CN112336723B - Method for preparing medicine for reducing coupling degree of TRPV4 and NOX2 - Google Patents

Abstract

The application discloses a method for preparing a drug for reducing the coupling degree of TRPV4 and NOX2, and belongs to the technical field of chemical medicine. The application takes the Entrictinib, L75507, gambogic acid and M12 compounds as active ingredients, and is applied to the preparation of medicaments for reducing the coupling degree of TRPV4 and NOX2, antioxidation stress and vascular permeability, so that the coupling degree of TRPV4 and NOX2 in obese patients is reduced, the excessively coupled two proteins are decoupled, the normal state of the two proteins is recovered, and the oxidative stress and vascular permeability of the obese patients are recovered to the normal level.

Description

Method for preparing medicine for reducing coupling degree of TRPV4 and NOX2

Technical Field

The application particularly relates to a method for preparing a drug for reducing the coupling degree of TRPV4 and NOX2, belonging to the technical field of chemical medicine.

Background

Reactive Oxygen Species (ROS) are a series of intermediates from which molecular oxygen is derived during the reduction process, mainly comprising superoxide anions (. O2-), hydrogen peroxide (H2O 2), hydroxyl radicals (. OH), hypochlorous acid (HOCl), etc. (Antioxid Redox Signal,2015,23,514-32). Free radicals are defined herein as any substance with unpaired electrons that react readily with healthy molecules, which can be produced in cells by different mechanisms, can proliferate indefinitely in the presence of oxygen, and once these free radicals reach high concentrations, can cause oxidative damage to tissues or organs (Biomed Res Int,2014, 831-841). In the mitochondrial respiratory chain, free radicals are produced as byproducts of the NADPH oxidase family or the double oxidase (DUOX) family. Among them, the main source of vascular endothelial ROS is the reducing nicotinamide purine dinucleotide oxidase (NOX), other enzymes (cyclooxygenase, cytochrome P450, mitochondrial electron transport chain) can produce ROS as byproducts, and dysfunctional nitric oxide synthase and xanthine dehydrogenase can also produce ROS (Trends Endocrinol Metab,2014,25,452-463). Under physiological conditions, the production and elimination of ROS in the body is in a dynamic equilibrium process, which is involved in signal transduction in the body, and is the basis of physiological functions of the body, regulating various signal transduction, modification of protein structures, transcription factors and genes by direct reaction, and regulating their functions (Circ J,2015,79,1145-1155) (Free Radic Biol Med,2014,76,208-226). Meanwhile, ROS are involved in the regulation of cell growth, differentiation, apoptosis signaling and enzymatic activity, participate in inflammatory reactions by stimulating the production of inflammatory factors, scavenge pathogenic microorganisms and foreign substances, and also can act as second messengers to sustain endothelial cell proliferation and differentiation and mediate stress and growth related concentration responses, including angiogenesis (Free Radic Biol Med,2014,69,278-88) (post Biochem,2009,55,145-152). However, under pathological conditions, such as when the NOX protein family is overexpressed, ROS homeostasis is imbalanced, ROS accumulate excessively in vascular endothelial cells, and high concentrations of ROS can peroxidate lipids, thereby decreasing the fluidity of the biofilm, increasing the permeability of the cell membrane (Arch international Med,2003,163,525-541), and causing abnormal expression of adhesion molecules, which eventually becomes an early pathological event leading to cardiovascular disease. In addition, there are related studies reporting that increased NADPH oxidase-dependent superoxide anion formation (J Cereb Blood Flow Metab,2011,31,991-993) (Diabetes, 2002,51,522-527), overproducing ROS, plays a key role in mediating endothelial dysfunction (Antioxid Redox Signal,2010,13,757-768) (Br J Phacol, 2011,164,598-606) can be observed in a high-fat diet-induced obesity model.

Reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase is a coenzyme for the reduction reaction, and is an enzyme complex composed of a membrane subunit gp91phox (Nox 2), a transmembrane subunit p22phox, a cytoplasmic subunit p47 phox, small molecule Guanosine Triphosphate (GTP), and p29 peroxidase (gp denotes glycoprotein, phox represents a phagocyte oxidase component), wherein gp91phox and a homolog p22phox are main functional subunits of the NADPH oxidase family. The activation mechanisms and profiles of the different members of the NADPH oxidase family are different, and the active oxygen produced by them can act as a second messenger, mediating the activation of the signaling pathways of various pro-fibrotic factors, including transforming growth factor receptors, platelet-derived growth factor receptors, angiotensin 2 receptors, etc. Mitochondrial NADPH oxidase can catalyze the generation of negative oxygen ions O2-, mitochondria can catalyze the generation of OH along with peroxidase, and monoamine oxidase outside the membrane activated by mitochondria and protein kinase C can catalyze the generation of H2O2. Among them, gp91phox (Nox 2), a main functional subunit of the NADPH oxidase family, belongs to one of Nox family members, and the Nox2 protein contains 570 amino acids and is expressed in phagocytes, but also in non-phagocytes such as cardiac muscle cells, neurons, endothelial cells and oriented hematopoietic stem cells. It has been shown that the main biological function of NOX2 is ROS production, which can maintain normal physiological activities of cells, but under abnormal conditions, such as when the body is stimulated by cytokines, inflammatory mediators, calcium ions, heavy metals, etc. in the environment, NOX2 protein is overexpressed to produce a large amount of ROS, which causes endothelial dysfunction, endothelial structural changes, causes increased cell permeability, causes extravasation of fluids, and seriously causes diseases such as tumors, atherosclerosis, post-reperfusion stenosis, thrombosis, hypoxia, multiple organ dysfunction syndrome, etc. and senile diseases such as alzheimer's disease, senile dementia, etc. (life sciences 2012, 568-577).

Vascular permeability changes are a common pathological feature of a series of inflammatory diseases, and thus, it becomes important to regulate and control oxidative stress, which is a causative factor of the inflammatory diseases. Several classes of anti-oxidative stress drugs have been discovered. Probucol, also known as probucol, is a lipid-regulating drug which has powerful antioxidant and anti-aging effects while reducing cholesterol, thereby playing roles in preventing restenosis after angioplasty, atherosclerosis and the like (Circulation, 2003,107,552-558). An application of acitretin to antitumor drugs, but recently, the Katerina Akassoglou subject group of the university of california san francisco wil neuroscience institute, journal Nature Immunology, has revealed potent anti-inflammatory, antioxidant stress and neuroprotection in neuroinflammatory diseases. Lipoic acid and B vitamins can play an antioxidant role through various mechanisms such as free radical removal, other antioxidant regeneration and the like, and a control experiment carried out by 5 research institutions such as Beijing hospital recently shows that: the lipoic acid tablet can treat diabetic peripheral neuropathy, and meanwhile, the lipoic acid tablet has good results in the fields of ophthalmology, heart, nerves, digestion and the like. The NOX inhibitor oleandrin, which is recently discovered by laboratory staff such as Dongzhimen Hospital, beijing university, ibrutinib, a drug for treating recurrent and refractory lymphomas, activates the myocardial cell oxidative stress signaling pathway to increase the susceptibility to atrial fibrillation, while the use of the NOX inhibitor oleandrin can inhibit the response and reduce the incidence and duration of atrial fibrillation (Redox Biol,2020,101432). All the drugs have experimental basis, and have a certain improvement effect on oxidative stress, but aiming at a single target, the drugs are developed, off-target is easy to generate, side effects are strong and the like, so that the novel efficient drugs for regulating and controlling oxidative stress are of great significance for TrpV4-NOX2 coupling complexes.

Disclosure of Invention

Technical problems:

provides a new target point for the regulation and control of oxidative stress, provides more drugs for patients with abnormal vascular permeability clinically, has stronger targeting, can realize local regulation, and has smaller side effect. So as to prevent the further development of cardiovascular diseases and prevent thrombosis, hypoxia, and multiple organ dysfunction syndrome.

The technical scheme is as follows:

through extensive screening studies, the following drugs, such as CB-839, were found: the current research focuses on the effects of resisting cell proliferation, treating leukemia and preventing, improving or treating obesity; l75507, a β3-AR agonist, has been less studied, focusing mainly on the role of L75507 in coupling with Gs and Gi to activate adenylate cyclase and MAPK signaling; entrictinib, a Trk, ROS1, and ALK inhibitor, has been studied primarily for its anticancer effect, and FDA approval has been obtained today for the treatment of ROS1, non-small cell lung cancer and NTRK, solid tumors, and the like; bohemine, a purine analog, is also a synthetic selective CDK inhibitor, and prior studies have focused on modulating cell cycle, anti-tumor proliferation; gambogic acid, bcl-XL, bcl-2, bcl-W, bcl-B, bfl-1 and Mcl-1 inhibitors, the prior researches focus on the effects of resisting cancer, resisting biomembrane related infection, regulating inflammation, vascular growth and the like; m12 has little current research and is primarily focused on protein kinase inhibitors. The above is the research direction of the medicines screened by us, but has no research in the aspect of cardiovascular, so the medicine has important significance as an antioxidant stress medicine for treating or delaying cardiovascular diseases.

The first object of the present application is to provide an application of a compound of the structure shown in formula (1) and pharmaceutically acceptable salts thereof in the preparation of a medicament for reducing the coupling degree of TRPV4 and NOX2, wherein the application utilizes the compound of the structure shown in formula (1) as an active ingredient of the medicament,

the second purpose of the application is to apply the compound with the structure shown in the formula (1) and pharmaceutically acceptable salts thereof to the preparation of antioxidant stress drugs for treating or delaying cardiovascular diseases.

The third object of the present application is to apply the compound of the structure shown in the formula (1) and pharmaceutically acceptable salts thereof in preparing medicines for treating or preventing atherosclerosis, restenosis, thrombosis, hypoxia, multiple organ dysfunction syndrome, alzheimer disease and senile dementia.

A fourth object of the present application is to provide an application of a compound having a structure represented by formula (2) and a pharmaceutically acceptable salt thereof in the preparation of a medicament for reducing the coupling degree of TRPV4 and NOX2, the application being to utilize the compound having a structure represented by formula (2) as an active ingredient of the medicament,

the fifth object of the present application is to apply the compound of the structure shown in the formula (2) and the pharmaceutically acceptable salt thereof in preparing the antioxidant stress drug for treating or delaying cardiovascular diseases.

The sixth object of the present application is to apply the compound of the structure shown in the formula (2) and pharmaceutically acceptable salts thereof to the preparation of a medicament for treating or preventing atherosclerosis, restenosis, thrombosis, hypoxia, multiple organ dysfunction syndrome, alzheimer disease and senile dementia.

A seventh object of the present application is to provide an application of a compound having a structure represented by formula (3) and a pharmaceutically acceptable salt thereof in the preparation of a medicament for reducing the coupling degree of TRPV4 and NOX2, the application being to utilize the compound having a structure represented by formula (3) as an active ingredient of the medicament,

an eighth object of the present application is to apply the compound of the structure shown in formula (3) and pharmaceutically acceptable salts thereof in preparing antioxidant stress drugs for treating or delaying cardiovascular diseases.

The ninth object of the present application is to apply the compound of the structure shown in the formula (3) and pharmaceutically acceptable salts thereof in preparing medicines for treating or preventing atherosclerosis, restenosis after reperfusion, thrombosis, hypoxia, multiple organ dysfunction syndrome, alzheimer disease and senile dementia.

In one embodiment of the application, the "pharmaceutically acceptable salts" are those salts which are meant to retain the biological effectiveness and properties of the parent compound. The term "salt" refers to any salt of a compound according to the application prepared from an inorganic or organic acid or base and an internally formed salt. Typically, such salts have a physiologically acceptable anion or cation.

In one embodiment of the present application, the pharmaceutically acceptable salt is an inorganic salt or an organic salt, and the inorganic salt includes inorganic acid salts such as: hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, phosphate, acid phosphate; the organic salt is selected from acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, sulfonate, benzenesulfonate, salicylate. Inorganic basic salts such as: potassium salt, calcium salt sodium salt, ferrous salt, ferric salt, zinc salt, barium salt, silver salt, copper salt, bismuth salt, antimony salt, aluminum salt, mercury salt, lead salt, bromide, iodide. Organic acid salt: citrate, malate, tartrate, quinite, acetate, succinate, oxalate, benzoate, salicylate, caffeate, and the like. Organic base salt: quinine salts, nicotine salts, t-butoxide, ethoxide, methoxide.

Further, all compounds of the present application or salts thereof may be isolated in the form of solvates, and thus any such solvates are within the scope of the present application.

In one embodiment of the present application, the medicament comprises a pharmaceutically acceptable carrier and/or pharmaceutical excipients in addition to the above-mentioned compound as active ingredient or a pharmaceutically acceptable salt thereof.

In one embodiment of the present application, the pharmaceutical excipients include any one or more of the following:

excipients, diluents, fillers, solubilizers, binders, humectants, disintegrants, slow solvents, absorption accelerators, wetting agents, adsorbents, lubricants.

In one embodiment of the application, in the preparation of a medicament, all compounds of the application or pharmaceutically acceptable salts thereof are typically admixed with a pharmaceutically acceptable carrier, excipient or diluent. Wherein the content of all the compounds of the present application or pharmaceutically acceptable salts thereof in a unit dosage form (e.g., a tablet or capsule) may be 0.01 to 1000mg, for example, 0.05 to 800mg, 0.1 to 500mg, 0.01 to 300mg, 0.01 to 200mg, 0.05 to 150mg, 0.05 to 50mg, etc.

In one embodiment of the present application, in the preparation of a medicament, the composition of the present application may be formulated into a conventional pharmaceutical formulation according to a conventional preparation method. Such as tablets, pills, capsules, powders, granules, emulsions, suspensions, dispersions, solutions, tinctures, syrups, ointments, drops, suppositories, inhalants, sprays and the like.

In one embodiment of the present application, the compounds of the present application or pharmaceutically acceptable salts thereof may be formulated as solid formulations for oral administration, including, but not limited to, capsules, tablets, pills, powders, granules, and the like.

In one embodiment of the application, it is admixed with at least one conventional inert excipient (or carrier), for example sodium citrate or dicalcium phosphate, or with one or more selected from the following:

(1) Fillers or solubilisers, for example starch, lactose, sucrose, glucose, mannitol, silicic acid and the like;

(2) Binders, for example, hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, acacia, and the like;

(3) Humectants, for example, glycerin, etc.;

(4) Disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, and the like;

(5) Slow solvents such as paraffin wax and the like;

(6) Absorption accelerators such as quaternary ammonium compounds and the like;

(7) Wetting agents such as cetyl alcohol and glycerol monostearate, and the like;

(8) Adsorbents such as kaolin and the like;

(9) Lubricants, for example, talc, calcium stearate, solid polyethylene glycol, sodium lauryl sulfate, and the like, or mixtures thereof. Buffers may also be included in capsules, tablets, pills.

In one embodiment of the present application, the solid dosage forms, such as tablets, dragees, capsules, pills and granules, may be provided with coatings and shell materials such as enteric coatings and other materials known in the art in the form of crystalline coatings or microencapsulations. They may contain opacifying agents and the release of the active ingredient in such a composition may be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. The active ingredient may also be in the form of microcapsules with one or more of the above excipients, if desired.

In one embodiment of the present application, the compounds of the present application or pharmaceutically acceptable salts thereof may be formulated into liquid dosage forms for oral administration, including, but not limited to, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, tinctures, and the like. In addition to all compounds of the application or pharmaceutically acceptable salts thereof as active ingredients, liquid dosage forms may contain inert diluents commonly used in the art such as water and other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular cottonseed, groundnut, corn, olive, castor, sesame oils and the like or mixtures of these substances and the like. In addition to these inert diluents, the liquid dosage forms of the present application can also include conventional adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents and the like.

Such suspending agents include, for example, ethoxylated stearyl alcohol, polyoxyethylene sorbitol, and sorbitan, microcrystalline cellulose, agar-agar, and the like, or mixtures of these.

In one embodiment of the application, the compounds of the application and pharmaceutically acceptable salts thereof may be formulated in dosage forms for parenteral injection, including, but not limited to, physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions and dispersions. Suitable carriers, diluents, solvents, excipients include water, ethanol, polyols and suitable mixtures thereof.

In one embodiment of the present application, the compounds of the present application or pharmaceutically acceptable salts thereof may be formulated into dosage forms for topical administration, including, for example, ointments, powders, suppositories, drops, sprays, inhalants and the like. All compounds of the application or pharmaceutically acceptable salts thereof as active ingredients are admixed under sterile conditions with a physiologically acceptable carrier and optionally with preservatives, buffers and, if necessary, propellants which may be required.

The compounds of the application or pharmaceutically acceptable salts thereof may be administered alone or in combination with other pharmaceutically acceptable therapeutic agents, particularly with other antihypertensive agents. Such therapeutic agents include, but are not limited to: antihyperlipidemic anti-atherosclerosis agents such as cholestyramine, colestipol, lovastatin, cervastatin, sitosterol, etc.; antioxidant anti-atherosclerosis agents such as procyanidine, vitamin E, etc.; polyene fatty acids such as mono-deca-penta-enoic acid, a-linolenic acid, etc.; the anti-atherosclerosis agents include polysaccharides and polysaccharides such as chondroitin sulfate, coronary heart disease relieving, etc. Diuretics such as thiazide hydrochlorothiazide, spirolactone of aldosterone, furosemide of loop diuretics, etc.; sympatholytic agents are mainly propranolol and the like, captopril, benazepril, which affects renin-angiotensin system inhibitory agents such as Angiotensin Converting Enzyme (ACE) inhibitory agents; valsartan as an angiotensin II receptor blocker; drugs that block calcium channel ions such as amlodipine and the like. Sodium channel drugs that block the cardiac muscle and cardiac conduction system: such as quinidine, procainamide, propidium, lidocaine, phenytoin sodium, mexiletine, propafenone, enrotinib, flurani, etc.; beta blocker such as propranolol, atenolol, metoprolol and the like; drugs for prolonging action potential time course, such as amiodarone, sotalol, benzalkonium bromide, ebutinib, dofetilide, and the like; calcium channel blockers such as verapamil and diltiazem. The individual components to be combined may be administered simultaneously or sequentially, in a single formulation or in different formulations. The combinations include not only combinations of one or more other active agents of the compounds of the present application, but also combinations of two or more other active agents of the compounds of the present application.

The term "disease" as used herein refers to any condition or disorder that impairs or interferes with the normal function of cells, organs or tissues.

The term "cardiovascular agent" as used herein refers to any agent useful in the treatment of atherosclerosis, hypertension, cardiac arrhythmias, myocardial ischemia/reperfusion injury, cardiomyopathy.

The term "pharmaceutically acceptable" as used herein refers to those compositions which are, within the scope of sound medicine, suitable for use in contact with the tissues of humans and other mammals without undue toxicity, irritation, allergic response and the like, commensurate with a reasonable benefit/risk ratio. By "pharmaceutically acceptable salt" is meant any non-toxic salt that, upon administration to a recipient, is capable of providing a compound or prodrug of a compound of the application, either directly or indirectly.

The term "effective amount" or "therapeutically effective amount" as used herein means an amount of a compound or pharmaceutical composition described herein sufficient to achieve the intended use, including, but not limited to, treatment of a disease.

In one embodiment of the application, the amount is a detected dose effective to attenuate coupling between TRPV4 and NOX 2; severity level, stage and progression of vascular permeability. The therapeutically effective amount may vary depending on the intended use, such as in vitro or in vivo, the condition and severity of the disease, the age, weight, or mode of administration of the subject, and the like. The term also applies to a dose that will induce electrophysiological activity of a target cell, e.g., a cell. The specific dosage will depend, for example, upon the particular compound chosen, the subject species and their age/existing health or risk of health, the route of administration, the severity of the disease, the administration in combination with other agents, the time of administration, the tissue to which it is administered, and the means of administration, etc.

In the present application, "administering" or "administering" a compound to an individual refers to providing a compound of the present application to an individual in need of treatment.

The compounds of the present application may contain one or more asymmetric centers and thus appear as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures.

Reference throughout this specification to "an embodiment" or "in another embodiment" or "in certain embodiments" or "in some embodiments" means that a particular reference element, structure, or feature described in connection with that embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" or "in another embodiment" or "in certain embodiments" or "in some embodiments" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular elements, structures, or features may be combined in any suitable manner in one or more embodiments.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be interpreted in an open-ended, inclusive sense, i.e. "including but not limited to.

It should be understood that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

The beneficial effects are that:

the application utilizes specific low-toxicity and high-activity small molecules to realize the reduction of the coupling degree of TRPV4 and NOX2, and obtains better experimental results on animal level, thereby providing a new target point for the regulation and control of oxidative stress, providing more medicine for patients with abnormal vascular permeability in clinic, having stronger targeting, realizing local regulation and having smaller side effect. So as to prevent the further development of cardiovascular diseases and prevent thrombosis, hypoxia, and multiple organ dysfunction syndrome.

Drawings

FIG. 1 is a graph showing comparison of the results of measurement of physical coupling degree of TRPV4-NOX2 complex between obese mice and wild-type mice.

FIG. 2 is a graph of an alignment of intracellular ROS level results from different mice.

FIG. 3 is a graph showing the comparison of the results of measurement of endothelial cell microtubule length in mice under different coupling conditions.

Fig. 4 is a graph of evans blue staining results for mice with different coupling status.

FIG. 5 is a graph comparing the results of coupling of various compounds to TRPV4 and NOX 2.

FIG. 6 is a graph of intracellular ROS levels following incubation of compounds.

FIG. 7 is a graph showing the result of staining the cell structure of obese mice with the compounds.

Detailed Description

In the compound shown in the structure of the formula (1), when R is H, the corresponding compound is emtrictinib; when R is-COCF 3, the corresponding compound is M12. The compound shown in the structure of the formula (2) is gambogic acid. The related compound shown in the structure of the formula (3) is L75507.

The application will now be described in detail by way of examples with reference to the accompanying drawings.

Example 1: compound in vitro coupling activity test

The experimental method comprises the following steps: HEK-293 cells co-transfected with TRPV4 and NOX2 or co-transfected with AQP1 and NOX2 were seeded in the center of confocal dishes after treatment, when cell confluence reached around 80%, respectively using DMSO (1%o), emtrictinib (10 nM), L755507 (10. Mu.M), gambogic acid (1. Mu.M), CB-839 (1. Mu.M), bohemine (1. Mu.M), M12 (10 nM) were incubated for 24 hours, then fixed with 4% paraformaldehyde for 20 minutes, permeabilized with PBS containing 0.1% Triton X-100 for 10 minutes, and blocked with PBS containing 5% BSA for half an hour.

Cells were then stained with TRPV4-550 and NOX2 as primary and conjugated secondary antibodies (invitrogen) and DAPI (Vector Laboratories, burlingame, CA, USA). Shooting is performed by using a confocal laser microscope, and Fret value is calculated. The specific results are shown in FIG. 5 and Table 1.

TABLE 1 influence of different compounds on the coupling of TRPV4 and NOX2

Compounds of formula (I)	Fret efficiency value (average)
		Positive control	5.75
Negative control	2.94
		Entrictinib	3.76
L755507	3.77
		Gambogic acid	3.28
CB-839	5.35
		Bohemine	4.95
M12	2.83

Wherein, the HEK-293 group co-transfected with TRPV4 and NOX2 incubated with DMSO is a positive control, the HEK-293 group co-transfected with AQP1 and NOX2 incubated with DMSO (there is no coupling of these two proteins) is a negative control; the remainder was named example name with the name of the incubation drug.

The structure of CB-839 and Bohemine is shown as follows:

the result shows that: FRET experiments demonstrated that the coupling of TRPV4 and NOX2 was significantly reduced in the drug-applied group compared to the positive control after 24 hours incubation with emtrictinib, L75507, M12 and gambogic acid, with a statistical difference compared to the positive control.

Example 2: investigation of the Effect of Compounds on intracellular ROS levels

The experimental method comprises the following steps: isolated obese mice were seeded in the center of confocal dishes after primary endothelial cells were treated, after incubation with 10Nm of M12 for 24 hours until cell confluence reached around 80%, the medium was discarded, washed three times with NPSS, and cells were incubated in a dark environment at 37 ℃ in an incubator with NPSS containing CM-H2 DCFDA. The images were then taken using a laser confocal microscope, fluorescence intensities were calculated using image J statistics, and intracellular ROS levels could be reflected. The specific results are shown in FIG. 6 and Table 2.

Effects of compounds of Table 2 on intracellular ROS levels

	CM-H2DCFDA fluorescence intensity (fold compared to DIO)
		DIO group without incubation treatment	1.000000
Incubation with M12	0.496864

Wherein: DIO represents a diet-induced obesity model. NPSS is a buffer solution. CM-H2DCFDA is a reactive oxygen species cell permeability indicator, and after staining, can generate fluorescence under confocal conditions, and the fluorescence intensity characterizes the intracellular ROS level, and is shown in the figure, and is a relative fluorescence intensity multiple obtained after normalization treatment by taking DIO group as a basic value.

The result shows that: as shown in FIG. 6, CM-H2DCFDA staining experiments: m12 proved to be effective in reducing ROS production compared to the obese model. After the treatment of the fat mouse cells with M12, the fluorescence intensity of CM-H2DCFDA produced by the cells is obviously reduced, compared with DIO cells, the fluorescence intensity of the M12 group is reduced with statistical difference, which indicates that the M12 can down regulate the intracellular ROS level, and the compound has obvious regulation and control effect on oxidative stress.

Example 3: the effect of compounds on the cellular structure of obese mice was investigated:

the experimental method comprises the following steps: primary endothelial cells of obese mice were isolated, seeded in confocal dishes after treatment, after incubation with 10nM M12 for 24 hours at about 80% confluence, medium was discarded, washed three times with NPSS, fixed with 4% paraformaldehyde for 20 min, permeabilized with PBS containing 0.1% triton X-100 for 10 min, and blocked with PBS containing 5% bsa for half an hour. Cells were incubated with NPSS mixed with rhodamine-phalloidin (Phllotoxins) for half an hour (Invitrogen) and stained with DAPI (Vector Laboratories, burlingame, calif., USA). Photographing was performed with a confocal laser microscope. The specific results are shown in FIG. 7

Wherein: BSA refers to bovine serum albumin. PBS is a buffer solution. Rhodamine-phalloidin is a high affinity probe of F-actin, useful for observing cytoskeletal structure changes. DAPI is a common dye that characterizes the nucleus.

As shown in fig. 7, the propiconazole staining experiment demonstrated that M12 significantly reversed the pathological changes in cytoskeletal structure compared to endothelial cells isolated from obese mice; the M12 effectively improves the cytoskeletal structure, so that the cytoskeleton after treatment becomes more compact.

Example 4: the relationship of obesity to physical coupling of TRPV4-NOX2 complex was explored.

The experimental method comprises the following steps: isolated obese mice (DIO) were aortic, fixed, dehydrated, sectioned, then blocked for 30 min with PBS containing 5% bsa and permeabilized for 10 min with PBS containing 0.1% triton X-100.

The treated tissue was stained with TRPV4-550 and NOX2 as primary and conjugated secondary antibodies (invitrogen) and DAPI (Vector Laboratories, burlingame, CA, USA). Shooting is performed with a confocal laser microscope, and the Fret value is directly calculated using software. The specific results are shown in Table 3 and FIG. 1.

TABLE 3 physical coupling degree measurement results of TRPV4-NOX2 Complex of obese mice and wild type mice

	Fret efficiency value (average)
		WT (wild type mouse)	9.993
DIO (obesity mouse)	14.42

Wherein: triton X-100 is a reagent for cell membrane perforation. Fret represents fluorescence resonance energy transfer, and can be used for biomacromolecule interaction analysis, cell physiology research, immunoassay and the like. Fret efficiency values were obtained directly from the confocal software.

As can be seen in conjunction with fig. 1 and table 3, the DIO group has significantly up-regulated Fret efficiency values, and there is a statistical difference compared to the WT group, which suggests significantly enhanced physical coupling of TRPV4-NOX2 complexes in obese mice compared to wild type mice.

Example 5: intracellular ROS level detection

The experimental method comprises the following steps: the isolated primary endothelial cells were seeded in the center of a confocal dish after treatment, and when the cell confluence reached about 80%, the medium was discarded, washed three times with NPSS, and cells were incubated in a dark environment at 37 ℃ in an incubator with NPSS containing CM-H2 DCFDA. The images were then taken using a confocal laser microscope, and the CM-H2DCFDA intensity was calculated and reflected in intracellular ROS levels. The specific results are shown in Table 4 and FIG. 2.

TABLE 4 intracellular ROS level results in different mice

Experimental group	CM-H2DCFDA fluorescence intensity (fold)
		WT	1.00000
DIO	2.59762
		TrpV4 KO	1.28529
TrpV4 KO DIO	1.49258

Wherein the TrpV4KO experimental group represents a group of ROS detection experiments performed using aortic endothelial cells isolated from TrpV4 knockout mice. The TrpV4KO DIO experimental group represents a group of ROS detection experiments performed using aortic endothelial cells isolated from TrpV4 knockout fed high fat diet mice. CM-H2DCFDA is a reactive oxygen species cell permeability indicator, and after staining, can fluoresce under confocal conditions, and the fluorescence intensity characterizes the intracellular ROS level, here, the relative fluorescence intensity fold obtained after normalization treatment with the WT group as the basis.

The experimental results show that: as shown in FIG. 2, the CM-H2DCFDA fluorescence intensity of DIO group cells was significantly increased, which showed that obesity up-regulated ROS expression compared to the WT group, while there was no statistical difference in the up-regulation of TrpV4KO DIO group compared to the TrpV4KO group, which indicated that obesity was up-regulated ROS levels by the TRPV4-NOX2 complex, thus affecting oxidative stress.

Example 6: effects of coupling on cellular structure:

the experimental method comprises the following steps: primary endothelial cells were isolated, seeded in the center of confocal dishes after treatment, and after cell confluence reached about 80%, cell obesity models were induced with FFA, and then the NOX2 inhibitor oleandrin (Apocynin) (5 μm) and TrpV4 inhibitor HC067047 (1 μm) were added, incubated overnight, medium was discarded, washed three times with NPSS, fixed with 4% paraformaldehyde for 20 min, permeabilized with PBS containing 0.1% triton X-100 for 10 min, and blocked with PBS containing 5% bsa for half an hour. Cells were incubated with NPSS mixed with rhodamine-phalloidin for half an hour (invitrogen) and stained with DAPI (Vector Laboratories, burlingame, CA, USA). The photographing was performed with a laser confocal microscope, and then the microtube length was counted using confocal software.

Wherein FFA is used as an in vitro induced hyperlipidemia cell model by using a mixed solution containing 0.2mM palmitic acid, 0.1mM oleic acid and 0.8% BSA; apocynin is the NOX2 inhibitor oleandrin; HC067047 is a specific TrpV4 inhibitor.

The specific results are shown in FIG. 3. The result shows that: compared with endothelial cells of wild-type mice, the endothelial cytoskeletal structures of obese mice are markedly rearranged, microtubule length is markedly increased, and after V4 or NOX2 is inhibited to break the coupling, the increase is insignificant.

Example 7: exploration of coupling and vascular permeability relationship

The experimental method comprises the following steps: obese mice (DIO), wild-type mice (WT), trpV4 knockout mice (TrpV 4 KO), trpV4 knockout mice fed with high fat mice (TrpV 4KO DIO), were anesthetized with 4% chloral hydrate, 100 μl of physiological saline containing 1% evans Blue (evans Blue) was injected from inferior vena cava after anesthesia, waiting for 30 minutes, separating the aorta, photographing under a light microscope, and then incubating the thoracic aorta in 4% paraformaldehyde solution at 55 ℃ for 24 hours, and the exudation evans Blue fluorescence intensity was detected with a wavelength of 610nm using an enzyme-labeled instrument, with specific results shown in fig. 4 and table 5.

TABLE 5 vascular permeability test results for mice with different coupling states

Implementation group	Absorbance (multiple)
		WT	1
DIO	2.6863
		TrpV4 KO	1
TrpV4 KO DIO	1.5857

Among them, evans blue is a commonly used azo dye preparation, and has high affinity with plasma albumin in blood, and plasma albumin cannot pass through the blood brain barrier under normal conditions, so that when staining, such as the nervous system is intact, evans blue combined with plasma albumin cannot stain. If, on the contrary, the nervous system blood brain barrier is disrupted, evans blue can enter the nervous system and color it. Therefore, it is often used in neuroscience research as a tracer to observe the integrity of the Blood Brain Barrier (BBB) and also to detect vascular permeability. After staining, the dye was eluted using 4% pfa, followed by detection of the fluorescent intensity of exuded evans blue with a wavelength of 610nm using a microplate reader.

WT group and DIO group are fluorescence intensity fold obtained by normalizing the WT group as a base value. TrpV4 knockout mice and TrpV4 knockout high fat diet groups were obtained as fold fluorescence intensity values after normalization treatment based on the TrpV4 knockout group.

As a result, it was found that there was a clear up-regulation of the Evansi's blue exudation value detected in the aorta of the obese mice compared to the wild-type mice, which showed a clear increase in the aortic permeability of the obese mice compared to the WT group, whereas the V4 knockout mice, due to the almost non-coupled state, showed no statistical difference in the elevation of the Evansi's blue exudation value compared to the TrpV4KO group, which showed that the effect of obesity on vascular permeability was exerted by TRPV4-NOX2 coupling.

In conclusion, by detecting the physical coupling state of the TRPV4-NOX2 complex of wild type, obese type and TRPV4 knockout mouse endothelial cells, ROS are generated, the skeleton structure is changed, and the aortic permeability is changed, the obesity is proved to be caused by up-regulating the physical coupling strength of the TRPV4-NOX2 complex, and excessive oxidative stress is induced, so that vascular permeability is up-regulated. This overcoupling state can be interrupted by the compounds of the present application, thereby down-regulating ROS production, improving cytoskeletal structure and aortic permeability, and thus reversing disease states and restoring health. The compound has better effect on resisting oxidative stress and can be used for preventing or treating cardiovascular diseases.

While the application has been illustrated by the foregoing specific examples, it should not be construed as being limited thereto; but rather the application encompasses the generic aspects previously disclosed. Various modifications and embodiments can be made without departing from the spirit and scope of the application.

Claims (2)

1. The application of the compound with the structure shown in the formula (1) and the pharmaceutically acceptable salt thereof in preparing the antioxidant stress medicine for treating or delaying cardiovascular diseases is that the compound with the structure shown in the formula (1) is used as an active ingredient of the medicine to reduce the coupling degree of TRPV4 and NOX2 and the antioxidant stress,

2. the use according to claim 1, wherein the pharmaceutical composition comprises, in addition to the active ingredient, a pharmaceutically acceptable carrier and/or pharmaceutical excipients.