Application of RUNX2 inhibitor in preparation of medicine for treating vascular calcification related diseases

Abstract

The invention discloses an application of an RUNX2 inhibitor in preparation of a medicine for treating vascular calcification related diseases, and the RUNX2 inhibitor comprises an ester compound which is selected from at least one of Corilagin, 1, 2, 3, 6-tetragalloylglucose, NADP (Nicotinamide Adenine Dinucleotide Phosphate), Stafib-2 and uridine triphosphate sodium salt. The ester compound provided by the invention is used as a drug for inhibiting vascular calcification and progression of the RUNX2 inhibitor, and provides a new thought, a target spot and a small molecule drug for treatment of vascular calcification. The five small molecule compounds have good drug safety and a strong vascular calcification inhibition effect, and can be used as candidate drugs for inhibiting vascular calcification.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of a RUNX2 inhibitor in the preparation of drugs for treating vascular calcification-related diseases. Background Technology

[0002] Reports indicate that the incidence of vascular calcification increases in healthy individuals over 60 years of age. Furthermore, a study based on coronary artery calcification scores and cumulative cardiovascular mortality showed that the 15-year cumulative cardiovascular mortality rate in individuals with low Framingham risk scores can be as high as 30%, suggesting that vascular calcification is a key risk factor for cardiovascular mortality, severely impacting quality of life and lifespan. Arterial calcification refers to the deposition of hydroxyapatite within the arterial wall and is a common complication of chronic kidney disease and diabetes. Approximately 47%–83% of patients with chronic kidney disease develop arterial calcification, and about 30% of diabetic patients present with high-scoring coronary artery calcification. Arterial calcification reduces vascular compliance and causes a series of adverse cardiovascular events, including myocardial ischemia. The independent association between all-cause mortality and the degree of calcification remains consistently significant. Currently, early diagnosis of arterial calcification is impossible; it can only be diagnosed through CT imaging when it has progressed to a more severe stage. More importantly, there is no effective treatment for arterial calcification after it is diagnosed. Multiple clinical studies have shown that calcium and phosphorus inhibitors cannot inhibit the progression of arterial calcification, which also suggests that the calcium and phosphorus deposition theory has limitations in explaining the pathogenesis of arterial calcification.

[0003] The transformation of vascular smooth muscle cells (VSMCs) into osteoblasts is a major cause of vascular calcification. During this transformation, RUNt-related transcription factor 2 (RUNX2) is an essential osteogenic transcription factor and an early regulator of VSMC osteogenic transformation. Studies have shown that RUNX2 is highly expressed in mammalian arterial calcification, and knocking out RUNX2 significantly reduces vascular calcification and the formation of osteogenic VSMCs. Therefore, RUNX2 is an important target for the treatment of vascular calcification. Exploring small molecule compounds that effectively target RUNX2 inhibition or degradation will provide new insights for the treatment of arterial calcification. Summary of the Invention

[0004] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide the application of a RUNX2 inhibitor in the preparation of a drug for treating vascular calcification-related diseases.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides the use of an ester compound or a pharmaceutically acceptable salt thereof in the preparation of a RUNX2 inhibitor; said ester compound is selected from the following compounds: , , , , .

[0006] In some embodiments, the RUNX2 inhibitor is used to prevent and / or treat vascular calcification-related diseases.

[0007] A second aspect of the present invention provides the use of an ester compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of vascular calcification-related diseases; said ester compound is selected from the following compounds: , , , , .

[0008] In this invention, the above-mentioned compound can promote the binding of RUNX2 to autophagosome LC3B, thereby targeting and degrading RUNX2, thereby inhibiting vascular calcification.

[0009] Among them, the compound of formula I is Corilagin, CAS number 23094-69-1; it is soluble in water or DMSO and has good stability; it can be flexibly administered through various routes such as oral administration. The hydroxyl group and the oxygen atom on the ester group in the Corilagin molecule can form hydrogen bonds with RUNX2; the hydroxyl group can form hydrogen bonds with LC3B; Corilagin has good drug safety and strong RUNX2 inhibitory effect and vascular calcification inhibitory effect.

[0010] Compound II is 1,2,3,6-Tetragalloylglucose, CAS number 79886-50-3, soluble in DMSO. It can be flexibly administered via oral and other routes. It exhibits good drug safety and strong inhibitory effects on vascular calcification. In the 1,2,3,6-Tetragalloylglucose molecule, the oxygen atoms on the hydroxyl and phosphate groups can form hydrogen bonds with RUNX2, and the benzene ring can form π interactions with RUNX2; the hydroxyl group forms hydrogen bonds with LC3B, and the benzene ring forms π interactions with LC3B. Therefore, 1,2,3,6-Tetragalloylglucose demonstrates good drug safety and strong inhibitory effects on RUNX2 and vascular calcification.

[0011] Compound III is nicotinamide adenine dinucleotide (NADP), CAS number 53-59-8; it is soluble in water. It has good drug safety and can be flexibly administered via various routes, including oral and intravenous injection. The hydroxyl group, the oxygen atom on the phosphate group and its anion, the oxygen atom on the amide group, and the oxygen atom on the furan ring in NADP can form hydrogen bonds with RUNX2; the hydroxyl group, the oxygen atom on the phosphate group, and the amino group form hydrogen bonds with LC3B; the oxygen anion on the phosphate group and the purine ring create salt bridges and π-cation interactions; NADP exhibits good drug safety and strong RUNX2 inhibitory and vascular calcification inhibitory effects.

[0012] Compound IV is Stafib-2, CAS number 2097938-74-2; it is a potent and selective inhibitor of the transcription factor STAT5b. It is soluble in DMSO. The hydroxyl group, oxygen atom, and -NH- group on the phosphate group of the Stafib-2 molecule can form hydrogen bonds with RUNX2; the N atom on the hydroxyl group, oxygen atom, and amide bond forms hydrogen bonds with LC3B; the benzene ring attached to the amide bond forms a π-interaction with LC3B; and the benzene ring attached to the phosphate group forms a π-cation interaction with LC3B. Stafib-2 exhibits good drug safety and strong RUNX2 inhibitory and vascular calcification inhibitory effects.

[0013] Compound V is Uridine triphosphate (UTP) trisodium salt, CAS number 19817-92-6; it is a pyrimidine nucleoside triphosphate, soluble in water. It has good drug safety and can be flexibly administered via oral and intravenous injection. The hydroxyl group and oxygen atom on the phosphate group in the uridine triphosphate sodium salt molecule can form hydrogen bonds with RUNX2; the oxygen antom in the phosphate group forms a salt bridge; the hydroxyl group, the oxygen atom on the carbonyl group of the pyrimidine ring, the oxygen atom on the phosphate group, and the -NH- group on the pyrimidine ring can form hydrogen bonds with LC3B, and LC3B forms a salt bridge with the fluoride ion on the phosphate group. Uridine triphosphate sodium salt exhibits good drug safety and strong RUNX2 inhibition and vascular calcification inhibition effects.

[0014] In some embodiments, the vascular calcification-related diseases include at least one of the following: cardiovascular disease, vascular calcification, cardiovascular disease associated with and / or caused by vascular calcification, cardiovascular disease associated with and / or caused by elevated arteriosclerosis levels, elevated arteriosclerosis levels, left ventricular hypertrophy, cardiovascular disease associated with and / or caused by left ventricular hypertrophy, and cardiovascular disease associated with and / or caused by kidney disease.

[0015] Vascular calcification (VC), or arterial calcium accumulation, is characterized by the deposition of calcium phosphate and other calcium phosphate salts in the form of hydroxyapatite crystals. VC is a complex and tightly regulated metabolic process with many characteristics shared with mineralization processes in bone or bone tissue. Both bone and VC development and metabolism involve an imbalance between local and systemic inhibitors and inducers of calcification. The main and critical steps involved in VC development include: (1) The transformation of normal / healthy vascular smooth muscle cells (VSMCs) and / or circulating stem cells into osteoblasts / osteomorphic cells, and (2) Calcium deposition in the form of hydroxyapatite crystals.

[0016] The first crucial and essential step in vascular calcification is the transformation of VSMCs and other cell types, such as circulating stem cells, into osteoblast-like cells. These are cells capable of forming bone-like structures. The ability to undergo reversible differentiation is characteristic of the VSMC phenotype; these cells are in their differentiated, contractile state at baseline, but respond to the aforementioned pathological stimuli by entering a proliferative, synthetic state, thereby producing extracellular matrix (ECM) and undergoing osteoblastic differentiation. This transformation is induced by, for example, repeated exposure to various chronic diseases, including hypertension, kidney disease, diabetes, inflammation, hyperphosphatemia / hypercalcemia, and oxidative stress. Under normal or healthy conditions, VSMCs are in their differentiated, contractile state; however, when repeatedly exposed to the aforementioned pathological inducers, they express proteins including bone morphogenetic proteins (BMPs: BMP2 and BMP4), osteogenic transcription factors (Runx2, also known as the core-binding factor α-1 subunit (Cbfα-1), Osterix, etc.), and signaling pathways (Wnt / β-linkin), all of which promote the differentiation of VSMCs into osteoblast-like cells. These osteoblast-like cells possess many properties similar to bone blasts, including alkaline phosphatase (ALP) activity and increased expression of osteocalcin, osteonectin, and osteopontin (OPN).

[0017] Extensive research reported in the literature provides evidence that VC is a tightly regulated process through competition between factors that induce or promote calcification and factors that inhibit mineralization. [See, for example, Zhu D, et al. Mechanisms and Clinical Consequences of Vascular Calcification[J]. Frontiers in Endocrinology, 2012, 3:95.DOI:10.3389 / fendo.2012.00095.; Johnson RC et al. (2006), Vascular Calcification Pathological Mechanisms] and Clinical Implications”. Circ. Research: 1044-1059. Positive regulators (i.e., inducers or inducers) of vitamin C are regulators that induce the formation of osteoblast-like cells in the vascular system and the upregulation of transcription factors that are crucial in the bone formation process. Reported inducers include high calcium, high phosphate, high glucose, uremia, osteoporosis, pro-inflammatory cytokines, lipids, macrophages, apoptosis, transglutaminase-2, high vitamin D, transforming growth factor β (TGF-β), and intravascular... Vein growth factor (VEGF), parathyroid hormone (PTH), glucocorticoids, and warfarin are all important regulators of blood vessel mineralization. Negative regulators (i.e., inhibitors or suppressors) are molecules normally expressed in blood vessels to inhibit mineralization, such as pyrophosphate and matrix Gla protein (MGP). A lack of these molecules means "loss of inhibition," which then leads to spontaneous vascular calcification. Other negative regulators include fetoglobulin-A, osteopontin, osteoprotegerin, statins, vitamin K, bisphosphonates, fibroblast growth factor 23 (FGF23) / Klotho protein, and insulin-like growth factor 1 (IGF-1).

[0018] In some embodiments, the vascular calcification includes at least one of calcified atherosclerosis, calcified medial vascular lesions (also known as Mönckeberg's medial calcification), medial calcification, elastocalcinosis, calcified uremic arterial lesions, calcified aortic valve stenosis, or portal vein calcification.

[0019] In some embodiments, the diseases associated with the calcified atherosclerosis include at least one of atherosclerosis, hyperlipidemia, osteoporosis, hypertension, inflammation, type 2 diabetes, end-stage renal disease, amputation, pseudoxanthoma elasticus, hyperlipidemia, congenital mitral valve, rheumatic heart disease, and liver disease.

[0020] In some embodiments, the cardiovascular disease is associated with at least one of the following conditions: vascular calcification leading to conditions such as atherosclerosis, hyperlipidemia, osteoporosis, hypertension, inflammation, type 2 diabetes, end-stage renal disease, amputation, pseudoxanthoma elasticus, congenital mitral valve, rheumatic heart disease, portal hypertension, or liver disease.

[0021] In some embodiments, the cardiovascular disease is secondary to chronic kidney disease.

[0022] In some embodiments, the chronic kidney disease is stage 3, 4, or 5 chronic kidney disease.

[0023] In some embodiments, the chronic kidney disease is a chronic kidney mineral and bone disease.

[0024] Non-contrast computed tomography (CT) for imaging the extent of coronary artery calcification (CAC) and contrast CT for non-invasive coronary angiography (CTA) are commonly used to diagnose the development of obstructive coronary artery disease. Radionuclide intensity testing, coronary calcium scanning, and non-invasive coronary angiography may also be used for diagnostic and prognostic cardiac assessment.

[0025] Possible methods for detecting and quantifying coronary artery calcification (CAC) include, but are not limited to, X-ray computed tomography and myocardial perfusion single-photon emission computed tomography (SPECT).

[0026] Another diagnostic method for vascular calcification is to combine positron emission tomography (PET) / computed tomography (CT) to detect fluorine-18-fluorodeoxyglucose (FDG) uptake in the thoracic aortic wall.

[0027] Ultra-fast CT scans can be used to detect the presence of atherosclerotic coronary heart disease.

[0028] Electron beam computed tomography (CT) can also be used to diagnose coronary artery disease.

[0029] Another test for vascular calcification involves plaque composition in plexogenic and thromboembolic pulmonary hypertension. Chronic thromboembolic pulmonary hypertension is associated with atherosclerotic plaques with a soft core rich in blood group glycoproteins, while plexogenic pulmonary hypertension is associated with fibrous plaques. Thromboembolic material plays a key role in the formation of the soft core, in which erythrocyte membrane-derived blood group glycoproteins are the main components. Therefore, chronic thromboembolic and plexogenic pulmonary hypertension (primary and secondary (Eisenmenger syndrome)) were investigated.

[0030] The Agatston score, a calcium scoring system based on the density of deposited calcium plaques, can be used to quantify vascular calcification. In this system, the level of vascular calcification can be measured using multi-detector computed tomography (MDCT), and a decrease in the rate of progression in the Agatston score can be assessed. (See, for example, Sharma et al., 2010, Vasc. Health Risk Manag. 6:603-611).

[0031] Furthermore, vascular calcification can be assessed using the methods described in Adragao et al., 2004, Nephrol. Dial. Transplant 19:1480-1488.

[0032] Another analysis used to quantify vascular calcification in subjects is lesion-specific calcium scoring, which includes calcium measurements generated from coronary artery calcification CT scans. This method is described, for example, by Akram and Voros, 2008, Int. J. cardiovac. Imaging 14:743-749.

[0033] A third aspect of the invention provides a pharmaceutical composition for the prevention and / or treatment of diseases related to vascular calcification, comprising the ester compound or a pharmaceutically acceptable salt thereof.

[0034] In some embodiments, the pharmaceutical composition includes a second pharmaceutically active agent.

[0035] In some embodiments, the second pharmaceutically active agent is an active agent for treating cardiovascular disease, vascular calcification, cardiovascular disease associated with and / or caused by vascular calcification, and / or cardiovascular disease associated with and / or caused by kidney disease, such as aldosterone signal transduction inhibitors, angiotensin II receptor blockers, beta-blockers, calcium channel blockers, cholesterol-lowering drugs, digoxin, diuretics, inotropic agents, potassium or magnesium, vasodilators, and / or warfarin.

[0036] In some embodiments, the second pharmaceutically active agent is an active agent for treating chronic kidney disease, such as angiotensin II receptor blockers, beta-blockers, calcium channel blockers, direct renin signaling inhibitors, diuretics, vasodilators, erythropoietin therapy, iron replacement therapy, and / or vitamin D.

[0037] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0038] In some embodiments, the choice of the pharmaceutically acceptable carrier varies depending on the route of administration and characteristics of action, and is typically at least one of a solvent, solubilizer, filler or swelling agent, diluent, binder, wetting agent, disintegrant, lubricant, emulsifier, colorant, stabilizer, flavoring agent, preservative, coating material, solution blocking agent, adsorbent, or suspending agent.

[0039] The pharmaceutical compositions of the present invention can be prepared using any method known to those skilled in the art, based on the disclosure. For example, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

[0040] The pharmaceutical compositions of this invention can be administered in any form, including mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, and intramuscular) administration. The pharmaceutical compositions of this invention can also be controlled-release or delayed-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, tablets, soft capsules, tablets, pills, drops, powders, lyophilized products, and granules. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, drops, emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of parenteral formulations include, but are not limited to, solutions for injection, dry formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and tablets.

[0041] In solid dosage forms intended for oral administration (capsules, tablets, pills, sugar-coated pills, powders, granules, etc.), one or more therapeutic compounds described herein may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) (2) Fillers or swelling agents, such as starch, lactose, sucrose, glucose, mannitol and / or silica; (3) binders, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and / or gum arabic; (4) wetting agents, such as glycerin, cetyl alcohol and glyceryl monostearate; (5) disintegrants, such as agar, calcium carbonate, potato or cassava starch, alginic acid, certain silicates and sodium carbonate; (6) solution blocking agents, such as paraffin; (7) absorption accelerators, such as quaternary ammonium compounds; (8) adsorbents, such as kaolin and bentonite; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate and mixtures thereof; and (in the case of capsules, tablets and pills, the pharmaceutical composition may also contain buffers. Similar types of solid compositions may also be used as fillers in soft and hard-filled capsules, using such excipients as lactose, and high molecular weight polyethylene glycol, etc.

[0042] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (especially cottonseed, peanut, corn, germ, olive, castor, and sesame oils), glycerin, tetrahydrofuran alcohols, fatty acid esters of polyethylene glycol and sorbitol, and mixtures thereof. Besides inert diluents, oral compositions may also include adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, coloring agents, fragrances, and preservatives.

[0043] In addition to the active compound, the suspension may contain suspending agents such as ethoxyisooctadecyl alcohol, polyoxyethylene sorbitol and sorbitan ester, microcrystalline cellulose, aluminum hydroxide, bentonite, agar and astragalus gum, and mixtures thereof.

[0044] A fourth aspect of the invention provides the use of the pharmaceutical composition described herein in the preparation of a medicament for the prevention and / or treatment of vascular calcification-related diseases.

[0045] A fifth aspect of the invention provides a method for preventing and / or treating vascular calcification-related diseases, comprising administering to a patient the said ester compound or a pharmaceutically acceptable salt thereof or the said pharmaceutical composition.

[0046] In some implementations, the patient has an elevated RUNX2 level compared to the RUNX2 level in the reference population.

[0047] In some implementations, the level of increase in RUNX2 is 10% to 500% higher than the level of RUNX2 in the reference population; such as about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 200%, or 500%.

[0048] In some embodiments, the pharmaceutically effective dose of the ester compound or a pharmaceutically acceptable salt thereof is 15 mg to 1 g or 0.1 to 1.5 mg / kg; such as about 15 mg, about 30 mg, about 45 mg, about 60 mg, about 75 mg, about 90 mg, or about 1 g, or about 0.1 mg / kg, about 0.13 mg / kg, about 0.2 mg / kg, about 0.26 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, or about 1.5 mg / kg.

[0049] In some embodiments, the pharmaceutically effective dose is administered orally or by injection.

[0050] In some embodiments, the pharmaceutically effective dose is administered (i) every 7 days sequentially; (ii) every 14 days; (iii) every 21 days; (iv) every 28 days; or (vi) every 42 days.

[0051] In some embodiments, the pharmaceutically effective dose is administered continuously and / or indefinitely.

[0052] In some implementations, the RUNX2 inhibitor reduces the patient's RUNX2 level.

[0053] In some implementations, the reduction includes bringing the patient's RUNX2 level closer to the RUNX2 level of the reference population.

[0054] In some embodiments, the dose of the ester compound or a pharmaceutically acceptable salt thereof administered to a patient according to the methods provided herein is sufficient to reduce the level and / or activity of RUNX2 in the patient by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 100%, or up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to 100%, compared to the level and / or activity of RUNX2 in a reference population.

[0055] In some embodiments, the RUNX2 inhibitor level and / or activity can be determined by any method known in the art or described herein. For example, Northern blotting, PCR analysis, real-time PCR analysis, or any other technique known in the art or described herein, can be used to determine the level by evaluating (e.g., quantifying) the transcribed RNA of RUNX2 in a sample.

[0056] The level of RUNX2 in tissue samples can also be determined by assessing (e.g., quantifying) the protein expression level of RUNX2 in the sample using, for example, immunohistochemical analysis, Western blot, ELISA, immunoprecipitation, flow cytometry analysis, or any other technique known in the art or described herein.

[0057] definition Reference Group In some implementations, data (e.g., biomarker levels or clinical symptoms) obtained from a reference population described herein are used to determine whether similar data obtained from subjects treated or to be treated according to the methods provided herein are pathologically high (e.g., elevated) or low (e.g., decreased).

[0058] In some embodiments, the size of the reference population can be 1, 5, 10, 25, 50, 75, 100, 200, 250, 300, 400, 500, or 1000 individuals. In some embodiments, the reference population consists of randomly selected volunteers. In some embodiments, the reference population consists of healthy individuals. In some embodiments, the reference population consists of individuals of the same age, weight, and / or sex as the patient population. In some embodiments, the reference population consists of individuals without cardiovascular disease. In some embodiments, the reference population consists of individuals without vascular calcification. In some embodiments, the reference population consists of individuals without cardiovascular disease. In some embodiments, the reference population consists of individuals without cardiovascular disease associated with and / or caused by vascular calcification. In some embodiments, the reference population consists of individuals without kidney disease. In some embodiments, the reference population consists of individuals without chronic kidney disease. In some embodiments, the reference population consists of individuals without a pathologically elevated level of arteriosclerosis. In some embodiments, the reference group consists of individuals without cardiovascular disease associated with and / or caused by elevated levels of atherosclerosis. In some embodiments, the reference group consists of individuals without left ventricular hypertrophy. In some embodiments, the reference group consists of individuals without cardiovascular disease associated with and / or caused by left ventricular hypertrophy.

[0059] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0060] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobroms, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetate, trifluoroacetate, propionates, hexanoates, octanoates, decanoates, undecenoates, glycolates, gluconates, lactates, sebates, adipates, glutarate, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginates, ascorbic acid salts, salicylates, 4-aminosalicylic acid salts, and naphthalene disulfonates. These salts can be prepared using methods known in this field.

[0061] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, and tertiary amines; substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0062] When used with the dosages provided in this article, the term “about” refers to any amount within 1%, 5%, or 10% of the reference amount.

[0063] The beneficial effects of this invention are: The ester compounds of this invention, as RUNX2 inhibitors, suppress vascular calcification and its progression, providing new ideas, targets, and small molecule drugs for the treatment of vascular calcification. These five small molecule compounds exhibit good drug safety and strong inhibitory effects on vascular calcification, and can serve as candidate drugs for inhibiting vascular calcification. Attached Figure Description

[0064] Figure 1 The results of SPR testing of Corilagin, TeGG and RUNX2 in Example 1 of this invention are shown below; where A is the affinity determination of RUNX2 and Corilagin; B is the affinity determination of LC3B and Corilagin; C is the affinity determination of RUNX2 and TeGG; and D is the affinity determination of LC3B and TeGG.

[0065] Figure 2 The results of detecting the inhibition of vascular calcification by ester compounds Corilagin, TeGG, NADP, Stafib-2, and uridine triphosphate sodium salt in Example 2 of the present invention are shown. Among them, A is the result of alizarin red staining after treating cells with different concentrations of TeGG; B is the result of alizarin red staining after treating cells with different concentrations of Corilagin; and C is the result of alizarin red staining after treating cells with NADP, Stafib-2, and uridine triphosphate sodium salt.

[0066] Figure 3 The results of the efficacy and safety evaluation of the ester compounds in Example 3 of this invention are shown, where A to E are the IC50 values ​​of Corilagin, TeGG, NADP, Stafib-2, and uridine triphosphate sodium salt, respectively. 50 Curve and EC 50 curve.

[0067] Figure 4 The results of the inhibition of vascular and cellular calcification by ester compounds in Example 4 of this invention are shown. A represents the aortic alizarin red staining results of three randomly selected mice from the control group, the VitD3 model group, and the VitD3 model group plus injection of each drug (scale bar 10mm). B represents the Western Blot detection results of RUNX2 protein levels in vascular tissue from the control group, the VitD3 model group, and the VitD3 model group plus injection of TeGG (numbered #1, #2, #3 corresponding to three randomly selected mice). C represents the Western Blot detection results of RUNX2 protein levels in vascular tissue from the control group, the VitD3 model group, and the VitD3 model group plus injection of Corilagin (numbered #1, #2, #3 corresponding to three randomly selected mice). The left and right images in D represent the quantitative calculation of grayscale values ​​for B and C. E represents the Western Blot detection of RUNX2 protein levels after ester drug treatment following induced calcification of human vascular smooth muscle cells, and F represents its quantitative results. Detailed Implementation

[0068] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0069] Reagents and materials: Human vascular smooth muscle cells (hVSMCs) were purchased from Cybio (Shanghai) Biotechnology Co., Ltd.; disodium hydrogen phosphate (Pi) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; calcium content reagent kits were purchased from Nanjing Jiancheng Bioengineering Institute; alizarin red dye was purchased from Beijing Solarbio Technology Co., Ltd.; RUNX2 primary antibody was purchased from Abcam; GAPDH primary antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd.; OPN and OCN primary antibodies were purchased from Wuhan Aibotek Biotechnology Co., Ltd.; goat anti-rabbit secondary antibody and goat anti-mouse secondary antibody were purchased from LI-COR (USA); 4% paraformaldehyde fixative and RIPA lysis buffer were purchased from Beyotime Biotechnology Co., Ltd.; Von Kossa staining solution was purchased from Wuhan Sewell Biotechnology Co., Ltd. Corilagin, 1,2,3,6-Tetragalloylglucose (hereinafter referred to as "TeGG"), NADP, Stafib-2, and uridine triphosphate sodium salt (hereinafter referred to as "UTP") were purchased from MCE. The SPR affinity testing instrument, biacore 8k, uses a CM5 chip from Cytiva.

[0070] Example 1 This embodiment uses surface plasmon resonance (SPR) to detect the affinity of ester compounds for RUNX2 and LC3B. The specific process is as follows: (1) Chip preparation: An activator consisting of 400 mM EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 100 mM NHS (N-hydroxysuccinimide) was injected before sample passage. The CM5 sensor chip was activated using this mixture for 420 seconds at a flow rate of 10 μL / min.

[0071] (2) Ligand immobilization: RUNX2 was diluted to 20 μg / mL with immobilization buffer (10 mM Sodium Acetate, pH 4.5) and then injected into sample channel Fc2 at a flow rate of 10 μL / min, typically achieving a immobilization level of 12600 RU. The reference channel Fc1 did not require ligand immobilization. The chip was deactivated by treating it with 1 M ethanolamine chloride solution at a flow rate of 10 μL / min for 420 seconds.

[0072] MAP1LC3B was diluted to 20 μg / mL with immobilization buffer (10 mM Sodium Acetate, pH 4.5) and then injected into sample channel Fc2 at a flow rate of 10 μL / min, typically achieving a immobilization level of 12600 RU. No ligand immobilization was required for reference channel Fc1. The chip was deactivated by treating it with 1 M ethanolamine chloride solution at a flow rate of 10 μL / min for 420 seconds.

[0073] (3) Analytical cycle method: Corilagin or TeGG was diluted to eight concentrations (0.08 μM, 0.16 μM, 0.31 μM, 0.62 μM, 1.25 μM, 2.50 μM, 5.00 μM, and 10.00 μM) using the same solution as the analyte buffer (1% DMSO PBST: 1×PBS, 1% DMSO, 0.005% Tween 20). Corilagin was injected into channels Fc1-Fc2 at a flow rate of 20 μL / min for a 100-second binding phase, followed by a 180-second dissociation phase. Both binding and dissociation processes were performed in the analyte buffer. The analyte cycle was repeated eight times in ascending order of concentration. After each interaction analysis cycle, the chip was regenerated (by injecting a low-pH reagent, such as 10 mM glycine, pH 1.5-2.5). Results are as follows: Figure 1 As shown in Table 1.

[0074] Table 1

[0075] Surface plasmon resonance (SPR) measurements showed that RUNX2 immobilized on the CM5 chip could bind to Corilagin with an affinity constant of 5.58 × 10⁻⁶. -7 M. LC3B, fixed to the CM5 chip, can bind to Corilagin with an affinity constant of 2.43 × 10⁻ 6 M. RUNX2, fixed on the CM5 chip, can bind to TeGG with an affinity constant of 4.43 × 10⁻ 6 M. LC3B, fixed on the CM5 chip, can bind to TeGG with an affinity constant of 9.37 × 10⁻ 6 M.

[0076] The results above show that the small molecule compounds Corilagin and TeGG have high affinity for RUNX2 and LC3B, respectively.

[0077] Example 2 This embodiment utilizes alizarin red to detect the anti-calcification effect of ester compounds. The specific process is as follows: 1. Calcification was induced in human vascular smooth muscle cells using inorganic phosphorus, while different concentrations of the small molecule compounds Corilagin and TeGG were administered. After 7 days, Alizarin Red staining was performed to evaluate the inhibitory effects of Corilagin and TeGG on vascular calcification. The specific procedures are as follows: Human vascular smooth muscle cells from passages 8-12 were used at a concentration of 0.5 × 10⁻⁶. 5 Seedlings were placed in 12-well plates at a density of cells / well, with 2 mL of DMEM complete medium added to each well. On day 1 after seeding, calcification was induced by 3.0 mM disodium hydrogen phosphate, and interventions were performed by adding 0.1 μM, 0.5 μM, 1.0 μM, 5.0 μM, 7.5 μM, 10 μM, and 15 μM Corilagin or TeGG. A control group was set up and stimulated with hyperbaric ultrapure water (equal volume to disodium hydrogen phosphate) and DMSO (equal volume to Corilagin or TeGG). On day 4, the DMEM complete medium was replaced, and calcification was induced again by 3.0 mM disodium hydrogen phosphate, while interventions continued with different concentration gradients of Corilagin or TeGG. The control group was treated the same as on day 1. Alizarin red staining was performed on day 7.

[0078] 2. Calcification was induced in human vascular smooth muscle cells using inorganic phosphorus induction, while simultaneously intervening with 10 μM NADP, Stafib-2, or uridine triphosphate sodium salt. Seven days later, Alizarin Red staining was performed to assess the inhibitory effects of NADP, Stafib-2, and uridine triphosphate sodium salt on vascular calcification. The specific procedures are as follows: Human vascular smooth muscle cells from passages 8-12 were used at a concentration of 0.5 × 10⁻⁶. 5 Six-well plates were seeded at a density of cells / well, with 2 mL of DMEM complete medium added to each well. On day 1 after seeding, calcification was induced by 3.0 mM disodium hydrogen phosphate, and intervention was performed by adding 10 μM NADP, Stafib-2, or uridine triphosphate, respectively. A control group was set up and stimulated with hyperbaric ultrapure water (equal volume to disodium hydrogen phosphate) and DMSO (equal volume to NADP, Stafib-2, or uridine triphosphate). On day 4, the DMEM complete medium was replaced, and calcification was induced again by 3.0 mM disodium hydrogen phosphate, while intervention continued with 10 μM NADP, Stafib-2, or uridine triphosphate trisodium salt. The control group was treated the same as on day 1. Alizarin red staining was performed on day 7. Results are as follows. Figure 2As shown, A represents the results of alizarin red staining after cell treatment with different concentrations of TeGG; B represents the results of alizarin red staining after cell treatment with different concentrations of Corilagin; and C represents the results of alizarin red staining after cell treatment with NADP, Stafib-2, and uridine triphosphate sodium salt.

[0079] It can be seen that after calcification modeling in human vascular smooth muscle cells induced by inorganic phosphorus, intervention with ester compounds Corilagin, TeGG, NADP, Stafib-2, and uridine triphosphate sodium salt showed that all five small molecule compounds had an antagonistic effect on calcification, as indicated by Alizarin Red staining.

[0080] In summary, the results show that Corilagin, TeGG, NADP, Stafib-2, and uridine triphosphate sodium salt can effectively inhibit the occurrence of vascular calcification and can be used as anti-vascular calcification drugs.

[0081] Example 3 This embodiment evaluates the efficacy and safety of ester compounds, and the specific process is as follows: 1. IC 50 Detection: VSMC cells were seeded in 2.0 × 10⁶ wells of a 96-well plate. 3 Cells were treated with concentration gradients of Corilagin, TeGG, NADP, Stafib-2, and uridine triphosphate (0 μM, 0.1 μM, 0.5 μM, 1.0 μM, 2.5 μM, 5.0 μM, 7.5 μM, 10.0 μM, 15.0 μM) per well, with DMSO treatment as a control. Three replicates were performed. Cell status was observed under a microscope after 7 days; cell viability was assessed using CCK8 assay. The IC50 values ​​were fitted based on the cell viability values ​​at the gradient concentrations. 50 The dose-response curve (by logarithmically transforming drug concentrations and using a nonlinear regression model) is used to calculate the IC50. 50 To clarify the safety of ester compounds in their effects on cells.

[0082] 2. EC 50 Detection: Seed VSMC cells at 2.0 × 10⁶ cells per well in a 12-well plate. 3 Cells were treated with 3.0 mM disodium hydrogen phosphate per well to induce VSMC calcification. Cells were treated with a concentration gradient of Corilagin, TeGG, NADP, Stafib-2, and uridine triphosphate (0 μM, 0.1 μM, 1.0 μM, 5.0 μM, 10.0 μM, 25.0 μM, 50.0 μM, 100.0 μM), with DMSO treatment as a control. The experiment was repeated three times. Cell density and state were observed under a microscope. Calcium ion content was then measured in each well using a calcium ion assay kit, and EC values ​​were fitted based on the calcium ion content at the gradient concentrations. 50The curve (by logarithmically transforming drug concentrations and using a nonlinear regression model to plot the dose-response curve) calculates EC. 50 To clarify the effectiveness of small molecule compounds in alleviating cell calcification.

[0083] The results are as follows Figure 3 As shown, A to E represent the IC50 values ​​of Corilagin, TeGG, NADP, Stafib-2, and uridine triphosphate sodium salt, respectively. 50 Curve and EC 50 curve.

[0084] It can be seen that the safe and effective concentration range (IC50) for Corilagin, TeGG, and Stafib-2 is... 50 The concentrations were 14.67 μM, 12.73 μM, and 9.259 μM, respectively, while NADP and uridine triphosphate significantly enhanced cell viability. Corilagin, TeGG, Stafib-2, and UTP were also observed in EC50. 50 The concentrations were 4.051 μM, 3.376 μM, 8.385 μM, and 3.186 μM, respectively, indicating that all five small molecule compounds had an antagonistic effect on calcification. These ester drugs can safely and effectively inhibit vascular calcification and can be used as anti-vascular calcification drugs.

[0085] Example 4 This embodiment utilizes an experiment on the inhibition of vascular and cellular calcification in mice using ester compounds. The specific procedure is as follows: 1. Six- to eight-week-old male C57BL / 6 mice, weighing approximately 25g, were selected. Vascular calcification was induced by intraperitoneal injection of Vitamin D3. Simultaneously, mice in each group were administered ester compounds. Ten days later, the mice were anesthetized and sacrificed. The aorta was isolated, fixed with paraformaldehyde, and stained with Alizarin Red to assess the effect of the ester compounds on vascular calcification in vivo. Specific procedures are as follows: (1) Six- to eight-week-old male C57bl / 6 mice were randomly divided into seven groups of 10 mice each. Group 1 was given the same dose of saline as the model group as the negative control group. Groups 2 to 7 were given 500 IU / g / day of vitamin D3 intraperitoneally for 3 consecutive days to model vascular calcification. At the same time, Group 3 was given 3.2 mg / kg of Corilagin intraperitoneally for intervention; Group 4 was given 7.9 mg / kg of TeGG intraperitoneally for intervention; Group 5 was given 77.5 mg / kg of TeGG intraperitoneally for intervention; Group 6 was given 6.5 mg / kg of Stafib-2 intraperitoneally for intervention; and Group 7 was given 5.5 mg / kg of uridine triphosphate sodium salt intraperitoneally for intervention. The volume of each group was controlled to be the same, and the intraperitoneal injection was carried out for one week. The experiment ended on day 7, three days after intraperitoneal injection of Vitamin D3. Mice were anesthetized and euthanized, and their aortas were harvested.

[0086] (1) Three animals were randomly selected from each group and fixed with 4% paraformaldehyde before being stained with angioalizaine red.

[0087] (2) The vascular tissue was ground using a tissue grinding machine to extract vascular tissue proteins. The level of RUNX2 protein was detected by Western blotting.

[0088] 2. Human vascular smooth muscle cells were induced to calcify using inorganic phosphorus. After seven days of intervention, cells were scraped and lysed to obtain protein samples, which were then analyzed by Western blot to assess changes in RUNX2 protein levels during calcification. The specific procedures are as follows: Human vascular smooth muscle cells from passages 8-12 were used at a concentration of 0.5 × 10⁻⁶. 5 Cells were seeded in 6-well plates at a density of cells / well, with 2 mL of DMEM complete medium added to each well. On day 1 after seeding, calcification was induced by 3.0 mM disodium hydrogen phosphate, while 10 μM of Corilagin, TeGG, NADP, Stafib-2, and uridine triphosphate were added for intervention. On day 4, the DMEM complete medium was replaced, and 3.0 mM disodium hydrogen phosphate was used again to induce calcification, while 10 μM of Corilagin, TeGG, NADP, Stafib-2, and uridine triphosphate were continued for intervention. On day 7, cells were lysed using lysis buffer, and proteins were collected. Western blot analysis was performed to detect changes in RUNX2 protein levels after drug treatment.

[0089] The results are as follows Figure 4As shown, A represents the results of Alizarin Red staining of the aorta in three mice from the control group, the Vitamin D3 model group, and the Vitamin D3 model group plus each drug injection group (scale bar 10 mm); B represents the Western Blot results of RUNX2 protein levels in vascular tissues of three random mice (numbered #1, #2, and #3) from each of the control group, the Vitamin D3 model group, and the Vitamin D3 model group plus TeGG injection group; C represents the Western Blot results of RUNX2 protein levels in vascular tissues of three random mice (numbered #1, #2, and #3) from each of the control group, the Vitamin D3 model group, and the Vitamin D3 model group plus Corilagin injection group; D shows the grayscale value quantitative calculations for B and C respectively; E represents the Western Blot detection of RUNX2 protein levels after ester drug treatment following induced calcification of human vascular smooth muscle cells, and F represents the quantitative results. "***" indicates p < 0.001; "**" indicates p < 0.01.

[0090] like Figure 4 As shown in Figure A, five small molecule compounds—Corilagin, TeGG, NADP, Stafib-2, and uridine triphosphate sodium salt—significantly slowed down arterial calcification in ViTD3-induced model mice. Figure 4 As shown in B, C, and D, Corilagin and TeGG have been clearly demonstrated to significantly reduce RUNX2 protein levels. Meanwhile, as... Figure 4 As shown in Figures E and F, cell experiments clearly demonstrated that the small molecule compounds Corilagin, TeGG, NADP, Stafib-2, and uridine triphosphate sodium salt significantly reduced RUXN2 protein levels.

[0091] In summary, the above results indicate that ester compounds Corilagin, TeGG, NADP, Stafib-2, and uridine triphosphate sodium salt can effectively inhibit the occurrence of RUNX2 protein and vascular calcification, and can be used as drugs for anti-vascular calcification.

[0092] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of an ester compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of vascular calcification; said ester compound being selected from the following compounds: 、 、 、 、 。 2. The application according to claim 1, characterized in that: The drug has the effect of inhibiting RUNX2 expression.

3. The use of an ester compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of vascular calcification-related diseases; said ester compound being selected from the following compounds: 、 、 、 、 。 4. The application according to claim 2 or 3, characterized in that: The vascular calcification-related diseases include at least one of the following: cardiovascular disease, vascular calcification, cardiovascular disease associated with and / or caused by vascular calcification, cardiovascular disease associated with and / or caused by elevated arteriosclerosis levels, elevated arteriosclerosis levels, left ventricular hypertrophy, cardiovascular disease associated with and / or caused by left ventricular hypertrophy, and cardiovascular disease associated with and / or caused by kidney disease.

5. The application according to claim 4, characterized in that: The vascular calcification includes at least one of the following: calcified atherosclerosis, calcified medial vascular lesions, medial calcification, elastic tissue calcification, calcified uremic arterial lesions, calcified aortic valve stenosis, or portal vein calcification.

6. The application according to claim 5, characterized in that: Diseases associated with the aforementioned calcific atherosclerosis include at least one of atherosclerosis, hyperlipidemia, osteoporosis, hypertension, inflammation, type 2 diabetes, end-stage renal disease, amputation, pseudoxanthoma elastica, hyperlipidemia, congenital mitral valve, rheumatic heart disease, and / or liver disease.

7. The application according to claim 4, characterized in that: The cardiovascular disease is associated with at least one of the following conditions: vascular calcification, such as atherosclerosis, hyperlipidemia, osteoporosis, hypertension, inflammation, type 2 diabetes, end-stage renal disease, amputation, pseudoxanthoma elasticus, congenital mitral valve, rheumatic heart disease, portal hypertension, or liver disease.

8. A pharmaceutical composition for the prevention and / or treatment of diseases related to vascular calcification, comprising the ester compound of claim 1 or a pharmaceutically acceptable salt thereof.

9. The pharmaceutical composition according to claim 8, characterized in that: The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 8, characterized in that: The formulation of the pharmaceutical composition includes any one of the following: a solid oral formulation, a liquid formulation for oral or mucosal administration, a topical formulation, or a parenteral formulation.

11. Use of the pharmaceutical composition according to any one of claims 8-10 in the preparation of a medicament for the prevention and / or treatment of vascular calcification-related diseases.

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