3-benzylamino coumarin compound and application thereof

Abstract

The invention belongs to the technical field of medicines, and relates to a 3-benzylamino coumarin compound and application thereof. Experimental results show that the compound provided by the invention can significantly increase the survival rate of HUVEC cells in a high glucose environment and resist vascular injury caused by high glucose. The compound provided by the invention can increase the release amount of NO in HUVEC cells damaged by high glucose, reduce the generation of malondialdehyde, and reduce the generation of tumor necrosis factors alpha.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, relates to 3-benzylaminocoumarin compounds and applications thereof, and specifically provides 3-benzylaminocoumarin compounds or pharmaceutically acceptable salts thereof and salts thereof and applications thereof in drugs for preventing, treating or improving high sugar-induced vascular damage. Background Art

[0002] Diabetes is a common metabolic disease characterized by high blood sugar, which can easily lead to vascular disease complications, including microvascular complications (retinopathy, nephropathy and neuropathy) and macrovascular complications (ischemic heart disease, stroke and peripheral vascular disease). Vascular disease is one of the important causes of death in diabetic patients. In the vascular damage caused by diabetes, intracellular high sugar can affect the exchange process of glucose and its intermediates through the polyol pathway, diacylglycerol protein kinase (PKC-Dag) pathway, advanced glycation end product (AGE) pathway and hexosamine pathway, thereby leading to excessive production of reactive oxygen species and inhibiting nitric oxide synthase to reduce the production of nitric oxide. Excessive production of reactive oxygen species will subsequently activate the misregulation of nuclear transcription factor NF-κB, promote the abnormal expression of multiple genes related to inflammatory response, such as inflammatory factors such as TNF-α and IL-6, etc. In addition, a large amount of ROS in the body will attack the lipid components in the biological membrane and generate the end product MDA, which will damage mitochondria and produce strong cytotoxicity. Ultimately, vascular endothelial cells are further damaged due to long-term abnormal inflammatory response, resulting in vascular dysfunction.

[0003] JAK2, Runx3 and STAT4 were elevated in human umbilical vein endothelial cells (HUVECs) induced by high glucose. The results showed that endothelial dysfunction could be alleviated by inhibiting the inactivation of the JAK2 / STAT4 pathway and the expression of Runx3 in diabetes. At the same time, TNF-a induced by high glucose upregulated Nrf2 / HO-1 expression by activating the JAK / STAT3 pathway, promoting the expression of adhesion molecules and inflammatory mediators in HUVECs, thereby producing atherosclerosis. Literature reports have shown that LncRNA SNHG1, which interacts with PSF, can regulate the JAK2 / STAT pathway. PSF PTB-associated splicing factor, also known as SFPQ Splicing factor proline-and glutamine-rich, is a protein containing tandem-specific DNA and RNA binding domains and is involved in a variety of nuclear processes. It plays a key role in gene expression, alternative splicing, DNA repair, mRNA and telomere stability, and other processes of nucleic acid metabolism in a variety of cells. Therefore, we want to design an inhibitor of PSF protein that can alleviate vascular damage by inhibiting the JAK2 / STAT pathway. Summary of the invention

[0004] The first object of the present invention is to provide a 3-benzylaminocoumarin compound or a pharmaceutically acceptable salt thereof, wherein the structural formula of the compound is:

[0005]

[0006] The compound represented by formula (I) is named as 3-(3'-fluorobenzylamino)-7-hydroxycoumarin, and the compound represented by formula (II) is named as 3-(4'-trifluoromethoxybenzylamino)-7-hydroxycoumarin.

[0007] As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by a compound of the present invention and an acid or base that is suitable for use as a drug. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is a salt formed by a compound of the present invention and an acid. Suitable acids for forming salts include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, and naphthalenesulfonic acid; and amino acids such as proline, phenylalanine, aspartic acid, and glutamic acid. Another preferred salt is a salt of the compound of the present invention and a base, such as an alkali metal salt (e.g., sodium salt or potassium salt), an alkaline earth metal salt (e.g., magnesium salt or calcium salt), an ammonium salt (e.g., lower alkanolammonium salt and other pharmaceutically acceptable amine salts), for example, methylamine salt, ethylamine salt, propylamine salt, dimethylamine salt, trimethylamine salt, diethylamine salt, triethylamine salt, tert-butylamine salt, ethylenediamine salt, hydroxyethylamine salt, dihydroxyethylamine salt, trihydroxyethylamine salt, and amine salts formed from morpholine, piperazine, and lysine, respectively.

[0008] The second object of the present invention is to provide the use of the above-mentioned 3-benzylaminocoumarin compounds or pharmaceutically acceptable salts thereof in the preparation of PSF protein inhibitors.

[0009] The third object of the present invention is to provide the use of the above-mentioned 3-benzylaminocoumarin compounds or pharmaceutically acceptable salts thereof in drugs for preventing, treating or improving high-glucose-induced vascular damage. The inventors have found through experiments that the compounds of formula (I) and formula (II) can significantly increase the survival rate of HUVEC cells in a high-glucose environment and resist vascular damage caused by high glucose. Preferably, the conditions for establishing the high-glucose model of human umbilical vein endothelial cells are a glucose concentration of 55 mmol / L and an injury time of 48 h.

[0010] The fourth object of the present invention is to provide the use of the above-mentioned 3-benzylaminocoumarin compounds or pharmaceutically acceptable salts thereof in drugs for preventing, treating or improving diabetes-induced vascular damage.

[0011] In the above applications, the compound can increase the amount of NO released, reduce the decrease in the release level of NO caused by high sugar stimulation, and effectively protect the structure and endothelial function of HUVEC under high sugar conditions. The compound can reduce the production of malondialdehyde and reduce oxidative stress. The compound reduces the production of tumor necrosis factor α and alleviates the damage of inflammatory mediators to endothelial cells.

[0012] The fifth object of the present invention is to provide a pharmaceutical composition, which contains a therapeutically effective amount of the compound according to claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0013] The dosage of the 3-benzylaminocoumarin compounds or pharmaceutically acceptable salts thereof of the present invention can vary according to the route of administration, the age and weight of the patient, the type and severity of the disease to be treated, etc. The dosage range for treating the disease can be 0.001-20 mg / kg / day / person, preferably 0.003-10 mg / kg / day / person, and can be administered once or multiple times.

[0014] The carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.

[0015] The pharmaceutical composition can be used orally or parenterally, such as by injection, spraying, nasal drops, eye drops, penetration, absorption, physical or chemical mediation, etc., into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue; or introduced into the body after being mixed or wrapped with other substances.

[0016] When used for oral administration, it can be made into conventional solid preparations, such as tablets, powders, granules, capsules, ointments, creams, etc.; it can be made into liquid preparations such as water or oil suspensions or other liquid preparations, such as oral liquids, etc. When used for parenteral administration, it can be made into injections, etc.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) Detect the inhibitory effect of different concentrations (20, 30, 40, 50, 55, 60, 65mM / L) and different times (24, 48h) on HUVEC. The results showed that glucose of different concentrations and times had different degrees of inhibitory effect on HUVEC cells. The inhibition rate of 55mmol / L glucose and incubation for 48h was about 50%, and the inhibitory effect was relatively strong. Therefore, 55mmol / L glucose and 48h incubation were selected as the damage concentration and damage time. 55mM / L glucose was used to carry out high-glucose oxidative damage for 48h, and the repair ability of compounds of formula (I) and (II) on cells was determined. 50μM / L aspirin was used as the positive control. Analysis of results: The cell viability of compounds of formula (I) and (II) was significantly higher than that of the model group and better than that of the positive control, indicating that they have a repair effect on HUVEC cells damaged by high glucose. 50 They were 4.77 μmol / L and 0.939 μmol / L respectively.

[0019] (2) The experimental results of the determination of nitric oxide (NO) release levels of model cells by the components of formula (I) and (II) compounds showed that high glucose stimulation caused a decrease in the release level of NO in HUVEC cells, while pretreatment with the compounds of formula (I) and (II) could increase the NO release amount to varying degrees, alleviate the decrease in NO release levels caused by high glucose stimulation, and effectively protect the structure of HUVEC and the stability of endothelial function under high glucose conditions.

[0020] (3) The experimental results of the effects of the components of the compounds of formula (I) and (II) on the secretion of TNF-α in model cells showed that the stimulation of high glucose conditions significantly increased the expression of the pro-inflammatory factor TNF-α in HUVECs cells, indicating that high glucose induced the occurrence of inflammation, and pretreatment with the compounds of formula (I) and formula (II) could reduce the levels of pro-inflammatory factors to varying degrees, alleviate the degree of inflammation, and play a protective role in endothelial function.

[0021] (4) The experimental results of the effects of the components of the compounds of formula (I) and (II) on the secretion of MDA in model cells showed that the MDA level of cells in the high glucose group increased significantly, and pretreatment with the compounds of formula (I) and (II) could significantly reduce the production of MDA, indicating that it can effectively reduce the oxidative stress damage of HUVEC caused by high glucose. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 2 is a Western blot protein band diagram. In the diagram, the three protein bands from left to right are blank control, formula (I), and formula (II).

[0023] Figure 2 is a protein quantitative graph; wherein 1 is a blank control, 2 is formula (I), and 3 is formula (II);

[0024] ****p<0.0001 compared with the blank group. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with specific embodiments, but this is not intended to limit the present invention.

[0026] Unless otherwise specified, the content of each component used below is weight percentage; the experimental methods used are conventional methods; the reagents, biological materials, etc. used can be obtained from commercial channels.

[0027] The compound numbers and structural formulas involved in the embodiments of the present invention are as follows:

[0028]

[0029]

[0030] Example 1. Molecular docking study

[0031] DS (Discovery Studio) was used to accurately dock the compounds with the PSF protein, and the compounds with better activity were screened out based on the docking score results.

[0032] (1) Preparation of protein

[0033] The required protein structure was downloaded from the PDB website (4wii). The proteins required for docking were pretreated using the CleanProtein and Prepare Protein methods built into DS. This operation can remove various conformations of proteins, supplement incomplete amino acid residues, and hydrogenate proteins. Water and endogenous ligands in the protein binding pocket were retained, and other parameter settings were all default parameters. The pretreated small molecule compound was used as a ligand, and the pretreated protein structure with a defined active site was used as a receptor. The rest of the settings remained default.

[0034] (2) Preparation of small molecules

[0035] The Prepare Ligands method built into DS was used, and tautomerism and isomers were not generated. The protonation pH value was set to 7, and the other parameters were the default parameters.

[0036] (3) Molecular docking

[0037] The docking of the compounds shown in Table 1 was performed using the LibDOCKER docking protocol of Accelrys Discovery Studio 2019 (DS, Accelrys Software Inc., San Diego, CA, USA).

[0038] The PSF protein was docked with the small molecule using Discovery Studio to obtain the docking score.

[0039] Table 1 Docking scores of compounds with PSF protein

[0040]

[0041]

[0042] Results analysis: The docking scores of formula (I) and formula (II) are better than those of formula (III) and formula (IV), indicating that they have better inhibitory effects on PSF than formula (III) and formula (IV). In addition, the survival rate determination in the following Example 4 also shows that the survival rates of formula (I) and formula (II) on HUVEC are higher than that of the positive control, while the survival rates of formula (III) and formula (IV) on HUVEC are lower than that of the positive control, so the compounds of formula (I) and formula (II) are selected for subsequent activity determination instead of formula (III) and formula (IV).

[0043] Example 2: RNA-pulldown experiment

[0044] Using Pierce TM The Magnetic RNA Protein Pull-Down Kit performs RNA pull-down experiments to verify the mechanism. The kit provides streptavidin magnetic beads and reagents that effectively enrich RNA-binding proteins. In the RNA pull-down experiment, the in vitro transcribed RNA is first bound to the magnetic beads, and then the RNA-magnetic bead complex is incubated with cell lysate, so that the proteins bound to the target RNA will be adsorbed on the magnetic beads together. After the RNA-protein complex is eluted from the magnetic beads with an elution buffer, the specific binding protein is detected by Western Blot.

[0045] 1. Experimental Protocol

[0046] Binding of magnetic beads to labeled RNA: resuspend 50 μL of magnetic beads in a sterile, enzyme-free 1.5 mL centrifuge tube, place on a magnetic separation rack, and discard the supernatant; wash the magnetic beads twice with 50 μL of 20 mM Tris; add 50 μL of 1× RNA Capture buffer to resuspend the magnetic beads, then add 50-100 pmol of avidin-labeled RNA (purchased from Gene Pharma), mix gently, and incubate at room temperature with shaking for 30 min.

[0047] Binding of labeled RNA to PSF protein: Place the above centrifuge tube on a magnetic separation rack, collect the magnetic beads on the side, and discard the supernatant; wash the magnetic beads twice with 50μL 20mM Tris; dilute 10×protein-RNA Binding buffer to 1× with ultrapure water, and resuspend the magnetic beads with 100μL 1×protein-RNA Binding buffer; prepare the premix for RNA-protein binding reaction according to the following table (Table 2); collect the magnetic beads on the side of the magnetic separation rack, discard the supernatant, take 100μL of the premix for RNA-protein binding reaction and add it to the magnetic beads, and incubate at 4°C for 60min. Drug group: In advance, the protein lysate was incubated with the compound (1nM) at room temperature for 1h.

[0048] Table 2 Premix formula for RNA-protein binding reaction

[0049]

[0050] Elution of RNA-protein binding complex: Place the centrifuge tube on the magnetic separation rack, collect beads on the side, and aspirate the supernatant for analysis; wash the magnetic beads twice with 100μL 1×Wash buffer, and keep the supernatant (for analysis); resuspend the magnetic beads with 50μL, mix gently, and incubate at 37℃ with shaking for 30min; place the centrifuge tube on the magnetic separation rack and keep the supernatant. Add an equal volume of 2×loading buffer (purchased from Solebol) to the supernatant of the Elution buffer in the last step, mix well, boil in a 95℃ metal bath for 10-15min, and detect the pulled-down PSF protein by Western blot.

[0051] 2. Results Analysis

[0052] Results Figure 1 and Figure 2 . Figure 1 and Figure 2 This indicates that the compounds represented by formula (I) and formula (II) have a greater ability to inhibit the binding of lncRNA to PSF protein. The inhibitory ability of formula (I) is slightly greater than that of formula (II). The inhibition rates of formula (I) and formula (II) are approximately 87% and 83%, respectively.

[0053] Example 3: Preparation of 3-benzylaminocoumarin compounds represented by formula (I) and (II)

[0054] (I) The specific preparation process of the compound 3-(3'-fluorobenzylamino)-7-hydroxycoumarin represented by formula (I) is as follows:

[0055] A 250mL single-mouth flask was placed on a magnetic stirrer, 60mL of methanol was added, and then 2,4-dihydroxybenzaldehyde (0.06mol), ethyl isocyanoacetate (0.06mol), pyridine (0.06mol), and cuprous iodide (0.005mol) were added in sequence under stirring. After all the raw materials were added, the flask was transferred to an oil bath pot, and the temperature of the oil bath pot was set to 40°C-60°C. The reaction was continued at this temperature and heated under oil bath reflux for 6 hours. During the reaction, a light pink solid gradually precipitated. When the amount of solid precipitation became less and less until no more precipitation occurred, the reaction was stopped. After the reaction was completed, the reaction solution was filtered while hot, and the filtered solid was eluted with methanol during the filtration process to obtain a pure yellow intermediate 3-formamido-7-hydroxycoumarin.

[0056] Take a 250mL single-mouth flask and fix it on an oil bath, add 75mL of methanol as a solvent, and start stirring. Add 3-formamido-7-methoxycoumarin (0.0063mol) obtained in the previous reaction to the solvent, then slowly drip 2.6mL of concentrated hydrochloric acid, set the oil bath temperature to 70°C, heat and reflux for 1 hour, and it can be found that the solid is dissolved. After the reaction is completed, use a rotary evaporator to remove the reaction solution, and then add 60mL of distilled water to dissolve. Use saturated sodium hydroxide solution to adjust the solution pH to 7-8, a large amount of yellow solid precipitates, and the precipitate is allowed to stand and filtered.

[0057] 3-amino-7-hydroxycoumarin (0.006 mol) and 3-fluorobenzyl bromide (0.0072 mol) were placed in a 100 mL three-necked flask with 30 mL DMF as the reaction solvent, and heated under reflux in an oil bath at 90°C-110°C with stirring for about 6 hours, during which the reaction was monitored by thin layer chromatography. After the reaction was completed, the reaction solution was poured into about 200 mL of ice water while hot, and solids precipitated. After standing and filtering, the crude product of the target compound was obtained, and the pure target product 3-(3'-fluorobenzylamino)-7-hydroxycoumarin was obtained by column chromatography.

[0058] (II) The specific preparation process of the compound 3-(4'-methoxybenzylamino)-7-hydroxycoumarin represented by formula (II) is as follows:

[0059] A 250mL single-mouth flask was placed on a magnetic stirrer, 60mL of methanol was added, and then 2,4-dihydroxybenzaldehyde (0.06mol), ethyl isocyanoacetate (0.06mol), pyridine (0.06mol), and cuprous iodide (0.005mol) were added in sequence under stirring. After all the raw materials were added, the flask was transferred to an oil bath pot, and the temperature of the oil bath pot was set to 50°C. The reaction was continued at 50°C for 6 hours in an oil bath heating reflux. During the reaction, a light pink solid gradually precipitated. When the amount of solid precipitation became less and less until no more precipitation occurred, the reaction was stopped. After the reaction was completed, the reaction solution was filtered while hot, and the filtered solid was eluted with methanol during the filtration process to obtain a pure yellow intermediate 3-formamido-7-hydroxycoumarin.

[0060] Take a 250mL single-mouth flask and fix it on an oil bath, add 75mL of methanol as a solvent, and start stirring. Add 3-formamido-7-methoxycoumarin (0.0063mol) obtained in the previous reaction to the solvent, then slowly drip 2.6mL of concentrated hydrochloric acid, set the oil bath temperature to 70°C, heat and reflux for 1 hour, and it can be found that the solid is dissolved. After the reaction is completed, use a rotary evaporator to remove the reaction solution, and then add 60mL of distilled water to dissolve. Use saturated sodium hydroxide solution to adjust the solution pH to 7-8, a large amount of yellow solid precipitates, and the precipitate is allowed to stand and filtered.

[0061] 3-amino-7-hydroxycoumarin (0.006 mol) and 4-trifluoromethoxybenzyl bromide (0.0072 mol) were placed in a 100 mL three-necked flask with 30 mL DMF as the reaction solvent, and the mixture was heated in an oil bath under reflux and stirred for about 6 hours at 100°C, during which the reaction was monitored by thin layer chromatography. After the reaction was completed, the reaction solution was poured into about 200 mL of ice water while hot, and solids were precipitated. After standing and filtering, the crude product of the target compound was obtained, and the pure target product 3-(4'-trifluoromethoxybenzylamino)-7-hydroxycoumarin was obtained by column chromatography.

[0062] Example 4: Determination of the anti-high glucose activity of the compounds of the present invention

[0063] 1. Cell Culture

[0064] HUVEC (human umbilical vein endothelial cells) were selected and cultured in endothelial cell growth medium.

[0065] 2. Determination of Glucose Damage Concentration

[0066] (1) Cell plating: Trypsinize the logarithmic phase cells, collect them by centrifugation after termination, prepare a cell suspension, and count the cells to adjust the concentration to 10×10 4 / ml.

[0067] (2) Preparation of cell plates: After the cell suspension is prepared, gently mix it and add it to a 96-well plate, adding 100 μL to each well, so that the density of the cells to be tested is 10,000 / well (the edge wells are filled with sterile PBS). The inoculated cell culture plate is placed in an incubator and cultured for 12 hours.

[0068] (2) Concentration gradient setting: Take out the prepared filtered and sterilized glucose solution (1 mol / L), dilute it with culture medium (serum-free) to a final concentration of 20, 30, 40, 50, 55, 60, 65 mM / L and a blank group (no cells, only culture medium), 100 μL / well, 5 replicate wells per group, and culture in a constant temperature incubator for 24 h and 48 h, respectively.

[0069] (4) MTT assay: Add MTT solution (10 μL / well) in a dark place, wrap the 96-well plate tightly with tin foil, and incubate in a constant temperature incubator for 4 h.

[0070] (5) Terminate the culture and dissolve the crystals: remove the supernatant, add 150 μl of dimethyl sulfoxide to each well, and shake at a low speed on a shaker for 10 min to fully dissolve the crystals. Use an ELISA reader to measure the absorbance at 490 nm, and calculate the cell inhibition rate according to the formula.

[0071] Calculation formula: Inhibition rate (%) = 1-OD value of drug-added group / OD value of control group. The experimental results are shown in Table 3.

[0072] Table 3 Inhibition rate of HUVEC at different concentrations of glucose and different time

[0073]

[0074]

[0075] Results: High concentration of glucose was used to induce oxidative stress damage, and the inhibitory effect of different concentrations (20, 30, 40, 50, 55, 60, 65mM / L) and different time (24, 48h) on HUVEC was detected. The results show that glucose of different concentrations and time had different degrees of inhibitory effect on HUVEC cells. The cell state at each concentration was observed under an inverted microscope. Compared with the normal control group, with the increase of glucose concentration, the number of rounded and shrunken cells increased. The glucose concentration of 20mmol / L had little effect on the cell morphology, and the change was not obvious compared with the normal group; however, in the glucose environment of 60, 65mmol / L, the number of spindle cells decreased significantly, the number of rounded and shrunken cells increased significantly, and almost no cobblestone-like growth was observed, and the cell state was poor; the inhibition rate of 55mmol / L glucose on HUVEC cells was about 50.8%, the number of rounded and shrunken cells increased, but there were still spindle cells, and some cells still grew like cobblestones. Therefore, 55mmol / L glucose and 48h incubation were selected as the injury concentration and injury time.

[0076] 3. High glucose resistance assay

[0077] 55 mM / L glucose was used to carry out high-glucose oxidative damage for 48 hours, and the cell repair ability of the compounds of formula (I), (II), (III) and (IV) was determined. 50 μM / L aspirin was used as the positive control.

[0078] (1) Cell plating: Trypsinize the logarithmic phase cells, collect them by centrifugation after termination, prepare a cell suspension, and count the cells to adjust the concentration to 10×10 4 / ml.

[0079] (2) High-glucose oxidative damage: The culture medium was discarded, and the normal group and the blank group were added with medium with normal sugar concentration, and the remaining groups were added with 55 mmol / L glucose for oxidative damage and incubated for 48 h.

[0080] (3) Preparation of solutions of the compounds of formula (I), (II), (III), (IV) and aspirin to be tested: Dissolve the compounds to be tested in DMSO to prepare a 0.1 mol / L stock solution, and dilute it with a culture medium containing 5% FBS to a concentration of 50 μM / L.

[0081] (4) Adding drugs: discard the culture medium, add culture medium with normal sugar concentration to the normal group and blank group, add sample with a concentration of 50 μm / L to the sample group, and add 0.05 mmol / L aspirin to the positive group, and incubate for 24 hours. Set up a 6-well experiment and repeat the experiment three times in parallel.

[0082] (5) Prepare MTT solution (final concentration of 5 mg / ml, using PBS as solvent). Keep the solution away from light and sterile during preparation and storage. Prepare it before use. Add 10 μL of MTT reagent to each well of the cell plate and incubate for 4 h.

[0083] (6) Terminate the culture and dissolve the crystals: remove the supernatant, add 150 μl of dimethyl sulfoxide to each well, and shake at a low speed on a shaker for 10 min to fully dissolve the crystals. Use an ELISA reader to measure the absorbance at 490 nm.

[0084] (7) The cell viability calculation formula for each group is as follows:

[0085] Cell viability of model group (%) = (OD model - OD blank) / (OD control - OD blank) × 100%

[0086] Cell viability of sample group (%) = (OD drug - OD blank) / (OD control - OD blank) × 100%

[0087] The results are shown in Table 4.

[0088] Table 4 Effects of different compounds on HUVEC cell survival under high glucose conditions

[0089]

[0090] Results: The cell viability of the compounds of formula (I) and (II) was significantly higher than that of the model group and better than that of the positive control, indicating that they have a repairing effect on HUVEC cells damaged by high glucose. 50 .

[0091] 4.EC 50 Determination

[0092] (1) Cell plating: Trypsinize the logarithmic phase cells, collect them by centrifugation after termination, prepare a cell suspension, and count the cells to adjust the concentration to 10×10 4 / ml.

[0093] (2) High-glucose oxidative damage: The culture medium was discarded, and the normal group and the blank group were added with medium with normal sugar concentration, and the remaining groups were added with 55 mmol / L glucose for oxidative damage and incubated for 48 h.

[0094] (3) Adding drugs: discard the culture medium, add culture medium with normal sugar concentration to the normal group and blank group, add the tested formula (I) and formula (II) at concentrations of 3.125, 6.25, 12.5, 25, 50, and 100 μM / L to the sample group, and add 50 μM / L aspirin to the positive group, 100 μL / well, and incubate for 24 h.

[0095] (4) Prepare MTT solution (final concentration of 5 mg / ml, using PBS as solvent). Keep the solution away from light and sterile during preparation and storage. Prepare it before use. Add 10 μL of MTT reagent to each well of the cell plate and incubate for 4 h.

[0096] (6) Terminate the culture and dissolve the crystals: remove the supernatant, add 150 μl of dimethyl sulfoxide to each well, and shake at a low speed on a shaker for 10 min to fully dissolve the crystals. Use an ELISA reader to measure the absorbance at 490 nm.

[0097] (7) The cell viability calculation formula for each group is as follows:

[0098] Cell viability of model group (%) = (OD model - OD blank) / (OD control - OD blank) × 100%

[0099] Cell viability of sample group (%) = (OD drug - OD blank) / (OD control - OD blank) × 100%

[0100] The results are shown in Table 5.

[0101] Table 5 EC values ​​of compounds of formula (I) and (II) 50

[0102]

[0103] Example 5 Determination of the release level of nitric oxide (NO) in model cells by the components of the compounds of formula (I) and (II)

[0104] The cells were inoculated in a 96-well plate, and the culture supernatant was collected after grouping and treatment to determine the NO level: (1) Reagents I and II in the detection kit were taken out from the storage environment in advance and placed at room temperature for a period of time to allow them to return to temperature; then, the cell culture fluid provided in the kit for diluting the standard was used to prepare a gradient concentration of the standard for making a standard curve, and the concentration range of the standard curve could be increased as needed; (2) Then, 50 μL of the standard and the sample to be tested were added to each well, and then 50 μL / well of reagents I and II were added in turn, mixed and allowed to stand for 2 minutes, and then the absorbance at 540 nm was measured, and the NO content in the sample was calculated according to the standard curve. The results are shown in Table 6.

[0105] Table 6 Effect of the compounds of formula (I) and (II) on the release of NO levels in HUVEC under high glucose conditions

[0106]

[0107] Analysis of results: High glucose stimulation decreased the release level of NO in HUVEC cells, while pretreatment with the compounds of formula (I) and (II) increased the release of NO to varying degrees, effectively protecting the structure and endothelial function of HUVEC under high glucose conditions.

[0108] Example 6 Effects of the components of the compounds of formula (I) and (II) on TNF-α secretion in model cells

[0109] (1) Cell plating: Logarithmic phase cells were inoculated into a 10 cm culture dish and cultured overnight.

[0110] (2) High-glucose oxidative damage: The culture medium was discarded, and the normal group and the blank group were added with medium with normal sugar concentration, and the remaining groups were added with 55 mmol / L glucose for oxidative damage and incubated for 48 h.

[0111] (3) Adding drugs: The culture medium was discarded, and the normal group and the blank group were added with a culture medium with normal sugar concentration, and the compound of formula (I) and (II) was added at a concentration of 25 μM / L, and incubated for 24 h.

[0112] (4) Equilibrate the TNF-α kit and IL-4 kit at room temperature for 20 minutes.

[0113] (5) Prepare 1X washing solution in advance at a ratio of washing solution (20X): double distilled water = 1:19 mL.

[0114] (6) Add the diluent to the standard bottle according to the required volume on the bottle wall and incubate for 15 minutes (at room temperature).

[0115] (7) The standard was diluted to obtain concentrations of 1000, 500, 125, 62.5, and 31.25 pg / mL, with 0 pg / mL being the standard dilution solution.

[0116] (8) Lyse the cells of each group and collect the supernatant (12000g, 10 min).

[0117] (9) Add the standard and sample to the reaction wells (100 μL / well), seal the plate with a sealing film, and incubate at room temperature for 2 h.

[0118] (10) Wash the plate five times with 1X washing buffer, 300-350 μL per well, and pat the plate dry on filter paper.

[0119] (11) Add biotinylated antibody (100 μL / well), seal the plate with film, and incubate at room temperature for 1 h.

[0120] (12) Wash the plate five times with 1X washing buffer, 300-350 μL per well, and pat the plate dry on filter paper.

[0121] (13) Add horseradish peroxidase-labeled streptavidin (100 μL / well), seal the plate with film, and incubate at room temperature for 20 min.

[0122] (14) Wash the plate five times with 1X washing buffer, 300-350 μL per well, and pat the plate dry on filter paper.

[0123] (15) Add the colorimetric reagent TMB solution (100 μL / well), seal the plate with a sealing film, and incubate at room temperature for 20 min.

[0124] (16) Add stop solution (50 μL / well), shake well, and measure the OD value at 450 nm.

[0125] (17) Calculate the sample concentration based on the standard curve.

[0126] The results are shown in Table 7.

[0127] Table 7 Effects of compounds of formula (I) and (II) on HUVEC TNF-α content under high glucose conditions

[0128]

[0129] Result analysis:

[0130] The stimulation of high sugar conditions significantly increased the expression of pro-inflammatory factor TNF-α in HUVECs cells, indicating that high sugar induced the occurrence of inflammation, and pretreatment with the compounds of formula (I) and formula (II) could reduce the levels of pro-inflammatory factors to varying degrees, alleviate the degree of inflammation, and play a protective role in endothelial function.

[0131] Example 7 Effects of the components of the compounds of formula (I) and (II) on the secretion of MDA in model cells

[0132] (1) Cell plating: Logarithmic phase cells were inoculated into a 10 cm culture dish and cultured overnight.

[0133] (2) High-glucose oxidative damage: The culture medium was discarded, and the normal group and the blank group were added with medium with normal sugar concentration, and the remaining groups were added with 55 mmol / L glucose for oxidative damage and incubated for 48 h.

[0134] (3) Adding drugs: The culture medium was discarded, and the normal group was added with a culture medium with normal sugar concentration, and the compound of formula (I) and (II) was added at a concentration of 25 μM / L, and incubated for 24 h.

[0135] (4) Lyse the cells of each group and collect the supernatant (12000g, 10min).

[0136] (5) Standard dilution: dilute the standard sample to 1, 2, 5, 10, 20, and 50 μmol / L for later use;

[0137] (6) Measure protein concentration by BCA method and draw a standard curve: In a 96-well plate, add and record the gradient concentration of protein standard solution according to the instructions, add samples to new wells, and then fill all wells with PBS to 20 μL; add the pre-prepared BCA working solution to each well, measure the absorbance, draw a standard curve, and calculate the protein concentration;

[0138] (7) Sample determination: PBS was used as a blank control group, and different concentrations of the standard substance in (1) were added to prepare a standard curve. Then, the sample and MDA working solution were added and mixed thoroughly, and the absorbance at 532 nm was measured;

[0139] (8) Calculation: MDA content = corresponding concentration of standard curve × dilution factor ÷ sample protein concentration. The results are shown in Table 8.

[0140] Table 8 Effects of compounds of formula (I) and (II) on HUVEC MDA content under high sugar conditions

[0141]

[0142] Result analysis: The results of MDA level detection are shown in the figure. It can be seen from the figure that the MDA level of cells in the high glucose group increased significantly, and compound pretreatment can significantly reduce the generation of MDA, indicating that it can effectively alleviate the oxidative stress damage of HUVEC caused by high glucose.

[0143] It can be seen from the above examples that the compounds of formula (I) and (II) have a certain repair effect on HUVEC cells damaged by high glucose, and can be used as a PSF inhibitor to treat vascular damage caused by high glucose.

[0144] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. 3-Benzylaminocoumarin compounds or pharmaceutically acceptable salts thereof, wherein the structural formula of the compound is 2. Use of the 3-benzylaminocoumarin compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a PSF protein inhibitor.

3. Use of the 3-benzylaminocoumarin compound or a pharmaceutically acceptable salt thereof according to claim 1 in a drug for preventing, treating or improving high glucose-induced vascular damage.

4. Use of the 3-benzylaminocoumarin compound or a pharmaceutically acceptable salt thereof according to claim 1 in a drug for preventing, treating or improving diabetes-induced vascular damage.

5. The use according to claim 3 or 4, characterized in that: The compounds can increase the amount of NO released.

6. The use according to claim 3 or 4, characterized in that: The compound can reduce the production of malondialdehyde.

7. The use according to claim 3 or 4, characterized in that: The compounds can reduce the production of tumor necrosis factor alpha.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition contains a therapeutically effective amount of the 3-benzylaminocoumarin compound or a pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable carrier.

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