CN116102573B - A thienopyrimidine compound containing a thienylene structure and its preparation method and application - Google Patents
A thienopyrimidine compound containing a thienylene structure and its preparation method and applicationInfo
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Abstract
The invention relates to a thienopyrimidine compound containing a thienylmethylene structure, and a preparation method and application thereof. The compound has a structure shown in a formula I. The invention also relates to a preparation method of the compound containing the structure shown in the formula I and a pharmaceutical composition. The invention also provides application of the compound in preparing uric acid reducing medicines.
Description
Technical Field
The invention belongs to the technical field of synthesis and medical application of organic compounds. In particular to a thienopyrimidine compound containing a thienylmethylene structure, a preparation method thereof or a pharmaceutical composition containing the thienopyrimidine compound and the thienopyrimidine compound, and application thereof in medicine.
Background
Gout is one of the most common chronic diseases in inflammatory arthritis, and hyperuricemia is a direct cause of gout formation. The diagnosis standard of hyperuricemia in the Chinese gout diagnosis and treatment guide is that the hyperuricemia in 2 times of fasting blood is more than 0.42mmol/L (7.0 mg/dL) on different days, and the standard of asymptomatic hyperuricemia in the U.S. gout guide is that the hyperuricemia is more than 0.408mmol/L (6.8 mg/dL). Under long-term hyperuricemia, needle-like crystal deposition of sodium urate slowly occurs in soft tissues or joints, and the sodium urate crystal activates NLRP3 inflammatory bodies in macrophages and monocytes, thereby causing inflammatory reaction and gout. The gout treatment medicines are mainly divided into three types according to action mechanisms, namely (1) xanthine oxidase inhibitors for inhibiting uric acid generation, marketed medicines comprise allopurinol and febuxostat, (2) URAT1 inhibitors for promoting uric acid excretion, marketed medicines comprise benzbromarone, levocinard and probenecid, and (3) uric acid oxidase analogues for decomposing uric acid, and marketed medicines comprise polyethylene glycol recombinant uricase. Lesinurad (Lesinurad) is a novel URAT1 inhibitor approved by the FDA in 2015, and the single dose of the novel URAT1 inhibitor is 200-600mg, and the novel URAT1 inhibitor is finally used for treating hyperuricemia with gout by combining 200mg with xanthine oxidase inhibitor due to serious renal toxicity of the novel URAT1 inhibitor. 200mg daily combined with allopurinol, only 55% of patients reached a treatment endpoint of less than 0.36mmol/L, and it was said that the efficacy was very poor compared to 43% of allopurinol alone. Therefore, the novel anti-hyperuricemia and anti-gout medicine with better activity, higher safety and independent intellectual property can be hopefully obtained by carrying out structural modification on the medicine.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a thienopyrimidine compound containing a thienomethylene structure and a preparation method thereof, and also provides an activity screening result of the compound serving as a URAT1 inhibitor and application thereof.
The technical scheme of the invention is as follows:
1. containing thiophene methylene structures thienopyrimidines
Thienopyrimidine compounds containing a thiomethylene structure or pharmaceutically acceptable salts thereof are characterized by having a structure shown in the following general formula I:
Wherein Ar 1、Ar2 is thiophene or thiophene substituted with one or more R 2, R is C 1-6 alkylene or C 1-6 alkylene substituted with one or more R 3, said R 2 is selected from H, C 1-6 alkyl, C 1-6 alkoxy, halogenated C 1-6 alkyl, halogenated C 1-6 alkoxy, C 3-6 cycloalkyl, halogen, -OH, -COOH, SH or two R 2 together with the two carbon atoms to which they are attached form an aromatic ring of C 5-12 optionally substituted by R 4, said R 3、R4 being selected from H, C 1-6 alkyl, C 1-6 alkoxy, halogenated C 1-6 alkyl, halogenated C 1-6 alkoxy, C 3-6 cycloalkyl, halogen, -OH, -COOH, -SH.
Preferably, R is C 1-3 alkylene or C 1-3 alkylene substituted with one or more R 3, said R 2 is selected from H, C 1-3 alkyl, C 1-3 alkoxy, halo C 1-3 alkyl, halo C 1-3 alkoxy, C 3-4 cycloalkyl, halogen, -OH, -COOH, -SH or two R 2 together with the two carbon atoms to which they are attached form a benzene ring, thiophene, pyridine or furan optionally substituted with R 4, said R 3、R4 is selected from H, C 1-3 alkyl, C 1-3 alkoxy, halo C 1 -3 alkyl, halo C 1-3 alkoxy, C 3-4 cycloalkyl, halogen, -OH, -COOH, -SH.
Further, R is C 1-3 alkylene or C 1-3 alkylene substituted with one or more R 3, R 2 is selected from H, C 1-3 alkyl, C 1-3 alkoxy, halogenated C 1-3 alkyl, halogen or two R 2 groups together with the two carbon atoms to which they are attached form a benzene ring optionally substituted with R 4, and R 3、R4 is selected from H, C 1-3 alkyl, C 1 -3 alkoxy, halogenated C 1 -3 alkyl, halogenated C 1-3 alkoxy, halogen.
Preferably, the thienopyrimidines containing a thienylmethylene structure or a pharmaceutically acceptable salt thereof is selected from the following formulas I-1 to I-5:
Further, thienopyrimidines containing a thiophene methylene structure or pharmaceutically acceptable salts thereof, preferably are represented by the following formulas I-1 'to I-5':
Preferably, R is selected from methylene, cyclopropyl substituted methylene, cyclobutyl substituted methylene, methyl methylene, dimethyl methylene, R 2 is selected from H, methyl, ethyl, propyl, cyclopropyl, isopropyl, methoxy, ethoxy, trifluoromethyl, difluoromethyl, fluoromethyl, fluoro, chloro, bromo, R 4 is selected from H, methyl, ethyl, propyl, isopropyl, cyclopropyl, methoxy, ethoxy, trifluoromethyl, difluoromethyl, fluoromethyl, fluoro, chloro, bromo.
Further, ar 2 and methylene are connected in a mode of thiophen-2-yl or thiophen-3-yl, and substituent R 2 is one or more of methyl, ethyl, cyclopropyl or bromine.
As a preferred scheme, the thienopyrimidine compound containing a thienylmethylene structure or a pharmaceutically acceptable salt thereof is selected from the following compounds:
TABLE 1 structural formulas of Compounds 1-18
2. Preparation method of thienopyrimidine compound containing thiophene methylene structure
The preparation method of the thienopyrimidine compound containing a thienylmethylene structure comprises the following steps:
(1) Reacting aminothiophene formate or aminothiophene formate containing substituent groups serving as a raw material with methylene dichloride serving as a solvent and triethylamine serving as an acid binding agent with thiophosgene to generate isothiocyanate (M1);
(2) Tetrahydrofuran is used as a solvent, M1 reacts with thiophene methylamine or substituted thiophene methylamine under the action of triethylamine to generate a thiourea intermediate, and then cyclized under the action of potassium hydroxide to generate a thienopyrimidinone intermediate (M2);
(3) Using DMF as a solvent, and reacting M2 with 2-bromocarboxylic acid under the action of potassium carbonate to generate a target compound;
the reaction conditions are (i) thiophosgene, methylene dichloride, triethylamine and 0-30 ℃, (ii) a thiophene methylamine, tetrahydrofuran and triethylamine, and (b) methanol, potassium hydroxide and room temperature, and (iii) N, N-dimethylformamide, potassium carbonate, 2-bromocarboxylic acid and room temperature.
The room temperature refers to 20-30 ℃.
3. Application of thienopyrimidine compound containing thiophene methylene structure
The invention discloses a screening result of inhibition effect of thienopyrimidine compounds containing a thiomethylene structure on uric acid transporter hURAT1 and application of the screening result as uric acid reducing drugs. The activity screening experiment proves that the thienopyrimidine compound containing the thiophene methylene structure has a certain inhibition effect on uric acid transporter hURAT 1.
The compound (numbered 1-12) synthesized by the invention is subjected to inhibition screening (single concentration experiment) of uric acid transporter hURAT1 by taking Lesinurad as a positive control drug, and the result shows that the 12 compounds all show a certain inhibition effect on uric acid transporter hURAT1, wherein the inhibition activity of 10 compounds is superior to or equivalent to that of the positive control Lesinurad. The inhibition IC 50 test is carried out on the compounds 3 and 4 with stronger single concentration inhibition activity, and the inhibition IC 50 of the compounds 3 and 4 is 3.097 and 1.514 mu mol/L respectively, and the activity is obviously superior to that of a positive control Lesinurad (IC 50 =7.3 mu mol/L).
Therefore, the thienopyrimidine compound containing the thiophene methylene structure has novel structure and strong inhibition effect on uric acid transporter hURAT1, can be used as a candidate compound for preparing uric acid lowering drugs, and is applied to the treatment of gout and hyperuricemia.
A uric acid reducing pharmaceutical composition comprises the thienopyrimidine compound containing a thienylmethylene structure or pharmaceutically acceptable salts thereof and one or more pharmaceutically acceptable carriers or excipients.
Drawings
FIG. 1 inhibition of hURAT1 by Compounds 1-12. Mu. Mol/L
FIG. 2 inhibition of hURAT1 by Compound 3
FIG. 3 inhibition of hURAT1 by Compound 4
Detailed Description
Embodiments of the present application will be described below by way of examples, and it will be appreciated by those skilled in the art that these specific examples merely illustrate embodiments selected for the purpose of the present application and are not limiting. Modifications to the teachings of the present application, in combination with the prior art, are apparent from the teachings of the present application, and all fall within the scope of the present application.
Synthetic route of the compound:
the reaction conditions are (i) thiophosgene, methylene dichloride, triethylamine and 0-30 ℃, (ii) a thiophene methylamine, tetrahydrofuran and triethylamine, and (b) methanol, potassium hydroxide and room temperature, and (iii) N, N-dimethylformamide, potassium carbonate, 2-bromocarboxylic acid and room temperature.
EXAMPLE 1 preparation of Compounds 1-M1
Methyl 2-aminothiophene-3-carboxylate (2.36 g,15 mmol) was dissolved in a 100mL round bottom flask with 30mL dichloromethane, cooled to 0℃and thiophosgene (2.07 g,18 mmol) was added dropwise. After stirring at this temperature for 10min, triethylamine (3.04 g,30 mmol) was added dropwise and gradually returned to room temperature after the addition was completed, monitored by TLC after 1 h. After the reaction was completed, the reaction system was poured into 100mL of water, extracted three times with methylene chloride (60 mL), the organic phases were combined, washed with a saturated aqueous NaCl solution (100 mL), and the separated organic phases were dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and subjected to column chromatography (EA: pe=1:20) to give intermediate compound 1-M1 (2.47 g) in 82.6% yield.
EXAMPLE 2 preparation of Compound 2-M1
The procedure of example 1 was followed except for reacting methyl 3-aminothiophene-2-carboxylate (2.36 g,15 mmol) with thiophosgene (2.07 g,18 mmol) to give intermediate compound 2-M1 (2.55 g) in 85.3% yield.
EXAMPLE 3 preparation of Compound 3-M1
The procedure used in example 1 was followed except for reacting methyl 2-amino-4-methyl-3-carboxylate (2.57 g,15 mmol) with thiophosgene (2.07 g,18 mmol) to give intermediate compound 3-M1 (2.80 g) in 87.4% yield.
EXAMPLE 4 preparation of Compound 4-M1
The procedure used in example 1 was followed except for reacting methyl 3-amino-4-methyl-2-carboxylate (2.57 g,15 mmol) with thiophosgene (2.07 g,18 mmol) to give intermediate compound 4-M1 (2.95 g) in 92.3% yield.
EXAMPLE 5 preparation of Compound 5-M1
The procedure of example 1 was followed except for reacting methyl 3-aminothiophene-4-carboxylate (2.36 g,15 mmol) with thiophosgene (2.07 g,18 mmol) to give intermediate compound 5-M1 (2.59 g) in 86.5% yield.
EXAMPLE 6 preparation of Compound 6-M1
The procedure was followed in example 1, except that methyl 3-aminobenzo [ b ] thiophene-2-carboxylate (3.11 g,15 mmol) was reacted with thiophosgene (2.07 g,18 mmol) to give intermediate compound 6-M1 (3.13 g), yield 83.7%.
EXAMPLE 7 preparation of Compounds 1-M2
Intermediate compound 1-M1 (0.70 g,3.5 mmol) was dissolved in tetrahydrofuran (30 ml) in a 100ml round bottom flask, 2-thiophenemethylamine (0.48 g,4.2 mmol), triethylamine (0.71 g,7 mmol) was added sequentially and stirred at room temperature. After 30min, TLC monitored the completion of the reaction and evaporated off tetrahydrofuran, methanol (30 ml) was added for dissolution, KOH (0.30 g,5.3 mmol) was added and stirring was continued at room temperature. After 30min, TLC monitored reaction was complete. The reaction system was poured into 100ml of water, ethyl acetate (50 ml) was added to extract three times, the organic phases were combined, the organic phases were washed with saturated NaCl solution (50 ml), dried over anhydrous sodium sulfate, and filtered. After that, the mixture was recrystallized from ethyl acetate to obtain intermediate compound 1-M2 (0.83 g) in a yield of 84.2%.
EXAMPLE 8 preparation of Compounds 2-M2
The procedure was as in example 7, except that intermediate compound 2-M1 (0.70 g,3.5 mmol) was reacted with 2-thiophenemethylamine (0.48 g,4.2 mmol), and ethyl acetate was recrystallized to give intermediate compound 2-M2 (0.81 g), yield 82.6%.
EXAMPLE 9 preparation of Compound 3-M2
The procedure was as in example 7, except that intermediate compound 3-M1 (0.75 g,3.5 mmol) was reacted with 2-thiophenemethylamine (0.48 g,4.2 mmol), and ethyl acetate was recrystallized to give intermediate compound 3-M2 (0.88 g) in 85.6% yield.
EXAMPLE 10 preparation of Compound 4-M2
The procedure was as in example 7, except that intermediate compound 4-M1 (0.75 g,3.5 mmol) was reacted with 2-thiophenemethylamine (0.48 g,4.2 mmol) and ethyl acetate was recrystallized to give intermediate compound 4-M2 (0.84 g) in 81.2% yield.
EXAMPLE 11 preparation of Compound 5-M2
The procedure was as in example 7, except that intermediate compound 5-M1 (0.70 g,3.5 mmol) was reacted with 2-thiophenemethylamine (0.48 g,4.2 mmol), and ethyl acetate was recrystallized to give intermediate compound 5-M2 (0.84 g) in 85.8% yield.
EXAMPLE 12 preparation of Compound 6-M2
The procedure was as in example 7, except that intermediate compound 6-M1 (0.87 g,3.5 mmol) was reacted with 2-thiophenemethylamine (0.48 g,4.2 mmol), and ethyl acetate was recrystallized to give intermediate compound 6-M2 (0.94 g) in 81.0% yield.
EXAMPLE 13 preparation of Compound 7-M2
The procedure was as in example 7, except that intermediate compound 1-M1 (0.70 g,3.5 mmol) was reacted with 2-thiophenemethylamine (0.48 g,4.2 mmol), and ethyl acetate was recrystallized to give intermediate compound 7-M2 (0.87 g) in 88.3% yield.
EXAMPLE 14 preparation of Compound 8-M2
The procedure was as in example 7, except that intermediate compound 2-M1 (0.70 g,3.5 mmol) was reacted with 2-thiophenemethylamine (0.48 g,4.2 mmol), and ethyl acetate was recrystallized to give intermediate compound 8-M2 (0.84 g) in 85.4% yield.
EXAMPLE 15 preparation of Compound 9-M2
The procedure was as in example 7, except that intermediate compound 3-M1 (0.75 g,3.5 mmol) was reacted with 2-thiophenemethylamine (0.48 g,4.2 mmol), and ethyl acetate was recrystallized to give intermediate compound 9-M2 (0.89 g) in 86.4% yield.
EXAMPLE 16 preparation of Compound 10-M2
The procedure was as in example 7, except that intermediate compound 4-M1 (0.75 g,3.5 mmol) was reacted with 2-thiophenemethylamine (0.48 g,4.2 mmol) and ethyl acetate was recrystallized to give intermediate compound 10-M2 (0.86 g) in 83.7% yield.
EXAMPLE 17 preparation of Compound 11-M2
The procedure was as in example 7, except that intermediate compound 5-M1 (0.70 g,3.5 mmol) was reacted with 2-thiophenemethylamine (0.48 g,4.2 mmol), and ethyl acetate was recrystallized to give intermediate compound 11-M2 (0.78 g) in 79.6% yield.
EXAMPLE 18 preparation of Compound 12-M2
The procedure was as in example 7, except that intermediate compound 6-M1 (0.87 g,3.5 mmol) was reacted with 2-thiophenemethylamine (0.48 g,4.2 mmol) and ethyl acetate was recrystallized to give intermediate compound 12-M2 (0.94 g) in 81.7% yield.
EXAMPLE 19 preparation of Compound 1
Compound 1-M2 (0.14 g,0.5 mmol) was dissolved in 10ml DMF in a 25ml round bottom flask and K 2CO3 (0.21 g,1.5 mmol) was added after trituration and stirred at room temperature. Bromoacetic acid (0.08 g,0.55 mmol) was slowly added to the system and monitored by TLC. After the reaction was completed, the reaction system was poured into 50ml of water, the system was adjusted to be weakly acidic with dilute hydrochloric acid, extracted three times with 50ml of ethyl acetate, the organic phases were combined, washed with saturated NaCl solution (50 ml), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and the product was obtained by column chromatography (MeOH: dcm=1:30) and then recrystallized from ethyl acetate to give compound 1 (0.13 g) in 76.9% yield. Spectroscopic data for Compound 1 ：1H NMR(400MHz,DMSO-d6)δ7.48(dd,J＝9.1,3.5Hz,1H),7.37(d,J＝5.8Hz,1H),7.23(d,J＝3.2Hz,1H),7.00(dd,J＝5.1,3.5Hz,1H),5.44(s,1H),4.08(s,1H).13C NMR(100MHz,DMSO-d6)δ169.19,162.66,157.39,156.96,136.99,128.47,126.73,126.60,122.89,121.76,119.79,42.28,35.97.
EXAMPLE 20 preparation of Compound 2
The same operations as in example 19 were conducted except that compound 2-M2 (0.14 g,0.5 mmol) was reacted with bromoacetic acid (0.08 g,0.55 mmol), and ethyl acetate was recrystallized to give compound 2 (0.13 g) in 78.7% yield. Spectroscopic data for Compound 2 ：1H NMR(400MHz,DMSO-d6)δ12.93(s,1H),8.18(d,J＝5.2Hz,1H),7.47(d,J＝5.1Hz,1H),7.24(d,J＝5.2Hz,1H),7.23(d,J＝3.1Hz,1H),6.99(dd,J＝5.0,3.6Hz,1H),5.46(s,2H),4.10(s,2H).13C NMR(100MHz,DMSO-d6)δ169.21,157.67,156.88,155.53,136.94,136.45,128.51,126.81,126.64,124.38,118.45,42.34,34.62.
EXAMPLE 21 preparation of Compound 3
The same operations as in example 19 were conducted except that compound 3-M2 (0.15 g,0.5 mmol) was reacted with bromoacetic acid (0.08 g,0.55 mmol), and ethyl acetate was recrystallized to give compound 3 (0.15 g), yield 86.2%. Spectroscopic data for Compound 3 ：1H NMR(400MHz,DMSO-d6)δ12.91(s,1H),7.47(dd,J＝5.1,1.2Hz,1H),7.22(d,J＝3.4Hz,1H),7.07(d,J＝1.2Hz,1H),7.00(dd,J＝5.1,3.5Hz,1H),5.41(s,2H),4.06(s,2H),2.46(d,J＝1.0Hz,3H).13C NMR(100MHz,DMSO-d6)δ169.16,163.11,157.56,156.64,137.02,133.61,128.47,126.74,126.61,118.23,117.33,42.11,34.54,16.09.
EXAMPLE 22 preparation of Compound 4
The same operations as in example 19 were conducted except that compound 4-M2 (0.15 g,0.5 mmol) was reacted with bromoacetic acid (0.08 g,0.55 mmol), and ethyl acetate was recrystallized to give compound 4 (0.15 g), yield 84.6%. Spectroscopic data for Compound 4 ：1H NMR(400MHz,DMSO-d6)δ12.84(s,1H),7.84(d,J＝1.1Hz,1H),7.48(dd,J＝5.1,1.2Hz,1H),7.23(dd,J＝3.5,1.0Hz,1H),7.00(dd,J＝5.1,3.5Hz,1H),5.45(s,2H),4.06(s,2H),2.25(d,J＝1.0Hz,3H).13C NMR(100MHz,DMSO-d6)δ169.37,157.46,157.03,154.42,136.99,132.92,131.06,128.50,126.81,126.64,118.13,42.28,34.82,12.08.
EXAMPLE 23 preparation of Compound 5
The same operations as in example 19 were conducted except that compound 5-M2 (0.14 g,0.5 mmol) was reacted with bromoacetic acid (0.08 g,0.55 mmol), and ethyl acetate was recrystallized to give compound 5 (0.15 g) in 88.7% yield. Spectroscopic data for Compound 5 ：1H NMR(400MHz,DMSO-d6)δ12.86(s,1H),8.54(d,J＝3.2Hz,1H),7.60(d,J＝3.2Hz,1H),7.46(dd,J＝5.1,1.2Hz,1H),7.20(d,J＝3.3Hz,1H),6.99(dd,J＝5.1,3.5Hz,1H),5.38(s,2H),4.04(s,2H).13C NMR(100MHz,DMSO-d6)δ169.34,157.01,153.80,145.91,137.88,129.26,128.04,126.64,126.53,123.13,116.50,41.59,34.50.
EXAMPLE 24 preparation of Compound 6
The same operations as in example 19 were conducted except that compound 6-M2 (0.17 g,0.5 mmol) was reacted with bromoacetic acid (0.08 g,0.55 mmol), and ethyl acetate was recrystallized to give compound 6 (0.17 g) in a yield of 85.3%. Spectroscopic data for Compound 6 ：1H NMR(400MHz,DMSO-d6)δ12.97(s,1H),8.18(dd,J＝17.4,7.9Hz,2H),7.71–7.64(m,1H),7.61(t,J＝7.5Hz,1H),7.50(dd,J＝5.1,1.1Hz,1H),7.28(d,J＝2.8Hz,1H),7.02(dd,J＝5.1,3.5Hz,1H),5.51(s,2H),4.16(s,2H).13C NMR(100MHz,DMSO-d6)δ169.56,159.03,157.38,150.85,140.71,136.61,133.34,129.51,128.77,126.99,126.69,125.56,123.98,123.33,117.91,42.79,35.06.
EXAMPLE 25 preparation of Compound 7
The same operations were conducted as in example 19 except that compound 7-M2 (0.14 g,0.5 mmol) was reacted with bromoacetic acid (0.08 g,0.55 mmol), and ethyl acetate was recrystallized to give compound 7 (0.15 g) in 89.3% yield. Spectroscopic data for Compound 7 ：1H NMR(400MHz,DMSO-d6)δ12.93(s,1H),7.51(d,J＝3.8Hz,1H),7.48(d,J＝5.7Hz,1H),7.44(s,1H),7.35(d,J＝5.7Hz,1H),7.13(d,J＝4.5Hz,1H),5.29(s,2H),4.06(s,2H).13C NMR(100MHz,DMSO-d6)δ169.18,162.51,157.20,157.08,135.75,127.40,126.69,123.75,122.89,121.84,119.96,42.82,34.56.
EXAMPLE 26 preparation of Compound 8
The same operations as in example 19 were conducted except that compound 9-M2 (0.15 g,0.5 mmol) was reacted with bromoacetic acid (0.08 g,0.55 mmol), and ethyl acetate was recrystallized to give compound 8 (0.15 g), yield 84.6%. Spectroscopic data for Compound 8 ：1H NMR(400MHz,DMSO-d6)δ12.91(s,1H),7.51(dd,J＝4.8,3.0Hz,1H),7.44(s,1H),7.13(d,J＝4.9Hz,1H),7.05(s,1H),5.26(s,2H),4.04(s,2H),2.45(s,3H).13C NMR(100MHz,DMSO-d6)δ169.19,163.10,157.69,156.98,135.88,133.66,127.43,126.64,123.66,118.27,117.13,42.57,34.49,16.09.
EXAMPLE 27 preparation of Compound 9
The same operations as in example 19 were conducted except that compound 11-M2 (0.14 g,0.5 mmol) was reacted with bromoacetic acid (0.08 g,0.55 mmol), and ethyl acetate was recrystallized to give compound 9 (0.15 g) in a yield of 90.3%. Spectroscopic data for Compound 9 ：1H NMR(400MHz,DMSO-d6)δ12.84(s,1H),8.50(d,J＝3.2Hz,1H),7.60(d,J＝3.2Hz,1H),7.50(dd,J＝4.9,3.0Hz,1H),7.41(d,J＝1.7Hz,1H),7.11(d,J＝4.9Hz,1H),5.23(s,2H),4.02(s,2H).13CNMR(100MHz,DMSO-d6)δ169.37,157.14,154.21,146.03,136.64,129.04,127.31,126.60,123.34,123.30,116.29,42.09,34.45.
EXAMPLE 28 preparation of Compound 10
The same operations were conducted as in example 19 except that compound 8-M2 (0.14 g,0.5 mmol) was reacted with bromoacetic acid (0.08 g,0.55 mmol), and ethyl acetate was recrystallized to give compound 10 (0.14 g), yield 82.5%. Spectroscopic data for Compound 10 ：1H NMR(400MHz,DMSO-d6)δ12.90(s,1H),8.19(d,J＝5.2Hz,1H),7.52(dd,J＝5.0,3.0Hz,1H),7.45(dd,J＝2.7,1.0Hz,1H),7.26(d,J＝5.2Hz,1H),7.13(dd,J＝5.0,1.2Hz,1H),5.31(s,2H),4.07(s,2H).13C NMR(100MHz,DMSO-d6)δ169.28,158.00,157.04,155.53,136.23,135.81,127.43,126.71,124.38,123.80,118.58,42.83,34.58.
EXAMPLE 29 preparation of Compound 11
The same operations as in example 19 were conducted except that compound 10-M2 (0.15 g,0.5 mmol) was reacted with bromoacetic acid (0.08 g,0.55 mmol), and ethyl acetate was recrystallized to give compound 11 (0.16 g) in 92.2% yield. Spectroscopic data for Compound 11 ：1H NMR(400MHz,DMSO-d6)δ12.83(s,1H),7.83(d,J＝1.1Hz,1H),7.52(dd,J＝5.2,3.1Hz,1H),7.44(dd,J＝3.1,1.3Hz,1H),7.12(dd,J＝5.2,1.4Hz,1H),5.29(s,2H),4.04(s,2H),2.26(d,J＝0.8Hz,3H).13C NMR(100MHz,DMSO-d6)δ169.42,157.77,157.18,154.40,135.83,132.88,130.82,127.43,126.69,123.81,118.25,42.74,34.78,12.08.
EXAMPLE 30 preparation of Compound 12
The same operations as in example 19 were conducted except that compound 12-M2 (0.17 g,0.5 mmol) was reacted with bromoacetic acid (0.08 g,0.55 mmol), and ethyl acetate was recrystallized to give compound 12 (0.17 g), yield 89.7%. Spectroscopic data for Compound 12 ：1H NMR(400MHz,DMSO-d6)δ8.21(d,J＝7.6Hz,1H),8.15(d,J＝8.1Hz,1H),7.71–7.64(m,1H),7.64–7.58(m,1H),7.54(dd,J＝4.9,3.0Hz,1H),7.50(dd,J＝2.7,1.1Hz,1H),7.17(dd,J＝5.0,1.3Hz,1H),5.35(s,2H),4.14(s,2H).13C NMR(100MHz,DMSO-d6)δ169.60,159.33,157.54,150.83,140.68,135.51,133.42,129.42,127.49,126.79,125.50,124.03,123.93,123.30,118.03,43.26,35.01.
EXAMPLE 31 screening assay for inhibition of uric acid transporter hURAT1 by Compounds
Test materials and methods:
(1) Cell strain is human uric acid transporter (hURAT 1) high expression cell strain (HEK 293-URAT 1).
(2) Experimental conditions:
(3) Preparing a test agent:
1) Preparation of 20mmol/L stock solution
Each compound was formulated as a 20mmol/L stock solution using DMSO as vehicle.
2) Preparation of working fluid
① Single concentration (1. Mu. Mol/L) stock solution 1-12 was diluted to 200. Mu. Mol/L as 200-fold working solution of the administration concentration using DMSO as vehicle, and Lesinurad (Les, positive control) stock solution was diluted to 200. Mu. Mol/L.
② IC 50. Stock solution No. 3 and No. 4 were diluted to 200 times of each of the administration concentrations using DMSO as a vehicle
The liquid is 6000,2000,600,200,60,20 mu mol/L.
③ And diluting the working solution 200 times in 1 and 2 by 100 times by using HBSS (no Cl-) buffer solution as a solvent to obtain the working solution 2 times of each dosing concentration.
④ Preparing 2 times working solution of standard substrate 14 C-Uric acid by using HBSS (no Cl-) buffer solution, and then respectively mixing with the 2 times working solution in 3 in equal volume to obtain the administration working solution.
(4) Administration method
After resuscitating and subculturing a cell line (HEK 293-URAT 1) expressing hURAT1, selecting well-grown adherent cells, using pancreatin to digest the cells into single cell suspension, regulating the cell density of the culture medium to about 1.5×105cells/mL, inoculating the cells into a 24-hole cell culture plate at1 mL/hole, and culturing the cells in an incubator with 37 ℃ and 5% CO2 and saturated air humidity for 2-3 days.
After cells were grown up in each well, the culture was removed and after 2 washes with 37 ℃ pre-warmed HBSS, 1mL of 37 ℃ HBSS buffer was added to each well for 10min incubation. Then, 500. Mu.L of the working solution containing the radiolabeled probe substrate was administered to replace HBSS, after 2min, the reaction was stopped with cold HBSS buffer, 300. Mu.L of 0.1mol/L NaOH was added to each well to lyse the cells, the cell lysate was taken in a scintillation vial, 1.5mL of scintillation solution was added, and the radioactivity in the samples was measured using a Tri-Carb 2910TR liquid scintillation device. 2 wells (n=2) were set for each compound, positive control, blank control.
(5) Data processing
The transport value Uc of cells of the administration group (control) containing only the labeling substrate is defined as 100%, and the percentage (In) of the transport value U to Uc of each administration group after the test compound is added, namely the transport ratio, is calculated by taking the transport value as a standard, and the inhibition effect of the compound on the transporter is represented by the transport ratio (the lower the transport ratio In is, the stronger the activity is), and the formula is as follows:
Each group was set with 2 wells (n=2), excel statistical formulas calculated mean±standard error (SD), GRAPHPAD PRISM software calculated IC 50.
Test results:
(1) Single concentration activity inhibition
As shown in FIG. 1, at a single concentration of 1. Mu. Mol/L, compounds 1-12 all produced different inhibitory effects on hURAT1, inhibiting the activity of hURAT1 to 82.30%, 81.15%, 61.97%, 58.89%, 76.13%, 78.90%, 66.77%, 67.63%, 79.93%, 65.81%, 72.15%, 87.47%, respectively. Compared with the positive control drug Lesinurad (80.03%) with the same concentration (1 mu mol/L), the inhibition activity of 10 compounds is superior to or equivalent to that of Lesinurad.
(2) IC 50 test results
Compounds 3 and 4 showed strong inhibitory activity against hURAT1, and were tested for inhibitory effect IC 50. As shown in fig. 2 and 3, compounds 3 and 4 showed significantly better activity against huat 1 inhibition IC 50 at 3.097 and 1.514 μmol/L, respectively, than the positive control Lesinurad (IC 50 =7.3 μmol/L).
The single-concentration activity inhibition test shows that the compounds 1-12 have different inhibition effects on the hURAT1, wherein the inhibition activity of 10 compounds is superior to or equivalent to that of Lesinurad, the inhibition activities of the compounds 3 and 4 are stronger, the inhibition effects on the hURAT1 are respectively 3.097 and 1.514 mu mol/L, and the activity is obviously superior to that of a positive control Lesinurad (IC 50 =7.3 mu mol/L), and the compound can be used as a candidate medicament for treating gout and hyperuricemia.
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