Medical application of 7-hydroxy-4-phenylcoumarin

Abstract

The invention relates to the field of compounds, in particular to pharmaceutical application of a compound with the following structural formula or pharmaceutically acceptable salt of the compound. The compound can be used for preparing medicines for preventing and treating diabetes.

Description

Technical Field

[0001] This invention relates to the field of medical technology, and in particular to the application of a coumarin compound. Background Technology

[0002] Currently, diabetes has become a major global public health challenge. From 1990 to 2022, the global prevalence of diabetes among adults rose from 7% to 14%, and the number of patients surged from approximately 198 million to over 800 million, an increase of nearly fourfold. Among those aged 30 and older with diabetes, approximately 59% did not receive routine treatment, indicating a significantly widening treatment gap. The largest increase in diabetes prevalence was observed in low- and middle-income countries, where 90% of untreated adults live, reflecting problems of unequal resource allocation and inadequate healthcare systems.

[0003] In China, the diabetes situation is even more severe. As of 2021, the number of diabetes patients in China reached 141 million, accounting for approximately 17% of the global total, making it the country with the largest number of diabetes patients in the world. Furthermore, about two-thirds of these patients remain undiagnosed, leading to a significant increase in the risk of disease progression and complications. Data from 2023 shows that the overall age-standardized prevalence of diabetes in China reached 13.7%, with a staggering 233 million patients, a 163% increase compared to 2005. If the current trend continues, the prevalence of diabetes is projected to rise to 16.15% by 2030, 21.52% by 2040, and 29.10% by 2050. Moreover, cities like Tianjin and Beijing may have prevalence rates exceeding 40%, becoming "hotspots" for diabetes. Summary of the Invention

[0004] The purpose of this invention is to provide a new use for coumarin compounds.

[0005] Specifically, this aspect provides the use of compounds of formula (I) or pharmaceutically acceptable salts thereof in the preparation of drugs for the prevention and treatment of diabetes:

[0006] (I).

[0007] In a preferred embodiment, the above-mentioned compound is used as the sole active ingredient in the preparation of a drug for the prevention and treatment of diabetes.

[0008] Details of various aspects of the invention will be described in detail in the following sections. The features, objects, and advantages of the invention will become more apparent from the following description and the claims. Attached Figure Description

[0009] Figure 1 Compound 7-hydroxy-4-phenylcoumarin lowers blood glucose levels in diabetic mice.

[0010] Results are expressed as Mean±SD; compared with the control group, **** p<0.0001; compared with the treatment group, ##P<0.01, ####p<0.0001.

[0011] Figure 2 Compound 7-hydroxy-4-phenylcoumarin reduced liver function ALT and AST in diabetic mice.

[0012] Results are expressed as Mean±SD; compared with the control group, ***p<0.001, ****p<0.0001; compared with the treatment group, ###p<0.001, #####p<0.0001.

[0013] Figure 3 Compound 7-hydroxy-4-phenylcoumarin reduces renal function (BUN) in diabetic mice.

[0014] Results are expressed as Mean±SD; compared with the control group, the model group showed ***p<0.001; compared with the model group, the drug-treated group showed ###p<0.001.

[0015] Figure 4 The protective effect of compound 7-hydroxy-4-phenylcoumarin on the liver and pancreas of diabetic mice.

[0016] Figure 5 Compound 7-hydroxy-4-phenylcoumarin lowers blood glucose levels in diabetic rats.

[0017] Results are expressed as Mean±SD; compared with the control group, **** p<0.0001; compared with the treatment group, ##P<0.01.

[0018] Figure 6 Compound 7-hydroxy-4-phenylcoumarin reduces glycated hemoglobin in diabetic rats

[0019] Results are expressed as Mean±SD; compared with the control group, ** p<0.01; compared with the treatment group, ## P<0.01. Detailed Implementation

[0020] This invention provides the use of compounds of formula (I) or pharmaceutically acceptable salts thereof in the preparation of medicaments for the prevention and treatment of diabetes:

[0021]

[0022] 7-Hydroxy-4-phenylcoumarin, molecular formula: C 15 H 11 O3, molecular weight 239.07.

[0023] Details of various aspects of the invention will be described in detail in the following sections. The features, objects, and advantages of the invention will become more apparent from the following description and the claims.

[0024] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0025] 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. The preferred embodiments and materials described herein are for illustrative purposes only.

[0026] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0027] Example 1: Preparation of 7-hydroxy-4-phenylcoumarin

[0028] In a dry round-bottom flask, add resorcinol (2.20 g, 2 mmol) and ethyl benzoyl (3.65 g, 1.9 mmol) dissolved in trifluoroacetic acid (50 mL). Under argon protection, stir and heat to 100 °C for 4 hours. Cool to room temperature, add 100 mL of water, and combine the organic phases with 100 mL × 3 ethyl acetate solutions. Backwash with saturated brine (50 mL). Dry the organic phase with anhydrous magnesium sulfate (5 g), filter and concentrate to obtain a pale yellow solid. Purify by column chromatography using 50 g of silica gel (200-300 mesh) and a gradient eluent of petroleum ether:ethyl acetate (10:1-3:1) to obtain 4.2 g of white solid, yield 93%, melting point 145-146 °C. The data for the synthesis of compounds using this method are largely consistent with those in the paper (Dejun Zhou, Youchao Zhuang and Zuntian Sheng. Study on effective synthesis of 7-hydroxy-4-substituted coumarins. Heterocyclic Communications, 2022, 28, 181-187.).

[0029] Example 2: Diabetic mice were constructed using a high-fat diet and streptozocin (STZ) to study the hypoglycemic effect of 7-hydroxy-4-phenylcoumarin

[0030] SPF-grade male C57BL / 6J mice, 8 weeks old, were purchased from Shanghai Slac Laboratory Animal Co., Ltd. Animal license: SCXK(Shanghai) 2022-0004. They were adaptively fed in the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine at a temperature of 20-24°C with good air circulation and a relative humidity of 40-70%. Five mice were randomly selected as the control group, 10 as the model group, and 10 as the administration group. Among them, the model group and the administration group were continuously fed with a high-fat diet for 28 days. The high-fat diet was purchased from Wuxi Fanbo Biotechnology, with the formula: 20% protein, 60% fat, and 20% carbohydrates. On the 7th to 12th days, the mice in the model group and the administration group were intraperitoneally injected with STZ (purchased from Shanghai Abbexa, 30 mg / kg, diluted with 0.1 mol / L citric acid). The mice in the administration group were intragastrically administered 7-hydroxy-4-phenylcoumarin (1 mg / kg) every day for 28 days. Blood was collected from the tail vein on days 1, 7, 14, 21, and 28, and blood glucose was measured using a Roche blood glucose meter according to the instructions.

[0031] The experimental results are as Figure 1 shown in and Table 1. During the high-fat diet feeding period, the body weight of the mice in the control group increased. After injecting STZ, the body weight of the mice in the model group tended to be stable, and the body weight of the mice in the administration group decreased compared with that in the model group. The blood glucose results showed that after the combination of high-fat diet and STZ, the blood glucose of the mice in the model group was significantly higher than that of the control group, indicating that the diabetes model was successfully established. And the administration group significantly reduced the blood glucose value of diabetic mice.

[0032] Table 1 Body weight and blood glucose values of mice after administration of 7-hydroxy-4-phenylcoumarin

[0033]

[0034] Example 3: Study the protective effect of 7-hydroxy-4-phenylcoumarin on the liver and kidney functions of diabetic mice

[0035] Diabetic patients often suffer from liver and kidney damage. On the 28th day, the mice were anesthetized and blood was collected from the heart. After centrifuging at 3000 rpm for 20 minutes, the supernatant was taken as serum. The liver function indexes ALT, AST and kidney function index BUN of the mice were detected according to the instructions. All reagents were purchased from Nanjing Jiancheng. An enzyme-labeled instrument was used to detect the absorbance of ALT and AST at a wavelength of 405 nm and the absorbance of BUN at 640 nm. The liver function results are as Figure 2 shown in and Table 2. The kidney function results are as Figure 3As shown in Table 3, 7-hydroxy-4-phenylcoumarin can significantly reduce ALT and AST activity in the liver and decrease BUN concentration in the kidneys of diabetic mice, suggesting that 7-hydroxy-4-phenylcoumarin can improve liver and kidney damage caused by diabetes.

[0036] Table 2. Effects of 7-hydroxy-4-phenylcoumarin administration on liver function in mice.

[0037]

[0038] Table 3. Effects of 7-hydroxy-4-phenylcoumarin administration on renal function in mice.

[0039]

[0040] Example 4: Study on the protective effect of 7-hydroxy-4-phenylcoumarin on the liver and pancreas of diabetic mice.

[0041] On day 28, mice were euthanized after anesthesia. The liver and pancreas were removed and placed in 10 ml of 4% neutral buffered formalin for fixation for 48 hours. The tissue was then subjected to a series of solutions: 70% ethanol → 80% ethanol → 95% ethanol I → 95% ethanol II → 100% ethanol I → 100% ethanol II, each step lasting approximately 1 hour. After dehydration, the tissue was transferred to xylene I → xylene II for clearing, approximately 30 minutes each time, until the tissue became transparent. The cleared tissue was then immersed in paraffin I → paraffin II at 56°C for approximately 1 hour each time. The paraffin-soaked tissue was placed in an embedding cassette, fresh paraffin was injected, and the mixture was allowed to cool and solidify into a paraffin block. After trimming the paraffin block, it was sliced ​​into continuous sections 4 micrometers thick using a microtome. The sections were flattened in a 42°C water bath and retrieved using a glass slide. The slides were then baked on a 60°C slide oven for at least 1 hour, or in a 62°C oven overnight, to ensure the sections adhered tightly to the slide and prevent detachment. Hematoxylin-eosin (HE) staining was performed. The procedure was as follows: Xylene I: 10 minutes → Xylene II: 10 minutes → 100% ethanol I: 5 minutes → 100% ethanol II: 5 minutes → 95% ethanol: 3 minutes → 85% ethanol: 3 minutes → Rinse with running water: 2 minutes. Immerse the sections in the hematoxylin staining solution for 8 minutes. Remove and rinse with running water to remove any excess stain. Immerse the sections in 1% hydrochloric acid-ethanol (1% hydrochloric acid + 70% ethanol) for a few seconds to a dozen seconds, lifting and inserting the sections several times until they turn red and the color lightens; stop immediately. Use weakly alkaline water (0.1% ammonia) to restore the blue color for a few seconds, turning the cell nuclei from red back to a bright blue. Immerse the sections in 1% eosin Y aqueous solution or ethanol solution for 3 minutes. Rapidly dehydrate the tissue using a gradient of alcohols: → 85% alcohol: rapid insertion and withdrawal several times (differentiation of eosin, removal of excess color) → 95% alcohol I: 1-2 minutes → 95% alcohol II: 1-2 minutes → 100% alcohol I: 2 minutes → 100% alcohol II: 2 minutes. Permanently preserve the tissue with neutral resin. Observe under an optical microscope.

[0042] The liver results of diabetic mice are as follows Figure 4 As shown, in the model group, the hepatocytes were filled with round vacuoles of different sizes, squeezing the cell nucleus to one side. Some hepatocytes showed ballooning degeneration (cell swelling and sparse and pale-stained cytoplasm) due to injury, and homogeneous pink-stained fibrous septa were visible. After administration of 7-hydroxy-4-phenylcoumarin, the round vacuoles in the hepatocytes almost disappeared, and the ballooning degeneration disappeared, indicating that 7-hydroxy-4-phenylcoumarin has a protective effect on the hepatocytes of diabetic mice.

[0043] The pancreatic results of diabetic mice are as follows Figure 4 As shown, in the model group, the islets of Langerhans showed compensatory hypertrophy and hyperplasia, with disordered structure. The β-cells showed vacuolar degeneration (intracytoplasmic vacuoles), and a large amount of homogeneous and pink-stained amyloid substances were deposited in the islets (especially around blood vessels). In the administration group, the volume and structure of the islets were restored to normal, and the amount of amyloid substances and the number of vacuoles in the β-cells were significantly reduced, indicating that 7-hydroxy-4-phenylcoumarin has a protective effect on the islet cells of diabetic mice.

[0044] Example 5: Using a high-fat diet and STZ to construct diabetic rats to study the hypoglycemic effect of 7-hydroxy-4-phenylcoumarin

[0045] SPF-grade male Wistar rats, 6 weeks old, were purchased from Vital River Laboratories in Beijing, and the animal license number was SCXK(Beijing) No. 2021-0006. They were adaptively fed in the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine at a temperature of 20-24°C, with good air circulation and a relative humidity of 40-70%. Five rats were randomly selected as the control group, 10 rats as the model group, and 10 rats as the administration group. Among them, the model group and the administration group were continuously fed a high-fat diet for 28 days. The high-fat diet was purchased from Wuxi Fanbo Biotechnology, and the formula was: 20% protein, 60% fat, and 20% carbohydrates. From the 7th to the 12th day, the rats in the model group and the administration group were intraperitoneally injected with STZ (purchased from Absin Shanghai, at a dose of 30 mg / kg, diluted with 0.1 mol / L citric acid). The rats in the administration group were intragastrically administered 7-hydroxy-4-phenylcoumarin (1 mg / kg) every day for 28 days. On the 28th day, blood was collected from the tail vein, and the blood glucose was measured using a Roche glucometer according to the instructions.

[0046] The experimental results are as follows Figure 5 and shown in Table 4. After a high-fat diet combined with STZ, the blood glucose of the rats in the model group was significantly higher than that of the control group rats, indicating that the diabetic model was successfully established. The administration group significantly reduced the blood glucose value of the rats. [[ID=A]] [[ID=B]]

[0047] Table 4 Blood glucose values of rats after administration of 7-hydroxy-4-phenylcoumarin

[0048]

[0049] Example 6: The effect of 7-hydroxy-4-phenylcoumarin on glycated hemoglobin

[0050] On day 28, after anesthetizing the rats, blood was collected from the abdominal aorta and added to heparin anticoagulant tubes. Centrifugation was performed at 1000 rpm for 5 minutes, the supernatant was discarded, and the precipitated red blood cells were retained. The cells were washed twice with physiological saline using the same method described above. Glycated hemoglobin was detected using a glycated hemoglobin assay kit (Nanjing Jiancheng) according to the manufacturer's instructions, and the absorbance was read using an ELISA reader. The experimental results are as follows: Figure 6 As shown in Table 5, 7-hydroxy-4-phenylcoumarin has a significant effect on reducing glycated hemoglobin.

[0051] Table 5. Percentage of glycosylated hemoglobin in rats after administration of 7-hydroxy-4-phenylcoumarin.

[0052]

[0053] Example 7: Comparison of the inhibitory effects of structurally similar compounds on DPPH

[0054] Coumarin derivatives a, b, c, d, e, and f were synthesized according to the literature (Hu Yuheng, Sun Jie, Yang Jie, Wang Xiaojing, Synthesis and in vitro hypoglycemic activity of 3-4'-benzoylamino-phenyl-coumarin derivatives, Journal of China Pharmaceutical University, 2019, 50, 168-174). Using a DPPH free radical scavenging kit (Nanjing Jiancheng), 20 μM of each derivative was added according to the instructions, mixed well, and incubated at 25°C in the dark for 30 minutes. Then, the mixture was centrifuged at 4000 rpm for 5 minutes. The absorbance was measured at 517 nm: 800 μL of supernatant was transferred to a cuvette, zeroed with 80% methanol, and the absorbance of each tube was measured sequentially. The calculation formula was: Sample DPPH free radical scavenging rate (%) = (1 - (A...) / ( ... 测定 -A 对照 ) / A 空白 (×100%). The experimental results are shown in Table 6. The inhibitory effect of 7-hydroxy-4-phenylcoumarin on DPPH was significantly higher than that of the other 6 similar compounds, suggesting that 7-hydroxy-4-phenylcoumarin has good antioxidant capacity.

[0055]

[0056]

[0057]

[0058]

[0059]

[0060] Table 6 Comparison of DPPH scavenging rates of various compounds (20 uM)

[0061]

[0062] Example 8: Comparison of the inhibitory activities of structurally similar compounds on glucosidase.

[0063] In a 96-well plate, add PBS buffer (pH 6.8), p-nitrophenyl-α-D-glucosidase, and 20 μM of the compound to each well sequentially. Mix well and incubate at 37°C for 10 minutes. Add α-glucosidase solution with an enzyme activity of 0.1 U / mL, mix well, and react at 37°C for 20 minutes. Finally, add 175 μL of 0.2 mol / L sodium carbonate to terminate the reaction. The inhibition rate of α-glucosidase is measured by detecting the absorbance of p-nitrophenol at 405 nm using a microplate reader. Calculation formula: Inhibition rate = [(A...]] C -A B )-(A S -A SB )] / (A C -A B ()×100%. The experimental results are shown in Table 7. The inhibitory effect of 7-hydroxy-4-phenylcoumarin on glucosidase was significantly higher than that of the other 6 similar compounds, suggesting that 7-hydroxy-4-phenylcoumarin has a greater potential to reduce postprandial blood glucose in in vitro experiments.

[0064] Table 7 Comparison of glucosidase inhibitory activities of each compound (20 uM)

[0065]

[0066] The various aspects of this invention have been described above. However, it should be understood that, without departing from the spirit of this invention, those skilled in the art can make equivalent changes and modifications, which also fall within the scope of the appended claims.

Claims

1. Use of compounds of formula (I) or pharmaceutically acceptable salts thereof in the preparation of medicaments for the prevention and treatment of diabetes: （Ⅰ）。 2. The application as described in claim 1, wherein the compound of formula (I) is used as the sole active ingredient in the preparation of a drug for the prevention and treatment of diabetes.

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