid derivative according to claim 3, wherein the alkyl is C.sub.1-C.sub.4 alkyl.

5. The thiazole-5-carboxylic acid derivative according to claim 1, wherein the Y and Z, together with R.sub.1 or R.sub.3, and R.sub.2 or R.sub.4, form a saturated six-membered ring structure or a derivative structure thereof.

6. The thiazole-5-carboxylic acid derivative according to claim 1, wherein the X, Y, and Z, together with R.sub.2 or R.sub.4, form a saturated five-membered ring structure or a derivative structure thereof.

7. The thiazole-5-carboxylic acid derivative according to claim 1, wherein the saturated six-membered ring structure or derivative structure thereof is a pyranose ring or a derivative structure thereof; and the saturated five-membered ring structure or derivative structure thereof is a pyrrole ring or a derivative structure thereof.

8. The thiazole-5-carboxylic acid derivative according to claim 1, wherein the compound represented by Formula (I) is selected from one or more of the following structures: ##STR00041## ##STR00042##

9. A method for preparing the thiazole-5-carboxylic acid derivative represented by Formula (I): ##STR00043## comprising using 2-[3-cyano-4-isobutoxyphenyl]-4-methylthiazole-5-carboxylic acid as a raw material, and adopting a compound containing a group ##STR00044## to perform a substitution reaction of carboxylhydroxyl, so as to obtain the thiazole-5-carboxylic acid derivative; wherein, X is selected from oxygen or nitrogen; both Y and Z are carbon; each of R.sub.1, R.sub.2, R.sub.3, R.sub.4 is independently selected from a hydrogen atom, halogen, oxo, and substituted or unsubstituted amino, alkylamino, aldehyde, alkyl, aminoalkyl, hydroxylalkyl, hydroxyl, alkoxyl, alkylcarbonyloxy, carboxyl, alkylcarbonyl or alkyloxycarbonyl; provided that not all R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are hydrogen atoms; or one or more of R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are optionally, together with one or more atoms of X, Y and Z, form a saturated five-membered ring structure, a saturated six-membered ring structure or a derivative structure thereof; provided that a saturated six-membered ring structure composed of R.sub.3 or R.sub.4, together with X, Y, and Z, is excluded.

10. A method for preventing or treating hyperuricemia and/or gout, comprising administrating an therapeutically effective amount of the thiazole-5-carboxylic acid derivative represented by Formula (I): ##STR00045## wherein: X is selected from oxygen or nitrogen; both Y and Z are carbon; each of R.sub.1, R.sub.2, R.sub.3, R.sub.4 is independently selected from a hydrogen atom, halogen, oxo, and substituted or unsubstituted amino, alkylamino, aldehyde, alkyl, aminoalkyl, hydroxylalkyl, hydroxyl, alkoxyl, alkylcarbonyloxy, carboxyl, alkylcarbonyl or alkyloxycarbonyl; provided that not all R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are hydrogen atoms; or one or more of R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are optionally, together with one or more atoms of X, Y and Z, form a saturated five-membered ring structure, a saturated six-membered ring structure or a derivative structure thereof; provided that a saturated six-membered ring structure composed of R.sub.3 or R.sub.4, together with X, Y, and Z, is excluded, and a stereoisomer and/or pharmaceutically acceptable salt thereof, to a subject in need thereof.

11. The method according to claim 10, wherein each of one or more hydrogen atoms of the above R.sub.1, R.sub.2, R.sub.3 and R.sub.4 groups is independently substituted by halogen, hydroxyl, alkoxyl, alkylcarbonyloxy, aldehyde, carboxyl, alkylcarbonyl, alkyloxycarbonyl, alkylaminoalkyloxycarbonyl, amino, alkylamino, alkyl, hydroxyalkyl, carboxyalkyl, aminoalkyl or alkylaminoalkyl.

12. The method according to claim 11, wherein each of the R.sub.1, R.sub.2, R.sub.3 and R.sub.4 is independently selected from a hydrogen atom, halogen, hydroxyl, oxo, aldehyde, carboxyl, amino, alkyl, halogenated alkyl, aminoalkyl, aminoalkylamino, alkoxyl, alkyloxyalkyl, alkyloxyalkyloxy, alkylcarbonyl, alkylcarbonyloxy, alkyloxycarbonyl, alkylamino, alkylaminoalkyl, hydroxylalkylamino, hydroxylalkylaminoalkyl, alkylaminoalkyloxycarbonyl, hydroxylalkyl, hydroxylalkyloxy, carboxylalkyl or aminoalkyl.

13. The method according to claim 12, wherein the alkyl is C.sub.1-C.sub.4 alkyl.

14. The method according to claim 10, wherein the Y and Z, together with R.sub.1 or R.sub.3, and R.sub.2 or R.sub.4, form a saturated six-membered ring structure or a derivative structure thereof.

15. The method according to claim 10, wherein the X, Y, and Z, together with R.sub.2 or R.sub.4, form a saturated five-membered ring structure or a derivative structure thereof.

16. The method according to claim 10, wherein the saturated six-membered ring structure or derivative structure thereof is a pyranose ring or a derivative structure thereof; and the saturated five-membered ring structure or derivative structure thereof is a pyrrole ring or a derivative structure thereof.

17. The method according to claim 10, wherein the compound represented by Formula (I) is selected from one or more of the following structures: ##STR00046## ##STR00047##

Description

DETAILED DESCRIPTION

[0050] The following examples are used to illustrate the present disclosure, but not to limit the scope of the present disclosure.

[0051] The raw materials involved in the following examples are all commercially available. Hereinafter, raw material 2-[3-cyano-4-isobutoxyphenyl]-4-methylthiazole-5-carboxylic acid will be referred to as compound F.

Example 1: Preparation of F-1A

[0052] ##STR00028##

[0053] Weigh 1 g of compound F into a round bottom flask, add 100 mL of dichloromethane, and stir for 2 min to obtain an insoluble suspension. Then, add 386 mg of DMAP (4-dimethylaminopyridine), 1.3 g of DCC (dicyclohexylcarbodiimide), 3.5 mL of ethylene glycol successively. After reacting at room temperature for 24 hours, the resulted white solid was removed by filtration. The filtrate was washed with water and saturated brine, dried over anhydrous sodium sulfate and concentrated to obtain a crude product, which was purified by column chromatography to obtain 640 mg of solid, with yield of 56.1% and HPLC purity of 96.2%.

[0054] The structure of the resulted compound F-1A was characterized by the followings:

[0055] .sup.1H-NMR (400 MHz, d.sup.6DMSO): 8.17 (s, 1H), 8.08-8.11 (d, 1H), 6.99-7.02 (d, 1H), 5.34 (s, 1H), 4.43-4.45 (t, 2H), 3.94-3.96 (d, 2H), 3.89-3.90 (t, 2H), 2.77 (s, 3H), 2.15-2.21 (m, 1H), 1.07-1.09 (d, 6H);

[0056] ESI-MS: 361.1 (M+1).

Example 2: Preparation of F-1B

[0057] ##STR00029##

[0058] Weigh 1 g of compound F into a round bottom flask, add 100 mL of dichloromethane, stir for 2 min to obtain an insoluble suspension. Then, add 386 mg of DMAP (4-dimethylaminopyridine), 1.3 g of DCC (dicyclohexylcarbodiimide) and 5 mL of glycerol successively, react at room temperature, TLC monitor. After the reaction was completed, the resulted white solid was removed by filtration. The filtrate was washed with water and saturated brine, dried over anhydrous sodium sulfate and concentrated to obtain a crude product, purified by column chromatography to obtain 510 mg of solid, with yield of 43.0% and HPLC purity of 98.4%.

[0059] The structure of the resulted compound F-1B was characterized by the followings:

[0060] .sup.1H-NMR (400 MHz, d.sup.6DMSO): 8.15 (s, 1H), 8.06-8.12 (d, 1H), 6.98-7.01 (d, 1H), 5.02 (s, 2H), 4.48-4.51 (m, 1H), 4.26-4.29 (m, 1H), 3.96-3.98 (d, 2H), 3.80-3.87 (m, 1H), 3.54-3.57 (m, 2H), 2.75 (s, 3H), 2.17-2.23 (m, 1H), 1.07-1.09 (d, 6H).

[0061] ESI-MS: 391.1 (M+1).

Example 3: Preparation of F-1C

[0062] ##STR00030##

[0063] Weigh 20 g of compound F into a round bottom flask, add 200 mL of dichloromethane, 1 drop of DMF (N,N-dimethylformamide), stir for 2 min to obtain an insoluble suspension. Then, add 15.8 ml of oxalyl chloride dropwise in an ice bath, react at 25 C. After 4.5 hours, the reaction system was pale yellow, and the solvent was evaporated to obtain a pale yellow solid. Replace air in the reaction flask with nitrogen for 5 minutes, then add 300 mL of anhydrous dichloromethane. Replace air with nitrogen again, then add 15 ml of acetaldehyde and 32 ml of 1 mol/L zinc chloride solution dropwise in an ice bath. As the addition of zinc chloride, floccule began to appear in the system. After the completion of the addition, the reaction was continued for additional 5 hours at room temperature and then finished. The reaction system was washed repeatedly with 5% sodium bicarbonate solution, and a large amount of floccule appeared, and was filtered, washed with water and saturated brine, dried, concentrated, purified to obtain 21.1 g of colorless solid, with yield of 88.3% and HPLC purity of 98.5%.

[0064] The structure of the resulted compound F-1C was characterized by the followings:

[0065] .sup.1H-NMR (400 MHz, d6DMSO): 8.19 (s, 1H), 8.09-8.12 (dd, 1H), 7.0-7.03 (d, 1H), 6.69-6.74 (d, 1H), 3.89-3.90 (d, 2H), 2.78 (s, 3H), 2.17-2.25 (m, 1H), 1.90-1.91 (d, 3H), 1.07-1.09 (d, 6H).

[0066] ESI-MS: m/z 378.9, 380.9 (M+1).

Example 4: Preparation of Compound F-2A

[0067] ##STR00031##

[0068] Weigh 3.16 g of compound F into a round bottom flask, add 25 mL of dimethylformamide, 0.75 g of glycine, 5.7 g of HATU [2-(7-azabenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate] and 1 ml of DIPA (diisopropylaniline), stir the reaction at room temperature, TLC monitor the reaction. After the reaction was completed, reduce pressure and evaporate the solvent, then add 100 ml of ethyl acetate and 1 ml of glacial acetic acid to the system, shake repeatedly. Then, the system was washed with saturated brine, purified by column chromatography to obtain 2.29 g product, with yield of 61.3% and HPLC purity of 98.9%.

[0069] The structure of the resulted compound F-2A was characterized by the followings: .sup.1H-NMR (400 MHz, d.sup.6DMSO): 12.78 (broad, 1H), 8.55-8.57 (d, 1H), 8.27 (s, 1H), 8.20-8.23 (d, 1H), 7.39-7.41 (d, 1H), 4.03-4.04 (d, 2H), 3.89-3.90 (d, 2H), 2.76 (s, 3H), 2.20-2.26 (m, 1H), 1.11-1.13 (d, 6H).

[0070] ESI-MS: m/z 372.1 (M-1).

Example 5: Preparation of Compound F-2B

[0071] ##STR00032##

[0072] Weigh 3.16 g of compound F into a round bottom flask, add 25 mL of dimethylformamide, 0.89 g of glycine, 5.7 g HATU and 1.5 ml of DIPA, stir at room temperature, TLC monitor the reaction. After the reaction was completed, reduce pressure and evaporate the solvent, then add 100 ml of ethyl acetate and 1 ml of glacial acetic acid to the system, and shake repeatedly. Then, the system was washed with saturated brine, purified by column chromatography to obtain 2.21 g product, with yield of 57.1% and HPLC purity of 98.7%.

[0073] The structure of the resulted compound F-2B was characterized by the followings:

[0074] .sup.1H-NMR (400 MHz, d.sup.6DMSO): 12.71 (broad, 1H), 8.53-8.55 (d, 1H), 8.27 (s, 1H), 8.19-8.22 (d, 1H), 7.37-7.40 (d, 1H), 4.64-4.66 (m, 1H), 4.01-4.02 (d, 2H), 2.79 (s, 3H), 2.19-2.25 (m, 1H), 1.44 (d, 3H), 1.11-1.13 (d, 6H).

[0075] ESI-MS: m/z 386.1 (M-1).

Example 6: Preparation of Compound F-2C

[0076] ##STR00033##

[0077] Weigh 3.16 g of compound F into a round bottom flask, add 25 mL of dimethylformamide, 1.33 g of aspartic acid, 5.7 g of HATU and 3 ml of DIPA, stir at room temperature, TLC monitor the reaction. After the reaction was completed, add 200 ml of ethyl acetate to the system, then add 4 ml of glacial acetic acid, and shake repeatedly. Then, the system was washed with saturated brine, purified by column chromatography to obtain 2.08 g, with yield of 48.2% and HPLC purity of 98.6%.

[0078] The structure of the resulted compound F-2C was characterized by the followings:

[0079] .sup.1H-NMR (400 MHz, d.sup.6DMSO): 12.74 (broad, 2H), 8.56-8.58 (d, 1H), 8.28 (s, 1H), 8.20-8.23 (d, 1H), 7.39-7.41 (d, 1H), 4.69-4.74 (m, 1H), 4.02-4.03 (d, 2H), 2.61-2.74 (m, 2H), 2.52 (s, 3H), 2.08-2.14 (m, 1H), 1.03-1.04 (d, 6H).

[0080] ESI-MS: m/z 430.1, 431.1 (M-1).

Example 7: Preparation of Compound F-2D

[0081] ##STR00034##

[0082] According to the above reactive equation, and referring to the method described in Example 6, 3.13 g of white powdery solid was obtained, with yield of 75.8% and HPLC purity of 97.6%.

[0083] The structure of the resulted compound F-2D was characterized by the followings:

[0084] .sup.1H-NMR (400 MHz, d.sup.6DMSO): 12.68 (broad, 1H), 8.28 (s, 1H), 8.20-8.23 (d, 1H), 7.39-7.41 (d, 1H), 4.67-4.71 (m, 1H), 4.0-4.01 (d, 2H), 2.71 (s, 3H), 2.01-2.08 (m, 1H), 1.87-1.90 (m, 1H), 1.67-1.70 (m, 1H), 1.63-1.66 (m, 2H), 1.43-1.46 (m, 2H), 1.0-1.01 (d, 6H).

[0085] ESI-MS: m/z 412.1 (M-1).

Example 8: Preparation of Compound F-3A

[0086] ##STR00035##

[0087] According to the above reactive equation and referring to the method described in Example 6, 2.84 g of white powdery solid F3A was obtained, with yield of 68.1%. Then, the white solid was dissolved in 15 ml of isopropanol under reflux, and then 1 ml of concentrated hydrochloric acid was added dropwise, stirred for 10 minutes, and stand overnight at room temperature to precipitate white solid. The resulted white solid was filtered, and washed with cold isopropanol to obtain 1.58 g product, with HPLC purity of 99.1%.

[0088] The structure of the resulted compound F-3A was characterized by the followings:

[0089] .sup.1H-NMR (400 MHz, d.sup.6DMSO): 10.26 (broad, 1H), 8.29-8.30 (d, 1H), 8.21-8.24 (s, 1H), 7.36-7.38 (m, 1H), 5.37 ((broad, 1H), 4.60 ((broad, 2H), 3.97-3.99 (d, 2H), 3.75-3.76 (d, 2H), 3.49-3.58 (broad, 2H), 3.20 (broad, 2H), 2.84 (s, 3H), 2.67 (s, 3H), 2.02-2.11 (m, 1H), 0.97-0.99 (s, 6H).

[0090] ESI-MS: m/z 418.2 (free base molecular weight M+1), 834.9 (2M+1).

Example 9: Preparation of Compound F-3B

[0091] ##STR00036##

[0092] According to the above equation and referring to the method described in Example 6, 2.91 g of white powdery solid F3B was obtained, with yield of 65.2% and HPLC purity of 98.7%.

[0093] The structure of the resulted compound F-3B was characterized by the followings:

[0094] .sup.1H-NMR (400 MHz, d.sup.6DMSO): 10.19 (broad, 1H), 8.23-8.24 (d, 1H), 8.17-8.20 (s, 1H), 7.30-7.32 (m, 1H), 5.30 ((broad, 1H), 4.52 ((broad, 2H), 3.91-3.93 (d, 2H), 3.70-3.71 (d, 2H), 3.42-3.51 (broad, 2H), 3.15 (broad, 2H), 2.80 (s, 3H), 2.61 (s, 3H), 1.98-2.07 (m, 1H), 1.80-1.84 (m, 2H), 1.51-1.54 (m, 2H), 0.96-0.98 (s, 6H).

[0095] ESI-MS: m/z 446.2 (free base molecular weight +1).

Example 10: Preparation of Compounds F-4A, F-4B

[0096] ##STR00037##

[0097] Weigh 3.16 g of compound F into a round bottom flask, add 15 mL of dimethylformamide and 15 ml of dimethylsulfoxide, 2.16 g of glucosamine hydrochloride, 5.7 g of HATU and 3 ml of DIPA, stir the reaction at room temperature for 24 hours, and then add 60 ml of ethyl ether to the system. The precipitated solid was purified by column chromatography to obtain 2.15 g product, with yield of 45.2% and HPLC purity of 98.1%.

[0098] The structure and characterization information of the resulted compound F-4A were as follows:

##STR00038##

[0099] .sup.1H-NMR (400 MHz, d.sup.6DMSO): 8.26-8.28 (d, 1H), 8.20-8.23 (dd, 1H), 7.88-8.10 (m, 1H), 7.40-7.42 (d, 1H), 6.60-6.70 (m, 1H), 5.11-5.13 (t, 1H), 5.01-5.05 (m, 1H), 4.87-4.88 (d, 1H), 4.59-4.64 (m, 1H), 4.49-4.52 (t, 1H), 4.03-4.04 (d, 2H), 3.36-3.75 (m, 5H), 2.56 (s, 3H), 2.04-2.15 (m, 1H), 1.09-1.10 (d, 6H).

[0100] ESI-MS: m/z:478.1 (M+1).

[0101] The structure and characterization information of tautomer F-4B of compound F-4A were as follows:

##STR00039##

[0102] .sup.1H-NMR (400 MHz, d.sup.6DMSO+D.sub.2O): 8.17 (s, 1H), 8.11-8.13 (d, 1H), 7.27-7.29 (d, 1H), 5.11-5.13 (d, 0.67H), 4.63-4.65 (d, 034H), 3.94-3.95 (d, 2H), 3.23-3.84 (m, 6H), 2.53 (s, 3H), 2.03-2.06 (m, 1H), 0.97-0.98 (d, 6H).

[0103] ESI-MS: m/z:478.1 (M+1).

Example 11

[0104] In order to verify the tolerance and safety of the compounds of the present disclosure, this example provides an oral acute toxicity experiment, and uses existing drugs for treating gout or lowering uric acid for a comparative study.

[0105] 1. Test Samples

[0106] Test samples: F-1A, F-1B, F-1C, F-2A, F-2B, F-2C, F-2D, F-3A, F-3B, F-4A prepared by the above examples 1-10, and febuxostat.

[0107] Formulation method: take each test sample and sodium carboxymethyl cellulose, grind to obtain 0.2% of sodium carboxymethyl cellulose suspension.

[0108] 2. Test anminals: ICR mice; body weight: 18-22 g.

[0109] 3. Dose setting:

[0110] Preliminary experiments were firstly conducted. The mice were intragastric administrated with each test sample at a dose of 1600 mg/kg, and toxic reactions were observed. Preliminary experiments showed that compounds F-1A, F-1B, F-1C, F-2A, F-2B, F-2C, F-2D, F-3A, F-3B, F-4A had less toxicity, and no obvious toxicity symptom was observed in the mice at a dose of 1600 mg/kg, and no animal died. Preliminary experiments showed that febuxotan had a certain toxicity, and caused death in several mice at a dose of 1600 mg/kg.

[0111] On the basis of the preliminary experiments, the dose of each test sample for formal test was set, as shown in table 1.

TABLE-US-00001 TABLE 1 Dose set for each test sample in toxicity test Dose (mg/kg) [Formulation Administration concentration volume Test sample (mg/ml)] (ml/kg) Remarks Febuxostat 800, 640, 512, 20 Determining 409.6, 327.7 LD.sub.50 or [40.0, 32.0, 25.6, maximum 20.48, 16.38] F-1A 3000[200*] 25 administration F-1B 3000[200*] 25 dose (mg/kg) F-1C 3000[200*] 25 F-2A 3000[200*] 25 F-2B 3000[200*] 25 F-2C 3000[200*] 25 F-2D 3000[200*] 25 F-3A 3000[200*] 25 F-3B 3000[200*] 25 F-4A 3000[200*] 25 * Maximum formulation concentration.

[0112] 4. Administration route: intragastric (ig) administration.

[0113] 5. Experiment methods:

[0114] Laboratory environment: room temperature 242 C., relative humidity 60-70%.

[0115] Observation indexes: According to the above doses and the administration volumes, each test sample was formulated into a solution having the corresponding concentration through geometric proportion dilution, and was ig administrated once with the same volume. Various toxic symptoms and death of the mice were recorded, and dead animals were examined by autopsy.

[0116] Observation period: 14 days.

[0117] 6. Test results

[0118] 6.1 Abnormal reactions: Within 12 hours from ig administration of F-1A, F-1B, F-1C, F-2A, F-2B, F-2C, F-2D, F-3A, F-3B, F-4A and febuxostat, only several animals from the high-dose group of febuxostat showed reduced activity, and no abnormalities were observed in the other groups.

[0119] Within 24 hours from the administration, no animal deaths were observed in all dose groups of F-1A, F-1B, F-1C, F-2A, F-2B, F-2C, F-2D, F-3A, F-3B, F-4A, and several animals of the high-dose group of febuxotan died. At Day 8 after the administration, no death was observed for the survival animals of all the groups. The survival animals only showed reduced activity and emaciation, and no other obvious abnormalities were observed.

[0120] 6.2 Autopsy results: The autopsy of dead animals of the high-dose group showed bilateral kidneys with pale color and urinary retention. No obvious abnormalities were observed in other organs. The autopsy of the survival animals showed that all the organs were normal. No obvious abnormalities were observed in the organs of all the survival animals of compounds F-1A, F-1B, F-1C, F-2A, F-2B, F-2C, F-2D, F-3A, F-3B, F-4A test groups.

[0121] 6.3 Cause of death: After administration of febuxostat, the mice might die due to its toxicity to the urinary system, which eventually lead to systemic failure.

[0122] 6.4 Table 2 shows the death and LD.sub.50 values of ig administration of the test samples in mice.

TABLE-US-00002 TABLE 2 LD.sub.50 values of ig administration of the test samples in mice (calculated by Bliss method) Total Number LD.sub.50/ Dose number of of dead Maximum dose Compound (mg/kg) animals animals (mg/kg) Febuxostat 800 10 6 701.29 47.6 640 10 3 512 10 1 409.6 10 0 327.7 10 0 F-1A 3000 10 0 3000 F-1B 3000 10 0 3000 F-1C 3000 10 0 3000 F-2A 3000 10 0 3000 F-2B 3000 10 0 3000 F-2C 3000 10 0 3000 F-2D 3000 10 0 3000 F-3A 3000 10 0 3000 F-3B 3000 10 0 3000 F-4A 3000 10 0 3000 Summary: Compounds F-1A, F-1B, F-1C, F-2A, F-2B, F-2C, F-2D, F-3A, F-3B, F-4A of the present disclosure are very well tolerated and safe, and the LD.sub.50 values are 3000 mg/kg or more, and thus are significantly superior to the existing drugs.

Example 12

[0123] In order to verify the pharmacological activities of the compounds of the present disclosure, this example provides a pharmacodynamic screening experiment based on a rat model, in which exogenous uric acid is administered and uric acid decomposition is inhibited simultaneously, and positive drugs are selected as control.

[0124] 1. Experimental animals: SD rats, free drinking and eating. Feed was sterilized by irradiation, and water was sterilized purified water. Adaptive feed for one week.

[0125] 2. Experimental drugs: F-1A, F-1B, F-1C, F-2A, F-2B, F-2C, F-2D, F-3A, F-3B and F-4A, 1.0 g for each compound; febuxostat 1.0 g; febuxostat ethyl ester (ethyl 2-[3-cyano-4-isobutoxyphenyl]-4-methylthiazole-5-carboxylate) 1.0 g.

[0126] 3. Drug formulation:

[0127] 3.1 Formulation of compounds F-1A, F-1B, F-1C, F-2A, F-2B, F-2C, F-2D, F-3A, F-3B, F-4A: For each compound, take 100 mg of the compound, add 20 ml of 0.5% sodium carboxymethyl cellulose and grind, and then dilute to 100 ml.

[0128] 3.2 Formulation of febuxostat and febuxostat ethyl ester: Take 140 mg of febuxostat or febuxostat ethyl ester, add 20 ml of 0.5% sodium carboxymethyl cellulose to grind, and then dilute to 100 ml respectively.

[0129] 4. Experimental reagents, drugs and instruments:

[0130] Oteracil potassium (Oxonic acid), Uric acid (Uric), sodium carboxymethyl cellulose, Uric acid (UA) assay kit. US Multiskan MK3 Microplate Reader. Thermo Labsystems Multiskan Ascent V1 Plate Reader, Finland.

[0131] 5. Grouping and administration

[0132] Except the blank group, the rest rats were modeled for 15 days, and then their serum uric acid levels were detected. The rats were equally divided into several groups according to the detected uric acid levels thereof.

[0133] Blank group: no drug administration, 10 animals

[0134] Model group: oral administration of 2 ml of 0.5% sodium carboxymethyl cellulose, QD, 10 animals.

[0135] Groups F-1A F-4A: each of 10 compounds was taken orally at 10 mg/kg, QD, and 10 animals per group.

[0136] Febuxostat group: oral administration of 14 mg/kg, QD, and 10 animals.

[0137] Febuxostat ethyl ester group: oral administration of 14 mg/kg, QD, and 10 animals.

[0138] 6. Experimental methods and operation processes.

[0139] The blank group was not administrated with any drug. Each of the rest groups was orally administrated with oteracil potassium 1.5 g/kg+uric acid 0.3 g/kg (dissolved in 0.5% sodium carboxymethyl cellulose), once a day on schedule, for 15 days. 0.5 ml of blood samples was drawn from orbit before the experiment and 13 days after the modelling, respectively, to separate serum and determine serum uric acid levels.

[0140] After successful modeling, the rats were equally divided into several groups according to the serum uric acid values thereof. After grouping, the rats were continued to be fed with the modeling drugs oteracil potassium 1.5 g/kg+uric acid 0.3 g/kg for additional two days. One hour after the last administration of the modelling drugs, the rats began to be intragastrically administrated with the test drugs. Three hours after the first administration, 0.5 ml of orbital venous blood was drawn from the orbit of the rats, centrifuged at 3000 RPM for 15 min, to obtain serum. The serum uric acid level of each rat was detected by using Uric acid (UA) assay kit, on Multiskan MK3 Microplate Reader (US), Thermo Labsystems Multiskan Ascent V1 Plate Reader (Finland).

[0141] 7. Statistical processing and experimental results.

[0142] Data were expressed by meanstandard deviation (xSD), and analyzed by Excel 7.0 and SPPS 13.0 for Windows software. Q-test was used for the comparison between groups, and a self-paired t-test was used for the comparison of a group before and after administration. It indicated significant difference when P<0.05. The mainly obtained experiment results are shown in table 3.

TABLE-US-00003 TABLE 3 Effects of the compounds of the present disclosure on serum uric acid level of hyperuricemia rats Administration dose Serum uric acid level (mol/L) (number of animals) Value after 13 days Value after 3 h Group (mg/kg) Normal value of modeling of drug administration Blank group (10) 45.1 4.0 50.6 8.7.sup. 53.9 12.8.sup. Model group (10) 47.0 7.5 157.9 22.7 .sup.c3 174.4 39.9.sup.c3 Febuxostat 14 (10) 45.6 7.6 153.1 22.0.sup.c3 86.6 17.5.sup.a3b3c3 Febuxostat ethyl ester 14 (10) 46.5 7.4 157.2 22.9.sup.c3 113.9 24.9.sup.a3b3c3 F-1A 10 (10) 47.4 6.6 156.1 27.0.sup.c3 71.0 24.4.sup.a3b3c1 F-1B 10 (10) 44.1 8.5 160.1 25.8 .sup.c3 67.5 19.4.sup.a3b3 F-1C 10 (10) 44.6 7.6 162.6 33.2.sup.c3 64.2 28.1.sup.a3b3 F-2A 10 (10) 43.8 8.4 163.0 25.5.sup.c3 78.3 25.1.sup.a3b3c2 F-2B 10 (10) 46.3 8.0 162.8 44.0.sup.c3 70.3 16.4.sup.a3b3c1 F-2C 10 (10) 45.4 6.7 158.9 31.0.sup.c3 68.6 19.2.sup.a3b3c1 F-2D 10 (10) 46.5 6.1 156.8 24.5.sup.c3 69.1 23.4.sup.a3b3c1 F-3A 10 (10) 45.9 5.7 160.4 27.7.sup.c3 64.1 16.1.sup.a3b3 F-3B 10 (10) 44.2 7.9 158.6 21.6.sup.c3 66.3 17.1.sup.a3b3 F-4A 10 (10) 45.5 7.3 160.8 40.5.sup.c3 71.4 19.6.sup.a3b3c1 Note: Administration dose is in mg/kg. Compared with the model group, a3: p < 0.001. Compared with 13 days of modeling, in each group, b3: p < 0.001. Compared with the blank group, c1: p < 0.05, c2: p < 0.01, c3: p < 0.001.

[0143] It can be seen from the results shown in table 3 that, in this experiment, the rat hyperuricemia model, in which exogenous uric acid was administered and uric acid decomposition was inhibited simultaneously, was successfully established. The rats were intragastrically administrated with a suspension of oteracil potassium 1.5 g/kg+uric acid 0.3 g/kg continuously for 15 days. There are significant differences in serum uric acid levels between the rats after and before the hyperuricemia modeling (p<0.001), indicating that the hyperuricemia model was successfully established.

[0144] Three hours after drug administration, the uric acid levels of the animals in each test drug group were significantly reduced as compared with those of the model group (a3, p<0.001), indicating that all the test drugs had uric acid-lowering effect. Nonetheless, there were still differences in specific effect of lowering uric acid among these groups. In particular, after the drug administration the uric acid levels of the animals administrated with compound F-1B, F-1C, F-3A or F-3B had no significant difference as compared with those of the blank group, and were very close to the normal value. Thus, compounds F-1B, F-1C, F-3A and F-3B have very obvious uric acid-lowering effects. The uric acid levels of the animals administrated with compounds F-1A, F-2B, F-2C, F-2D, F-4A were significantly different from those of the blank group (c1, p<0.05). The uric acid level of the group administrated with Compound F-2A was significantly different from the blank group (c2, p<0.01). It indicates that, though there was a certain difference between the uric acid level of the administrated animals with the normal value, its uric acid-lowering effect was still excellent. The positive control, febuxostat, had good effect on lowering uric acid. However, there was an extremely significant difference (c3,p<0.001) between the uric acid level of the group administrated with febuxostat and that of the blank group. It indicates that the uric acid levels of the animals administrated with febuxostat is quite different from the normal value, and the efficacy of febuxostat is inferior to the compounds of the present disclosure. Further, the efficacy of febuxostat ethyl ester, an intermediate of febuxostat, was inferior to febuxostat. This study indicates that the compounds of the present disclosure have excellent uric acid-lowering effect, which is better than the existing uric acid lowering drugs.

Example 13

[0145] In order to further study the pharmacological activity of the compounds of the present disclosure, as well as the relationship between the activity and the dose, this example selected several compounds of the present disclosure to conduct a dose-effect relationship study in a rat model, in which exogenous uric acid was administered and uric acid decomposition was inhibited simultaneously. A positive drug was used as a control.

[0146] 1. Experimental animals: Male SPF grade SD rats weighing 110-150 grams. The animals were fed adaptively for 1 week, and were observed for body surface signs. The animals were free to drink and eat. All cages were sterilized at 121 C., feed was sterilized by irradiation, and water was sterilized purified water.

[0147] 2. Experimental drugs: F-1C, F-2C, F-3A, 1.0 g for each compound; febuxostat (positive drug) 1.0 g; febuxostat ethyl ester (ethyl 2-[3-cyano-4-isobutoxyphenyl]-4-methylthiazole-5-carboxylate) 1.0 g;

[0148] 3. Drug formulation:

[0149] 3.1 Formulation of F-1C, F-2C, F-3A: Tor each of the compounds, take 100 mg, add 20 ml of 0.5% sodium carboxymethyl cellulose and grind, and then dilute to 100 ml.

[0150] 3.2 Take 100 mg of febuxostat, add 20 ml of 0.5% sodium carboxymethyl cellulose and grind, and then dilute to 100 ml.

[0151] 4. Reagents, drugs and instruments for testing:

[0152] Oteracil potassium (Oxonic acid), Uric acid (Uric), sodium carboxymethyl cellulose, Uric acid (UA) assay kit. Automatic Bio-Chemistry Analyzer: AU480 automatic Bio-Chemistry Analyzer, Olympus corporation, Japan.

[0153] 5. Grouping and administration:

[0154] Except the blank group, the rest rats were modeled for 15 days and then their serum uric acid levels were detected. The rats were equally divided into several groups according to the detected uric acid levels thereof.

[0155] Blank group: no drug administration, 10 animals

[0156] Model group: oral administration of 2 ml of 0.5% sodium carboxymethyl cellulose, QD, 10 animals.

[0157] F-1C, F-2C and F-3A groups: each compound was taken orally at 3 doses, i.e., 2.5, 5 and 10 mg/kg; QD; and 10 animals for each dose group.

[0158] Febuxostat group: oral administration of 10 mg/kg, QD, and 10 animals.

[0159] Febuxostat ethyl ester group: oral administration of 10 mg/kg, QD, and 10 animals.

[0160] 6. Experimental methods and operation processes:

[0161] The blank group was not administrated with any drug. Each of the rest groups was orally administrated with oteracil potassium 1 g/kg and uric acid 0.3 g/kg and intraperitoneally injected with 0.1 g/kg uric acid (dissolved in 0.5% sodium carboxymethyl cellulose), once a day on schedule, for 15 days. 0.5 ml of blood samples was drawn from orbit before the experiment and 13 days after the modelling, respectively, to separated serum for determining serum uric acid levels.

[0162] After successful modeling, the rats were equally divided into several groups according to the serum uric acid values thereof, and then were intragastrically administrated with the test drugs. Three hours after the first administration, 0.5 ml of orbital venous blood was drawn from the orbit of the rats, centrifuged at 3000 RPM for 15 min, to obtain for determining the serum uric acid level of each rat.

[0163] 7. Statistical processing and experimental results:

[0164] Data were expressed by meanstandard deviation (xSD), and analyzed by Excel 7.0 and SPPS 13.0 for Windows software. Q-test was used for the comparison between groups, and the self-paired t-test was used for the comparison of a group before and after administration. It indicated significant difference when P<0.05. The mainly obtained experiment results are shown in table 4.

TABLE-US-00004 TABLE 4 Effects of different doses of the compounds of the present disclosure on serum uric acid level in hyperuricemia rats Administration dose Serum uric acid content (mol/L) (number of animals) Value after 13 days Value after 3 h Group (mg/kg) Normal value of modeling of drug administration Blank group (10) 30.2 2.9 35.5 4.0.sup. 37.4 5.1.sup. Model group (10) 30.5 2.7 159.5 22.4.sup.a3 226.2 35.2.sup.a3 Febuxostat 10 (10) 31.7 1.9 159.1 30.9.sup.a3 140.8 33.5.sup.a3, b3 Febuxostat ethyl ester 10 (10) 30.9 1.5 163.2 29.8.sup.a3 177.3 38.6.sup.a3 F-1C 10 (10) 32.1 3.0 161.0 21.5.sup.a3 .sup.100.1 25.8.sup.a3, b3, c3 5 (10) 31.9 2.7 161.2 27.6.sup.a3 146.9 19.5.sup.a3, b3 2.5 (10) 31.5 3.3 160.5 34.9.sup.a3 189.8 25.3.sup.a3 F-2C 10 (10) 32.3 1.5 160.0 29.3.sup.a3 .sup.107.3 21.2.sup.a3, b3, c3 5 (10) 29.6 2.5 159.7 23.4.sup.a3 131.9 29.7.sup.a3, b3 2.5 (10) 31.6 2.6 161.3 26.8.sup.a3 172.7 33.9.sup.a3 F-3A 10 (10) 33.0 4.1 161.5 34.1.sup.a3 .sup.82.3 16.8.sup.a3, b3, c3 5 (10) 30.8 3.2 159.5 25.6.sup.a3 137.6 24.3.sup.a3, b3 2.5 (10) 30.6 2.9 159.1 21.0.sup.a3 173.4 32.7.sup.a3, b3 Note: Compared with the blank group, a3: p < 0.001; Compared with the model group, b3: p < 0.001; Compared with the febuxostat group, c3: p < 0.001.

[0165] It can be seen from the results shown in table 4 that after the rats were continuously intragastrically administrated with oteracil potassium 1 g/kg+uric acid 0.3 g/kg suspension, and intraperitoneally injected with 0.1 g/kg uric acid (dissolved in 0.5% sodium carboxymethyl cellulose) for 15 days, there are significant differences (p<0.001) in serum uric acid levels between the rats after and before the hyperuricemia modeling. In this experiment, the rat uricemia model, in which exogenous uric acid was administered and uric acid decomposition was inhibited simultaneously, was successfully established.

[0166] Three hours after the administration of the test drugs, the uric acid level of each test drug group was lower than that of the model group, indicating that each test drug had a certain effect on lowering uric acid. With comparing each dose group, the compounds of the present disclosure showed a good dose-dependent effect, the higher the dose, the better the effect of lowering uric acid will be. In addition, the uric acid-lowering effect of the compounds of the present disclosure, at the same dose, is obviously better than that of the positive drug (p<0.001).

[0167] The above examples are only for illustrating the preferred embodiments of the present disclosure, and are not intended to limit the scope of the present disclosure. Without departing from the spirit of the present disclosure, various modifications and improvements can be made by those skilled in the art to the technical solutions of the present disclosure, and all shall fall within the scope of protection defined by the claims of the present disclosure.