CN112168817A

CN112168817A - Application of 3-aryl coumarin compound

Info

Publication number: CN112168817A
Application number: CN202011135279.6A
Authority: CN
Inventors: 孙捷; 李雨霏; 牟艳玲; 王晓静; 王兵
Original assignee: Institute Of Materia Medica Shandong Academy Of Medical Sciences (shandong Anti-Aging Research Center Shandong New Technology Pharmaceutical Research Institute)
Current assignee: Institute Of Materia Medica Shandong Academy Of Medical Sciences (shandong Anti-Aging Research Center Shandong New Technology Pharmaceutical Research Institute)
Priority date: 2020-10-21
Filing date: 2020-10-21
Publication date: 2021-01-05

Abstract

The invention discloses application of 3-aryl coumarin compounds shown in structural formula I and/or structural formula II in preparation of a medicine for preventing and/or treating vascular calcification of a diabetic patient, and belongs to the field of medicine production.

Description

Application of 3-aryl coumarin compound

Technical Field

The invention relates to the field of medicine production, and particularly provides an application of 3-aryl coumarin compounds in preparation of medicines for treating diabetes and related vascular calcification.

Background

Diabetes mellitus is a chronic, multifactorial metabolic disorder characterized by hyperglycemia, insulin resistance, and islet beta cell dysfunction. The diabetic patients are very easy to have cardiovascular diseases, the cardiovascular diseases are the most main death reasons of the diabetic patients, and the cardiovascular diseases are greatly related to vascular calcification diseases, and the patients have extensive vascular calcification. Compared with the common population, the diabetic patients have obviously raised vascular calcification index which is mainly generated in coronary artery and lower limb blood vessels, and the vascular calcification is closely related to the prognosis of atherosclerosis, heart failure and the like. Vascular calcification refers to the hardening of the medial layer of the artery by depositing hydroxyapatite mineral into the extracellular matrix. In contrast to the traditional belief that vascular calcification is a progressive passive process with increasing age, dominated by disorders of mineral metabolism, it is now found that vascular calcification formation is an active, highly regulated complex pathophysiological process of diverse cellular origin and similar to bone development.

The molecular regulatory mechanisms of high sugar on endothelial cell phenotypic changes are not well understood at present. According to the existing literature, important metabolites of hyperglycemia, namely glycosylation end products (AGEs), can induce calcification cascade through various ways and modes to further cause vascular calcification, which is probably the most important reason for high-glucose induced vascular calcification.

In the aspect of vascular calcification treatment, no means for reversing vascular calcification exists at present, but various intervention measures can be used for slowing or delaying the progress of vascular calcification, such as controlling traditional risk factors such as hyperglycemia, blocking AGEs generation, AGEs/RAGE interaction, reducing oxidative stress and the like, but the intervention measures still have the defects of single action target, large side effect, poor treatment effect and the like, so that the vascular calcification treatment is a difficult point and a hot point of current research. In recent years, various natural products have been reported to have a vascular protection effect, and in recent years, coumarin and flavonoid compounds have attracted attention because of good AGEs production inhibition, antioxidant and anti-inflammatory activities, and in particular, umbelliferone, quercetin, soybean isoflavone and the like are considered to have good vascular protection effects and are researched and reported many times, but the direct drug formation of the side effects such as cytotoxicity and carcinogenesis is difficult because of weak activity.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the application of two 3-aryl coumarin compounds.

The technical scheme adopted by the invention for solving the technical problems is as follows:

the application of the 3-aryl coumarin compound shown in the structural formula I and/or the structural formula II in preparing the medicine for preventing and/or treating vascular calcification of diabetes patients,

through a large number of experiments, the applicant finds that the 3-aryl coumarin compounds shown in the structural formula I and the structural formula II can well prevent and treat vascular calcification caused by diabetes while reducing blood sugar.

Preferably, the compounds of formula I and/or formula II are useful in the preparation of a medicament for multi-target anti-diabetes induced vascular calcification.

The anti-diabetes-induced vascular calcification drug takes 3-aryl coumarin compounds shown in structural formula I and/or structural formula II as effective components.

Preferably, the compounds of formula I and/or formula II are used to prepare inhibitors of alkaline phosphatase activity.

The alkaline phosphatase activity inhibitor takes 3-aryl coumarin compounds shown in structural formula I and/or structural formula II as effective components.

Preferably, the compounds of formula I and/or formula II are useful in the preparation of AGEs formation inhibitors.

The AGEs formation inhibitor takes 3-aryl coumarin compounds shown in structural formula I and/or structural formula II as effective components.

The composition for preventing and/or treating diabetes and complications thereof comprises 3-aryl coumarin compounds shown in structural formula I and/or structural formula II as effective components.

The compound of formula I, the compound of formula II, or the composition can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spray, nasal drop, eye drop, osmotic, absorption, physical or chemical mediated method; or mixed or coated with other materials and introduced into body.

When necessary, one or more pharmaceutically acceptable carriers can be added into the compound of the structural formula I, the compound of the structural formula II or the composition to prepare medicines and/or health care products in various forms such as injection, tablets, powder, granules, capsules, oral liquid, ointment, cream and the like. The carrier comprises diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants and the like which are conventional in the pharmaceutical field. The medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field.

Furthermore, the 3-aryl coumarin compound shown in the structural formula I and/or the structural formula II can also be applied to in vitro tests on the calcification level of vascular smooth muscle cells.

The applicant evaluates and screens the activity of slowing down or delaying the vascular calcification process of the 3-aryl coumarin compounds on a cellular level, and the two related 3-aryl coumarin compounds have high activity and low toxicity and can obviously inhibit vascular calcification.

Drawings

FIG. 1 shows the ALP results of AGEs induced calcification in example III;

FIG. 2 shows the results of ALP induction of calcification by AGEs in example four.

Detailed Description

The anti-diabetic activity of the two 3-arylcoumarins of the present invention will be described in detail below with reference to specific examples, but the present invention is not limited thereto. Various modifications made by those skilled in the art in light of the present patent disclosure are intended to be within the scope of the appended claims.

The test methods described in the following examples are all conventional methods unless otherwise specified; the reagents and biomaterials, if not specifically indicated, are commercially available.

The structural formulae and the numbering of the two compounds used in the examples are as follows:

the first embodiment is as follows: determination of the toxicity of two Compounds on cells

(1) 2-8 generation human aortic vascular smooth muscle cells were cultured, single cell suspension was prepared with culture medium containing 10% fetal bovine serum, 5000 cells per well were inoculated into 96-well plates, with 200ul per well volume.

(2) Set up 7 groups, do respectively: normal group, I-6.25 ug, I-12.5 ug, I-25 ug, II-6.25 ug, II-12.5 ug, II-25 ug. Each group of the medicine is provided with 6 compound holes. Culturing for 24h and 48h under the same general culture conditions.

(3) After the incubation, 20ul of MTT solution (5mg/ml, prepared in PBS < ph ═ 7.4 >) was added to each well.

Incubation was continued for 4 hours, the culture was terminated, and the culture supernatant in the wells was carefully aspirated, after centrifugation was required for suspension cells, and the culture supernatant in the wells was aspirated. Add 150ul DMSO/well and shake for 10 minutes to fully melt the crystals.

(4) The 490nm wavelength is selected, the light absorption value of each pore is measured on an enzyme linked immunosorbent instrument, and the result is recorded.

(5) The results are shown in the following table:

group of	Normal group	Ⅰ-6.25ug	Ⅰ-12.5ug	Ⅰ-25ug	Ⅱ-6.25ug	Ⅱ-12.5ug	Ⅱ-25ug
								OD mean value	0.573	0.565	0.601	0.554	0.564	0.599	0.578

From the test results, it can be seen that both structural formula I and structural formula II are non-toxic to cells at different concentration gradients.

Example two: calcification induction of vascular smooth muscle cells in humans

(1) Preparation of calcification-inducing liquid

20mg of bovine serum albumin was weighed and dissolved in 50mmol/L PBS (pH7.4) to prepare PBS (pH7.4) buffer solution of bovine serum albumin at a final concentration of 10 mg/mL. 79.2mg of glucose was weighed and dissolved in 2ml of distilled water to prepare a glucose solution having a final concentration of 0.2 mol/L. The two solutions were mixed and filtered, and 0.02% double antibody was added to prevent bacterial growth. The above mixed solution was incubated at 37 ℃ for 14 days, and the excitation wavelength and emission wavelength were set to 350nm and 420nm, respectively, using a fluorescence spectrophotometer, and the fluorescence intensity of AGE was measured as follows:

(2) calcification induction of aortic vascular smooth muscle in humans

Human Aortic Vascular Smooth Muscle Cells (HAVSMCs) were purchased. Cells from 2-8 passages were used for the experiment. After induction of cells with AGEs calcification medium, the cells were divided into 6 groups, including normal control group, calcification model group, vehicle control group, positive control group, sample I treatment group, and sample II treatment group. Wherein the sample treatment groups were further divided into 3 groups by concentration: 6.25ug/ml, 12.5ug/ml, 25 ug/ml.

At 37 deg.C, 5% CO₂The alkaline phosphatase activity was measured after 4 days of incubation under the conditions.

Example three: determination of the inhibitory Effect of two Compounds on the alkaline phosphatase Activity of calcified cells

1. Principle for measuring alkaline phosphatase activity in vitro

The alkaline phosphatase is widely distributed in bones, intestines, kidneys, livers, placentas and other tissues of human bodies, and is a detection index for screening and diagnosing bone diseases which is commonly used clinically. Under alkaline conditions, Para-nitrophenyl phosphate (pNPP) can generate Para-nitrophenylol under the action of alkaline phosphatase. para-nitrophenol is a yellow product under alkaline conditions, and the absorbance can be detected at 400-415 nm. The darker the product yellow, the higher the alkaline phosphatase activity and the higher the absorbance value, and vice versa the lower the enzyme activity.

2. Preparation of reaction solution

(1) And (4) reagent preparation, namely taking out all reagents and returning to room temperature for use.

(2) Preparing a chromogenic substrate solution, namely dissolving a tube of chromogenic substrate in 2.5ml of detection buffer solution, dissolving the chromogenic substrate in 1ml of detection buffer solution, fully dissolving and uniformly mixing the chromogenic substrate, transferring the chromogenic substrate to a 15ml centrifuge tube, adding 1.5ml of detection buffer solution, fully dissolving and uniformly mixing the chromogenic substrate and the centrifuge tube, and placing the chromogenic substrate on ice. The freshly prepared chromogenic substrate solution was used within 6 hours.

(3) The working solution for the standard was prepared by diluting 10ul of pnitrophenol solution (10mM) to 0.2ml with the detection buffer to a final concentration of 0.5 mM.

(4) Preparation of cell lysate, cells after induction of calcification were freed from the culture medium and washed once with PBS. Add 80. mu.l of lysis solution to each well of the 24-well plate. The lysate was brought into full contact with the cells by several blows from a gun. The supernatant was then centrifuged at 10000g for 3min and used for the detection of alkaline phosphatase.

(5) Blank control wells, standard wells, and sample wells were set using 96-well plates as follows. The amounts of standards were 4, 8, 16, 24, 32 and 40 microliters, respectively, with 50 microliters of sample added.

(6) Lightly blowing and beating the mixture by using a gun head and uniformly mixing the mixture.

(7) Incubate at 37 ℃ for 15 minutes.

(8) The reaction was stopped by adding 100u1 of a reaction stop solution to each well. At this point, the standard or wells with alkaline phosphatase activity will appear in different shades of yellow.

(9) The absorbance was measured at 405 m.

(10) The results are shown in FIG. 1 and the following table. From the test results it can be seen that: the structural formula I and the structural formula II can inhibit the activity of calcified cell alkaline phosphatase, wherein the structural formula I has the best effect, and the inhibition effect of the structural formula II on the activity of alkaline phosphatase is less than that of the structural formula I and a positive control drug, but the activity of the alkaline phosphatase can be still obviously inhibited compared with a calcified group.

Group of	Normal low sugar	Normal high sugar	Calcification induction	Negative control	Positive control	Ⅰ-6.25ug	Ⅰ-12.5ug	Ⅰ-25ug
									OD mean value	0.148	0.254	0.477	0.467	0.323	0.323	0.348	0.36
						Ⅱ-6.25ug	Ⅱ-12.5ug	Ⅱ-25ug
															0.331	0.419	0.418

Example four: comparison of the inhibitory Effect of two Compounds on the alkaline phosphatase Activity of calcified cells with other Compounds of the same framework

Four compounds were randomly selected for comparative experiments of alkaline phosphatase activity inhibition.

(1) The preparation of the calcification-inducing solution and the induction of calcification in human aortic vascular smooth muscle cells were carried out according to the method of example two.

(2) The working solution for the standard was prepared by diluting 10ul of pnitrophenol solution (10mM) to 0.2ml with the detection buffer to a final concentration of 0.5 mM.

(3) The inhibition of the alkaline phosphatase activity of calcified cells by six compounds of the structural formulae I, II, III, IV, V and VI was determined according to the method of example III.

(4) The results are shown in FIG. 2 and the following table. It can be seen that the compound of formula I, II has a more significant inhibitory effect on the activity of alkaline phosphatase in calcification-induced cells than other similar compounds.

Group of	Normal low sugar	Normal high sugar	Calcification induction	Negative control	Positive control
							OD mean value	0.216	0.327	0.619	0.607	0.384
Group of	Ⅰ-12.5ug	Ⅱ-12.5ug	III-12.5ug	IV-12.5ug	V-12.5ug	VI-12.5ug
							OD mean value	0.369	0.452	0.578	0.604	0.539	0.577

In summary, it can be seen from all the above embodiments that the two compounds have good calcium-reducing activity, and play roles in reducing vascular calcification and protecting cardiovascular complications caused by diabetes through the ways of inhibiting alkaline phosphatase activity and the like. The invention provides an important idea for developing a multi-target medicament for treating the diabetic vascular complications and has important research value and application potential.

Claims

1. The application of the 3-aryl coumarin compound shown in the structural formula I and/or the structural formula II in preparing the medicine for preventing and/or treating vascular calcification of diabetes patients,

2. the use of 3-arylcoumarins according to claim 1, wherein: is used for preparing a medicament for treating multi-target diabetes-induced vascular calcification.

3. The use of 3-arylcoumarins according to claim 2, wherein: used for preparing alkaline phosphatase activity inhibitor.

4. The use of 3-arylcoumarins according to claim 2, wherein: used for preparing AGEs formation inhibitor.

5. The medicine for preventing and/or treating vascular calcification of diabetics is characterized by comprising the following components in percentage by weight: the effective component is 3-aryl coumarin compound shown in structural formula I and/or structural formula II.

6. An inhibitor of alkaline phosphatase activity, characterized by: the effective component is 3-aryl coumarin compound shown in structural formula I and/or structural formula II.

An inhibitor of AGEs formation, characterized by: the effective component is 3-aryl coumarin compound shown in structural formula I and/or structural formula II.

8. The application of the 3-aryl coumarin compound shown in the structural formula I and/or the structural formula II in-vitro vascular smooth muscle cell calcification level tests.