3-benzylamino coumarin compounds and uses thereof

Abstract

The application belongs to the technical field of medicine, and relates to 3-benzylamino coumarin compounds and application thereof. Experimental results show that the compounds can significantly increase the survival rate of HUVEC cells in a high-sugar environment, and resist blood vessel injury caused by high sugar. The compounds can increase the release amount of NO in HUVEC cells damaged by high sugar, reduce the generation of malondialdehyde, and reduce the generation of tumor necrosis factor alpha.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and relates to 3-benzylamino coumarin compounds and application thereof, and specifically provides 3-benzylamino coumarin compounds or pharmaceutically acceptable salts thereof and application thereof in preventing, treating or improving high-glucose-induced vascular injury. BACKGROUND

[0002] Diabetes is a common metabolic disease characterized by high blood sugar, which is prone to cause vascular disease complications, including microvascular complications (retinopathy, nephropathy and neuropathy) and macrovascular complications (ischemic heart disease, stroke and peripheral vascular disease), and vascular lesions are one of the important causes of death in diabetic patients. In the vascular damage caused by diabetes, intracellular high glucose can affect the exchange process of glucose and intermediates through the polyol pathway, the diacylglycerol protein kinase (PKC-Dag) pathway, the advanced glycosylation end product (AGE) pathway and the hexosamine pathway, thereby causing excessive production of reactive oxygen species and inhibiting the production of nitric oxide by nitric oxide synthase. The excessive production of reactive oxygen species also subsequently activates the nuclear transcription factor NF-κB to cause misregulation and promote the abnormal expression of various genes related to inflammatory reactions, such as inflammatory factors like TNF-α, IL-6 and the like, in addition, a large amount of ROS in the body also attacks the lipid components in the biological membrane to generate end products MDA, which damages mitochondria and produces strong cytotoxicity. Ultimately, the vascular endothelial cells are further damaged due to long-term abnormal inflammatory reactions, causing vascular dysfunction.

[0003] JAK2, Runx3 and STAT4 were elevated in high glucose-induced human umbilical vein endothelial cells HUVEC. It was found that the purpose of relieving endothelial dysfunction could be achieved by inhibiting the inactivation of JAK2 / STAT4 pathway and the expression of Runx3 in diabetes, and TNF-a caused by high glucose could up-regulate the expression of Nrf2 / HO-1 by activating the JAK / STAT3 pathway, promote the expression of adhesion molecules and inflammatory mediators in HUVEC, and thus produce atherosclerosis. Literature has reported that LncRNA SNHG1 which interacts with PSF can regulate JAK2 / STAT pathway. Polypyrimidine tract binding protein associated splicing factor (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 domain, which is involved in various nuclear processes. It can play a key role in gene expression, alternative splicing, DNA repair, mRNA and telomere stability, and other processes of nucleic acid metabolism in various cells. Therefore, an inhibitor of PSF protein can be designed to achieve the purpose of relieving vascular injury by inhibiting the JAK2 / STAT pathway. SUMMARY

[0004] The first object of the present application is to provide a 3-benzylamino coumarin compound or a pharmaceutically acceptable salt thereof, the structural formula of the compound being:

[0005]

[0006] Among them, 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 of a compound of the present application which is suitable for use as a medicament. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is salts of the compounds of the present application with acids. Suitable acids for salt formation include, but are not limited to, hydrochloric, hydrobromic, hydrofluoric, sulfuric, nitric, phosphoric, and the like inorganic acids; formic, acetic, trifluoroacetic, propionic, oxalic, malonic, succinic, fumaric, maleic, lactic, malic, tartaric, citric, picric, benzoic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, benzenesulfonic, naphthalenesulfonic, and the like organic acids; and amino acids such as proline, phenylalanine, aspartic acid, glutamic acid, and the like. Another preferred class of salts is salts of the compounds of the present application with bases, such as alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (e.g., lower alkylammonium salts and other pharmaceutically acceptable amine salts), such as methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, t-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, and trihydroxyethylamine salts, and amine salts of morpholine, piperazine, lysine, respectively.

[0008] A second object of the present application is to provide use of the above-mentioned 3-benzylamino coumarin compound or a pharmaceutically acceptable salt thereof in the preparation of a PSF protein inhibitor.

[0009] A third object of the present application is to provide use of the above-mentioned 3-benzylamino coumarin compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing, treating or ameliorating high glucose-induced vascular injury. The inventors have found through experiments that both the compound of formula (I) and the compound of formula (II) can significantly increase the survival rate of HUVEC cells in a high glucose environment and resist high glucose-induced vascular injury. As a preference, the established condition of the human umbilical vein endothelial cell high glucose model is a glucose concentration of 55 mmol / L and an injury time of 48 h.

[0010] A fourth object of the present application is to provide use of the above-mentioned 3-benzylamino coumarin compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing, treating or ameliorating diabetes-induced vascular injury.

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

[0012] A fifth object of the present application is to provide a pharmaceutical composition containing a therapeutically effective amount of the compound or a pharmaceutically acceptable salt thereof according to claim 1 and a pharmaceutically acceptable carrier.

[0013] The amount of the 3-benzylamino coumarin compound or its pharmaceutically acceptable salt used in the present application can vary according to the administration route, the age and weight of the patient, the type and severity of the disease to be treated, etc. The therapeutic dose can range from 0.001 to 20 mg / kg / day per person, preferably from 0.003 to 10 mg / kg / day per person, which can be administered in one or more doses.

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

[0015] The pharmaceutical composition can be used in the form of oral or non-oral, etc. It can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spraying, nose drops, eye drops, penetration, absorption, physical or chemical mediated methods; or it can be introduced into the body after being mixed or wrapped with other substances.

[0016] For oral use, it can be made into conventional solid preparations such as tablets, powders, granules, capsules, ointments, creams, etc.; liquid preparations such as water or oil suspensions or other liquid preparations such as oral liquids, etc. For non-oral use, it can be made into injection solutions, etc.

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

[0018] (1) The inhibitory effect of different concentrations (20, 30, 40, 50, 55, 60, 65 mM / L) and different times (24, 48 h) on HUVEC was detected. The results showed that glucose of different concentrations and times had different degrees of inhibition on HUVEC cells. The inhibition rate of 55 mmol / L glucose incubated for 48 h was about 50%, and the inhibition was strong. Therefore, 55 mmol / L glucose and 48 h incubation were selected as the damage concentration and damage time. 55 mM / L glucose was used for high glucose oxidative damage for 48 h, and the repair ability of compounds of formula (I) and (II) on cells was determined. The positive control used 50 μM / L aspirin. The results showed that 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 had a repair effect on HUVEC cells damaged by high glucose, and their EC 50 were 4.77 μmol / L and 0.939 μmol / L, respectively.

[0019] (2) The results of the experiment of measuring the release level of model cell nitric oxide (NO) by the components of formula (I) and (II) compounds show that the stimulation of high glucose condition decreases the release level of NO in HUVEC cells, and the pre-treatment of formula (I) and formula (II) compounds can increase the release amount of NO to different degrees, reduce the decrease of the release level of NO caused by high glucose stimulation, and effectively protect the structure and endothelial function stability of HUVEC under high glucose condition.

[0020] (3) The results of the experiment of the influence of the components of formula (I) and (II) compounds on the secretion of model cell TNF-α show that the stimulation of high glucose condition significantly increases the expression of pro-inflammatory related factor TNF-α in HUVECs, which indicates that high glucose induces the occurrence of inflammation, and the pre-treatment of formula (I) and formula (II) compounds can reduce the level of pro-inflammatory factor to different degrees, reduce the degree of inflammation, and play a protective role of endothelial function.

[0021] (4) The results of the experiment of the influence of the components of formula (I) and (II) compounds on the secretion of model cell MDA show that the MDA level of the high glucose group is significantly increased, and the pre-treatment of formula (I) and formula (II) compounds can significantly reduce the generation of MDA, which indicates that it can effectively reduce the oxidative stress damage of HUVEC caused by high glucose. BRIEF DESCRIPTION OF DRAWINGS

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

[0023] Figure 2 It is a protein quantification diagram; wherein 1 is blank control, 2 is formula (I), and 3 is formula (II).

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

[0025] The application will be further described below in combination with specific examples, but not as a limitation to the application.

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

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

[0028]

[0029]

[0030] Example 1. Molecular docking study

[0031] The compounds were respectively precisely docked with PSF protein using DS (Discovery Studio), and the compounds with better activity were screened according to the docking score results.

[0032] (1) Preparation of protein

[0033] The required protein structure was downloaded from the PDB website (4wii), and the CleanProtein and Prepare Protein methods built-in DS were used for pretreatment docking required protein. The operation can remove various conformations of the protein, supplement incomplete amino acid residues, and add hydrogen to the protein, etc. The water in the protein binding pocket and the endogenous ligand were retained, and other parameter settings were default parameters. The pretreated compound small molecules were used as ligands, and the protein structure pretreated and defined active site was used as the receptor, and the remaining settings remained default.

[0034] (2) Preparation of small molecules

[0035] The Prepare Ligands method built-in DS was used, and the settings were not to generate tautomer and isomer, and the protonation pH value was set to 7, and the remaining parameters were default parameters.

[0036] (3) Molecular docking

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

[0038] The PSF protein and small molecules were docked by Discovery Studio to obtain the docking score.

[0039] Table 1 Docking score of compounds and PSF protein

[0040]

[0041]

[0042] Result analysis: The docking scores of formula (I) and formula (II) are better than formula (III) and formula (IV), which indicates that their inhibitory effect on PSF is better than formula (III) and formula (IV). In addition, the survival rate determination in Example 4 below also shows that the survival rate of HUVEC of formula (I) and formula (II) is higher than that of the positive control, while the survival rate of HUVEC of formula (III) and formula (IV) is lower than that of the positive control, so formula (I) and formula (II) compounds are selected for subsequent activity determination, and formula (III) and formula (IV) are not used.

[0043] Example 2: RNA-pulldown experiment

[0044] RNA pulldown experiment was performed using Pierce TM Magnetic RNA protein pull-down kit to verify the mechanism of RNA pull-down experiment. The kit provides streptavidin magnetic beads and reagents for efficient enrichment of RNA-binding proteins. RNA pull-down experiment first binds the in vitro transcribed RNA to the magnetic beads, then incubates the RNA-magnetic bead complex with cell lysate, so that the proteins bound to the target RNA will be adsorbed on the magnetic beads. After eluting the RNA-protein complex from the magnetic beads with eluent, Western Blot is used to detect specific binding proteins.

[0045] 1. Experimental protocol

[0046] Binding of magnetic beads to labeled RNA: Take 50 μL magnetic beads and resuspend them in a sterile 1.5 mL centrifuge tube without enzyme, place them in a magnetic separation rack, and discard the supernatant; wash the magnetic beads with 50 μL of 20 mM Tris twice; 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 for 30 min.

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

[0048] Table 2 Formulation of RNA-protein binding reaction premix

[0049]

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

[0051] 2. Result analysis

[0052] Results are shown in Figure 1 and Figure 2 . Figure 1 and Figure 2 It is shown that the ability of the compounds represented by formula (I) and formula (II) to inhibit the binding of lncRNA to PSF protein is increased, and the inhibitory ability of formula (I) is slightly greater than that of formula (II), and the inhibition rates of formula (I) and formula (II) are about 87% and 83%, respectively.

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

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

[0055] Place a 250 mL single-necked flask on a magnetic stirrer, add 60 mL of methanol, and then add 2,4-dihydroxybenzaldehyde (0.06 mol), ethyl isocyanoacetate (0.06 mol), pyridine (0.06 mol), and cuprous iodide (0.005 mol) in sequence under stirring. After all the raw materials are added, transfer the flask to an oil bath, set the temperature of the oil bath to 40-60°C, and allow the reaction to continue under oil bath heating reflux at this temperature for 6 hours. During the reaction, light pink solids are gradually precipitated. When the amount of precipitated solids becomes less and less until no more solids are precipitated, stop the reaction. After the reaction is completed, hot filtration is performed on the reaction liquid. During the filtration process, the precipitated solids are washed with methanol to obtain pure yellow intermediate 3-formamido-7-hydroxycoumarin.

[0056] A 250 mL single-necked flask was fixed on an oil bath, 75 mL of methanol was added as solvent, and stirring was started. The 3-formylamino-7-methoxycoumarin (0.0063 mol) obtained in the previous reaction was added to the solvent, and 2.6 mL of concentrated hydrochloric acid was slowly added dropwise. The temperature of the oil bath was set to 70°C, and the reaction was heated to reflux for 1 hour. It was found that the solid was dissolved. After the reaction was completed, the reaction solution was removed using a rotary evaporator, and then 60 mL of distilled water was added for dissolution. The pH of the solution was adjusted to 7-8 using a saturated sodium hydroxide solution, and a large amount of yellow solid was precipitated. The precipitate was allowed to stand and was filtered.

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

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

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

[0060] A 250 mL single-necked flask was fixed on an oil bath, 75 mL of methanol was added as solvent, and stirring was started. The 3-formylamino-7-methoxycoumarin (0.0063 mol) obtained in the previous reaction was added to the solvent, and 2.6 mL of concentrated hydrochloric acid was slowly added dropwise. The temperature of the oil bath was set to 70°C, and the reaction was heated to reflux for 1 hour. It was found that the solid was dissolved. After the reaction was completed, the reaction solution was removed using a rotary evaporator, and then 60 mL of distilled water was added for dissolution. The pH of the solution was adjusted to 7-8 using a saturated sodium hydroxide solution, and a large amount of yellow solid was precipitated. The precipitate was allowed to stand and was filtered.

[0061] In a 100 mL capacity three-necked flask, 3-amino-7-hydroxycoumarin (0.006 mol) and 4-trifluoromethoxybenzyl bromide (0.0072 mol) were placed in 30 mL of DMF as the reaction solvent, and stirred at 100°C under oil bath heating for about 6 hours, during which thin layer chromatography was used to monitor the reaction. After the reaction was completed, the reaction solution was poured into about 200 mL of ice water while hot, and a solid was precipitated. After standing and suction filtration, the crude product 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 compound of the present application

[0063] 1. Cell culture

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

[0065] 2. Determination of the glucose damage concentration

[0066] (1) Cell plating: The logarithmic phase cells were trypsinized, centrifuged after termination, and prepared into a cell suspension. The cell concentration was adjusted to 10 x 10 4 / ml.

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

[0068] (2) Concentration gradient setting: The prepared glucose solution (1 mol / L) and culture medium (serum-free) were diluted 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 replicates per group, and incubated in a constant temperature incubator for 24 h and 48 h, respectively.

[0069] (4) MTT detection: MTT solution (10 μL / well) was added in the dark, and the 96-well plate was wrapped tightly with tin foil. Incubation was carried out in a constant temperature incubator for 4 h.

[0070] (5) Termination of culture and dissolution of crystals: The supernatant was removed, 150 ul of dimethyl sulfoxide was added to each well, and the mixture was shaken on a shaker for 10 min to fully dissolve the crystals. The absorbance at 490 nm was measured using a microplate reader, and the cell inhibition rate was calculated according to the formula.

[0071] The calculation formula is: inhibition rate (%) = 1 - OD value of the drug-treated group / OD value of the control group. The experimental results are shown in Table 3.

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

[0073]

[0074]

[0075] Results analysis: High concentration of glucose was selected to cause oxidative stress damage, and the inhibitory effect of different concentrations (20, 30, 40, 50, 55, 60, 65 mM / L) and different time (24, 48 h) on HUVEC was detected. The results showed that different concentrations and time of glucose had different degrees of inhibition on HUVEC cells. Under the inverted microscope, the cell state under each concentration was observed. Compared with the normal control group, the number of rounded and shriveled cells increased with the increase of glucose concentration. The 20 mmol / L glucose concentration had little effect on cell morphology, and the change was not obvious compared with the normal group. However, in the 60 and 65 mmol / L glucose environment, the number of rounded and shriveled cells increased significantly, and almost no brick-like growth was observed. The inhibition rate of 55 mmol / L glucose on HUVEC cells was about 50.8%, and the number of rounded and shriveled cells increased, but there were still some cells in the shape of a shuttle, and some cells still grew in a brick-like manner. Therefore, 55 mmol / L glucose was selected for 48 h incubation as the damage concentration and damage time.

[0076] 3. Anti-high glucose assay

[0077] 55 mM / L glucose was selected for high glucose oxidative damage for 48 h, and the repair ability of compounds of formula (I), (II), (III), (IV) on cells was determined. The positive control was selected as 50 μM / L aspirin.

[0078] (1) Cell plating: trypsinize the logarithmic phase cells, centrifuge and collect after termination, prepare cell suspension, and adjust the concentration to 10 x 10 4 / ml.

[0079] (2) High glucose oxidative damage: discard the culture medium, add normal sugar concentration medium to the normal and blank groups, and add 55 mmol / L glucose to the remaining groups for oxidative damage, and incubate for 48 h.

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

[0081] (4) Drug addition: Discard the culture solution, add the normal sugar concentration medium to the normal group and the blank group, add the 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 h. Set 6 replicate wells, and repeat the experiment in parallel 3 times.

[0082] (5) Prepare MTT solution (final concentration of 5 mg / ml, using PBS as solvent), avoid light and maintain sterility during preparation and storage, and prepare and store it immediately 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 ul of dimethyl sulfoxide to each well, and place it on a shaking bed for low-speed shaking for 10 min to fully dissolve the crystals. Use the enzyme marker to measure the absorbance at 490 nm.

[0084] (7) Calculate the cell viability of each group according to the following formula:

[0085] Model group cell viability (%) = (OD model-OD blank) / (OD control-OD blank) x 100%

[0086] Sample group cell viability (%) = (OD drug-OD blank) / (OD control-OD blank) x 100%

[0087] The results are shown in Table 4.

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

[0089]

[0090] Result analysis: The cell viability of formula (I) and formula (II) compounds is 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, and the EC 50 .

[0091] 4. EC 50 determination

[0092] (1) Cell plating: trypsinize the logarithmic phase cells, centrifuge and collect them after termination, prepare a cell suspension, and adjust the concentration to 10 x 10 4 / ml.

[0093] (2) High glucose oxidative damage: discard the culture solution, add the normal sugar concentration medium to the normal group and the blank group, and add 55 mmol / L glucose to the remaining groups for oxidative damage, and incubate for 48 h.

[0094] (3) Drug administration: Discard the culture solution, add the normal sugar concentration medium to the normal group and the blank group, and add the measured formula (I) and formula (II) with concentrations of 3.125, 6.25, 12.5, 25, 50, 100 μm / L to the sample group, 100 μL per well, and incubate for 24 h.

[0095] (4) Prepare MTT solution (final concentration 5 mg / ml, using PBS as solvent), avoid light and maintain sterility during preparation and storage, and prepare and store as needed. 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 ul of dimethyl sulfoxide to each well, and place it on a shaking bed at low speed for 10 min to dissolve the crystals. Use the enzyme marker to measure the absorbance at 490 nm.

[0097] (7) Calculate the cell viability of each group according to the following formula:

[0098] Model group cell viability (%) = (OD model-OD blank) / (OD control-OD blank) x 100%

[0099] Sample group cell viability (%) = (OD drug-OD blank) / (OD control-OD blank) x 100%

[0100] The results are shown in Table 5.

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

[0102]

[0103] Example 5 Determination of the effect of formula (I) and formula (II) compound components on the release level of model cell nitric oxide (NO)

[0104] The cells were seeded in a 96-well plate, and the culture supernatant was collected after grouping treatment to determine the NO level: (1) The 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 recover the temperature; then the cell culture medium provided in the kit for diluting the standard was used to prepare gradient concentration standards for making a standard curve, which could be increased in concentration range as needed; (2) Then add 50 μL of standard and sample to be tested per well, then add 50 μL of reagent I and II per well, mix well, and measure the absorbance at 540 nm after standing for 2 min, and calculate the NO content in the sample according to the standard curve. The results are shown in Table 6.

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

[0106]

[0107] Results analysis: The stimulation of high glucose condition decreased the release level of NO in HUVEC cells, while the pre-treatment of compounds of formula (I) and formula (II) could increase the release amount of NO to different degrees, effectively protecting the structure and endothelial function stability of HUVEC under high glucose condition.

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

[0109] (1) Cell plating: logarithmic phase cells were inoculated in 10 cm culture dishes and cultured overnight.

[0110] (2) High glucose oxidative damage: discard the culture solution, add normal sugar concentration medium to the normal group and the blank group, and add 55 mmol / L glucose to the remaining groups for oxidative damage, and incubate for 48 h.

[0111] (3) Drug addition: discard the culture solution, add normal sugar concentration medium to the normal group and the blank group, and add compounds of formula (I) and formula (II) at a concentration of 25 μm / L, and incubate for 24 h.

[0112] (4) TNF-α kit and IL-4 kit were equilibrated at room temperature for 20 min.

[0113] (5) Prepare 1X washing solution in advance, and prepare according to the ratio of washing solution (20X): double distilled water = 1:19 mL.

[0114] (6) In the standard bottle, add diluent according to the required volume on the bottle wall, and incubate for 15 min (room temperature).

[0115] (7) Dilute the standard to obtain concentrations of 1000, 500, 125, 62.5, 31.25 pg / mL, and 0 pg / mL is the standard diluent.

[0116] (8) After lysing the cells in each group, take the supernatant (12000g, 10 min).

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

[0118] (10) Wash the plate with 1X washing solution 5 times, 300-350 μL per well, and dry the plate on filter paper.

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

[0120] (12) Wash the plate 5 times with 1X wash buffer, 300-350 μL per well, and tap the plate on paper towels to dry.

[0121] (13) Add horseradish peroxidase-labeled streptavidin (100 μL per well). Cover the plate with adhesive plate sealers and incubate at room temperature for 20 min.

[0122] (14) Wash the plate 5 times with 1X wash buffer, 300-350 μL per well, and tap the plate on paper towels to dry.

[0123] (15) Add color developing solution TMB (100 μL per well). Cover the plate with adhesive plate sealers and incubate at room temperature for 20 min.

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

[0125] (17) Calculate the concentration of the sample according to the standard curve.

[0126] The results are shown in Table 7.

[0127] Table 7 Effect of compounds of formula (I) and (II) on the content of TNF-α in HUVEC under high glucose condition

[0128]

[0129] Result analysis:

[0130] The stimulation of high glucose condition significantly increased the expression of TNF-α, a pro-inflammatory related factor, in HUVECs, indicating that high glucose induced inflammation, and the pre-treatment of compounds of formula (I) and (II) reduced the level of pro-inflammatory factors to different degrees, alleviated the degree of inflammation, and played a protective role in endothelial function.

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

[0132] (1) Cell plating: logarithmic phase cells were inoculated in 10 cm culture dishes and cultured overnight.

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

[0134] (3) Drug addition: the culture solution was discarded, normal sugar concentration medium was added to the normal group, and compounds of formula (I) and (II) were added at a concentration of 25 μm / L, and incubated for 24 h.

[0135] (4) The supernatant (12000 g, 10 min) was taken after lysing the cells in each group.

[0136] (5) Standard dilution: Gradient dilution of standard to 1, 2, 5, 10, 20, 50 μmol / L, ready for use;

[0137] (6) BCA method for measuring protein concentration and drawing standard curve: In the 96-well plate, according to the instructions, the protein standard solution of gradient concentration was added and recorded, and the sample was added in a new well, followed by adding PBS to all wells to 20 μL; the BCA working solution prepared in advance was added to each well to measure the absorbance and draw the standard curve, and the protein concentration was calculated;

[0138] (7) Sample determination: PBS was used as a blank control group, different concentrations of standard in (1) were added for making standard curve, and then the sample and MDA working solution were added and mixed well to measure the absorbance at 532 nm;

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

[0140] Table 8 Effect of compounds of formula (I) and formula (II) on MDA content of HUVEC under high glucose condition

[0141]

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

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

[0144] Finally, it should be explained that the above examples are only used to illustrate the technical solutions of the present application, but not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A 3-benzylamino coumarin compound or a pharmaceutically acceptable salt thereof, the compound having a structural formula of 2. Use of the 3-benzylamino coumarin compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a PSF protein inhibitor.

3. Use of the 3-benzylamino coumarin compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for preventing, treating or ameliorating high glucose-induced vascular injury.

4. Use of the 3-benzylamino coumarin compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for preventing, treating or ameliorating diabetes-induced vascular injury.

5. Use according to claim 3 or 4, characterized in that, The compound can increase the amount of NO release.

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

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

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

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