Hydroxyl chalcone carboxyl derivatives, methods of making and using the same

Abstract

The application discloses a hydroxyl chalcone carboxyl derivative and a preparation method thereof, and provides use of the compound, a pharmaceutically acceptable salt thereof or a mixture thereof in preparation of a medicine for preventing and / or treating diabetes and complications thereof. The compound is used as an aldose reductase selective inhibitor, an alpha-glucosidase inhibitor, a hypoglycemic agent, an antioxidant and a lipid metabolism improver, and has the effects of preventing and / or treating diabetes and complications thereof. The application further provides a pharmaceutical composition containing the compound and having the effects of preventing and / or treating diabetes and complications thereof. In the application, R1 is a hydroxyl group or an alkoxy group, the alkoxy group is preferably a methoxy group or an ethoxy group; R2, R3, R4, R5 and R6 are independently selected from hydrogen, halogen, a hydroxyl group, a methoxy group or a trifluoromethyl group, and at least one of R2, R3, R4, R5 and R6 is a hydroxyl group; and the halogen is preferably fluorine.

Description

Technical Field

[0001] This invention relates to the fields of organic chemistry, medicinal chemistry, pharmacology, and pharmaceuticals, specifically to a series of hydroxychalcone carboxyl derivatives and their preparation methods, to the application of hydroxychalcone carboxyl derivatives as aldose reductase inhibitors, α-glucosidase inhibitors, antioxidants, lipid metabolism modifiers, and hypoglycemic agents, and to the use of hydroxychalcone carboxyl derivatives in the preparation of drugs for the prevention and / or treatment of diabetes and its complications. Background Technology

[0002] Diabetes is a prevalent disease second only to cancer in its severity. Over the past decade, while the overall mortality rates of the four major non-communicable chronic diseases—cancer, cardiovascular disease, diabetes, and chronic respiratory diseases—have been declining year by year, the mortality rate from diabetes has been on the rise. Due to its high incidence and mortality rates, diabetes has become a serious health problem worldwide. Population growth and aging have exacerbated the rising prevalence of diabetes.

[0003] Diabetes mellitus is a chronic metabolic disease, most of which are accompanied by serious degenerative complications such as peripheral neuropathy, nephropathy, retinopathy, cataracts, atherosclerosis, and myocardial infarction. Due to the high rates of disability and mortality associated with diabetes complications, it has become one of the major threats to the health and lifespan of residents in most countries worldwide. In fact, the prevention and control of diabetes, especially its complications, remains a challenging problem due to the lack of targeted medications.

[0004] In tissues with insulin-independent glucose transport (retina, lens, kidneys, and nerves), increased glucose metabolic flux via the polyol pathway under hyperglycemic conditions is a well-known and important factor associated with secondary diabetic complications. Aldose reductase (ALR2) is a key rate-limiting enzyme in the polyol pathway. Studies have shown that enhanced polyol pathway activity, abnormal accumulation of its intermediate sorbitol, and increased oxidative stress downstream of this pathway are closely related to diabetic complications. Numerous animal and clinical trials have demonstrated that aldose reductase inhibitors are a promising approach for treating diabetic complications. Over the past 30 years, at least 14 aldose reductase inhibitors have entered clinical trials, but all have failed due to insufficient clinical efficacy. Analysis suggests that this is because single aldose reductase inhibition is insufficient to address the multifactorial causes of diabetic complications. Epalrestat, an aldose reductase inhibitor, is currently the only marketed drug for diabetic complications, specifically targeting peripheral neuropathy associated with diabetes. It was first marketed in Japan and has recently entered the Chinese and Indian markets.

[0005] Therefore, developing multifunctional aldose reductase inhibitors with hypoglycemic and antioxidant activities could simultaneously control blood sugar, inhibit the abnormal accumulation of sorbitol and oxidative stress, and thus have the potential to develop into highly effective or specific drugs for the prevention and treatment of diabetes and its complications.

[0006] Furthermore, α-glucosidase is a membrane-bound enzyme located on the brush border membrane of the small intestine. It catalyzes the hydrolysis of the non-reducing terminal glycosidic bonds of polysaccharides, breaking them down into the monosaccharide glucose. α-glucosidase inhibitors, by inhibiting this enzyme's catalytic action in hydrolyzing polysaccharides into monosaccharides, can slow down the absorption of carbohydrates in the intestine, thereby controlling blood glucose. Therefore, integrating α-glucosidase inhibition into aldose reductase inhibitors to control blood glucose is also an important aspect of developing the aforementioned multifunctional aldose reductase inhibitors. Summary of the Invention

[0007] This invention provides a compound or a pharmaceutically acceptable salt thereof, characterized in that the compound has the following structural formula.

[0008]

[0009] Wherein, R1 is a hydroxyl or alkoxy group, preferably a methoxy or ethoxy group; R2, R3, R4, R5, and R6 are independently selected from hydrogen, halogen, hydroxyl, methoxy, or trifluoromethyl, and at least one of R2, R3, R4, R5, and R6 is a hydroxyl group; the halogen is preferably fluorine.

[0010] This invention provides a compound of formula I above or a pharmaceutically acceptable salt thereof, characterized in that the compound is one of the following compounds:

[0011]

[0012] This invention provides a method for preparing the compound of formula I above or a pharmaceutically acceptable salt thereof, characterized in that the method comprises the following steps:

[0013] (i) Using formula II as a starting material, react with acylating agent 1,1′-carbonyldiimidazole to generate compound III;

[0014]

[0015] (ii) Using a compound of formula III as a starting material, reacting it with an alkylating agent in the presence of a base such as potassium carbonate, and coupling an alkyl acetate to the N at the 1-position through the formation of a CN bond, thereby forming a compound of formula IV. The alkylating agent is an alkyl bromoacetate, or it may be an alkyl chloroacetate or an alkyl iodoacetate, and the alkyl acetate is methyl acetate or ethyl acetate, etc.; wherein, R in formula IV is an alkyl group, and the alkyl group is methyl or ethyl, etc.

[0016]

[0017] (iii) Using compound IV as a starting material, in the presence of a dehydrating agent or a dehydroxylating agent, reacting it with benzaldehyde containing the corresponding substituents, wherein the corresponding substituents are R2, R3, R4, R5 and R6 in formula I, to construct an aryl-substituted α,β-unsaturated ketone at the 7-position C through the formation of a carbon-carbon double bond, thereby generating the carboxylic acid ester form of compound I, or subsequently generating the carboxylic acid form of compound I through a hydrolysis reaction in the presence of a base; wherein the dehydrating agent or dehydroxylating agent is preferably sulfoxide dichloride, and the base is preferably sodium hydroxide;

[0018]

[0019] The present invention provides the use of the compound of Formula I above or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of diabetes and its complications.

[0020] The present invention provides an aldose reductase inhibitor, an α-glucosidase inhibitor, a hypoglycemic agent for diabetic animals or human patients, an antioxidant, and a lipid metabolism improver for diabetic animals or human patients, characterized in that it comprises a compound as described in Formula I above or a pharmaceutically acceptable salt thereof.

[0021] The present invention provides a pharmaceutical composition for the prevention and / or treatment of diabetes and its complications, characterized in that the pharmaceutical composition comprises: a therapeutically effective amount of the compound of formula I above, a pharmaceutically acceptable salt as the active ingredient; and a pharmaceutically acceptable carrier, excipient or sustained-release agent.

[0022] This invention provides the above-described pharmaceutical composition, characterized in that the pharmaceutical composition is a tablet, capsule, granule, syrup, solution, suspension, or aerosol, and the active ingredient therein accounts for 0.01-99.9% of the total weight of the pharmaceutical composition. Active ingredient:

[0023] As used herein, the terms “active ingredient,” “active compound,” “compound of the present invention,” “aldose reductase inhibitor,” “α-glucosidase inhibitor,” “antioxidant,” “hypoglycemic agent,” and “lipid metabolism improver” are used interchangeably and refer to the hydroxychalcone carboxyl derivatives of the present invention having the structural formula I and their pharmaceutically acceptable salts.

[0024] Pharmaceutical composition:

[0025] The present invention also provides pharmaceutical compositions for the prevention and / or treatment of diabetes and its complications, comprising:

[0026] (a) The active ingredient is a preventive and / or therapeutically effective amount of the hydroxychalcone carboxyl derivative of Formula I and its pharmaceutically acceptable salts.

[0027] (b) Pharmaceutically acceptable carriers, excipients or sustained-release agents.

[0028] In this invention, the term "containing" indicates that various ingredients may be used together in the mixtures or compositions of this invention. Therefore, the terms "consistent with..." and "composed of..." are both included in the term "containing".

[0029] In this invention, a "pharmaceuticalally acceptable" ingredient is a substance that is suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., has a reasonable benefit / risk ratio.

[0030] In this invention, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, suspending agent, or excipient used to deliver the active substance of this invention or its physiologically acceptable salt to humans and / or animals. The carrier may be solid or liquid.

[0031] The pharmaceutical composition of the present invention contains an active ingredient comprising 0.01-99.9% 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%.

[0032] The present invention has the following beneficial effects:

[0033] The hydroxychalcone carboxyl derivatives of structural formula I provided by this invention have a simple structure, are easy to synthesize, use inexpensive and readily available raw materials, have a short synthetic route, mild reaction conditions, and are easy to implement. In vitro experiments have confirmed that these compounds have excellent inhibitory effects on aldose reductase, α-glucosidase, free radicals, and lipid peroxidation, and exhibit weak inhibitory effects on aldehyde reductase (ALR1), indicating that the hydroxychalcone carboxyl derivatives of this invention are highly efficient, highly selective, and low-toxicity multifunctional aldose reductase inhibitors, as well as potent α-glucosidase inhibitors. Furthermore, experiments using diabetic rats as an animal model showed that these compounds significantly reduced blood glucose levels, improved glucose tolerance (GTT), inhibited aldose reductase activity and the production of its product sorbitol, reduced the content of lipid peroxides (MDA), increased glutathione (GSH) content, enhanced superoxide dismutase (SOD) activity, and improved triglyceride and total cholesterol levels in diabetic rats. Therefore, these compounds possess multi-target effects, including hypoglycemic activity, inhibition of aldose reductase activity and the accumulation of its product sorbitol, inhibition of α-glucosidase, antioxidant effects against oxidative stress, and improvement of lipid metabolism. These compounds are suitable for use in the preparation of highly effective drugs for the prevention and / or treatment of diabetes and its complications. Furthermore, unlike traditional aldose reductase inhibitors which are primarily carboxylic acids, the hydroxychalcone carboxyl derivatives provided in this invention, in a non-free carboxylic acid state, can exert potent aldose reductase inhibitory effects both in vitro and in vivo, thus also exhibiting the aforementioned multifunctional effects. Detailed Implementation

[0034] The present invention will be further described below through specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not depart from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0035] In order to address the problems existing in the prior art as stated in the background section, and to develop a highly efficient, selective, and low-toxicity multifunctional aldose reductase and α-glucosidase inhibitor that combines hypoglycemic and antioxidant properties, and which is useful for preparing a drug for the prevention and / or treatment of diabetes and its complications, the inventors of this invention have innovatively proposed a compound of structural formula I, thereby obtaining a drug for the prevention and / or treatment of diabetes and its complications.

[0036] Based on the above concept, the present invention provides compounds represented by Formula I and their pharmaceutically acceptable salts.

[0037]

[0038] In formula I, R1 is a hydroxyl or alkoxy group, preferably a methoxy or ethoxy group; R2, R3, R4, R5, and R6 are independently selected from hydrogen, halogen, hydroxyl, methoxy, or trifluoromethyl, and at least one of R2, R3, R4, R5, and R6 is a hydroxyl group; the halogen is preferably fluorine.

[0039] When R2, R3, R4, R5, and R6 are all hydroxyl groups, the compound exhibits strong inhibitory effects on aldose reductase and α-glucosidase in in vitro experiments, as well as strong antioxidant effects. In diabetic animal models, this class of hydroxychalcone carboxyl derivatives demonstrates potent aldose reductase inhibition and antioxidant capacity, excellent hypoglycemic ability, and the ability to improve glucose tolerance, and also shows the ability to improve lipid metabolism. The presence of polyphenolic hydroxyl groups endows this compound with the ability to scavenge free radicals, thus producing antioxidant effects.

[0040] The present invention also provides a method for preparing the compound shown in Formula I above, the method comprising the following three steps S1 to S3 in the following synthetic formula: 3-amino-2-hydroxyacetophenone II reacts with N,N′-carbonyldiimidazole to generate compound III (S1); compound III reacts with an alkylating agent such as alkyl bromoacetate in the presence of a base to generate compound IV (S2); compound IV reacts with a correspondingly substituted benzaldehyde in the presence of a dehydrating agent or a dehydroxylating agent such as sulfoxide to generate the target compound I of the alkyl carboxylic acid ester type, which can also undergo further ester hydrolysis reaction in the presence of a base to generate the target compound I of the carboxylic acid type (S3).

[0041]

[0042] In the synthetic formula, R is an alkyl group; R1 is hydrogen or an alkyl group; R2, R3, R4, R5, and R6 are independently selected from hydrogen, halogen, hydroxyl, methoxy, or trifluoromethyl, and at least one of R2, R3, R4, R5, and R6 is a hydroxyl group; the halogen is preferably fluorine.

[0043] S1: The acylation reagent used in the reaction is 1,1′-carbonyldiimidazole; the solvent can be N,N-dimethylformamide, acetonitrile, tetrahydrofuran, etc.; the reaction temperature is 60℃-90℃.

[0044] S2: The alkylating agent used in the reaction is an alkyl bromoacetate, or an alkyl chloroacetate or an alkyl iodoacetate, wherein the alkyl group can be methyl or ethyl, etc.; the base can be potassium carbonate, sodium carbonate, cesium carbonate, etc., in addition to potassium carbonate; the solvent can be acetonitrile, N,N-dimethylformamide, tetrahydrofuran, dioxane, etc., in addition to N,N-dimethylformamide; the reaction temperature is 60℃-90℃.

[0045] S3: The dehydrating or dehydroxylating reagent used in the reaction is sulfoxide, which can also be sulfuric acid, oxaloyl chloride, phosphorus trichloride, Burgess reagent (i.e., methyl N-(triethylammonium sulfonyl)carbamate), or Martinsulfurane reagent (i.e., bis[a,a-bis(trifluoromethyl)phenylethanol]-diphenylsulfonate); the reaction temperature is 10℃-100℃; the solvent, in addition to ethanol, can also be acetonitrile, tetrahydrofuran, dioxane, methanol, etc. The base used in the hydrolysis reaction of the alkyl carboxylic acid ester can be sodium hydroxide, lithium hydroxide, potassium hydroxide, etc.; the solvent used, in addition to tetrahydrofuran, can also be dioxane, ethanol, methanol, water, etc.; the reaction temperature is 0℃-30℃.

[0046] Furthermore, this invention provides the in vitro and in vivo biological effects of these compounds in diabetic rats. In vitro, these compounds significantly inhibit the activities of both aldose reductase and α-glucosidase, while exhibiting weak inhibition of aldose reductase. They also inhibit the formation of lipid peroxides (MDA) and quench DPPH free radicals. In diabetic rats, these compounds significantly reduce blood glucose levels, enhance glucose tolerance (GTT), inhibit aldose reductase activity, decrease lipid peroxide and sorbitol levels, increase glutathione (GSH) levels and superoxide dismutase (SOD) activity, and improve lipid metabolism.

[0047] Example

[0048] The present invention is further illustrated below with examples. These examples are for illustrative purposes only and are not intended to limit the invention in any way. All parameters and statements in the examples are based on quality, unless otherwise stated. Test methods not specifically described in the examples are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0050] Example 1: Preparation of (E)-2-(7-(3-(3,4-dihydroxyphenyl)acryloyl)-2-oxobenzo[d]oxazol-3(2H)-yl)methyl acetate (compound 1)

[0051] 3-Amino-2-hydroxyacetophenone (1.0 mol) and N,N′-carbonyldiimidazole (1.6 mmol) were dissolved in 3 mL of N,N-dimethylformamide, and the solution was heated to 60 °C and stirred for 2 h. After the reaction was complete, the mixture was poured into 50 mL of water, the precipitate was filtered and dried under vacuum to give the product 7-acetylbenzo[d]oxazol-2(3H)-one. Yellow solid. Yield: 0.150 g (85%); 1H NMR (400 MHz, Chloroform-d) δ 8.22 (s, 1H), 7.71 (dd, J = 5.3, 4.0 Hz, 1H), 7.25 (d, J = 4.2 Hz, 2H), 2.77 (s, 3H).

[0052] The above product, 7-acetylbenzo[d]oxazol-2(3H)-one (1.0 mol), methyl bromoacetate (1.2 mmol), and Na₂CO₃ (2.0 mmol), were dissolved in 5 mL of acetonitrile, and the solution was heated to 65 °C and stirred for 2 h. After the reaction was complete, the mixture was filtered and the solvent was evaporated under vacuum. The product, methyl 2-(7-acetyl-2-oxobenzo[d]oxazol-3(2H)-yl)acetate, was recrystallized from ethyl acetate and petroleum ether. It was a yellow solid. Yield: 0.216 g (87%). 1 H NMR (400 MHz, Chloroform-d) δ 7.71 (dd, J = 8.2, 1.2 Hz, 1H), 7.26 (t, J = 8.0 Hz, 1H), 7.08 (dd, J = 7.7, 1.2 Hz, 1H), 4.63 (s, 2H), 3.82 (s, 3H), 2.76 (s, 3H).

[0053] The above-mentioned methyl 2-(7-acetyl-2-oxobenzo[d]oxazol-3(2H)-yl)acetate (1 mmol), SOCl2 (0.1 mL), and 3,4-dihydroxybenzaldehyde (1.2 mmol) were added to 1 mL of ethanol. The mixture was stirred at room temperature for 2 h. After the reaction was complete, the reaction mixture was poured into 50 mL of water and extracted three times with 30 mL of ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, filtered, the solvent was removed by distillation, and purified by column chromatography (petroleum ether:ethyl acetate = 4:1-2:1) to give the target product (compound 1). Yellow solid. Yield: 0.261 g (71%); Melting point: 225-228 °C; Purity: 100%; 1H NMR (400MHz, DMSO-d6) δ9.52 (d, J=145.8Hz, 2H), 7.72-7.63 (m, 2H), 7.57 (dd, J=7.8, 1.3Hz, 1H), 7.48-7.36 (m, 2H), 7.22 (d, J=2.1Hz, 1H), 7.13 (dd, J= 8.2, 2.1Hz, 1H), 6.83 (d, J=8.2Hz, 1H), 4.86 (s, 2H), 3.74 (s, 3H). 13 C NMR (101MHz, DMSO) δ186.21, 168.24, 153.86, 149.78, 146.24, 146.07, 141.04, 132.34, 126.32, 124.54, 123.35, 123.13, 122.18, 120.90, 116.36, 115.24, 113.81, 53.08, 43.49; HRMS(ESI)m / z calcd for[M+H] + 370.0927, found 370.0918.

[0054]

[0055] Example 2: Preparation of (E)-2-(7-(3-(3,5-difluoro-4-hydroxyphenyl)acryloyl)-2-oxobenzo[d]oxazol-3(2H)-yl)methyl acetate (compound 2)

[0056] The product was prepared according to the preparation method described in Example 1, replacing 3,4-dihydroxybenzaldehyde in Example 1 with 3,5-difluoro-4-hydroxybenzaldehyde. It was a yellow solid. Yield: 0.116 g (30%); Melting point: 208-210 °C; Purity: 96.72%. 1 H NMR (400MHz, DMSO-d6) δ10.96 (s, 1H), 7.75 (dd, J=8.1, 1.2Hz, 1H), 7.67 (s, 2H), 7.65 (dd, J=8.0, 1.8Hz, 2H), 7.61 (d, J=1.2Hz, 1H), 7.39 (t, J=7.9Hz, 1H), 4.87 (s, 2H), 3.75 (s, 3H); 13C NMR (101MHz, DMSO) δ186.83, 168.23, 153.96, 151.50, 143.33, 141.09, 136.82, 132.40, 125.64, 124.50, 123.91, 123.31, 121.97, 114.09, 113.15, 53.08, 43.45; HRMS(ESI)m / z calcd for[M+H] + 390.0789, found 390.0786.

[0057]

[0058] Example 3: Preparation of (E)-2-(7-(3-(4-hydroxyphenyl)acryloyl)-2-oxobenzo[d]oxazol-3(2H)-yl)methyl acetate (compound 3)

[0059] The product was prepared according to the method described in Example 1, replacing 3,4-dihydroxybenzaldehyde with 4-hydroxybenzaldehyde. It was a yellow solid. Yield: 0.268 g (76%); Melting point: 191-193 °C; Purity: 100%. 1 H NMR (400 MHz, DMSO-d6) δ10.16 (s, 1H), 7.75-7.67 (m, 4H), 7.61-7.53 (m, 2H), 7.38 (t, J=7.9 Hz, 1H), 6.88 (s, 1H), 6.85 (s, 1H), 4.86 (s, 2H), 3.74 (s, 3H); 13 C NMR (101MHz, DMSO) δ186.58, 168.24, 160.99, 153.90, 145.62, 141.01, 132.35, 131.54, 125.89, 1 24.52, 123.14, 122.24, 121.09, 116.48, 113.80, 53.08, 43.47; HRMS (ESI) m / z calcd for[M+H] + 354.0977, found 354.0968.

[0060]

[0061] Example 4: Preparation of (E)-2-(7-(3-(4-hydroxyphenyl)acryloyl)-2-oxobenzo[d]oxazol-3(2H)-yl)acetic acid (compound 4)

[0062] Hydrolysis of the acetate ester in the structure of compound 3 yielded the corresponding carboxylic acid compound, compound 4. Methyl (E)-2-(7-(3-(4-hydroxyphenyl)acryloyl)-2-oxobenzo[d]oxazol-3(2H)-yl)acetate (compound 3) (1 mmol) and saturated LiOH (5 mL) were added to THF and stirred at room temperature for 2 h. After the reaction was complete, 0.1 N HCl was added to acidify to pH 3. The suspension was extracted with EtOAc (3 × 15 mL), the organic phase was collected, dried over MgSO4, and evaporated under vacuum. The residue was recrystallized from ethyl acetate to give (E)-2-(7-(3-(4-hydroxyphenyl)acryloyl)-2-oxobenzo[d]oxazol-3(2H)-yl)acetic acid (compound 4). Yellow crystals. Yield: 72% (0.244 g); 1 H NMR (400MHz, DMSO-d): δ10.12 (s, 1H), 8.09 (d, J=1.7Hz, 1H), 8.07-8.00 (m, 1H), 7.79-7.70 (m, 4H), 7.56 (dd, J=8.4, 5.1Hz, 1H), 6.91-6.82(m, 2H), 4.78(s, 2H); 13 C NMR (101 MHz, DMSO): δ 192.67, 173.93, 165.46, 159.10, 150.29, 139.54, 136.29, 131.00, 129.31, 123.38, 121.07, 114.70, 48.35. HRMS(ESL)m / z calcd for[M+H] + 340.0797, found 339.0743.

[0063]

[0064] Example 5: Compound 1 and Compound 2 were subjected to hydrolysis reactions according to the method in Example 4 to obtain the corresponding carboxylic acid compounds.

[0065] Example 6: In vitro inhibitory effects of the compound on ALR2 and ALR1

[0066] The experiment used phosphate buffer solution for ALR2 determination, sodium phosphate buffer 1 and sodium phosphate buffer 2 for ALR1 determination, NADPH solution, D,L-glyceraldehyde solution, and D-glucuronide solution. Their preparation methods are as follows:

[0067] (1) Prepare a 0.1M phosphate buffer solution for ALR2 determination at pH 6.2.

[0068] Solution A: Dissolve 3.12g NaH2PO4·2H2O in 100ml of water to prepare a 0.2M solution;

[0069] Solution B: Dissolve 3.58g Na2HPO4·12H2O in 50ml of water to prepare a 0.2M solution.

[0070] Take 81.5 ml of A and 18.5 ml of B, dilute with water to a final volume of 200 ml, and adjust the pH to 6.2.

[0071] (2) Prepare a 10 mM phosphate buffer solution at pH 7.2 for ALR1 determination.

[0072] Dissolve 0.3801g sodium phosphate, 8.5513g sucrose, 0.0809g dipotassium EDTA, and 0.0175mL β-mercaptoethanol in 100mL of water and adjust the pH to 7.2 to obtain the final product.

[0073] (3) Prepare a 10 mM phosphate buffer solution at pH 7.2 for ALR1 determination.

[0074] Dissolve 0.3801g sodium phosphate, 0.0809g dipotassium EDTA, and 0.0140mL β-mercaptoethanol in 100mL of water and adjust the pH to 7.2 to obtain the solution.

[0075] (4) Prepare a 0.104 mM NADPH solution (using a buffer solution as the solvent).

[0076] It was prepared by dissolving 0.0043g of NADPH in 50ml of buffer solution.

[0077] (5) Prepare a 10 mM D, L-glyceraldehyde solution (using a buffer solution as the solvent).

[0078] It was prepared by dissolving 0.045g of D,L-glyceraldehyde in 50ml of buffer solution.

[0079] (6) Prepare a 20mM sodium D-glucuronide solution (using a buffer solution as the solvent).

[0080] 0.2341g of sodium D-glucuronide was dissolved in 50ml of buffer solution 1 to prepare the solution.

[0081] (7) Processing dialysis bags:

[0082] ① Cut the dialysis bag into three sections of appropriate length (10-20cm). Boil the dialysis bag in a large volume of 2% (w / v) NaHCO3 and 1mM EDTA dipotassium salt (pH=8.0) for 10 minutes.

[0083] ② Thoroughly rinse the dialysis bag with distilled water, place it in 1mM EDTA dipotassium salt (pH=8.0), and boil for 10 minutes.

[0084] ③ After cooling, store at 4℃, ensuring the dialysis bag remains submerged in the solution at all times. Gloves must be worn when handling the dialysis bag at this temperature. Before use, fill the dialysis bag with water, then drain and clean it thoroughly.

[0085] (8) Extraction of ALR2: Lenses were rapidly extracted from the eyeballs of normally euthanized mice, and then 3 times their volume (0.4 ml / lens) of cold deionized water (0-4℃) was added. The mixture was then homogenized using a Glas-Potter homogenizer. The homogenate was centrifuged at 12000×g at 0-4℃ for 30 min in a low-temperature centrifuge. The supernatant was collected as the aqueous solution of ALR2 for enzyme activity testing.

[0086] (9) Extraction of ALR1: Rat euthanized by cervical dislocation, and the kidney was quickly removed. Three times (3 ml / g kidney) of the kidney volume of cooled 10 mM sodium phosphate buffer 1 (pH = 7.2, containing 0.25 M sucrose, 2.0 mM EDTA dipotassium salt, and 2.5 mM β-mercaptoethanol) (0-4℃) was added, and the mixture was homogenized using a Glas-Potter homogenizer. The homogenate was centrifuged at 12000 × g at 0-4℃ for 30 min. The supernatant was collected, and saturated ammonium sulfate solution was added to form a 40% saturated ammonium sulfate solution. The solution was stirred at 0-4℃ for 30 min and then centrifuged at 12000 × g for 15 min. The supernatant was used to repeat the above steps, first to achieve 55% ammonium sulfate saturation, and then to achieve 75% salt solution. The precipitate obtained after centrifugation of a 75% saturated ammonium sulfate solution was dissolved in 50 volumes of 10 mM sodium phosphate buffer 2 (pH 7.2, containing 2.0 mM EDTA dipotassium salt and 2.0 mM β-mercaptoethanol), and dialyzed overnight with this buffer. The resulting aqueous solution of ALR1 was used for enzyme activity testing.

[0087] (10) Determination of enzyme activity: At 30°C, add 0.25 mL of 0.104 mM NADPH, 0.25 mL of 0.1 M phosphate buffer solution (pH = 6.2), 0.1 mL of the extracted enzyme solution, and 0.15 mL of deionized water to a 1 mL test cuvette. Add 0.25 mL of 0.104 mM NADPH, 0.50 mL of 0.1 M phosphate buffer solution (pH = 6.2), 0.1 mL of the extracted enzyme solution, and 0.15 mL of deionized water to a reference cuvette. Then, place both cuvettes containing the above mixtures at 30°C for 10 min. Finally, add 0.25 mL of 10 mM substrate to the test cuvette to start the reaction, and monitor the reaction at 340 nm using a UV spectrophotometer for 5 min. From the obtained data, a straight line can be obtained by plotting absorbance on the vertical axis and time on the horizontal axis. The slope of this line is calculated and denoted as I0, representing enzyme activity. The optimal enzyme activity is within the range of NADPH absorbance variation of 0.01 ± 0.0010 (ALR2) or 0.015 ± 0.0010 (ALR1) absorbance units / min. If it is not within this range, the enzyme solution should be diluted to bring it into this range. A control cuvette should be added to the test cuvette to correct for NADPH oxidation caused by non-enzymatic factors (such as oxidation of NADPH by oxygen in the air).

[0088] (11) Test for the percentage inhibition of a compound: Similar to the method for measuring enzyme activity, except that 5 μL of the test compound solution is added to both the test cuvette and the reference cuvette before adding the substrate. The slope of the resulting straight line is denoted as Ix. The percentage inhibition at that concentration can then be calculated using the formula below.

[0089] I%=(|I0-I x | / |I0|)×100%

[0090] By repeatedly measuring compound solutions of different concentrations and calculating the inhibition percentage at each concentration, the relationship between the "inhibition percentage" and the "concentration logarithm" can be obtained. Then, the concentration logarithm and antilogarithm corresponding to a 50% inhibition percentage can be calculated to obtain the IC50. 50 .

[0091] Table 1. In vitro inhibitory activity of compound I against ALR2 and ALR1.

[0092]

[0093] In the above table, (95% CL) is the value measured in the experimental system implemented in this invention.

[0094] The results showed that the test compounds had a significant inhibitory effect on ALR2 in vitro, especially compound 4, which had an IC50 value of 4. 50 The values ​​can reach 0.13 nM, and these compounds have only a weak inhibitory effect on ALR1, indicating that these compounds have high selectivity.

[0095] Example 7: Determination of the in vitro antioxidant activity of compounds by DPPH method

[0096] (1) Preparation of 0.025 mg / mL DPPH solution

[0097] Dissolve 0.025g of DPPH in 1000mL of methanol and stir until completely dissolved.

[0098] (2) Preparation of methanol solution of compound

[0099] Different compounds were prepared into different concentrations of 100 μM, 50 μM, 10 μM, 5 μM, and 1 μM.

[0100] (3) Determination of the free radical scavenging rate of compound DPPH

[0101] The procedure for determining the DPPH free radical scavenging ability of the compound is shown in Table 2. 0.1 mL of the compound solution was added to 1 mL of DPPH solution, and methanol was added to bring the total volume to 3 mL. The mixture was shaken well and reacted at room temperature for 2 hours. The absorbance of each sample was measured at 517 nm. Each sample was measured in triplicate, and the average value was used to calculate the inhibition rate. Trolox was used as a control. The results of the DPPH free radical scavenging rate determination are shown in Table 3.

[0102] The formula for determining the DPPH free radical scavenging rate is as follows:

[0103]

[0104] Where A i A represents the absorbance value after the addition of the compound. j Absorbance value of the compound without DPPH; A c The absorbance values ​​are those without any added compounds, only with added DPPH.

[0105] Table 2. Procedure for determining the DPPH free radical scavenging rate of compound (unit: mL)

[0106]

[0107] Table 3 Results of DPPH free radical scavenging rate determination of compounds

[0108]

[0109] The results showed that the test compounds had a significant inhibitory effect on DPPH free radicals in vitro, especially compound 1, which had a DPPH free radical scavenging rate of 41.2% at a concentration of 1 μM, and had strong in vitro antioxidant activity.

[0110] Example 8: Determination of in vitro antioxidant activity of compounds by MDA method

[0111] 1. Solution preparation

[0112] (1) Preparation of 20 μM / mL ferric chloride solution

[0113] Dissolve 0.0027g of ferric chloride (FeCl3·6H2O) in 10mL of double-distilled water, then take 1mL of the diluted solution, add 4mL of double-distilled water, and stir until completely dissolved to obtain the final product.

[0114] (2) Preparation of 100 μM / mL Vitamin C solution

[0115] Dissolve 0.0088g of vitamin C in 10mL of double-distilled water. Take 1mL of this solution, add 4mL of double-distilled water, and stir until completely dissolved.

[0116] (3) Preparation of methanol solution of compound

[0117] Different compounds were prepared into methanol solutions with concentrations of 100 μM and 50 μM, and then stirred until completely dissolved.

[0118] 2. Preparation of brain homogenate

[0119] (1) Rats were euthanized by perfusion, and brain tissue was quickly removed. After being moistened with filter paper, the wet weight was measured. 2 grams were weighed for each experiment and added to a manual homogenizer.

[0120] (2) Add a certain volume of cold physiological saline to the homogenizer and manually homogenize for 10 minutes in an ice bath atmosphere;

[0121] (3) Pour the homogenate into a centrifuge tube and centrifuge at 4℃ and 3000r / min for 10min. Take the supernatant and store it at -20℃.

[0122] 3. Co-incubation of the compound with brain homogenate

[0123] Take out the prepared homogenate and other solutions, and add 0.5 mL of brain homogenate, drug solution, ferric chloride, vitamin C, etc. into a 1.5 mL centrifuge tube according to Table 4. Mix well. For blank tubes and control tubes, methanol or double-distilled water should be added to make up the volume of the reaction system to 0.5 mL.

[0124] Place the centrifuge tube in a 37°C water bath and incubate for 30 minutes, shaking the centrifuge tube 2-3 times during the incubation period to ensure that the compound reacts fully with the brain homogenate. Remove the centrifuge tube and place it in ice water. Proceed with the next steps according to the kit instructions.

[0125] Table 4. Lipid peroxidation in brain homogenate induced by the ferric chloride-vitamin C system (unit: mL)

[0126]

[0127] 4. Determination of MDA (lipid peroxide) content in brain homogenate

[0128] (1) Kit composition and solution preparation:

[0129] Reagent 1: Liquid, 20ml per bottle, store at room temperature for later use;

[0130] Reagent 2: This is a 12ml liquid. When using, add a certain amount of double-distilled water to each vial according to the kit instructions, mix thoroughly, and store in a refrigerator at 4℃.

[0131] Reagent 3: This is a powder. When using it, add the powder to hot double-distilled water at 90-100℃, dissolve it completely, and then make up the volume with double-distilled water according to the kit instructions. Then add glacial acetic acid according to the instructions, mix thoroughly, protect from light, and store in a refrigerator at 4℃.

[0132] Standard: 10 nmol / mL tetraethoxypropane, stored at 4°C.

[0133] (2) Experimental steps

[0134] 1) Take several centrifuge tubes, make three parallel tubes for each sample, and add the samples according to Table 5;

[0135] Table 5. Sample loading table for the determination of lipid peroxide (MDA) content in brain homogenate (unit: mL)

[0136]

[0137] 2) Shake the test tube rack a few times to mix well. Then add the other reagents according to Table 6;

[0138] Table 6. Sample loading table for the determination of lipid peroxide (MDA) content in brain homogenate (unit: mL)

[0139]

[0140] 3) Mix well with a vortex mixer, seal the mouth of the test tube with plastic wrap, poke a small hole with a needle, and place in a 95-degree water bath or boil for 40 minutes.

[0141] 4) Remove, cool with running water, centrifuge at 3500-4000 rpm for 10 minutes, then take the supernatant, and measure the absorbance of each tube at 532 nm with distilled water as the zero point.

[0142] 5) MDA content calculation formula:

[0143]

[0144] 5. The inhibition rate of the compound on lipid peroxides in brain homogenate was measured, and the results are shown in Table 7.

[0145] Table 7. Inhibition rate of compounds on lipid peroxides in brain homogenate.

[0146]

[0147]

[0148] The results showed that the tested compounds had a significant inhibitory effect on lipid peroxides in brain homogenate. Compound 1 showed an inhibition rate of up to 36% against lipid peroxides at a concentration of 1 μM, indicating strong antioxidant activity.

[0149] Example 9: STZ-induced diabetic rats in in vivo animal experiments

[0150] (1) Laboratory animals and grouping

[0151] Streptozotocin (STZ), as a broad-spectrum antibiotic, not only has antibacterial and neurotoxic effects, but is also widely used in induction experiments in diabetic mice.

[0152] Because STZ selectively destroys pancreatic β-cells in certain animal species, it has been used to induce many animal models of diabetes. Rats and mice are commonly used, and repeated verifications have shown that the model establishment rate in male mice is significantly higher than in female mice. This method has advantages such as short model establishment time, stable and persistent symptoms, and minimal damage to the animal's body. The diabetic animal model used in this invention is STZ-induced diabetic rats. Rats were administered a multiple-dose regimen at a dose of 35 mg / kg, once daily for three consecutive days.

[0153] Healthy adult male SD rats (200±5g) were used. The room temperature was controlled at 22-25℃, humidity at 30%, with ventilation, and alternating lighting for 12 hours. Rats had free access to food and water, and their cages were kept dry and clean. After 3 days of acclimatization, rats were fasted for 12 hours and then intraperitoneally injected with STZ (35mg / kg) dissolved in 0.1mM citrate buffer to induce diabetes. Rats serving as the normal control group were injected with citrate buffer. Ten days later, fasting blood glucose was measured; rats with blood glucose levels exceeding 16.7mmol / L were considered diabetic. After removing rats with unacceptable blood glucose levels, the diabetic rat model was established. Diabetic rats were randomly divided into five groups and administered the drugs accordingly: STZ model control group (STZ, n=12), epalrestat group (epalrestat, 80 mg / kg / d, n=10), metformin group (metformin, 100 mg / kg / d, n=10), high-dose compound 1 group (1-H, 160 mg / kg / d, n=10), and low-dose compound 1 group (1-L, 80 mg / kg / d, n=10). These five groups, along with the normal control group (normal, n=6), totaled six groups. The test drugs were suspended in 0.5% sodium carboxymethyl cellulose (CMCNa) solution and administered by gavage (0.5 ml / 100 g) once daily at a fixed time for 28 consecutive days. The STZ group and the normal group were administered 0.5% CMCNa solution (0.5 ml / 100 g) by gavage daily. On day 28, after a 12-hour fast, retroorbital blood samples were collected to determine biochemical parameters. The rats were euthanized, and their livers and lenses were separated and stored at -80°C.

[0154] Drugs and reagents

[0155] 1) 0.5% CMCNa solution: Weigh 0.5g of CMCNa and slowly add it to 100mL of deionized water. Heat at 90℃ and stir until dissolved, then filter. Cool to room temperature, seal, and store in a refrigerator at 4℃ until use.

[0156] Metformin CMCNa solution: Weigh 500 mg of metformin, add 25 mL of CMCNa solution, grind evenly in a mortar, and store in a refrigerator at 4°C for later use.

[0157] 3) Epalrestat CMCNa solution: Weigh 400 mg of epalrestat, add 25 mL of CMCNa solution, grind evenly in a mortar, and store in a refrigerator at 4°C for later use.

[0158] 4) High-dose CMCNa solution of compound 1: Weigh 800 mg of compound 1, add 25 mL of CMCNa solution, grind evenly in a mortar, and store in a refrigerator at 4°C for later use.

[0159] 5) CMCNa solution for low-dose group of compound 1: Weigh 400mg of compound 1, add 25mL of CMCNa solution, grind evenly in a mortar, and store in a refrigerator at 4℃ for later use.

[0160] 6) Preparation of citric acid buffer solution: First, weigh 2.1g of citric acid, add 100mL of deionized water, and dissolve by sonication; then weigh 2.94g of sodium citrate, add 100mL of deionized water, and dissolve by sonication. Mix the two solutions in a 1:1 ratio and shake well. Adjust the pH of the solution to 4.2-4.5 to obtain the target solution, and store it in a refrigerator at 4℃ for later use.

[0161] 7) Preparation of STZ citric acid solution: In an ice bath environment, quickly weigh 100mg STZ and dissolve it in 100mL of citric acid buffer solution. Wrap it in aluminum foil to protect it from light and store it at 0℃ for later use. Filter it with a 0.22μm filter membrane when using it.

[0162] Example 10: Blood Glucose Test (Glu)

[0163] Common portable blood glucose meters employ three analytical methods: glucose oxidase electrochemical method, glucose oxidase photochemical method, and glucose dehydrogenase electrochemical method. This experiment used a blood glucose meter employing the glucose dehydrogenase electrochemical method. Glucose dehydrogenase specifically catalyzes the reaction of β-D-glucose and NAD+ (NADP+) to produce D-gluconolactone and NADH (NADPH). The change in current before and after the reaction reflects the blood glucose level.

[0164] Diabetic rats were administered the drug via gavage for 28 consecutive days in different groups. Blood glucose concentrations were measured using a glucometer-test strip method. On days 0, 7, 14, 21, and 28 after administration, the diabetic rats were fasted for 12 hours (water was allowed but not food), and blood was collected by tail clipping for glucometer measurement. Care was taken not to squeeze the blood excessively to prevent tissue fluid from entering the bloodstream, diluting the blood and causing inaccurate measurements. The tail clipping site was disinfected with povidone-iodine to prevent infection in the rats.

[0165] Table 8. Blood glucose data of diabetic rats after 28 days (unit: mmol / L)

[0166]

[0167] The blood glucose test results are shown in Table 8. In the initial observation period after modeling, the fasting blood glucose values ​​of diabetic rats in all groups were similar, with no significant differences. After 7 days of gavage, the blood glucose values ​​showed a certain upward trend, indicating that the STZ-induced hyperglycemia model damage was ongoing. Comparing the effects of compound 1 on blood glucose in diabetic rats, after 21 days of gavage, the positive control metformin group and the high-dose compound 1 group showed significant inhibition of blood glucose elevation, which was 40.2% and 32.4% lower than the STZ model group, respectively. After another 7 days of gavage, the average blood glucose in the model group continued to rise, while the upward trend of blood glucose in other groups and the positive control group slowed down. In particular, the blood glucose values ​​in the high-dose compound 1 group and the metformin group were significantly lower than those in the model group. Compared with the model group, the high-dose compound 1 group reduced blood glucose by 30.4%, which was close to that of the metformin group (38.5%). In addition, within 28 days, the blood glucose reduction in the high-dose compound 1 group was consistently better than that in the low-dose compound 1 group. This indicates that compound 1 can inhibit the rise of blood glucose in diabetic rats, effectively alleviate diabetic symptoms, and reduce the fasting blood glucose value of diabetic rats in a dose-dependent manner. At the end of day 28, the high-dose group of compound 1 showed a 20.9% decrease. Therefore, compound 1 significantly inhibited the increase of fasting blood glucose in diabetic rats, with the high-dose group showing the best effect, similar to that of metformin.

[0168] Example 11: Glucose Tolerance Test (GTT)

[0169] (1) Blood glucose measurement using glucose oxidase method

[0170] When the blood glucose concentration in diabetic rats is high and exceeds the detection limit of a blood glucose meter, a glucose assay kit can be used to detect the blood glucose concentration in the serum.

[0171] (2) Specific procedures for the rat glucose tolerance test

[0172] After fasting for 12 hours following 28 days of drug administration, patients were administered a 20% glucose solution by gavage at a dose of 2 g / kg body weight. Serum blood glucose levels were measured at 0, 30, 60, and 120 minutes after gavage.

[0173] Rat serum was collected as follows: At the test time point, blood was collected from the orbital vein of the rat. The collected blood was then placed in a 1 mL centrifuge tube and stored at 4°C until the serum separated. After centrifugation, the serum was centrifuged at 3000 rpm for 10 min at 4°C. The supernatant serum was collected, and the blood glucose level was measured according to the glucose assay kit.

[0174] Table 9 Results of STZ-induced glucose tolerance test in rats (unit: mmol / L)

[0175]

[0176] Glucose tolerance (GTT) is the body's ability to regulate blood glucose levels. Under normal circumstances, the body has a strong tolerance to glucose, meaning that even with large amounts of glucose ingested, it can still regulate and maintain blood glucose balance. This is because when glucose enters the body, the elevated blood glucose level stimulates pancreatic β-cells to secrete insulin, thereby promoting glycogen synthesis and inhibiting glycogen breakdown. However, in diabetic patients, due to damage to pancreatic β-cells or insulin resistance, the body cannot effectively regulate blood glucose levels when they rise, resulting in persistently high blood glucose levels, i.e., impaired glucose tolerance. Therefore, under certain circumstances, glucose tolerance can reflect the condition and treatment status of diabetic patients. In the oral glucose tolerance test (OGTT), it can be seen that the blood glucose levels of the control group rats were significantly higher than those of normal rats at all time points after the glucose load, indicating that their glucose tolerance was impaired, which is a typical characteristic of diabetes. As shown in Table 9, the blood glucose level of the control group remained high 60 minutes after the glucose load, while the metformin group and the high-dose compound 1 group showed a significant reduction in blood glucose levels 60 minutes after the glucose load. The intervention effect of the high-concentration group of compound 1 was better than that of the low-concentration group. Therefore, compound 1 and metformin have a good effect on blood glucose recovery in diabetic rats and significantly improve glucose tolerance, indicating that compound 1 and metformin have a repairing effect on impaired glucose tolerance in rats.

[0177] Example 12: Determination of glycated hemoglobin (HbA1c) in diabetic rats

[0178] (1) Pretreatment of red blood cells

[0179] One mL of blood was collected from the orbital vein of rats and placed in a centrifuge tube containing 20 μL of heparin solution (1000 u / mL). The blood sample should be placed on wet ice immediately and centrifuged within 2 hours of collection at 3000 rpm for 10 min at 4°C. The supernatant was discarded, and three volumes of physiological saline at 4°C were added to the red blood cells. The mixture was gently inverted and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the red blood cells were collected as a pellet. This process was repeated 2–3 times until the supernatant became colorless. The red blood cells were then quantitatively collected and stored at -80°C.

[0180] (2) Reagent kit for measuring HbA1c content

[0181] Preparation of hemolysed blood: (I) Take 2 mL of the above red blood cells and add 3 mL of cold double-distilled water, mix thoroughly to prepare hemolysed blood, store at -40℃, and it can be stored for two months. (II) The method for determining the hemoglobin (Hb) concentration of hemolysed blood is as follows: Take 20 μL of hemolysed blood and add 5 mL of reagent IV, mix well, let stand at room temperature for 10 min, and measure the absorbance of each tube at 540 nm using a spectrophotometer; the Hb content (g / mL) is obtained by multiplying the obtained absorbance by 0.3677.

[0182] Acidification: Take a centrifuge tube, add 1 mL of double-distilled water to the blank tube, add 1 mL of hemolyzed blood to the test tube, and then add 0.5 mL of reagent to each tube for acidification.

[0183] Hydrolysis: Wrap the centrifuge tubes tightly with plastic wrap, poke small holes in them, and place them in a boiling water bath for 1 hour.

[0184] Colorimetric analysis: Add 0.5 mL of reagent II to each tube, mix well, and centrifuge at 3000 r / min for 10 min. Take 1 mL of the supernatant, add 0.25 mL of reagent III to each tube, and incubate in a 40℃ water bath for 30 min. After cooling, zero the instrument with double-distilled water, set the wavelength to 443 nm and the optical path length to 1 cm, and measure the absorbance of each tube.

[0185] The formula for calculating HbA1c content is as follows:

[0186]

[0187] Note: Results are expressed as absorbance per 10g of hemoglobin.

[0188] During diabetes, excess glucose in the blood reacts with hemoglobin to form HbA1c. Since hemoglobin has a lifespan of 120 days, it reflects the average blood glucose level over the past two to three months, and the test results are unaffected by temporary fluctuations in blood glucose, making it an important indicator for the clinical diagnosis of diabetes. As shown in Table 10, compared to the normal group, the control group showed a significant increase in HbA1c. After 28 days of oral administration, HbA1c in diabetic rats at all dosage groups was downregulated to varying degrees compared to the control group, with metformin showing a significant difference.

[0189] Table 10 Data on HbA1c, glycogen, ALR2, and sorbitol

[0190]

[0191] Example 13: Determination of liver glycogen in diabetic rats

[0192] A liver sample (approximately 20 mg) was cleaned with physiological saline, blotted dry with filter paper, and weighed. The sample weight (mg) and alkali solution volume (μL) were added to a centrifuge tube at a ratio of 1:3. The centrifuge tube was tightly wrapped with plastic wrap, a small hole was poked in it, and the mixture was heated in a boiling water bath for 20 minutes, followed by cooling in an ice water bath to obtain glycogen hydrolysate. Then, double-distilled water (16 times the weight of the liver) was added to further prepare a 5% glycogen detection solution.

[0193] Perform the assay according to the instructions of the glycogen assay kit (see table below).

[0194] Table 11. Sample loading table for liver glycogen measurement (unit: mL)

[0195]

[0196] The formula for calculating rat liver glycogen content is as follows:

[0197]

[0198] Glycogen is the body's primary storage form of blood glucose; glucose polymers are stored in the liver in the form of glycogen. The synthesis and breakdown of glycogen play a crucial role in controlling blood glucose balance. It absorbs glucose into liver cells and converts it into glycogen to lower blood glucose, and when needed, it degrades stored glycogen into glucose to raise blood glucose. Therefore, the body can directly regulate blood glucose concentration through breakdown and synthesis. Experiments have shown that in a diabetic state, diabetes impairs the liver's normal ability to synthesize glycogen, leading to hyperglycemia. Therefore, glycogen content is closely related to the development of diabetes. As shown in Table 10, compared with the normal group, the liver glycogen content in the control group was significantly reduced, less than one-third of that in the normal group. Compared with the control group, treatment with compound 1 increased liver glycogen content to varying degrees, with the high-dose group showing the most significant effect, increasing liver glycogen content by 36.58% (P < 0.01), far exceeding the effect of metformin. It is evident that compound 1 may lower blood glucose levels in diabetes through pathways such as promoting glycogen synthesis and reducing glycogen breakdown.

[0199] Example 14: Determination of ALR2 activity in vivo

[0200] Take the lens of a rat, add 0.8 mL of pre-cooled deionized water to each lens, and grind in an ice bath. Centrifuge the homogenate and use the supernatant for enzyme activity determination. First, determine the protein content in the lens according to the protein content determination method in Example 16 below. Then, determine the ALR2 activity according to the method described in Example 7. The change in NADPH absorbance represents enzyme activity, and the ratio of enzyme activity to protein content is the desired result.

[0201] Since ALR2 is the first rate-limiting enzyme in the glucose polyol pathway, and sorbitol is an important metabolite of the glucose polyol metabolic pathway and also a product of ALR2-catalyzed glucose metabolism, the concentrations of enzyme protein and sorbitol in vivo are measured to determine the activity of ALR2 in vivo.

[0202] As shown in Table 10, at the end of administration, ALR2 activity of 0.119 nmol / min / mg prot was detected in the lens of diabetic rats, while that of 0.041 nmol / min / mg prot was detected in the lens of normal rats. This indicates that ALR2 activity in diabetic rats was very high, significantly exceeding that in normal rats by more than 2.9 times. This suggests that the polyol pathway is significantly activated in vivo under hyperglycemic conditions. ALR2 activity was normalized by 52.1% in the high-dose group of compound 1 and by 60.8% in the low-dose group of compound 1. These results indicate that ALR2 in the lens of diabetic rats was largely activated and then almost completely restored to normal through treatment with compound 1.

[0203] Example 15: Determination of sorbitol content in red blood cells and lens in vivo

[0204] Quantitatively measure the red blood cell and lens homogenate processed according to the method in Example 12 above, precipitate the protein, and perform the determination according to the sorbitol assay kit method specifications (as shown in the table below).

[0205] Table 12 Sorbitol Content in Rat Erythrocytes (Unit: μL)

[0206]

[0207] First, a standard curve for sorbitol content needs to be determined. Standard solutions of sorbitol are prepared by diluting it with distilled water to concentrations of 4, 2, 1, 0.5, 0.25, 0.125, and 0 mg / mL. Reagents and the sorbitol solution to be tested are added sequentially as shown in Table 5.10. Let y be the standard concentration and x be the absorbance value. A linear regression equation is then plotted, which forms the standard curve. The sorbitol content of the sample can then be calculated.

[0208] Example 16: Determination of GSH, SOD and MDA content in liver tissue

[0209] (1) Pretreatment of rat liver tissue

[0210] Preparation of 10% liver tissue homogenate: After euthanizing diabetic rats by cervical dislocation, liver tissue (approximately 1g) was quickly dissected and removed. The tissue was cleaned with pre-cooled physiological saline, blotted dry with filter paper, and weighed. It was then placed in a dry, clean glass homogenizer. Nine times its weight of physiological saline was pipetted in, and the homogenate was manually ground in an ice-water bath for 15-20 minutes until no obvious liver tissue was visible. Finally, the homogenate was centrifuged at 3000 rpm and 4°C for 10 minutes, and the supernatant was collected to obtain the 10% liver tissue homogenate, which was stored at -40°C for later use.

[0211] (2) Determination of protein content in liver homogenate supernatant

[0212] According to the kit instructions, the optimal concentration for assay is 1%-2% for the tissue homogenate. Therefore, the 10% liver tissue homogenate supernatant was diluted to a 1% liver tissue homogenate at a tissue:physiological saline ratio of 1:9. The assay was performed according to the standard procedure of the protein quantification kit (see table below).

[0213] Table 13. Sample loading table for measuring protein in liver homogenate supernatant using the Coomassie Brilliant Blue method (unit: mL)

[0214]

[0215] The formula for calculating protein content is as follows:

[0216]

[0217] (3) Determination of MDA content in rat liver tissue

[0218] Take 0.1 mL of 10% liver tissue supernatant and perform the assay according to the MDA assay kit specifications (similar to the in vitro MDA assay method for compound antioxidants).

[0219]

[0220] (4) Determination of SOD activity in rat liver tissue

[0221] Take 0.1 mL of liver tissue homogenate supernatant and dilute to the required concentration. Perform the assay according to the superoxide dismutase assay kit specifications (see table below).

[0222] Table 14 SOD Activity Sample Loading Table for Liver Homogenate Supernatant (Unit: μL)

[0223]

[0224] The calculation formula is as follows: first, the inhibition rate needs to be calculated, and then the activity of SOD enzyme can be calculated.

[0225]

[0226]

[0227] Note: The definition of SOD enzyme activity unit adopted in this paper is based on the definition in the kit instructions, that is, the amount of enzyme corresponding to an SOD inhibition rate of 50% in this reaction system is one SOD activity unit (U).

[0228] (5) The kit was used to measure the GSH content in rat liver tissue.

[0229] Take 0.1 mL of liver tissue homogenate supernatant and dilute it to the required concentration. Perform the assay according to the GSH and GSSG detection kit instructions (see table below).

[0230] Table 15. Sample loading table for measuring GSH content in liver homogenate supernatant (unit: μL)

[0231]

[0232] First, a standard curve is prepared: A 10 mM total glutathione stock solution is diluted to a 15 μM GSH scavenging solution using protein removal reagent M. Then, it is diluted sequentially to 10, 5, 2, 1, and 0.5 μM GSSG solutions to create a standard curve. The total glutathione concentration in the sample can be determined using the absorbance measured from the test sample and the standard curve. After measuring the total glutathione concentration in 100 μL of the test sample, 20 μL of GSH scavenging auxiliary solution is added and mixed. Then, 4 μL of GSH scavenging reagent working solution is added and mixed. The mixture is incubated at 25°C for 60 min. By following these steps, the content of oxidized glutathione (GSSG) can be determined, and the GSH content of the test sample can be calculated.

[0233] (6) Analysis of measurement results

[0234] The role of oxidative stress in the pathogenesis of diabetes has been extensively studied, and the role of antioxidants in alleviating diabetes has been confirmed in experimental and clinical studies. Diabetes can produce various manifestations of oxidative stress, including increased lipid peroxidation products and DNA base oxidative damage, and decreased protective antioxidants; oxidative stress can cause and aggravate the occurrence and exacerbation of diabetes and its complications. An imbalance between the body's oxidative and antioxidant systems leads to oxidative stress, which in turn causes cellular lipid peroxidation and ultimately damages lysosomes and mitochondria.

[0235] Table 16 Specific contents of GSH, SOD and MDA in liver tissue

[0236]

[0237] Glutathione (GSH) and the antioxidant enzyme superoxide dismutase (SOD) are key factors in protecting tissues from oxidative damage. GSH participates in the elimination of reactive intermediates by reducing hydrogen peroxide in the presence of glutathione peroxidase. SOD catalyzes the conversion of superoxide radicals into hydrogen peroxide and molecular oxygen, playing a crucial role in resisting cell damage caused by oxygen free radicals, and also inhibiting apoptosis in some cells. MDA is one of the end products of lipid peroxidation produced by free radicals and unsaturated fatty acid metabolism in the body. Increased intracellular free radicals lead to excessive production of MDA in the body, damaging biomembrane structures and thus affecting normal physiological and biochemical reactions. The level of MDA can not only reflect the rate and intensity of lipid peroxidation in the body, but also indirectly reflect the degree of cell damage. Therefore, compound 1 was evaluated by combining MDA, SOD, and GSH as markers of oxidative stress.

[0238] As shown in Table 16, the levels of SOD and GSH in the liver of the Stz control group were less than half that of the normal group, while the MDA level was more than twice that of the normal group, indicating that the diabetic rats experienced an imbalance in the liver's antioxidant system and oxidative stress.

[0239] The effects of the compound on GSH activity in the liver of diabetic rats are shown in Table 16. Under hyperglycemic conditions, abnormal glucose metabolism leads to a significant decrease in GSH levels in the body. Compound 1 can effectively increase liver GSH levels. Compared with the Stz control group, the high-dose and low-dose groups of compound 1 increased by 84.4% and 43.5%, respectively, showing a certain dose-dependent effect. Simultaneously, compound 1 can significantly increase liver SOD activity. Compared with the Stz control group, the high-dose and low-dose groups of compound 1 increased by 76.9% and 33.6%, respectively, demonstrating a strong antioxidant effect.

[0240] Furthermore, compound 1 significantly reduced liver MDA levels (Table 16), with a 47.9% reduction in the high-dose group and a 66.9% reduction in the low-dose group compared to the Stz control group. The results indicate that compound 1 can effectively restore liver MDA levels to normal.

[0241] In addition, while metformin and epalrestat also showed a slight increase in GSH, no significant antioxidant activity was observed.

[0242] Example 16: Determination of triglyceride (TG) and total cholesterol (TC) content in tissues

[0243] 1. The kit measures the TG content in rat liver and serum.

[0244] Take 0.1 mL of liver tissue homogenate supernatant or 2.5 μL of serum and dilute to the required concentration. Perform the assay according to the triglyceride assay kit instructions (see table below).

[0245] Table 17 Sample loading table for TG content determination in rats (unit: μL)

[0246]

[0247] The calculation formula is as follows:

[0248]

[0249] 2. The kit measures the TC content in rat liver and serum.

[0250] Take 0.1 mL of liver tissue homogenate supernatant or 2.5 μL of serum and dilute to the required concentration. Perform the test according to the instructions of the total cholesterol test kit (see table below).

[0251] Table 18 Sample loading table for TC content determination in rats (unit: μL)

[0252]

[0253] The calculation formula is as follows:

[0254]

[0255] Lipid metabolism disorders are an independent damaging factor in various complications of diabetes. Besides hyperglycemia, dyslipidemia is also a risk factor associated with diabetes, and the two are mutually causal. Diabetic patients with glucose metabolism disorders often also have lipid metabolism disorders. Studies have confirmed that lipid metabolism disorders and increased lipid peroxides in the body are closely related to the formation of atherosclerosis. The experimental results of this invention are shown in Table 19. Compared with the normal group, the serum and liver levels of total cholesterol (TC) and triglycerides (TG) in the Stz control group were significantly increased (P < 0.01), indicating that the rats had lipid metabolism disorders. After 28 days of treatment, compared with the Stz control group, the serum TC levels in the high-dose and low-dose groups of compound 1 decreased by 44.1% and 46.0%, respectively, while the TG levels decreased by 56.5% and 44.0%, respectively. The results show that administration for 28 days effectively reduced the serum TC and TG levels. In liver lipid metabolism, the TC levels in the high-dose and low-dose groups of compound 1 decreased by 3.1% and 32.1%, respectively, and the TG levels decreased by 33.8% and 30.6%, respectively.

[0256] These results indicate that the levels of total cholesterol (TC) and triglycerides (TG) in diabetic rats were significantly elevated; compound 1 significantly reduced the levels of triglycerides and cholesterol in the serum of diabetic rats. Therefore, compound 1 can improve hyperlipidemia, a complication of diabetes, and correct lipid metabolism disorders in diabetic rats by reducing serum TC and TG levels.

[0257] Table 19. Specific levels of TG and TC in liver and serum (unit: mmol / L)

[0258]

[0259] Explanation and Analysis of Experimental Methods and Results

[0260] The hydroxychalcone carboxyl derivative of this invention is a highly efficient and selective aldose reductase inhibitor with hypoglycemic and antioxidant effects, and is also an α-glucosidase inhibitor, possessing the potential use in the preparation of drugs for the prevention and / or treatment of diabetes and its complications. In vivo experiments of compound 1 in diabetic rats in the examples clearly showed that this type of compound can reduce blood glucose levels in diabetic patients; improve the polyol glucose metabolism pathway, including inhibiting aldose reductase activity and sorbitol production; exhibit significant antioxidant effects, including reducing the content of the lipid peroxide malondialdehyde (MDA), increasing glutathione (GSH) content and superoxide dismutase (SOD) activity; and improve lipid metabolism.

[0261] Furthermore, it should be understood that after reading the above description of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. In addition, those skilled in the art can make other changes within the spirit of this invention; of course, all such changes made in accordance with the spirit of this invention should be included within the scope of protection claimed by this invention.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that, The compound is one of the following compounds:

2. A method for preparing the compound as described in claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The method includes the following steps: S1: In formula Using it as a raw material, it reacts with the acylating agent 1,1′-carbonyldiimidazole to generate compound III; S2: Using compound III as a starting material, react with an alkylating agent in the presence of sodium carbonate to couple an alkyl acetate onto the N at the 1-position through the formation of a CN bond, thereby forming compound IV. The alkylating agent is an alkyl bromoacetate, and the alkyl acetate is methyl acetate; wherein, R in formula IV is methyl. S3: Using compound IV as a starting material, in the presence of a dehydrating agent or a dehydroxylating agent, reacts benzaldehyde containing the corresponding substituent in ethanol, wherein the benzaldehyde containing the corresponding substituent is 3,4-dihydroxybenzaldehyde, 3,5-difluoro-4-hydroxybenzaldehyde, or 4-hydroxybenzaldehyde, and constructs an aryl-substituted α,β-unsaturated ketone at the 7-position C through the formation of a carbon-carbon double bond, thereby generating compounds 1 to 3, or subsequently generating carboxylic acid compound 4 through the hydrolysis reaction of compound 3 in the presence of a base; wherein the dehydrating agent or dehydroxylating agent is sulfoxide, and the base is saturated LiOH; 3. Use of a compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of diabetes and its complications.

4. An aldose reductase inhibitor, characterized in that, It contains the compound as described in claim 1 or a pharmaceutically acceptable salt thereof.

5. An α-glucosidase inhibitor, characterized in that, It contains the compound as described in claim 1 or a pharmaceutically acceptable salt thereof.

6. A hypoglycemic agent for diabetic animals or human patients, characterized in that, It contains the compound as described in claim 1 or a pharmaceutically acceptable salt thereof.

7. An antioxidant, characterized in that, It contains the compound as described in claim 1 or a pharmaceutically acceptable salt thereof.

8. A lipid metabolism modifier for diabetic animal or human patients, characterized in that, It contains the compound as described in claim 1 or a pharmaceutically acceptable salt thereof.

9. A pharmaceutical composition for the prevention and / or treatment of diabetes and its complications, characterized in that, The pharmaceutical composition comprises: a therapeutically effective amount of the compound of claim 1, a pharmaceutically acceptable salt as the active ingredient; and a pharmaceutically acceptable carrier, excipient, or sustained-release agent.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition is in the form of tablets, capsules, granules, syrups, solutions, suspensions, or aerosols, and the active ingredient therein accounts for 0.01-99.9% of the total weight of the pharmaceutical composition.

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