CN112574136A - Structure, preparation method and application of a series of benzoxazolone derivatives - Google Patents
Structure, preparation method and application of a series of benzoxazolone derivatives Download PDFInfo
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Abstract
The invention discloses a structure, a preparation method and application of a series of benzoxazolone derivatives, namely provides a benzoxazolone compound structure shown in a compound I, a synthesis method, pharmaceutically acceptable salts thereof or application of a mixture thereof in preparation of medicines for preventing and/or treating diabetic complications. The compound is used as aldose reductase inhibitor and antioxidant, and has hypoglycemic effect. The compounds can reduce blood sugar, inhibit activity of aldose reductase, scavenge free radical and inhibit generation of lipid peroxide, and increase glutathione content and superoxide dismutase activity, thereby reducing urine protein level, and preventing and/or treating diabetic complications, especially diabetic nephropathy. The invention also provides a compound containing the compound and having prevention and/or treatment effectsA pharmaceutical composition for treating diabetic complication.
Wherein, X is-, or-CH ═ CH-; r1Is hydrogen, hydroxy, methoxy, or trifluoromethyl, R2Is hydrogen, methoxy, or hydroxy.
Description
Technical Field
The invention relates to the fields of organic chemistry, medicinal chemistry and pharmacology, in particular to a series of benzoxazolone derivatives, a benzoxazolone derivative serving as an aldose reductase inhibitor, a preparation method of the benzoxazolone derivative and application of the benzoxazolone derivative in preparing medicines for preventing or treating diabetic complications.
Background
Diabetes Mellitus (DM) is a common chronic metabolic disease associated with serious degenerative complications such as neuropathy, nephropathy, retinopathy, cataracts, atherosclerosis and myocardial infarction. Because of its high disability rate and high mortality rate, diabetic complications have become one of the major threats to the health and longevity of the inhabitants in most countries of the world. Indeed, their prevention and control remains a challenging problem. Epalrestat (Epalrestat) is currently the only diabetes complication treatment drug on the market, but is limited to the japanese market and has recently been introduced in china and india.
In tissues with insulin-independent glucose transport (retina, lens, kidney and nerves), increased glucose flux through the polyol pathway that occurs under hyperglycemic conditions is a well-known factor associated with secondary diabetic complications. Aldose reductase (ALR2) is a key rate-limiting enzyme in the polyol pathway. Studies have shown that the polyol pathway and its downstream oxidative stress are closely related to diabetic complications. Therefore, development of an aldose reductase inhibitor having antioxidant activity can suppress both abnormal accumulation of sorbitol and oxidative stress, and thus, development of a therapeutic agent for diabetic complications is expected.
Numerous animal and clinical trials have demonstrated that aldose reductase inhibitors are very effective in treating diabetic complications. Over the last 30 years, at least 14 aldose reductase inhibitors have proven to be very effective and have passed phase II clinics. Among them, epalrestat marketed in Japan, and Fidarestat (Fidarestat) and AS-3201 that have entered or passed through phase III clinical trials are the most effective, and Fidarestat has been shown to be more effective than epalrestat marketed, and its inhibitory effect in vivo is much higher than epalrestat. Meanwhile, antioxidants that directly inhibit active oxygen or oxidative stress may also be effective in treating diabetic complications. Flavonoids as natural antioxidants have been found to have aldose reductase inhibitory activity, but their activity intensity is insufficient and no further development has been achieved. In addition, the composition can reduce the blood sugar level in a diabetic organism and improve the regulation capability of the diabetic organism on the blood sugar concentration while preventing or treating diabetic complications, and is also an important approach for treating diabetes. Therefore, it is urgent to develop a safer and more effective drug for the treatment of diabetic complications.
Disclosure of Invention
The invention aims to provide a novel compound which has stronger inhibition capability on ALR2, stronger inhibition capability on free radicals and lipid peroxides and has a blood sugar reducing effect and a preparation method thereof, wherein an inhibitory activity experiment of the compound on ALR2 is carried out by extracting aldose reductase of rat lens, and a free radical removing experiment of the compound in vitro of the rat is carried out by preparing 1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH) solution and extracting brain homogenate of the rat, so that the compound is proved to be a high-efficiency aldose reductase inhibitor, can obviously reduce blood sugar, inhibit the activity of ALR2 and simultaneously effectively inhibit the generation of the free radical, thereby achieving the effect of preventing and/or treating diabetic complications, particularly diabetic nephropathy.
Therefore, the invention provides the application of the compound shown in the formulas I, II and III, the structure, the synthesis, the pharmaceutically acceptable salt or the mixture thereof in the preparation of the medicine for preventing and/or treating diabetic complications,
in the formula I, X is-, -CH ═ CH-;
r is hydrogen, hydroxyl, methoxy, or trifluoromethyl.
The present invention also provides a pharmaceutical composition for preventing and/or treating diabetic complications, comprising: a therapeutically effective amount of benzoxazolone compounds represented by formula I, pharmaceutically acceptable salts or mixtures thereof as an active ingredient; and a pharmaceutically acceptable carrier, excipient or sustained release agent.
In a second aspect of the invention, there is provided a process for the preparation of a compound having the structure of formula i, comprising the steps of:
(i) taking a formula Ia as a raw material, taking 1,1' -carbonyldiimidazole as an acylation reagent, and forming a compound shown as a formula Ib through one-step operation;
(ii) coupling methyl acetate at the 1-position N by formation of a C-N bond in the presence of a base starting from formula Ib to form a compound of formula ic:
(iii) coupling a substituted or unsubstituted aryl, aralkyl group at the 7-position C by formation of a C-C bond under inert conditions in the presence of a base and a metal catalyst starting from formula ic to form a compound of formula id:
(iv) demethylating the compound of formula id to form a compound of formula ie;
wherein R in the compound I is as defined above.
In a third aspect of the present invention, there is provided a pharmaceutical compound for preventing and/or treating diabetic complications, comprising: a therapeutically effective amount of the use according to any of the preceding claims, characterized in that: the compound, pharmaceutically acceptable salt or mixture thereof can be used as aldose reductase inhibitor in the preparation of medicines for preventing or treating diabetic complications and reducing the blood sugar content of diabetic organisms.
The invention has the main advantages that:
through extensive and intensive research, a novel benzoxazolone derivative with a structural formula I is synthesized, and in-vitro experiments prove that the compound has a good inhibition effect on ALR2 and free radicals and only has a weak inhibition effect on Aldehyde Reductase (Aldehyde Reductase, ALR1), so that the benzoxazolone derivative is a high-efficiency, high-selectivity and low-toxicity multifunctional aldose Reductase inhibitor and has an application in preparing medicines for preventing and/or treating diabetic complications, especially diabetic nephropathy. The compound is found to have the function of reducing blood sugar of mice with diabetes in vivo, the function of removing lipid peroxide and the function of inhibiting active oxygen in vivo through a sugar tolerance test (GTT) of the compound in the mice with diabetes, and the content of lipid peroxide (MDA) and Glutathione (GSH) and the activity of superoxide dismutase (SOD) in the mice.
Active ingredient
As used herein, the terms "active ingredient", "active compound", "compound of the invention", "novel aldose reductase inhibitor" are used interchangeably and refer to benzoxazolones of the invention having the structure i, pharmaceutically acceptable salts or mixtures thereof.
Pharmaceutical composition
The present invention also provides a pharmaceutical composition for preventing and/or treating diabetic complications and for hypoglycemic effects in diabetic subjects, comprising:
(a) a benzoxazolone compound shown as a formula I, a pharmaceutically acceptable salt or a mixture thereof which is used as an active ingredient in an effective amount for prevention and/or treatment;
(b) a pharmaceutically acceptable carrier, excipient or sustained release agent.
In the present invention, the term "comprising" means that various ingredients can be used together in the mixture or composition of the present invention. Thus, the terms "consisting essentially of …" and "consisting of …" are both encompassed by the term "comprising".
In the context of the present invention, a "pharmaceutically acceptable" component is a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.
In the present invention, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, suspending agent or excipient for delivering the active substance of the present invention or a physiologically acceptable salt thereof to a human and/or an animal. The carrier may be solid or liquid.
The active ingredients contained in the pharmaceutical composition of the invention account for 0.01 to 99.9 percent of the total weight of the pharmaceutical composition; and a pharmaceutically acceptable carrier, excipient or sustained release agent, wherein the total weight of the composition is 100%.
Detailed Description
The present invention is further described in the following description of the embodiments with reference to the drawings, which are not intended to limit the invention, and those skilled in the art may make various modifications or improvements based on the basic idea of the invention, but within the scope of the invention, unless departing from the basic idea of the invention.
In order to solve the problems in the prior art, the invention provides a highly effective, highly selective, and low-toxicity multifunctional aldose reductase inhibitor, and the multifunctional aldose reductase inhibitor has the application of preparing a medicament for preventing and/or treating diabetic complications, especially diabetic nephropathy.
Based on the ingenious conception, the invention provides a compound shown as a formula I or pharmaceutically acceptable salt thereof or a mixture of the compound and the pharmaceutically acceptable salt,
wherein, X is-, or-CH ═ CH-;
R1is hydrogen, hydroxy, methoxy, or trifluoromethyl, R2Is hydrogen, methoxy, or hydroxy.
When R is1Is hydroxy, R2When the compound is methoxy, the compound has the strongest inhibitory effect on aldose reductase and has stronger antioxidant effect. When hydroxyl and methoxyl exist, the hydroxyl and methoxyl of the compound can form hydrogen bonds with a specific pocket of aldose reductase, and the inhibitory effect of aldose reductase is enhanced. Meanwhile, the existence of phenolic hydroxyl groups enables the compound to have the property of intermolecular Diels-Alder generation, so that free radicals are captured, and the antioxidant effect is generated.
The present invention also provides a process for preparing a compound of formula I as described above, comprising the steps of:
wherein, X is-, or-CH ═ CH-;
r1 is hydrogen, hydroxy, methoxy, or trifluoromethyl, R2 is hydrogen, methoxy, or hydroxy
FIG. 1 is a series of structures of benzoxazolone derivatives and preparation method thereof
S1: the acylating agent used in the reaction is 1,1' -carbonyldiimidazole; the solvent can be acetonitrile, tetrahydrofuran and the like besides N, N-dimethylformamide; the reaction temperature is 60-90 ℃.
S2: the alkali used in the reaction can be sodium carbonate, cesium carbonate and the like besides potassium carbonate; the solvent can be N, N-dimethylformamide, tetrahydrofuran, dioxane and the like besides acetonitrile; the reaction temperature is 60-90 ℃.
S3: the catalyst used in the reaction can be palladium tetratriphenylphosphine, palladium acetate and triphenylphosphine, palladium chloride and trinitromethylphenyl phosphorus and the like besides palladium acetate and tri-o-methylphenyl phosphine; the alkali used in the reaction can be diethylamine, sodium tert-butoxide and the like besides triethylamine; the solvent used in the reaction may be toluene, dimethyl sulfoxide, etc. in addition to N, N-dimethylformamide; the reaction temperature is 90-120 ℃;
s4: the alkali used in the reaction can be sodium hydroxide, potassium hydroxide and the like besides lithium hydroxide; the solvent can be dioxane, methanol, water and the like besides tetrahydrofuran; the reaction temperature is 0-30 ℃.
In the step S1, 1' -carbonyl diimidazole is used as an acylating reagent, so that the construction of a benzoxazolone parent nucleus can be completed in one step, and in addition, the lithium hydroxide is used for hydrolysis in S4, so that a carboxylic acid side chain can be generated while the structure of the benzoxazolone is not damaged.
In addition, the invention researches the inhibiting effect of the compound on ALR2 and free radicals, and performs a sugar tolerance test (GTT) on the compound in a diabetic mouse, and determines the content of lipid peroxide (MDA), sorbitol and Glutathione (GSH) and the activity of superoxide dismutase (SOD) in the mouse, thereby finding out the effect of the compound on reducing blood sugar of the diabetic mouse in vivo, the removing effect of the lipid peroxide and the inhibiting effect of active oxygen, and the effective inhibition of aldose reductase in vivo.
The experimental activity proves that the carboxylic acid side chain and the aryl side chain in the compound can effectively enter an anion pocket and a specificity pocket of aldose reductase respectively, so that the aldose reductase inhibiting effect is generated, the metabolism of glucose through a polyol passage is reduced, the content of sorbitol is reduced, the loss of glutathione is reduced, and the content of glutathione is increased. And the existence of the phenolic hydroxyl in the aryl side chain can effectively reduce the free radicals in the trapped mouse, thereby reducing the generation of lipid peroxide and improving the activity of superoxide dismutase. The overall structure of the compound lowers blood glucose in diabetic mice.
Examples
The invention will be further illustrated by the following examples. These examples are intended to illustrate the invention, but not to limit it in any way. All parameters and descriptions in the examples are based on mass unless otherwise specified. The test methods in the examples, in which the specific conditions are not specified, are generally carried out under the conventional conditions or under the conditions recommended by the manufacturers.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention.
Example 1: methyl (E) -2- (7- (4-hydroxy-3-methoxystyryl) -2-benzoxazolone-3 (2H) -alkyl) acetic acid (Compound 1)
1,1' -carbonyldiimidazole (1.6mmol) was added to a solution of 2-amino-6-bromophenol (1.0mmol) in DMF (3mL), heated at 60 ℃ for 2h, the reaction mixture was poured into water (15mL) and extracted with EtOAc (3X 15 mL). The organic layer was collected, washed with brine (15mL), MgSO was added4Drying and rotary steaming. Recrystallizing from ethyl ethoxyacetate to obtain the desired product 7-bromo-2-benzoxazolone. (yellow crystals, yield 46%, 115 mg):1H NMR(400MHz,DMSO-d6)δ11.81ppm(s,1H),7.59ppm(d,J＝1.6 Hz,1H),7.33ppm(dd,J＝8.3,1.6Hz,1H),7.05ppm(dd,J＝8.3,1.2Hz,1H)。
mixing 7-bromo-2-benzoxazolone (1mmol) and K2CO3(1mmol), methyl bromoacetate (1.2mmol) was added to CH3CN (10mL), and stirred at 65 ℃ for 3 h. After the reaction was completed, the mixture was filtered through a celite pad and evaporated by rotary evaporation. The residue was recrystallized from ethyl acetate to give the compound methyl 2- (7-bromo-2-benzoxazolone-3 (2H) -alkyl) acetate. (white crystals, yield 74%, 211 mg):1H NMR(400MHz, Chloroform-d)δ7.30–7.26ppm(m,1H),7.11ppm(d,J＝8.5Hz,1H),7.04ppm(d, J＝1.9Hz,1H),4.55ppm(s,2H),3.82ppm(s,3H)。
mixing methyl 2- (7-bromo-2-benzoxazolone-3 (2H) -alkyl) acetate, Pd (OAc)2(0.03mmol) and P (o-tolyl)3(0.07mmol) was added to DMF (10mL) and stirred at room temperature for 30min under argon. 2-methoxy-4-vinylphenol (1.5mmol), Et was added3N (0.30g,3mmol), DMF (1 ml). The reaction mixture was heated at 100 ℃ for 12h and after completion of the reaction, the mixture was poured into water and extracted with EtOAc (3X 15 ml). Collecting the organic layer, adding MgSO4Dried and evaporated in vacuo. The mixture is then subjected to column chromatography to yield the desired product methyl (E) -2- (7- (4-hydroxy-3-methoxystyryl) -2-benzoxazolone-3 (2H) -alkyl) acetate. (yellow crystals, yield 35%, 118 mg):1H NMR(400MHz, DMSO-d6)δ9.26ppm(d,J＝1.7Hz,1H),7.38ppm(s,1H),7.35ppm(d,J＝7.0Hz, 1H),7.24ppm(d,J＝13.0Hz,2H),7.21–7.15ppm(m,1H),7.12ppm(d,J＝16.5 Hz,1H),7.06ppm(d,J＝8.2Hz,1H),6.80ppm(d,J＝8.0Hz,1H),4.81ppm(s, 2H),3.86ppm(s,3H),3.73ppm(s,3H)。
methyl (E) -2- (7- (4-hydroxy-3-methoxystyryl) -2-benzoxazolone-3 (2H) -alkyl) acetate (1mmol) and saturated LiOH (5mL) were added to THF, stirred at rt for 2H, and after completion of the reaction, acidified to pH 3 by addition of 0.1N HCl. The suspension was extracted with EtOAc (3X 15mL), the organic phase was collected and MgSO was added4Dried and evaporated in vacuo. The residue was recrystallized from ethyl acetate to give methyl (E) -2- (7- (4-hydroxy-3-methoxystyryl) -2-benzoxazolone-3 (2H) -alkyl) acetic acid. (yellow crystals, yield 48%, 149 mg):1H NMR(400MHz,DMSO-d6)δ9.30ppm(s,1H),7.39–7.30 ppm(m,2H),7.25ppm(s,1H),7.20ppm(t,J＝7.9Hz,1H),7.13ppm(d,J＝8.2Hz, 1H),7.10ppm(s,1H),7.05ppm(d,J＝8.2Hz,1H),6.80ppm(d,J＝8.1Hz,1H), 4.54ppm(s,2H),3.86ppm(s,3H)；13C NMR(101MHz,DMSO-d6)δ169.36, 154.43,148.38,147.74,139.24,133.22,132.13,128.68,124.48,121.11,121.00, 118.75,116.07,110.44,108.19,56.13,44.04ppm；HRMS(ESI)m/z calcd for[M-H] -340.0827,found 340.0816.
example 2: methyl (E) -2- (7- (4-methoxystyryl) -2-benzoxazolone-3 (2H) -alkyl) acetic acid (Compound 2)
Prepared as described in example 1, substituting methyl (E) -2- (7- (4-hydroxy-3-methoxystyryl) -2-benzoxazolone-3 (2H) -alkyl) acetate for methyl (E) -2- (7- (4-hydroxy-3-methoxystyryl) -2-benzoxazolone-3 (2H) -alkyl) acetate in example 1.
1H NMR(400MHz,DMSO-d6)δ7.61ppm(d,J＝8.2Hz,2H),7.45–7.34ppm(m, 2H),7.22ppm(dd,J＝13.2,5.2Hz,2H),7.19–7.12ppm(m,1H),6.98ppm(d,J＝ 8.1Hz,2H),4.68ppm(s,2H),3.79ppm(s,3H)；13C NMR(101MHz,DMSO-d6)δ 169.20,159.91,154.30,139.31,132.54,131.88,129.69,128.59,124.58,121.33, 121.00,119.39,114.74,108.37,55.66,43.45ppm；HRMS(ESI)m/z calcd for[M-H] -324.0877,found 324.0876。
Example 3: methyl (E) -2- (7- (4-hydroxy-3-methoxystyryl) -2-benzoxazolone-3 (2H) -alkyl) acetic acid (Compound 3)
Prepared as described in example 1, substituting methyl (E) -2- (7- (4-hydroxy-3-methoxystyryl) -2-benzoxazolone-3 (2H) -alkyl) acetate for methyl (E) -2- (7- (4-hydroxy-3-methoxystyryl) -2-benzoxazolone-3 (2H) -alkyl) acetate in example 1.
1H NMR(400MHz,DMSO-d6)δ7.36ppm(dd,J＝7.8,1.5Hz,2H),7.28ppm(d,J ＝16.4Hz,1H),7.24–7.14ppm(m,2H),7.07–6.98ppm(m,1H),6.97–6.90ppm (m,1H),6.81–6.73ppm(m,1H),4.67ppm(s,2H)；13C NMR(101MHz,DMSO-d6) δ169.21,154.31,146.72,146.01,139.21,133.25,131.85,128.63,124.57,121.15, 121.10,119.55,118.07,116.27,113.76,108.11,43.45ppm；HRMS(ESI)m/z calcd for[M-H]-326.0670,found 326.0680。
Example 4: 2- (2-carbonyl-7-benzoxazole-3 (2H) -yl) acetic acid derivative (Compound 4)
Methyl 2- (7-bromo-2-benzoxazolone-3 (2H) -alkyl) acetate (1mmol), (PPh)3)4Pd (0.03mmol), 4-trifluoromethylphenylboronic acid (1.1mmol), K2CO3(0.65g,2mmol) and H2O (1mL) was added to DMF (10mL) and stirred at 100 ℃ for 12 h. After completion of the reaction, the mixture was poured into water, extracted with EtOAc (3X 15mL), and the organic phase was collected and MgSO was added4Dried and evaporated in vacuo. The mixture is subjected to column chromatography to obtain a compound methyl 2- (2-oxygen-7- (4- (trifluoromethyl) phenyl) benzoxazolone-3 (2H) -alkyl) acetate. (white crystals, yield 47%, 161 mg):1H NMR(400MHz,DMSO-d6)δ8.01 ppm(d,J＝8.1Hz,2H),7.91ppm(d,J＝8.0Hz,2H),7.49ppm(dd,J＝7.1,2.3Hz, 1H),7.44–7.33ppm(m,2H),4.86ppm(s,2H),3.75ppm(s,3H)。
methyl 2- (2-oxo-7- (4- (trifluoromethyl) phenyl) benzoxazolone-3 (2H) -alkyl) acetate (1mmol) and saturated LiOH (5mL) were added to THF (5mL) and mixed, stirring at rt for 2H. After completion of the reaction, 0.1N HCl was added to acidify to pH 3. The suspension was extracted with EtOAc (3X 15mL), the organic phase was collected and MgSO was added4Dried and evaporated in vacuo. The residue was recrystallized from ethyl acetate to give 2- (2-oxo-7- (4- (trifluoromethyl) phenyl) benzoxazolone-3 (2H) -alkyl) acetic acid. (white crystals, yield 37%, 92 mg):1H NMR(400MHz,DMSO-d6)δ13.20ppm(s,1H),8.01ppm(d,J＝8.0 Hz,2H),7.91ppm(d,J＝8.1Hz,2H),7.47ppm(d,J＝7.4Hz,1H),7.43–7.33ppm (m,2H),4.72ppm(s,2H)；13C NMR(101MHz,DMSO-d6)δ169.15,154.11, 139.58,138.76,132.33,129.31,126.28,126.25,125.08,122.43,121.84,110.19, 49.06,43.54ppm；HRMS(ESI)m/z calcd for[M-H]-336.0489,found 336.0482.
example 5: 2- (2-oxo-7- (3, 4-dihydroxyphenyl) benzoxazolone-3 (2H) -alkyl) acetic acid (Compound 5)
Prepared as described in example 1, substituting methyl 2- (2-oxo-7- (4- (trifluoromethyl) phenyl) benzoxazolone-3 (2H) -alkyl) acetate in example 4 with methyl 2- (2-oxo-7- (3, 4-dihydroxyphenyl) benzoxazolone-3 (2H) -alkyl) acetate.
1H NMR(400MHz,DMSO-d6)δ8.86ppm(s,1H),7.99ppm(s,1H),6.90 ppm(s,1H),6.74ppm(q,J＝10.1,9.4Hz,3H),6.39ppm(dd,J＝34.5,7.8Hz, 2H),3.84ppm(s,2H)；13C NMR(101MHz,DMSO-d6)δ169.24,154.38, 146.30,145.89,138.98,132.13,125.67,124.78,123.82,121.63,119.70, 116.45,115.81,108.00,43.43ppm；HRMS(ESI)m/z calcd for[M-H]- 300.0514,found 300.0517.
Example 5.Test of in vitro inhibitory Effect of Compounds on ALR2 and ALR1
The phosphate buffer solution for ALR2 measurement, the sodium phosphate buffer solution 1 for ALR1 measurement, the sodium phosphate buffer solution 2, the NADPH solution, the D, L-glyceraldehyde solution and the D-sodium glucuronate solution are used in the experiment, and the preparation method comprises the following steps:
(1) a phosphate buffer solution was prepared for 0.1M ALR2 assay at pH 6.2
Solution A: 3.12g NaH2PO4·2H2Dissolving O in 100ml of water to prepare 0.2M solution;
solution B: 3.58g Na2HPO4·12H2O was dissolved in 50ml of water to prepare a 0.2M solution.
And (3) taking 81.5ml of A81.5ml and 18.5ml of B, diluting with water to a final volume of 200ml, and adjusting the pH value to 6.2 to obtain the compound.
(2) Phosphate buffer solution 1 for 10mM ALR1 assay at pH 7.2 was prepared
0.3801g of sodium phosphate, 8.5513g of cane sugar, 0.0809g of EDTA dipotassium salt and 0.0175mL of beta-mercaptoethanol are dissolved in 100mL of water, and the pH value is adjusted to 7.2, thus obtaining the sodium glutamate.
(3) Phosphate buffer solution 2 for 10mM ALR1 assay at pH 7.2 was prepared
0.3801g of sodium phosphate, 0.0809g of EDTA dipotassium salt and 0.0140mL of beta-mercaptoethanol are dissolved in 100mL of water, and the pH is adjusted to 7.2, thus obtaining the sodium glutamate.
(4) A0.104 mM NADPH solution (buffer solution as solvent) was prepared
0.0043g of NADPH was dissolved in 50ml of buffer solution to prepare a solution.
(5) Preparing 10mM D, L-glyceraldehyde solution (using buffer solution as solvent)
0.045g D, L-glyceraldehyde was dissolved in 50ml of buffer solution to prepare.
(6) Preparing 20mM D-sodium glucuronate solution (taking buffer solution as solvent)
0.2341g D-sodium glucuronate was dissolved in 50ml of buffer solution 1 to prepare a solution.
(7) Treating the dialysis bag:
firstly, cutting the dialysis bag into small sections with proper length (10-20cm), and cutting into three sections. In a large volume of 2% (W/V) NaHCO3And 1mM dipotassium EDTA (pH 8.0) the dialysis bag was boiled for 10 min.
② the dialysis bag was thoroughly washed with distilled water, and placed in 1mM dipotassium EDTA (pH 8.0) and boiled for 10 min.
③ after cooling, storage at 4 ℃ it is necessary to ensure that the dialysis bag is always immersed in the solution, from which time it must be taken out with gloves. Before use, the dialysis bag is filled with water, then drained and cleaned.
(8) Extraction of ALR 2: the lens was quickly extracted from a normally killed mouse eyeball, then 3 times (0.4ml/lens) cold deionized water (0-4 ℃) in its volume was added, and homogenized with a Glas-Potter homogenizer. The homogenate was centrifuged in a low temperature centrifuge at 12000 Xg at 0-4 ℃ for 30 min. And finally, taking the supernatant, namely the aqueous solution of ALR2, and using the supernatant for enzyme activity test.
(9) Extraction of ALR 1: rats were sacrificed by cervical dislocation, the kidneys were removed rapidly, and 3-fold (3ml/g kidney) volume-cooled 10mM sodium phosphate buffer 1(pH 7.2 containing 0.25M sucrose, 2.0mM dipotassium EDTA, 2.5mM β -mercaptoethanol) (0-4 ℃) was added and homogenized with a Glas-Potter homogenizer. The homogenate was centrifuged in a low temperature centrifuge at 12000 Xg at 0-4 ℃ for 30 min. Collecting supernatant, adding saturated ammonium sulfate solution to obtain 40% ammonium sulfate solution, stirring at 0-4 deg.C for 30min, and centrifuging at 12000 Xg speed for 15 min. The supernatant was taken and the above steps were repeated to achieve 55% saturation of ammonium sulfate and then 75% salt solution, respectively. The precipitate after centrifugation of a 75% saturated ammonium sulfate solution was dissolved in 50 volumes of 10mM sodium phosphate buffer 2(pH 7.2 containing 2.0mM dipotassium EDTA, 2.0mM β -mercaptoethanol) and dialyzed against this buffer overnight. The dialyzed solution is an aqueous solution of ALR1 and is used for enzyme activity test.
(10) Testing of enzyme Activity: 0.25mL of 0.104mM NADPH,0.25mL of 0.1M phosphate buffer solution (pH 6.2),0.1mL of the extracted enzyme solution, and 0.15mL of deionized water were added to a 1mL test cuvette at 30 ℃. To the reference cuvette, 0.25mL of 0.104mM NADPH,0.50mL of 0.1M phosphate buffer solution (pH 6.2),0.1mL of the extracted enzyme solution, and 0.15mL of deionized water were added. Then, two cuvettes containing the mixed solution were placed at 30 ℃ and incubated for 10 min. Finally, 0.25mL of 10mM substrate was added to the test cuvette to start the reaction, which was monitored with an ultraviolet spectrophotometer at 340nm for 5 min. From the obtained data, a straight line was obtained with the absorbance as the vertical axis and time as the horizontal axis, and the slope of the straight line was obtained and was designated as I0And represents enzyme activity. The optimum value of the activity of the enzyme is in the range where the variation in NADPH absorbance is 0.01. + -. 0.0010(ALR2) or 0.015. + -. 0.0010(ALR1) absorbance units/min, and if not, this range is reached by diluting the enzyme solution. The control cuvette was added to the test cuvette to correct for the oxidation of NADPH due to non-enzymatic factors (e.g., oxygen in air also oxidizes NADPH).
(11) Test of percent inhibition of compounds: similar to the assay method, except that 5. mu.L of each test compound solution was added to the test and reference cuvettes without substrate. The slope of the resulting line is denoted as Ix. The percent inhibition at this concentration was then calculated according to the following equation.
I％＝(|I0-Ix|/|I0|)×100％
Repeatedly measuring compound solutions with different concentrations, respectively calculating the inhibition percentage of corresponding concentrations, and obtaining the inhibition percentage versus concentration logarithm "Then reading the logarithm of concentration corresponding to 50% of the percent inhibition from the graph, and obtaining the IC by inverse logarithm50。
TABLE 1 inhibitory Activity of Compound I against ALR2 and ALR1 in vitro
aIC50(nM) (95% C.L.) is the value measured in the experimental system in which the invention was carried out
bIC50(. mu.M) (95% C.L.) is the value measured in the experimental system in which the present invention was carried out
Experiments prove that part of compounds have obvious inhibiting effect on ALR2 in vitro, especially compound 1, IC50The value was 6.9. + -. 2.1nM and these compounds had only weak inhibitory effect on ALR1, indicating high selectivity of these compounds.
Determination of in vitro antioxidant activity of compound by DPPH method
(1) Preparation of 0.025mg/mLDPPH solution
Dissolving 0.025g of DPPH in 1000mL of methanol, and stirring to completely dissolve the DPPH.
(2) Preparation of Compound methanol solution
Different compounds were formulated at different concentrations of 100. mu.M, 50. mu.M, 10. mu.M, 5. mu.M, 1. mu.M, respectively.
(3) Determination of DPPH radical scavenging Rate of Compounds
Procedure for determining DPPH radical scavenging ability of Compounds As shown in Table 2, 0.1mL of compound solution was added to 1mL of DPPH solution, and the solution was made up to 3mL with methanol. Shaking the mixed solution uniformly, reacting at room temperature for 2h, measuring the absorbance value of each sample at the wavelength of 517nm, measuring each sample in parallel for 3 times, taking the average value, calculating the inhibition rate, taking Trolox as a control sample, and measuring the DPPH free radical clearance rate of the compound, wherein the result is shown in Table 3.
The DPPH free radical clearance determination formula of the compound is as follows:
wherein A isiIs the absorbance value after the compound is added; a. thejAdding the absorbance value of the compound without adding DPPH; a. thecFor no compound, only the absorbance of DPPH was added.
TABLE 2 determination procedure for DPPH radical scavenging of Compounds (Unit mL)
TABLE 3 measurement of DPPH radical scavenging ratio of compound
Experiments prove that part of compounds have obvious inhibition effect on DPPH free radicals in vitro, and especially the DPPH free radical clearance rate of the compound 1 is 94.5% under the concentration of 100 mu M, which indicates that the compounds have strong in vitro antioxidant activity.
Example 6 MDA assay of Compounds for in vitro antioxidant Activity
1. Preparation of the solution
(1) Preparation of 20 mu M/mL ferric trichloride solution
0.0027g of ferric chloride (FeCl)3·6H2O), dissolving in 10mL of double distilled water, then taking 1mL of the diluent, adding 4mL of double distilled water, and stirring to completely dissolve to obtain the compound.
(2) Preparation of 100. mu.M/mL vitamin C solution
Dissolving 0.0088g of vitamin C in 10mL of double distilled water, taking 1mL of the solution, adding 4mL of double distilled water, and stirring to completely dissolve the solution to obtain the vitamin C.
(3) Preparation of Compound methanol solution
Preparing methanol solution with concentration of 100 μ M and 50 μ M from different compounds, and stirring for dissolving completely.
2. Preparation of brain homogenate
(1) Rat perfusion is sacrificed, brain tissues are quickly taken out, after the brain tissues are absorbed by filter paper, wet weight is weighed, 2g of the brain tissues are weighed in each experiment, and the brain tissues are added into a manual homogenizer;
(2) adding a certain volume of cold normal saline into a homogenizer, and manually homogenizing for 10 minutes in an ice bath atmosphere;
(3) pouring the homogenate into a centrifuge tube, centrifuging for 10min at 4 ℃ and 3000r/min, taking supernatant, and storing at-20 ℃.
3. Co-incubation of Compounds with brain homogenates
Taking out the prepared homogenate and other solutions, adding 0.5mL of the brain homogenate, the liquid medicine, ferric trichloride, vitamin C and the like into a 1.5mL centrifuge tube according to the table 4 in sequence, mixing uniformly, and adding methanol or double distilled water to the blank tube and the reference tube to complement the volume of the reaction system to be 0.5 mL.
Placing the centrifuge tube in a 37 deg.C water bath, incubating for 30min, and continuously shaking the centrifuge tube for 2-3 times to make the compound and brain homogenate fully act; the centrifuge tube was taken out and placed in ice water, and the following operations were performed according to the kit.
TABLE 4 lipid peroxidation in brain homogenate induced by ferric trichloride vitamin C System (Unit mL)
4. Determination of lipid peroxide MDA content in brain homogenate
(1) The kit comprises the following components in part by weight:
a first reagent: storing the liquid in a bottle of 20ml at room temperature for later use;
and a second reagent: adding a certain amount of double distilled water into each bottle according to the kit specification when the liquid is 12ml liquid, fully and uniformly mixing, and storing in a refrigerator at 4 ℃;
and (3) reagent III: adding the powder into hot double distilled water of 90-100 deg.C, dissolving completely, adding double distilled water to complement volume according to kit instruction, adding glacial acetic acid according to instruction, mixing completely, keeping out of the sun, and storing in 4 deg.C refrigerator;
and (3) standard substance: tetraethoxypropane of 10n mol/mL, stored in a refrigerator at 4 ℃.
(2) Experimental procedure
1) Taking a plurality of centrifuge tubes, making three parallel tubes for each sample, and adding samples according to the table 5;
TABLE 5 determination of lipid peroxide MDA content in brain homogenate sample application table (Unit mL)
2) Shake the tube rack several times and mix well. Then adding other reagents according to the table 6;
TABLE 6 determination of lipid peroxide MDA content in brain homogenate sample application table (Unit mL)
3) Mixing with vortex mixer, tightening test tube with preservative film, pricking a small hole with needle, and boiling in 95 deg.C water bath for 40 min;
4) taking out, cooling by running water, centrifuging for 10 minutes at the speed of 3500-;
5) MDA content calculation formula:
5. the inhibitory rate of the compounds against lipid peroxides in brain homogenates was determined as shown in table 7.
TABLE 7 inhibition of lipid peroxides in brain homogenates by compounds
Experiments prove that part of compounds have obvious inhibition effect on lipid peroxides in brain homogenate, particularly the inhibition percentage of the compounds 1 and 3 on the lipid peroxides is as high as 50 percent, and 53.8 percent of the compounds have strong antioxidant activity.
Example 7 in vivo animal experiments (STZ-induced mice)
(1) Laboratory animals and groups
Streptozotocin (STZ) as a broad-spectrum antibiotic not only has antibacterial and toxic effects on endocrine tumor cells in nerves, but also is widely applied to induction experiments of diabetic mice. The diabetic mouse model used in this experiment was an STZ-induced diabetic mouse.
According to the principle of random grouping, the diabetic mice successfully molded are averagely divided into four groups, wherein each group comprises 10 mice, and the groups comprise a blank group, a low-dose drug group, a high-dose drug group, a metformin positive control group and an epalrestat positive control group.
During the experiment, the mice are raised in the pharmaceutical engineering professional chemical experiment center of Beijing university of science and engineering, and the instruments and food of the experiment are sterilized. The method is characterized in that SPF animal feeding conditions are followed, the temperature is set at 24 ℃, ventilation is carried out, 12 hours of alternate illumination is carried out, mice freely feed and feed water, and the mice cage is kept dry and clean.
(2) Drugs and agents
Preparation of 0.5% CMCNa solution: accurately weighing 0.05g of CMCNa, slowly adding into 100ml of deionized water at 90 ℃ for multiple times, and continuously stirring for 5 hours under heating until the sodium carboxymethylcellulose is completely dissolved. When the solution is at room temperature, pouring the solution into a centrifugal tube of 150ml, sealing the centrifugal tube by using a sealing film, and placing the centrifugal tube in a refrigerator at 4 ℃ for cooling for later use. The gavage amount of the mice is in the volume range of 0.1-0.3ml/10g, the experiment selects 0.15ml/kg for gavage, and the dosage is selected as follows: the low dose group is 80mg/kg, the high dose group is 160mg/kg, the positive control group is 80mg/kg, and the blank group is gavaged according to the corresponding body weight and has different volumes of CMCNa.
Low dose group: accurately weighing 0.267g of compound 1, placing in a mortar, grinding to obtain uniform fine powder without obvious large particles, slowly adding compound into 50ml CMCNa solution, and stirring to obtain 5.33mg/ml solution (80mg/kg mouse weight)
High dose group: accurately weighing 0.534g of compound 1, placing in a mortar, grinding to obtain uniform fine powder without obvious large particles, slowly adding the compound into 50ml of CMCNa solution, and stirring to obtain solution with concentration of 10.66mg/ml (weight of 160mg/kg mouse)
Metformin positive control group: placing two pieces of 0.5 g/piece of metformin into a mortar, grinding to obtain uniform fine powder without obvious large particles, slowly adding the metformin powder into 188ml of CMCNa solution, and stirring to obtain solution with concentration of 5.33mg/ml (80mg/kg mouse weight)
Epalrestat positive control group: accurately weighing metformin 0.267g, placing in mortar, grinding to obtain uniform fine powder without obvious large particles, slowly adding compound into 50ml CMCNa solution, and stirring to obtain solution with concentration of 10.66mg/ml (80mg/kg mouse weight)
Example 8 blood glucose test (Glu)
The common portable glucometer has three analysis methods, namely a glucose oxidase electrochemical method, a glucose oxidase photochemical method and a glucose dehydrogenase electrochemical method, and the glucometer selected in the experiment is the glucose dehydrogenase electrochemical method. The glucose dehydrogenase can specifically catalyze beta-D glucose and NAD+(NADP+) D-gluconolactone and NADH (NADPH) are generated, and the magnitude of the blood sugar value is reflected according to the change of current before and after the reaction.
For the diabetic mice successfully modeled, blood glucose was measured by group administration at 2, 4, 6, 8 weeks after 10 hours of fasting (fasting without water prohibition) and by tail-cutting. When squeezing blood, the patient does not need to exert excessive force so as to prevent partial interstitial fluid from entering the blood, diluting the blood and measuring data in a misaligned mode. And (4) disinfecting the tail shearing part by iodophor to prevent the mouse from being infected.
The data obtained by the above method are as follows:
TABLE 8 results of glucose content experiment in STZ-induced mice
After the mice are successfully modeled, namely at 0 th week, the blood sugar values of five groups of diabetic mice are all around 21.3 mmol/L. The experiment was carried out until the first week, and the blood glucose values of mice in the high, low dose and metformin groups were all decreased to some extent compared to the Ctrl group, in which the blood glucose values of the L-dose and H-dose groups were decreased by 17.7% and 34.9%, respectively. The data analysis shows that the metformin and the compound 1 have obvious blood sugar reducing efficacy (P is less than 0.05). At week 4, mice were still significantly lower in blood glucose compared to Ctrl for the metformin, L-dose and H-dose groups. In week 6, the blood sugar values of all groups are increased compared with day 7, the initial analysis on the blood sugar increase is related to the deterioration of the diabetes of the mice, and with the progress of the experiment, the damage degree of islet beta cells of the mice is deepened, and the utilization rate of glucose by organisms is reduced. The metformin group, the L-dose group and the H-dose group still have obvious effect of reducing the blood sugar of the model mice. The P of the metformin group and the high-low dose group is less than 0.05 by SPSS.17 test. Further illustrating the hypoglycemic effect of compound 1 and metformin. At week 8, metformin still exhibited significant hypoglycemic effects with compound 1. Through 8-week administration and blood sugar tests, the compound 1 has an obvious blood sugar reducing effect on diabetes model mice, and the metformin has a good blood sugar reducing effect.
Example 9.Glucose Tolerance Test (GTT)
(1) Mice were fasted overnight for 16 hours.
(2) Weighing, and taking 0 point blood.
(3) Intraperitoneal injection of 20% glucose 2g/Kg body weight, the specific time of injection for each animal was recorded separately.
(4) Blood glucose measurements were performed at time points 0, 15, 30, 60, 90, 120min, respectively, according to the specific injection time of different animals. The results are shown in Table 5.
TABLE 9 results of STZ-induced glucose tolerance test in mice
As can be seen from the table, blood glucose values of five groups of diabetes model mice are increased rapidly within 30min after glucose injection, and when the blood glucose values of the Metformin group are up to 28.6mmol/L, the L-dose group is up to 46.2mmol/L, the H-dose group is up to 36.5mmol/L, and the Ctrl group is up to 45.9mmol/L at 30 min. The H-dose group reached 38.6mmol/L at 60 min. 30min-60min, except H-dose group and metformin, the blood sugar of other three groups is reduced to different degrees, when 60min, Ctrl group is reduced to 44.5mmol/L, and L-dose group is reduced to 39.6 mmol/L. The peak value of H-dose group is reached at 60min, and blood sugar is reduced at 60-120 min. Blood sugar of the Metformin group reaches a peak value in 90min, and is reduced in 90-120 min. And compared with Ctrl group, the compound 1 and metformin have obvious effect on regulating blood sugar, can promote the absorption and decomposition of glucose and reduce the glucose content in blood.
Analysis of the data shows that the compound 1 and metformin have a good effect of restoring blood sugar of diabetes model mice, and the glucose tolerance is obviously improved, which indicates that the compound 1 and metformin have a certain repairing effect on impaired glucose tolerance of mice.
Example 10 determination of the amount of lipid peroxide MDA in liver
1) The kit comprises the following components in part by weight:
a first reagent: storing the liquid in a bottle of 20ml at room temperature for later use;
and a second reagent: adding a certain amount of double distilled water into each bottle according to the kit specification when the liquid is 12ml liquid, fully and uniformly mixing, and storing in a refrigerator at 4 ℃;
and (3) reagent III: adding the powder into hot double distilled water of 90-100 deg.C, dissolving completely, adding double distilled water to complement volume according to kit instruction, adding glacial acetic acid according to instruction, mixing completely, keeping out of the sun, and storing in 4 deg.C refrigerator;
and (3) standard substance: tetraethoxypropane of 10n mol/mL, stored in a refrigerator at 4 ℃.
2) Experimental procedure
(1) Taking a plurality of centrifuge tubes, making three parallel tubes for each sample, and adding samples according to the table 6;
TABLE 10 determination of lipid peroxide MDA content in plasma sample application Table (Unit mL)
(2) Shake the tube rack several times and mix well. Then adding other reagents according to the table 6;
TABLE 11 measurement of lipid peroxide MDA content in plasma sample application Table (Unit mL)
(3) Mixing with vortex mixer, tightening test tube with preservative film, pricking a small hole with needle, and boiling in 95 deg.C water bath for 40 min;
(4) taking out, cooling by running water, centrifuging for 10 minutes at the speed of 3500-;
(5) MDA content calculation formula:
the data obtained by the above method are as follows:
TABLE 12 STZ Induction of mouse MDA Experimental data
In the experiment, the MDA kit is used for testing the MDA content of the liver tissue of the diabetic mouse after administration for 8 weeks, and the antioxidant capacity and the treatment effect of the medicine are reflected by the MDA content in the liver. The measurement values of the above groups were those after 8 weeks of administration, wherein the normal group and the blank group represent the corresponding doses of the sodium carboxymethylcellulose solution for intragastric administration, respectively. The analysis table shows that at 8 weeks, the MDA value of the normal group mice is 0.557 nmol/mgprot, the Ctrl group is 1.407nmol/mgprot, the L-dose group is 0.468nmol/mgprot, and the H-dose group is 0.774 nmol/mgprot. The L-dose group and the H-dose group have significant difference compared with the Ctrl group (p is less than 0.05) through analysis of statistical software SPSS.17.0; the metformin group was 0.797nmol/mgprot, and the epalrestat group was 0.504 nmol/mgprot. The aldose reductase inhibitor has obvious inhibiting effect on malondialdehyde in the process of oxidative stress.
Example 11 detection of Total SOD Activity in Kidney
1) The kit comprises the following components in part by weight:
a first reagent: preparing a SOD sample preparation solution;
and a second reagent: SOD detection buffer solution;
and (3) reagent III: WST-8;
and (4) reagent IV: an enzyme solution;
and a fifth reagent: a reaction starting liquid (40X);
2) experimental procedure
(1) Preparation of tissue samples: the animals were perfused with normal saline (0.9% NaCl, containing 0.16mg/ml heparin sodium) to remove blood and obtain tissue samples. Taking a proper amount of tissue sample, and homogenizing at 4 deg.C or ice bath (glass homogenizer or various common electric homogenizers can be used) according to the proportion of adding 100 microliters of SOD sample preparation solution into every 10mg of tissue. Centrifuging at 4 deg.C for 3-5min at about 12,000g, and collecting supernatant as sample to be tested.
(2) Preparation of WST-8/enzyme working solution: an appropriate amount of WST-8/enzyme working solution was prepared in a volume of 160. mu.l per reaction. 151. mu.l of SOD detection buffer, 8. mu.l of WST-8 and 1. mu.l of enzyme solution were mixed uniformly to prepare 160. mu.l of WST-8/enzyme working solution.
Preparing a reaction starting working solution: and (3) melting the reaction starting solution (40X) in the kit, uniformly mixing, diluting according to the proportion that 39 mul of SOD detection buffer solution is added into every 1 mul of reaction starting solution (40X), and uniformly mixing to obtain the reaction starting working solution.
(3) The sample wells and various blank control wells were set using a 96-well plate with reference to the following table. And the sample to be tested and the other various solutions were added in the order according to the following table. Adding the reaction starting working solution and fully and uniformly mixing.
(4) Incubate at 37 ℃ for 30 minutes. Absorbance was measured at 450 nm.
(5) SOD activity calculation formula: percent inhibition ═ [ (a blank 1-a blank 2) - (a sample-a blank 3) ]/(a blank 1-a blank 2) × 100%
SOD enzyme activity unit in the sample to be detected is SOD enzyme activity unit in the detection system is inhibition percentage/(1-inhibition percentage) units
TABLE 13 Total SOD Activity measurement sample application Table in Kidney (Unit mL)
The data obtained by the above method are as follows:
TABLE 14 Total SOD Activity test data of Kidney
The experiment utilizes the SOD activity kit to carry out total SOD activity test on kidney tissues of diabetic mice after 8 weeks of administration, and the oxidation resistance and the treatment effect of the medicament are embodied through the total SOD activity of the kidney. The measurement values of the above groups were those after 8 weeks of administration, wherein the normal group and the blank group represent the corresponding doses of the sodium carboxymethylcellulose solution for intragastric administration, respectively. The above table shows that, at 8 weeks, the SOD activity value of the normal group mice is 20.2U/mgprot, Ctrl group is 9.6U/mgprot, L-dose group is 26.7U/mgprot, and H-dose group is 13.3U/mgprot. The statistical software SPSS.17.0 analysis shows that the L-dose group and the H-dose group have significant difference (p is less than 0.05) compared with the Ctrl group; it is shown that the aldose reductase inhibitor can effectively increase SOD activity in oxidative stress process.
Example 12.Determination of Glutathione (GSH) content in kidney
1) The kit comprises the following components in part by weight:
a first reagent: a total glutathione detection buffer solution;
and a second reagent: glutathione reductase;
and (3) reagent III: oxidized glutathione (GSSG);
and (4) reagent IV: DTNB;
and a fifth reagent: a protein-removing agent M;
reagent six: NADPH;
a seventh reagent: DMSO;
and eighth reagent: GSH removal aid;
and a reagent nine: a GSH-clearing agent.
2) Experimental procedure
(1) Preparation of tissue samples: the tissue is taken and quick-frozen by liquid nitrogen, and then ground into powder. For every 10mg of ground tissue powder, 30. mu.l of protein removal reagent M solution was added, and Vortex was performed sufficiently. Then, 70. mu.l of the protein-removing agent M solution was added thereto, and the mixture was thoroughly homogenized with a glass homogenizer (for a tissue which is relatively easy to homogenize, a suitable amount of the protein-removing agent M solution may be directly added thereto for homogenization without subjecting the tissue to treatments such as quick freezing with liquid nitrogen, etc.). After standing at 4 ℃ for 10min, 10,000g of the supernatant was centrifuged at 4 ℃ for 10min, and the supernatant was used for measurement of total glutathione.
(2) Preparing a GSSG content sample to be detected: and taking part of the prepared sample to be tested for the total glutathione content, adding the diluted GSH removal auxiliary liquid according to the proportion of adding 20 mul of the diluted GSH removal auxiliary liquid into each 100 mul of the sample, and immediately mixing by vortex. Then adding GSH removing working solution according to the proportion of adding 4 mul GSH removing reagent working solution into each 100 mul sample, mixing evenly by vortex immediately, and reacting for 60min at 25 ℃. GSH up to 50 μ M can be removed by the above reaction, and if the content of GSH in the sample is too high, the GSH is removed after proper dilution. The above-described processing can be used for subsequent measurement.
(3) Referring to the table below, samples or standards were added sequentially using 96-well plates and mixed well. Adding 150 μ l total glutathione detection working solution, mixing, and incubating at 25 deg.C or room temperature for 5 min.
TABLE 15 GSH content determination in Kidney sample application Table (μ L)
(3) After reacting for 25min (or 30-60min), measuring the absorbance at 412 nm.
The data obtained by the above method are as follows:
TABLE 16 Experimental data for the GSH content in STZ-induced mice
The experiment utilizes the GSH kit to test the GSH content of the kidney tissues of the diabetic mice after 8 weeks of administration, and the antioxidant capacity and the treatment effect of the medicament are embodied through the GSH content in the kidney. The measurement values of the above groups were those after 8 weeks of administration, wherein the normal group and the blank group represent the corresponding doses of the sodium carboxymethylcellulose solution for intragastric administration, respectively. The analysis of the table shows that at 8 weeks, the GSH value of the normal group of mice is 0.158 nmol/mgprot, the Ctrl group is 0.085nmol/mgprot, the L-dose group is 0.227nmol/mgprot, and the H-dose group is 0.263 nmol/mgprot. The L-dose group and the H-dose group have significant difference compared with the Ctrl group (p is less than 0.05) through analysis of statistical software SPSS.17.0; the metformin group was 0.170nmol/mgprot, and the epalrestat group was 0.174 nmol/mgprot. It is shown that the aldose reductase inhibitor can increase glutathione content in kidney tissue during oxidative stress.
Example 13.Urine protein/creatinine assay
1. Urine protein content detection
1) The kit comprises the following components in part by weight:
a first reagent: CBB reagents
And a second reagent: 563mg/L Standard protein solution
2) Experimental procedure
(1) Referring to the table below, samples or standards were added sequentially using 96-well plates and mixed well.
TABLE 17 measurement of urine protein content sample application Table (Unit mL)
(2) The calculation formula of the urine protein content is as follows:
2. urinary creatinine content detection
1) The kit comprises the following components in part by weight:
a first reagent: enzyme solution A
And a second reagent: enzyme solution B
And (3) reagent III: standard substance (442 mu mol/L)
2) Experimental procedure
(1) Referring to the table below, samples or standards were added sequentially using 96-well plates and mixed well.
TABLE 18 measurement of urine creatinine content sample table (Unit 10 μ L)
(2) Formula for calculating creatinine content
Note:
the data obtained by the above method are as follows:
TABLE 12 STZ induced mouse urokinase protein/creatinine experimental data
The experiment utilizes a urine protein content and creatinine content detection kit to measure the urine protein/creatinine level of a diabetic mouse after 8 weeks of administration, and embodies the treatment and protection effects of the drug on the kidney under the hyperglycemia state. The measurement values of the above groups were those after 8 weeks of administration, wherein the normal group and the blank group represent the corresponding doses of the sodium carboxymethylcellulose solution for intragastric administration, respectively. The analysis above shows that at 8 weeks, the urine protein/creatinine value of the normal group mice is 0.546, Ctrl group is 1.012, L-dose group is 0.477nmol/mgprot, and H-dose group is 0.584. The statistical software SPSS.17.0 analysis shows that the L-dose group and the H-dose group have significant difference (p is less than 0.05) compared with the Ctrl group; the metformin group was 0.832, and epalrestat group was 0.893. The medicine can effectively protect and treat the kidney and reduce the level of urine protein under the condition of high blood sugar.
Experimental methods and Experimental results description analysis
The benzoxazolone derivative is a high-efficiency and high-selectivity aldose reductase inhibitor with blood sugar reducing and antioxidant effects, and has application in preparing medicines for preventing and/or treating diabetic complications, especially diabetic nephropathy. In vivo experiments in STZ diabetic mice all demonstrated that Compound 1 is reducing glucose levels in diabetic subjects; meanwhile, the lipid peroxide Malondialdehyde (MDA) content is reduced, the Glutathione (GSH) content and the activity of superoxide dismutase (SOD) are improved, and the obvious antioxidant effect is shown; in addition, the compound 1 effectively reduces the urinary protein level of diabetic mice, and shows the protective and therapeutic effects of the compound 1 on the kidney in a hyperglycemic state.
All references mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the above teachings of the present invention, and such equivalents also fall within the scope of the appended claims. In addition, other modifications within the spirit of the invention will occur to those skilled in the art, and it is understood that such modifications are included within the scope of the invention as claimed.
Claims (7)
1. A compound of formula I or a pharmaceutically acceptable salt thereof or a mixture thereof,
wherein, X is-, or-CH ═ CH-;
R1and R2Each independently selected from hydrogen, hydroxy, methoxy, or trifluoromethyl.
2. The compound of claim 1, wherein the compound is one of the following:
3. a process for preparing a compound of claim 1, a pharmaceutically acceptable salt thereof, or a mixture thereof, comprising the steps of:
(i) taking a compound Ia as a raw material, and taking 1,1' -carbonyldiimidazole as an acylating reagent for reaction, thereby obtaining a compound Ib;
(ii) starting from compound Ib, in the presence of a base, coupling methyl acetate in position 1N by formation of a C-N bond, thereby obtaining compound ic:
(iii) coupling a substituted or unsubstituted aryl, or aralkyl group at the 3-position C by formation of a C-C bond under inert conditions in the presence of a base and a metal catalyst starting from compound ic to give compound id:
(iv) subjecting the compound id as a starting material to hydrolysis reaction to obtain a compound ie;
4. use of the compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof or a mixture thereof for the manufacture of a medicament for the prevention or treatment of diabetic complications.
5. An aldose reductase inhibitor comprising the compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof or a mixture thereof.
6. A pharmaceutical composition for preventing or treating diabetic complications, comprising: a therapeutically effective amount of a compound, pharmaceutically acceptable salt or mixture thereof according to claim 1 or 2 as an active ingredient; and a pharmaceutically acceptable carrier, excipient or sustained release agent.
7. The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition is in the form of a tablet, capsule, granule, syrup, solution, suspension or aerosol, and contains the active ingredient in an amount of 0.01 to 99.9% by weight based on the total weight of the pharmaceutical composition.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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