KR102367101B1 - New compounds having cardiac protection effect and a medium composition for culturing stem cell using thereof - Google Patents

Abstract

The present invention relates to a novel compound having a cardioprotective effect and a medium composition for culturing stem cells using the same.

Description

New compounds having cardiac protection effect and a medium composition for culturing stem cell using thereof

The present invention relates to a novel compound having a cardioprotective effect and a stem cell culture medium composition using the same, and more particularly, to inhibit senescent activity of stem cells and promote cell differentiation, and to have a cardioprotective effect, in particular, prevention or treatment of cardiomyopathy It shows an effect on and relates to a novel compound usable as a stem cell culture medium composition, a stem cell culture medium composition using the same, and a stem cell culture method using the same.

Although cardiac stem cells exist in very small numbers in the heart, they play a key role in maintaining homeostasis of the heart, and can differentiate into three types of cells: blood vessels, smooth muscle cells, and myocardium. Accordingly, related research is being actively conducted as an excellent source of stem cell-based cells for the treatment of cardiomyopathy and cardiovascular regeneration. Until now, various studies on myocardial regeneration using cardiac stem cells have been conducted, and clinical studies for the development of stem cell therapeutics using these are being actively conducted.

In order to apply stem cell therapy to clinical practice, a large amount of cells is required, and the process of amplification is absolutely necessary to secure the number of cells. That is, for treatment using stem cells, an amplification process is absolutely necessary when culturing cells, but the amplification ability of the cells is limited, and senescence of cells is irreversibly progressed in the process of subculture for cell amplification. Aged cells have very low cell functions such as proliferative capacity and differentiation capacity, which may reduce their value as a therapeutic agent. Therefore, there is a need for the development of a novel compound for enhancing the function of stem cells and alleviating the aging of cells, a culture medium composition, and a culture method.

KR10-2019-0003871 KR10-2015-0018203 KR10-1358777

An object of the present invention is to provide a pharmaceutical composition for preventing or treating cardiomyopathy comprising a novel compound.

The problem to be solved by the present invention is to provide a stem cell culture medium composition that can enhance the function of stem cells, inhibit cellular aging, and promote cell proliferation.

Another object to be solved by the present invention is to provide a method for culturing stem cells using the medium composition for culturing stem cells.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating cardiomyopathy comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In the formula,

Z ₁ and Z ₂ are the same as or different from each other and are each independently O, NH or S,

R ₁ to R ₄ are the same as or different from each other, _{and each independently any one selected from the group consisting of H, OH, C 1-4 alkoxy} , halogen, acetoxy and benzyloxy.

According to the present invention, the compound of Formula 1 may be a compound represented by Formula 2 below.

[Formula 2]

In the formula,

R ₁ to R ₄ are the same as or different from each other, and each independently any one selected from _{the group consisting of H, OH and C 1-4 alkoxy} .

According to the present invention, the compound of Formula 2 may be a compound in which R ₁ , R ₂ , and R ₄ are each H, and R ₃ is OH.

Preferably, it may be a compound represented by the following formula (3).

[Formula 3]

In addition, the present invention provides a medium composition for culturing stem cells comprising the compound represented by Formula 1 as an active ingredient.

In the stem cell culture medium composition of the present invention, the compound represented by Formula 1 may be a compound represented by Formula 2 above.

In the stem cell culture medium composition of the present invention, the compound represented by Formula 2 is preferably R ₁ , R ₂ and R ₄ are each H, and R ₃ may be OH.

In the present invention, the stem cells may be mammalian stem cells.

In the present invention, the stem cells may be selected from vascular endothelial progenitor cells, mesenchymal stem cells and myocardial progenitor cells.

In the present invention, cellular senescence of stem cells may be inhibited and cell differentiation may be promoted.

In the present invention, the stem cell culture medium composition may be for using the cultured stem cells as a cell therapy agent.

In the present invention, the stem cell culture medium composition is cardiomyopathy, ischemic disease, stroke, coronary artery disease, retinopathy, muscular dystrophy, neuromuscular disease, myasthenia gravis, muscle damage, multiple sclerosis, amyotrophic lateral sclerosis and age-related macular degeneration It may be for use as a cell therapy agent for a disease selected from the group consisting of.

In addition, the present invention provides a stem cell culture method comprising; culturing the stem cells in a culture medium containing the compound represented by the formula (1).

The compound represented by Formula 1 according to the present invention helps cardiovascular regeneration and has excellent cardiomyopathy, so it not only has an effect in the prevention or treatment of cardiomyopathy, but also inhibits cellular aging of stem cells and promotes cell differentiation. Therefore, it is useful as a medium composition for stem cell culture because it can alleviate senescence of cells that can be irreversibly progressed in the process of subculture for clinical stem cell amplification and promote cell differentiation. In addition, the stem cell culture method using the stem cell culture medium composition according to the present invention suppresses cellular aging of the amplified stem cells, and can provide stem cells with improved functionality, and the stem cells obtained by the method of the present invention is industrially advantageous as a cell therapy product.

1 shows the verification of cytotoxicity through the confirmation of cell viability by treating cardiac stem cells with the compound of Formula 1 by concentration.
Figure 2 schematically shows the design of the in-vitro experiment for the culture and verification method of cardiac stem cells.
3a is the result of quantification of the viability of cardiac stem cells cultured in the medium with and without the compound of Example 1 according to the present invention at passage 16 by CCK-8 assay, and FIG. 3b shows the cells in real time. This is the result of verifying the proliferative ability. Figure 3c is a result of comparing and verifying the proliferative capacity of cells through the BrdU assay. Figure 3d is a result of verifying the expression of the cell proliferation cycle-related markers through western blot assay.
Figure 4a is the result of quantifying the degree of intracellular reactive oxygen species through H2-DFFDA assay after inducing oxidative stress through H ₂ O ₂ treatment, Figure 4b is the oxidative stress induced through H ₂ O ₂ treatment It is the result of quantifying the degree of reactive oxygen species in mitochondria through mitosox staining, and FIG. 4c is the result of confirming the degree of apoptosis through Annexin V-7AAD staining after inducing oxidative stress through H ₂ O ₂ treatment.
5 is a morphological analysis result of cardiac stem cells cultured according to the use of a medium with and without the compound of Example 1 according to the present invention.
6 is a SA-β-gal assay result for confirming the degree of senescence of cardiac stem cells cultured in the same passage according to the use of a medium with and without the compound of Example 1 according to the present invention. .
7 is a result of quantifying the division time for each passage of cardiac stem cells according to the use of a medium with and without the compound of Example 1 according to the present invention.
8 is an evaluation of the expression of senescence-related markers in cardiac stem cells according to the use of a medium in which the compound of Example 1 according to the present invention is present and a medium in which it does not exist (vehicle). Figure 8a is a heat map showing the FPKM values of the aging markers based on the RNA-seq results, Figure 8b is the result of confirming the expression levels of the aging markers p16, p53, p27 through western blot, Figure 8c is the aging markers p16, This is the result of confirming the expression level of p53 at the mRNA level.
9 is a result of verifying the vascular differentiation capacity of aged vascular endothelial stem cells through the matrigel tube formation assay.
10a is a heat map of the gene expression of endothelial cells and angiogenesis-related markers through RNA-seq results, and FIG. 10b is a table showing an ontology analysis of genes that are significantly different in relation to vascular differentiation. and FIG. 10c is a result of verifying the gene expression of endothelial markers after induction of differentiation into endothelial cells compared with before induction of differentiation.
11A is a result of verifying cardiac stem cells cultured according to the use of a medium with and without the compound of Example 1 according to the present invention by a-SMA staining. 11B is a result of comparing the genetic differentiation of smooth muscle cell markers with those before differentiation induction. 11C is a table showing differences in ontology of genes related to differentiation into smooth muscle cells and muscles.
12a is a result of evaluating the colony-forming ability in the culture medium. 12B is a result of evaluating the gene expression of stem cell markers by qRT-PCR.
13 is a schematic view illustrating an experimental method of an animal model of myocardial infarction in order to confirm the effect of the compound according to the present invention on cardiomyopathy and myocardial infarction.
14 shows the cell viability of the stem cell transplantation group subcultured in the infarct area and the stem cell transplantation group subcultured in a medium supplemented with the compound according to the present invention to the infarct area 4 weeks after induction of myocardial infarction in an animal model of myocardial infarction; This is the evaluation result.
15 is a result of confirming the apoptosis rate in the infarct region in an animal model of myocardial infarction by TUNEL staining.
16 shows a control group, a group directly administered with the compound according to the present invention to the infarct site, a stem cell transplant group subcultured at the infarct site, and a compound according to the present invention added to the infarct site 4 weeks after myocardial infarction in an animal model of myocardial infarction These are the blood vessel density evaluation results for the stem cell transplantation group subcultured in the cultured medium.
17 shows the vascular differentiation ability of the stem cell transplantation group subcultured to the infarct site and the stem cell transplantation group subcultured in a medium supplemented with the compound according to the present invention to the infarct area 4 weeks after induction of myocardial infarction in an animal model of myocardial infarction. This is the evaluation result.
18 is a myocardial infarction animal model 4 weeks after induction of myocardial infarction, myocardial differentiation ability in the stem cell transplantation group subcultured to the infarct area and the stem cell transplantation group subcultured in the medium to which the compound according to the present invention was added to the infarct area. This is the evaluation result.
19 is a control, direct administration group of the compound according to the present invention to the infarct site, stem cell transplantation group subcultured at the infarct site, and the compound according to the present invention added to the infarct site 4 weeks after induction of myocardial infarction in an animal model of myocardial infarction These are the results of evaluation of the fibrosis inhibitory ability of the stem cell transplantation group subcultured in the treated medium.
20 is a control group, the direct administration group of the compound according to the present invention to the infarct site, the stem cell transplantation group subcultured at the infarct site, and the compound according to the present invention added to the infarct site 4 weeks after myocardial infarction in an animal model of myocardial infarction These are the results of echocardiography of the stem cell transplantation group subcultured in the cultured medium.

For the commercialization of adult stem cell therapy, it is necessary to secure a large amount of cells, and for this, an in vitro cell amplification culture method is absolutely necessary. In the process of in vitro cell amplification culture, several passages are necessarily performed, and in this process, aging and functional deterioration of cells occur.

The present invention is meaningful to develop a compound having a myocardial protective effect, and to provide a novel compound having a preventive or therapeutic effect on cardiomyopathy. In addition, it is suggested that the composition for culturing stem cells using the compound according to the present invention can inhibit cellular aging of stem cells and enhance cellular functions.

Hereinafter, the present invention will be described in more detail.

The present invention provides a pharmaceutical composition for preventing or treating cardiomyopathy comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In the formula,

Z ₁ and Z ₂ are the same as or different from each other and are each independently O, NH or S,

R ₁ to R ₄ are the same as or different from each other, _{and each independently any one selected from the group consisting of H, OH, C 1-4 alkoxy} , halogen, acetoxy and benzyloxy.

According to the present invention, the preferred compound of Formula 1 may be a compound represented by Formula 2 below.

[Formula 2]

In the formula,

R ₁ to R ₄ are the same as or different from each other, and each independently any one selected from _{the group consisting of H, OH and C 1-4 alkoxy} .

According to the present invention, the more preferred compound of Formula 1 may be a compound represented by Formula 3 below.

[Formula 3]

According to the present invention, when the compound of Formula 1 was directly administered in an animal model for inducing myocardial infarction, a significant decrease in wounds in the infarct region and a significant increase in myocardium were observed. Cardiac fibrosis was reduced. This suggests that the composition comprising the compound of Formula 1 according to the present invention is effective for preventing and treating cardiomyopathy and has a cardioprotective effect.

The pharmaceutically acceptable salts thereof include hydrochloride, bromate, sulfate, phosphate, nitrate, citrate, acetate, lactate, tartrate, maleate, gluconate, succinate, formate, trifluoroacetate, oxalic acid It may be any one selected from the group consisting of salt, fumarate, methanesulfonate, benzenesulfonate, para-toluenesulfonate, and camphorsulfonate.

The pharmaceutical composition may further include an appropriate carrier, excipient or diluent commonly used in the manufacture of the pharmaceutical composition.

Carriers, excipients and diluents usable in the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, mineral oil, and the like.

The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. there is.

In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and in such solid preparations, the compound may contain at least one excipient, for example, starch, calcium carbonate, sucrose ( sucrose) or lactose, gelatin, etc. can be mixed to prepare it.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The pharmaceutical composition of the present invention is an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human as considered by a researcher, veterinarian, physician or other clinician, that is, the amount of the symptom of the disease or disorder being treated. It can be administered in a therapeutically effective amount, which is an amount inducing remission. It is apparent to those skilled in the art that the therapeutically effective dosage and frequency of administration for the pharmaceutical composition of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of active ingredients and other ingredients contained in the composition, the type of formulation, the age, weight, and general health of the patient. , sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and various factors including concurrently used drugs. The pharmaceutical composition of the present invention may be administered to an individual by various routes. For example, intravenous, intraperitoneal, intramuscular, intraarterial, buccal, intracardiac, intramedullary, intrathecal, transdermal, enteral, subcutaneous, sublingual or topical administration may be administered, but not limited thereto.

The pharmaceutical composition of the present invention may be administered in an amount of 1 to 10,000 mg/kg/day, preferably 1 to 200 mg/kg/day, and may be administered once a day or divided into several administrations. .

In addition, the dosage of the compound according to the present invention may be increased or decreased depending on the route of administration, the degree of disease, sex, weight, age, and the like. Accordingly, the above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, and humans by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, endobronchial inhalation, intrauterine dural or intracerebrovascular injection.

The compound according to the present invention has a 50% lethal dose (LC50) of 2 g/kg or more, which ensures stability, and can be used in the pharmaceutical composition of the present invention.

In addition, the present invention provides a medium composition for culturing stem cells comprising the compound represented by the following formula (1) as an active ingredient.

[Formula 1]

In the formula,

Z ₁ and Z ₂ are the same as or different from each other and are each independently O, NH or S,

R ₁ to R ₄ are the same as or different from each other, _{and each independently any one selected from the group consisting of H, OH, C 1-4 alkoxy} , halogen, acetoxy and benzyloxy.

According to the present invention, the preferred compound of Formula 1 may be a compound represented by Formula 2 below.

[Formula 2]

In the formula,

R ₁ to R ₄ are the same as or different from each other, and each independently any one selected from _{the group consisting of H, OH and C 1-4 alkoxy} .

According to the present invention, the more preferred compound of Formula 1 may be a compound represented by Formula 3 below.

[Formula 3]

In the present invention, the stem cells may be mammalian stem cells.

According to the present invention, the stem cells may be mammalian cardiac stem cells.

Although cardiac stem cells exist in very small numbers in the heart, they play a key role in maintaining homeostasis of the heart, and can differentiate into vascular cells, smooth muscle cells, and cardiomyocytes. In the present invention, the stem cells may be preferably selected from vascular endothelial progenitor cells, mesenchymal stem cells and myocardial progenitor cells.

According to the present invention, compared to the conventionally known culture medium, the culture medium composition comprising the compound of Formula 1 according to the present invention suppresses cellular senescence of stem cells, improves cellular activity, and the cultured cardiac stem cells It is effective as a medium composition for stem cell culture because it can overcome the aging of stem cells in myocardial infarction and promote cardiovascular regeneration.

In the present invention, cellular senescence of stem cells may be inhibited and cell differentiation may be promoted.

In the present invention, the stem cell culture medium composition may be for using the cultured stem cells as a cell therapy agent.

In the present invention, the stem cell culture medium composition is cardiomyopathy, ischemic disease, stroke, coronary artery disease, retinopathy, muscular dystrophy, neuromuscular disease, myasthenia gravis, muscle damage, multiple sclerosis, amyotrophic lateral sclerosis and age-related macular degeneration It may be for use as a cell therapy agent for a disease selected from the group consisting of.

According to the present invention, the stem cells cultured in the medium composition for stem cell culture containing the compound represented by Formula 1 has a significant increase in cell proliferation rate compared to the medium composition for stem cell culture that does not contain the compound represented by Formula 1 There was a positive increase, and the active parathyroid and mitochondrial reactive oxygen species were decreased in the oxidative stress situation, and the cell viability increased.

In addition, aging markers such as p53, p16 and β-GAL were significantly reduced, and morphological changes due to aging were reduced, qRT-PCR, RNA-seq, tube formation assay, immunocytochemistry, etc. As a result of verification by various methods, stem cells cultured in the medium composition for stem cell culture containing the compound represented by Formula 1 according to the present invention are in the medium composition for stem cell culture that does not contain the compound represented by Formula 1 above. Compared to that, it was confirmed that the gene expression levels of OCT4, NANOG, and KLF4 were high. Therefore, the stem cell culture medium composition comprising the compound of Formula 1 according to the present invention is a stem cell, preferably a cardiac stem cell, to improve biological activities such as alleviation of aging and maintenance of stem cell ability, cell proliferation ability, differentiation ability. can

On the other hand, in the myocardial infarction-induced animal model transplanted with stem cells, preferably cardiac stem cells, cultured in a stem cell culture medium composition comprising the compound of Formula 1 according to the present invention, alleviation of cardiomyopathy and cell viability in the infarct region , increased vascular density in the infarcted region, increased vascular differentiation capacity, decreased cardiac fibrosis, and significantly increased left ventricular ejection fraction and cardiac output did

In addition, the present invention provides a stem cell culture method comprising; culturing the stem cells in a culture medium containing the compound represented by Formula 1 above.

According to the present invention, the stem cells may be selected from vascular endothelial progenitor cells, mesenchymal stem cells, neural progenitor cells, and myocardial progenitor cells.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example

Synthesis Example 1. Preparation of 5-(substituted benzylidene)pyrimidine-2,4,6(1H,3H,5H)trione derivative

A suspension of substituted benzaldehyde (1.44-2.60 mmol) and barbituric acid (0.7-1.2 equiv. (eq.)) in EtOH (4 mL) and H ₂ O (4 mL) solvent is heated to 80 °C. heated. Before the reaction temperature reached 80° C., in most cases the reaction mixture became a clear solution. During further heating (1-18 hours), a precipitate formed and after cooling the precipitate was filtered off. In consideration of the characteristics of the remaining substituted benzaldehyde, the filter cake was washed with ethanol and/or methylene chloride and water to obtain the desired product (yield: 60.3 to 99.3%).

Example 1. Synthesis of 5-(4-hydroxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)trione

A yellow solid was obtained by the method of Synthesis Example. reaction time, 6 hours; yield, 826%; melting point, >300 °C; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1123 (s, 1 H), 1110 (s, 1 H), 1079 (s, 1 H), 829 (d, 2 H, J=88 Hz), 817 ( s, 1H), 684 (d, 2H, J=88 Hz); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 1648, 1637, 1630, 1561, 1509, 1390, 1244, 1162, 1149; LRMS(ES) m/z

231 (MH) ^- .

Example 2. Synthesis of 5-(3,4-dihydroxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione

orange solid; reaction time, 8 hours; yield, 993%; melting point, >300 °C; ¹ H NMR (500 MHz, DMSO-d ⁶ ) δ 1114 (br s, 2H), 976 (br s, 1 H), 818 (s, 1 H), 810 (s, 1 H), 761 (d, 1 H, J = 80 Hz), 683 (d, 1 H, J = 75 Hz); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 1649, 1629, 1567, 1530, 1509, 1455, 1320, 1249, 1220, 1160, 1143; LRMS (ES) m/z 247 (MH) ^- .

Example 3. Synthesis of 5-(4-hydroxy-3-methoxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione

yellow solid; reaction time, 10 hours; yield, 603%; melting point, >300 °C; ¹ H NMR (500 MHz, D ₂ O+NaOH) δ 807 (s, 1 H), 726 (d, 1 H, J=85 Hz), 644 (dd, 1H, J=20, 90 Hz), 624 (s, 1H); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 1685, 1667, 1622, 1572, 1568, 1457, 1340, 1165, 1131, 1098, 1033; LRMS (ES) m/z 247 (MH) ^- .

Example 4. Synthesis of 5-(4-hydroxy-3-methoxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione

dark yellow solid; reaction time, 18 hours; yield, 97%; Melting point, 2886-2907 °C; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1123 (s, 1 H), 1111 (s, 1 H), 1054 (s, 1 H), 844 (d, 1 H, J=20 Hz), 818 (s, 1H), 777 (dd, 1H, J=20, 84 Hz), 686 (d, 1H, J=84 Hz), 379 (s, 3 H); ¹³ C NMR (100 MHz, DMSO-d6) δ 1648, 1632, 1566, 1537, 1509, 1476, 1332, 1249, 1186, 1160, 1146, 562; LRMS (ES) m/z 261 (MH) ^- .

Example 5. Synthesis of 5-(3-ethoxy-4-hydroxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione

orange solid; reaction time, 15 hours; yield, 77%; Melting point, 2447-2461 ° C; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 1124 (s, 1 H), 1111 (s, 1 H), 1046 (s, 1 H), 848 (s, 1 H), 820 (s, 1 H) ), 774 (d, 1 H, J = 85 Hz), 690 (d, 1H, J = 85 Hz), 408 (q, 2H, J = 70 Hz), 136 (t, 3H, J = 70 Hz); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 1648, 1631, 1566, 1540, 1508, 1468, 1332, 1249, 1197, 1161, 1146, 645, 152; LRMS (ES) m/z 275 (MH) ^- .

Example 6. Synthesis of 5-(3-hydroxy-4-methoxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione

dark yellow solid; reaction time, 17 hours; yield, 93%; melting point, 2793-2814 °C; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 1126 (s, 1 H), 1113 (s, 1 H), 942 (s, 1 H), 814 (s, 1 H), 810 (s, 1 H) ), 770 (d, 1 H, J = 85 Hz), 703 (d, 1 H, J = 90 Hz), 387 (s, 3H); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 1647, 1628, 1562, 1536, 1509, 1464 1310, 1261, 1211, 1157, 1120, 564; LRMS (ES) m/z 261 (MH) ^- .

Example 7. Synthesis of 5-(4-methoxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione

yellow solid; reaction time, 13 hours; yield, 93%; Melting point, 2924-2943 °C; ¹ H NMR (400 MHz, DMSO-d6) δ 1127 (s, 1 H), 1114 (s, 1 H), 833 (d, 2 H, J=92 Hz), 821 (s, 1H), 702 (d, 2H, J=88Hz), 383(s, 3H); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 1646, 1641, 1628, 1556, 1509, 1381, 1258, 1162, 1146, 564; LRMS (ES) m/z 245 (MH) ^- .

Example 8. Synthesis of 5-(3,4-dimethoxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione

yellow solid; reaction time, 9 hours; yield, 966%; melting point, >300 °C; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1127 (s, 1 H), 1115 (s, 1 H), 837 (d, 1 H, J=20 Hz), 821 (s, 1H), 786 (dd , 1H, J = 20,84 Hz), 707 (d, 1H, J = 84 Hz), 384 (s, 3H), 377 (s, 3H); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 1647, 1630, 1561, 1543, 1509, 1485, 1324, 1259, 1174, 1159, 1118, 565, 561; LRMS (ES) m/z 275 (MH) ^- .

Example 9. Synthesis of 5-(2,4-dimethoxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione

orange solid; reaction time, 8 hours; yield, 97%; Melting point, 2911-2917 °C; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 1121 (s, 1 H), 1106 (s, 1 H), 861 (s, 1 H), 853 (d, 1 H, J=85 Hz), 663 ( d, 1H, J=20 Hz), 661 (dd, 1H, J=20,90 Hz), 390 (s, 3H), 387 (s, 3H); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 1665, 1648, 1631, 1628, 1509, 1497, 1361, 1153, 1149, 1065, 981, 569, 565; LRMS (ES) m/z 275 (MH) ^- .

Example 10. Synthesis of 5-(3,4,5-trimethoxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione

yellow solid; reaction time, 1 hour; yield, 840%; Melting point, 2748-2754 °C; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1133 (s, 1 H), 1120 (s, 1 H), 822 (s, 1 H), 780 (s, 2 H), 378 (s, 6 H) ), 375 (s, 3 H); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 164.4, 162.8, 155.9, 152.6, 150.8, 142.6, 128.2, 117.9, 113.3, 61.0, 56.7; LRMS (ES) m/z 305 (MH) ^- .

Example 11. Synthesis of 5-(4-hydroxy-3,5-dimethoxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione

orange solid; reaction time, 2 hours; yield, 994%; melting point, >300 °C; ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 1125 (s, 1 H), 1112 (s, 1 H), 997 (br s, 1 H), 824 (s, 1 H), 800 (s, 2 H), 382 (s, 6 H); ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 1648, 1632, 1570, 1509, 1478, 1431, 1235, 1149, 1146, 567; LRMS (ES) m/z 291 (MH) ^- .

production example

A composition for culturing stem cells was prepared including the compound of Example 1, and stem cells were cultured using this.

Test Example 1. Cytotoxicity

Cardiac stem cells were cultured in a 96-well cell culture plate at 37°C, 5% CO ₂ environment for one day, and DMSO and the compound of Example 1 were treated by concentration. After exposure to the drug for 24 hours, it was exchanged for a medium in which the D-Plus CCK kit (Dongin, DI1701-01) solution and the cell culture solution were mixed in a 1:10 ratio. After reacting for 1 hour at 37°C and 5% CO2, absorbance was measured at 450 nm to measure CCK activity, and cytotoxicity compared to DMSO control was confirmed.

1 shows the verification of cytotoxicity through the confirmation of cell viability by treating cardiac stem cells with the compound of Formula 1 by concentration. It was confirmed that there was no significant change in cardiac stem cell viability at a concentration of 10 μM or less. Thereafter, the experiment was conducted within a concentration that did not cause toxicity.

Test Example 2. Evaluation of biological activity of cells of the same passage

An in-vitro experiment was designed for the culture and verification of cardiac stem cells, and it is schematically shown in FIG. 2 . According to the design, cardiac stem cells were continuously subcultured using the culture medium according to the prior art and the cardiac stem cell culture medium prepared in Preparation Example 1, and the biological activity of the cells of the same passage was compared.

3A is a result of quantifying the viability of cardiac stem cells cultured in a medium in which the compound of Example 1 according to the present invention is present and a medium in which it does not exist (vehicle) at passage 16 by CCK-8 assay. It was found that the medium composition according to the present invention improved the cell viability more than twice as compared to the vehicle.

Figure 3b is the result of assaying the cell proliferation ability in real time. It can be seen that the medium in which the compound of Example 1 according to the present invention is present improves the proliferative ability of cardiac stem cells compared to the vehicle.

Figure 3c is a result of comparing and verifying the proliferative capacity of cells through the BrdU assay. It can be seen that the cell proliferation ability of cardiac stem cells cultured in the medium in which the compound of Example 1 according to the present invention is present is enhanced compared to that of the vehicle.

Figure 3d is a result of verifying the expression of the cell proliferation cycle-related markers through western blot assay. It was confirmed that the expression of the markers related to the cell proliferation cycle of cardiac stem cells cultured in the medium containing the compound of Example 1 according to the present invention was significantly enhanced compared to that of the vehicle.

Test Example 3. Evaluation of intracellular reactive oxygen species

Intracellular reactive oxygen species evaluation was performed according to the use of a medium in which the compound of Example 1 according to the present invention is present and a medium in which it does not exist (vehicle).

4a is a result of quantifying the level of intracellular reactive oxygen species through H2-DFFDA assay after inducing oxidative stress through H ₂ O ₂ treatment. The cardiac stem cells cultured in the medium containing the compound of Example 1 according to the present invention had reduced oxidative stress compared to the vehicle.

Figure 4b is a result of quantifying the level of reactive oxygen species in mitochondria through mitosox staining after inducing oxidative stress through H ₂ O ₂ treatment. Cardiac stem cells cultured in the medium containing the compound of Example 1 according to the present invention significantly reduced reactive oxygen species in mitochondria compared to vehicle.

Figure 4c is a result of confirming the degree of cell death through Annexin V-7AAD staining after inducing oxidative stress through H ₂ O ₂ treatment. Cardiac stem cells cultured in the medium containing the compound of Example 1 according to the present invention decreased apoptosis compared to vehicle, and the cell viability increased in a compound concentration-dependent manner.

Test Example 4. Morphological analysis of cardiac stem cells

Morphological analysis of cardiac stem cells cultured according to the use of a medium in which the compound of Example 1 according to the present invention exists and a medium in which the compound of Example 1 does not exist (vehicle) was used.

As shown in FIG. 5 , in the cardiac stem cells cultured in the medium containing the compound of Example 1 according to the present invention, the length and area of the cells were significantly reduced, so the morphological change due to cellular aging was alleviated. was evaluated to have been

Test Example 5. Evaluation of the ability to inhibit the expression of aging markers

SA-β-gal assay

The degree of senescence of cardiac stem cells cultured in the same passage according to the use of a medium with and without the compound of Example 1 according to the present invention was evaluated by SA-β-gal assay.

As shown in FIG. 6 , it can be confirmed that the cardiac stem cells cultured in the medium containing the compound of Example 1 according to the present invention have significantly reduced β-gal positive cells compared to the vehicle.

Evaluation of doubling time

Fig. 7 shows the quantification of the division time of cardiac stem cells for each passage according to the use of a medium with and without the compound of Example 1 according to the present invention.

It was found that the cardiac stem cells cultured in the medium containing the compound of Example 1 according to the present invention had a significantly shorter division time than that of the vehicle.

Evaluation of expression of aging-related markers

The expression of senescence-related markers in cardiac stem cells according to the use of a medium in which the compound of Example 1 according to the present invention is present and a medium in which the compound is not present (vehicle) was evaluated.

Figure 8a is based on the RNA-sequence (RNA-seq) results of cardiac stem cells according to the use of a medium in which the compound of Example 1 according to the present invention is present and a medium in which it does not exist (vehicle). Figure 8b is the result of confirming the expression levels of the aging markers p16, p53, p27 through western blot, Figure 8c is the result of confirming the expression level of the aging markers p16, p53 at the mRNA level. As shown in FIGS. 8A to 8C , it was found that the compound according to the present invention significantly reduced the expression of aging markers.

Test Example 6. Vascular differentiation capacity evaluation

matrigel tube formation assay

In order to confirm the vascular differentiation ability according to the use of the medium with and without the compound of Example 1 according to the present invention, the vascular endothelial progenitor cells treated with DMSO and the compound of Example 1 were treated with Matrigel GFP (BD). ) was added, 7,500 cells per well were cultured, and the number of formed tubes was counted to confirm the angiogenesis performance.

9 is a result of verifying the vascular differentiation ability of aged vascular endothelial stem cells through a matrigel tube formation assay. Cardiac stem cells cultured in the medium containing the compound of Example 1 according to the present invention have higher vascular differentiation ability than the vehicle. It can be seen that excellent

Assessment of expression of markers related to vascular differentiation

The expression of markers related to vascular differentiation of cardiac stem cells according to the use of a medium in which the compound of Example 1 according to the present invention exists and a medium in which the compound of Example 1 does not exist (vehicle) was evaluated.

10a is a heat map of the gene expression of endothelial cells and angiogenesis-related markers through RNA-seq results, and FIG. 10b is a table showing the ontology analysis of genes that are significantly different in relation to vascular differentiation. and FIG. 10c is a result of verifying the gene expression of endothelial markers after induction of differentiation into endothelial cells compared with before induction of differentiation. These results suggest that the compound according to the present invention enhances the ability to differentiate into blood vessels.

to smooth muscle cells pluripotency evaluation

The differentiation ability of cardiac stem cells into smooth muscle cells according to the use of a medium in which the compound of Example 1 according to the present invention exists and a medium in which it does not exist (vehicle) was evaluated.

Figure 11a is the verification of cardiac stem cells cultured according to the use of a medium with and without the compound of Example 1 according to the present invention by a-SMA staining, wherein the compound of Example 1 is in a vehicle In comparison, it shows that it is effective in differentiation into smooth muscle cells.

11B is a result of comparing the gene differentiation of smooth muscle cell markers with those before differentiation induction. The cardiac stem cells cultured in the medium containing the compound of Example 1 according to the present invention significantly increased the expression level of smooth muscle markers compared to the vehicle. it can be seen that

11c is a table showing the differences in ontology of genes related to differentiation into smooth muscle cells and muscles. Muscle cell proliferation and muscle structure in cardiac stem cells cultured in a culture medium containing the compound of Example 1 according to the present invention. It can be seen that the gene ontology related to development, muscle tissue development, and muscle contraction regulation was significantly changed.

Test Example 7. Evaluation of single cell-derived colony forming ability

The single cell-derived colony-forming ability of cardiac stem cells according to the use of a medium with and without the compound of Example 1 according to the present invention was evaluated, and this is shown in FIG. 12 .

12a shows that the passage #16 cardiac stem cells cultured in the medium containing the compound of Example 1 significantly improved the ability to form colonies in the culture medium compared to the vehicle. Figure 12b is a result of evaluating the gene expression of stem cell markers by qRT-PCR, the cardiac stem cells cultured in the medium containing the compound of Example 1 according to the present invention significantly increased the gene expression of the stem cell markers. it can be confirmed that

Test Example 8. In-vivo evaluation

In order to confirm the effect of the compound according to the present invention on cardiomyopathy and myocardial infarction, an animal model of myocardial infarction was constructed, which is shown in FIG. 13 .

The myocardial infarction animal model was divided into four groups, each injected with PBS buffer solution into the infarct site (group 1), injection of 10 mg/kg of the compound of Example 1 into the infarct site (group 2), stem cells aged by subculture (hCPCs) Transplantation (group 3) and senescent stem cells (hCPCs + Example 1) cultured in a culture medium containing the compound of Example 1 were transplanted (group 4).

Cell viability evaluation

To evaluate the cell viability following transplantation of senescent stem cells cultured in an animal model of myocardial infarction, the transplanted cells of groups 3 and 4 engrafted in the infarct area at the 4th week of transplantation were quantified, and the results are shown in FIG. 14 . indicated.

It was confirmed that the cell viability after myocardial infarction was increased in the 4 groups transplanted with senescent stem cells cultured in a culture medium containing the compound of Example 1 according to the present invention.

Apoptosis Assessment

After 4 weeks of induction of myocardial infarction, the apoptosis rate in the infarcted region was confirmed by TUNEL staining, which is shown in FIG. 15 .

The 4 group transplanted with the senescent stem cells cultured with the culture medium containing the compound of Example 1 according to the present invention had a significant decrease in TUNEL-positive cells compared to the 3 group.

Blood vessel density evaluation

In order to confirm the effect in the myocardial infarction animal model according to the supply form of the compound of Example 1, PBS buffer injection into the infarct site (group 1), injection of the compound of Example 1 10 mg/kg into the infarct site (group 2) , transplanted senescent stem cells (hCPCs) cultured in a general culture medium with the same subculture (group 3) and senescent stem cells (hCPCs + Example 1) cultured in a culture medium containing the compound of Example 1 (group 4) After 4 weeks, the capillaries were stained, and the results are shown in FIG. 16 .

As shown in FIG. 16, in the group (group 2), in which the compound according to the present invention was administered directly into the heart, the vascular density of the border zone was significantly improved, and the aged group cultured in a culture medium containing the compound of Example 1 Even in the group (group 4) transplanted with stem cells (hCPCs + Example 1), the vascular density was significantly improved in the infact zone and the border zone. Accordingly, the compound according to the present invention is useful for both a pharmaceutical composition for preventing or treating cardiomyopathy and a medium composition for culturing cardiac stem cells.

blood vessel pluripotency evaluation

The blood vessels proliferating in the infarcted tissue and the transplanted cells were stained together to evaluate the vascular differentiation ability, which is shown in FIG. 17 .

It can be seen that the vascular differentiation ability of cardiac stem cells was enhanced in the group (group 4) transplanted with senescent stem cells (hCPCs + Example 1) cultured in a culture medium containing the compound of Example 1.

myocardium pluripotency evaluation

The myocardial differentiation capacity in the heart tissue was evaluated, and this is shown in FIG. 18 .

It was confirmed that the differentiation ability into myocardium was enhanced in the group (group 4) transplanted with senescent stem cells (hCPCs + Example 1) cultured in a culture medium containing the compound of Example 1. These results suggest that the compound according to the present invention is useful as a pharmaceutical composition for preventing or treating cardiomyopathy.

fibrosis inhibitory ability evaluation

Injection of PBS buffer into the infarct site (group 1), injection of 10 mg/kg of the compound of Example 1 into the infarct site (group 2), and transplantation of senescent stem cells (hCPCs) cultured in general culture medium with the same subculture (group 3) And the degree of myocardial fibrosis in an animal model of myocardial infarction transplanted (4 groups) of aged stem cells (hCPCs + Example 1) cultured in a culture medium containing the compound of Example 1 was confirmed through masson's trihorme stain, which is shown in FIG. indicated.

A significant decrease in wound size was observed in group 2 in which the compound of Example 1 was injected directly into the heart, and a significant increase in myocardium in the infarcted region was observed compared to group 1 as a control group. On the other hand, in the group in which the stem cells of the same passage were transplanted, compared to the group (group 3) in which the senescent stem cells cultured in a general culture medium were transplanted, the aged stem cells cultured in the culture medium containing the compound of Example 1 were transplanted. In the group (Group 4), it was observed that the myocardium in the infarct area was significantly increased, and the wound size and the rate of fibrosis were decreased.

echocardiography evaluation

Four weeks after the induction of myocardial infarction, echocardiography was taken for groups 1 to 4, and the results are shown in FIG. 20 . Compared to the group (group 3) transplanted with senescent stem cells cultured in a general culture medium, the left ventricular ejection fraction and The contractile force was significantly improved.

Claims (12)

A pharmaceutical composition for preventing or treating cardiomyopathy comprising a compound represented by the following formula (3) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 3]

.



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