KR20140046808A - Compositions for prevention or treating hyperproliferative vascular disorders - Google Patents

Abstract

The present invention relates to a composition for preventing or treating heperproliferative vascular disorders including arteriosclerosis or angiostenosis. A compound disclosed in the present invention can be used as an effective therapeutic composition for treating diseases such as arteriosclerosis and angiostenosis by significantly inhibiting proliferation of vascular smooth muscle cells which plays a key role in a progression of hyperproliferative vascular disorders.

Description

Compositions for Prevention or Treating Hyperproliferative Vascular Disorders [

The present invention relates to a composition for treating various abnormal proliferative vascular diseases caused by excessive proliferation of vascular smooth muscle cells.

The number of patients with metabolic syndrome is rapidly increasing due to changes in eating habits and population aging, and cerebral cardiovascular complications are the most common cause of death among metabolic syndrome patients. Cerebral cardiovascular complications are caused by blood vessels narrowed by thrombus formation and arteriosclerosis, and cause stroke due to myocardial infarction and carotid stenosis. In general, atherosclerosis is known to be a major cause of myocardial infarction, cerebral hemorrhage and peripheral vascular disease, and more than half of the deaths in developed countries are due to arteriosclerosis.

Vascular smooth muscle cells (VSMCs) are one of the major components of blood vessel walls and have important functions to maintain blood vessel shape and physiology (Vinters HV, Berliner JA, The blood vessel wall as an insulin target tissue. Diabete Metab. Jul 1987; 13 (3 Pt 2): 294-300). The quantitative and qualitative increase and migration of vascular smooth muscle cells play a key role in causing atherosclerosis.

Various factors such as growth factors, oxidative stress, and physical share stress are associated with the development of atherosclerosis. The platelet-derived growth factor is a dimer that is linked by a disulfide bond and is a protein tyrosine kinase known to be involved in the regulation of various cellular functions including gene expression, growth, differentiation and cell migration. kinase) receptors.

It is already known that the platelet-derived growth factor has the proliferative effect of various cells, especially vascular smooth muscle cells. Therefore, much research has been conducted to inhibit the progression of atherosclerosis by inhibiting the proliferation of vascular smooth muscle cells by the platelet-derived growth factor.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made intensive researches in order to discover effective therapeutic compositions for abnormal proliferative vascular diseases including arteriosclerosis and vascular stenosis. As a result, compounds represented by the following formulas (1) to (3) effectively inhibit the proliferation of vascular smooth muscle cells (VSMC), which provide a major cause of atherosclerosis due to excessive proliferation or migration, The present inventors have completed the present invention by discovering that various abnormal proliferative vascular diseases based on smooth muscle cell abnormalities can be effectively prevented and treated.

Accordingly, an object of the present invention is to provide a composition for preventing or treating abnormal proliferative vascular diseases including arteriosclerosis or vascular stenosis.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a composition for preventing or treating hyperproliferative vascular disorders comprising, as an active ingredient, a compound represented by the following formula (1), (2) or (3)

Formula 1

In the above formula, R ₁ To R ₆ are each independently hydrogen or C ₁ -C ₃ alkoxy; R ₇ and R ₈ are each independently hydrogen or C ₁ -C ₅ Alkyl;

(2)

In the above formula, A ₁ is C ₁ -C ₅ Alkyl; A ₂ to A ₆ are each independently hydrogen or C ₁ -C ₃ alkoxy; A ₇ is hydrogen or C ₁ -C ₅ Alkyl; n is an integer of 0-2;

(3)

In the above formulas, B ₁ to B ₄ and B ₉ to B ₁₂ are each independently hydrogen or C ₁ -C ₃ alkoxy; B ₅ to B ₇ are each independently hydrogen or C ₁ -C ₅ Alkyl.

The present inventors have made intensive researches in order to discover effective therapeutic compositions for abnormal proliferative vascular diseases including arteriosclerosis and vascular stenosis. As a result, it has been found that the compounds represented by the above formulas (1) to (3) effectively inhibit the proliferation of vascular smooth muscle cells (VSMC), which provide a major cause of atherosclerosis due to excessive proliferation or migration, It is possible to effectively prevent and treat various abnormal proliferative vascular diseases caused by cell abnormalities.

As used herein, the term " hyperproliferative vascular disorders " refers to all diseases or pathologic conditions caused by overgrowth of cells present in blood vessels, particularly vascular smooth muscle cells, Arteriosclerosis or vascular stenosis.

Atherosclerosis is a disease in which lipids are deposited or fibrosis in the inner layer of the arteries, resulting in narrowing of the blood vessel walls or loss of elasticity. Vascular stenosis means a disease in which blood vessels become abnormally narrowed due to various causes. Vascular stenosis is the main cause of vascular damage due to abnormal blood lipid concentration. However, there is also a case where the vascular passage is narrowed (restenosis) after the mechanical damage (traumatization) of the vessel wall due to angioplasty. It is known that vascular restenosis after arteriosclerosis and stenosis and stenting is due to vascular smooth muscle cell proliferation, migration and secretion of extracellular matrix (Circulation, 1997, 95, 1998-2002 J. Clin. Invest., 1997, 99, 2814-2816; Cardiovasc. Res., 2002, 54, 499-502). In order to prevent progression of arteriosclerosis and prevent vascular stenosis, researches on drugs that inhibit the proliferation of vascular smooth muscle cells have been widely conducted, and some drugs are currently used for treating patients (J. Am. Coll Cardiol , 2002, 39, 183-193). According to the present invention, the composition of the present invention can arrest the cell cycle of vascular smooth muscle cells, inhibit DNA synthesis, increase the formation of intracellular reactive oxygen species, and inhibit the proliferation of vascular smooth muscle cells very efficiently. It was confirmed through experiments. Therefore, the composition of the present invention can be usefully used for the treatment of abnormal proliferative vascular diseases.

The term " alkoxy " in the specification means a radical formed by removing hydrogen from an alcohol, and when C ₁ -C ₃ alkoxy is substituted, the number of carbon atoms of the substituent is not included.

As used herein, the term " alkyl " means a straight or branched saturated hydrocarbon group, including, for example, methyl, ethyl, propyl, isobutyl, pentyl or hexyl. C ₁ -C ₅ Alkyl means an alkyl group having an alkyl unit having 1 to 5 carbon atoms, and when C ₁ -C ₅ alkyl is substituted, the number of carbon atoms of the substituent is not included. In Chemical Formulas 1 to 3, C ₁ -C ₅ Alkyl is preferably C ₁ -C ₃ Alkyl, more preferably C ₁ -C ₂ alkyl.

According to a preferred embodiment of the invention, R ₁ , R _{5 of the} compound of formula 1 of the present invention And R ₇ is hydrogen; R ₂ To R ₄ and R ₆ are C ₁ -C ₃ alkoxy; R ₈ is C ₁ -C ₅ Alkyl. More preferably, the compound of formula (1) of the present invention is represented by the following formula (4).

Formula 4

According to a preferred embodiment of the present invention, A ₁ of the compound of formula (II) of the present invention is C ₁ -C ₅ alkyl; A ₂ , A ₃ , A ₅ and A ₆ are hydrogen; A ₄ is C ₁ -C ₃ alkoxy; A ₇ is hydrogen. More preferably, the compound of formula (2) of the present invention is represented by the following formula (5).

Formula 5

According to a preferred embodiment of the present invention, B ₁ , B ₄ , B ₅ , B ₇ , B ₉ and B ₁₂ are hydrogen, and B ₂ , B ₃ , B ₁₀ and B ₁₁ are C ₁ -C ₃ alkoxy and B ₆ is C ₁ -C ₅ Alkyl. More preferably, the compound of formula (III) of the present invention is represented by the following formula (6).

6

The compound of the present invention can be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. As the free acid, inorganic acid and organic acid can be used.

Preferably, the pharmaceutically acceptable salts of the compounds of the present invention are selected from the group consisting of hydrochloride, bromate, sulfate, phosphate, citrate, acetate, trifluoroacetate, lactate, tartrate, maleate, fumarate, But are not limited to, methanesulfonate, glycolate, succinate, 4-toluenesulfonate, gluturonate, ebonate, glutamate, or aspartate salts, And salts formed using various inorganic acids and organic acids. The compounds of the present invention may also exist in the form of solvates (e.g., hydrates).

The composition of the present invention may be provided in the form of a pharmaceutical composition for preventing or treating dysplastic vascular disease. When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations include, but are not limited to, Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration or the like.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . The daily dosage of the pharmaceutical composition of the present invention is, for example, 0.001-100 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a conventional preparation using pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs May be prepared in unit dosage form or may be manufactured by intrusion into a multi-dose container. Typical formulations are, for example, oral (tablets, capsules, powders), intraoral, sublingual, rectal, vaginal, intranasal, topical or parenteral (intravenous, intracameral, intramuscular, subcutaneous And intravenous) administration formulations. For example, a compound according to the present invention may be in the form of tablets containing starch or lactose, in the form of capsules containing the active ingredient alone or in an excipient, or in the form of an elixir or suspension containing a chemical that tastes or colors For example, orally, buccally or sublingually. Liquid preparations may contain suspending agents (e. G., A mixture of a semisynthetic glyceride such as methylcellulose, witepsol or apricot kernel oil and a PEG-6 ester, or a mixture of PEG-8 and caprylic / Such as a glyceride mixture, such as a mixture of glycerides (e.g., a mixture of glycerides). It is also most preferred to use it as a sterile aqueous solution form when injected parenterally, for example, intravenously, intraperitoneally, intramuscularly, subcutaneously, and intratracheally, wherein the solution is administered in order to have isotonicity with the blood It may contain other substances (for example, salts or monosaccharides such as mannitol, glucose).

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a composition for preventing or treating abnormal proliferative vascular diseases including arteriosclerosis or vascular stenosis.

(b) The compounds disclosed in the present invention can be effectively used as an effective therapeutic composition composition for diseases such as arteriosclerosis and vascular stenosis by greatly inhibiting vascular smooth muscle cell proliferation which plays a key role in the progression of abnormal vascular disease have.

FIG. 1 shows the results of stimulation with 25 ng / ml of PDGF-BB after 24 hour exposure to variously increased concentrations of the cell line after culturing the cells for 18 hours in serum-free DMEM medium (CCK- 8 analysis and BrdU analysis.
FIG. 2 is a graph showing the results of FACs analysis after culturing rat aortic smooth muscle cells at various concentrations for 1 hour, followed by addition of 25 ng / ml of PDGF-BB and co-culture for 24 hours .
FIG. 3 is a graph showing the results of exposure of the rat aortic smooth muscle cells to various concentrations of the callide for 30 minutes, followed by addition of 25 ng / ml of PDGF-BB and co-cultivation for 30 minutes. Western blotting The results are shown in Fig.
FIG. 4 shows that rat aortic smooth muscle cells were exposed to variously increased concentrations of collidine for 1 hour, then 25 ng / ml of PDGF-BB was added and co-cultured for 24 hours. Then, protein disruption was prepared and Western blotting The results are shown in Fig.
Figure 5 is a graph showing the results of western blotting of cells transfected with si-ERK for 48 hours. After 48 h of transfection, cells were exposed to 0.3 [mu] M of bone meal and PDGF-BB for 24 h and cell viability was determined by CCK-8 assay.
FIG. 6 shows the results of stimulation with PDGF-BB at 25 ng / ml after culturing the cells in serum-free DMEM medium for 18 hours, exposing the cells to anisomycin at various concentrations for 24 hours, -8 analysis and BrdU analysis.
FIG. 7 shows that rat aortic smooth muscle cells were exposed to various concentrations of anismaicin for 30 minutes, followed by addition of 25 ng / ml of PDGF-BB, followed by co-cultivation for 30 minutes. Western blotting The results are shown in Fig.
FIG. 8 shows the results of FACS analysis of rat aortic smooth muscle cells exposed to various concentrations of anismaicin for 1 hour, followed by addition of 25 ng / ml of PDGF-BB and co-culture for 24 hours It is a picture.
FIG. 9 shows that rat aortic smooth muscle cells were exposed to various concentrations of anismaicin for 1 hour, then 25 ng / ml of PDGF-BB was added and co-cultured again for 24 hours. Then, protein fragments were prepared and subjected to western blotting The results are shown in Fig.
FIG. 10 shows that rat aortic smooth muscle cells were exposed to various concentrations of anismaicin for 1 hour, then 25 ng / ml of PDGF-BB was added, co-cultured for 24 hours, and then exposed to 5 μM of DCFHDA for 30 minutes FACS analysis (left panel) and Western blotting (right panel).
FIG. 11 shows the results of stimulation with 25 ng / ml of PDGF-BB after 24 hours of exposure to variously increased concentrations of emetin after culturing the cells in serum-free DMEM medium for 18 hours (CCK- 8 analysis and BrdU analysis.
FIG. 12 shows that the rat aortic smooth muscle cells were exposed to variously increased concentrations of emertin for 1 hour, then added with 25 ng / ml of PDGF-BB and co-cultivated for 30 minutes, followed by western blotting Fig.
FIG. 13 is a graph showing the results of FACs analysis after exposing the rat aortic smooth muscle cells to various concentrations of emertin for 1 hour, adding 25 ng / ml of PDGF-BB and co-culturing for 24 hours again to be.
FIG. 14 is a graph showing the results of exposure to emittin of various concentrations of rat aortic smooth muscle cells for 1 hour, adding 25 ng / ml of PDGF-BB and co-culturing for 24 hours. Western blotting The results are shown in Fig.
FIG. 15 shows that the rat aortic smooth muscle cells were exposed to variously increased concentrations of emertin for 1 hour, then 25 ng / ml of PDGF-BB was added and co-cultured for 24 hours. Then, the cells were exposed to 5 μM DCFHDA for 30 minutes, The results of the analysis (left panel) and the results of western blotting (right panel) are shown in preparation of protein fragments.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

Measurement of arterial vascular smooth muscle cell proliferation inhibitory effect

1. Vascular smooth muscle primary culture

Rat aortic vascular smooth muscle cells (VSMCs) were cultured using enzymatic dispersion. The cells were cultured in a medium containing DMEM (10% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin, 8 mM HEPES and 2 mM L-glutamine) in a 5% CO ₂ incubator at 37 ° C. The passage of VSMC used in the present invention is 5-15.

2. Cell proliferation assay (CCK-8 assay)

VSMCs were seeded in 96-well plates and 70% confluence. The medium was replaced with serum-free (containing 0.1% FBS) medium and treated 18 hours later. One hour after the compound treatment, PDGF-BB was added, CCK-8 was treated with medium for 22 hours, incubated for 2 hours, and optical density (OD) was measured at 450 nm.

3. Cell proliferation assay (BrDU assay)

VSMC were seeded in a 96-well plate and 70% confluent, and the medium was changed to a serum-free (containing 0.1% FBS) medium and the compound was treated after 18 hours. One hour later, PDGF-BB was added and after 20 hours, the BrDU was treated in the medium and incubated for 4 hours. The optical density was then measured at 450 nm.

4. Cell cycle progression analysis

VSMC were seeded in 6-well plates and 70% confluent, and the medium was changed to a serum-free (containing 0.1% FBS) medium and treated with compound after 18 hours. One hour after the compound treatment, PDGF-BB was added. After 24 hours, the cells were fixed with 70% EtOH for 30 minutes. After 30 minutes, 500 μL of PI / RNase was treated. The treated samples were incubated at room temperature for 30 minutes and then measured by FACs.

5. Active Oxygen species ( ROS Generation analysis

VSMCs were seeded in 6-well plates and 70% confluent. After 18 hours, the medium was replaced with serum-free (medium containing 0.1% FBS). After 1 hour, PDGF-BB was added and treated with 5 μM of DCFA-DH for 24 hours, followed by incubation for 30 minutes. After 30 minutes, the cells were trypsinized and measured by FACs.

Arterial vascular smooth muscle cell Thickening  And mechanisms of anti-proliferative effect drugs target  Identification

1. Protein Extraction and Western Blot  analysis

VSMC were cultured in 6-well plates in serum-free medium and the compounds were treated at different concentrations and then incubated for 18 hours. After 24 hours, the cells were stimulated with PDGF-BB for 5 minutes, 15 minutes and 30 minutes. After the medium was removed, the cells were suspended in SDS lysis buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS % Glycerol and 50 mM dithiothreitol). The lysate was centrifuged at 15,000 rpm for 15 minutes, and the supernatant was transferred to a new tube, and the protein was quantified using BCA reagent. Protein (10 μg) was analyzed by Mini Gel Protein system (Bio-Rad) using SDS-PAGE with 10% acrylamide. The loaded protein was transferred to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Korea) and transfected in 250 mM transition buffer (PH 8.3) (25 mM Tris-HCl, 192 mM glycine and 20% methanol) at 250 mA. After transfection, the PVDF membrane was blocked with blocking reagent (containing 5% non-fat dry oil (Amersham Pharmacia Biotech, Korea) and TBS-T at room temperature for 1 hour), washed with primary antibody (p44 / 42MAPK (ERK1 / , PDGFRbeta, p-pRb, E2F-1, CDK2, CDK4, cyclin E, cyclin D1, p21, p27, p53, p-p53, ATM, ATR, p- MDM2 primary antibody) was diluted in BSA / TBS- 1: 1000, mixed and incubated with O / N at 4 ° C. After washing, the secondary antibody (horse radish peroxidase (HRP) -conjugated IgG secondary antibody (New England Biolabs, Boston, Mass.)) Was also diluted 1: 5000 in BSA / TBS-T buffer Lt; 0 > C or at room temperature for 3 hours or more. The membranes were then washed and the membranes were detected with a chemiluminescent reagent (ECL plus kit, Amersham Pharmacia Biotech, KOREA) followed by exposure of the membrane with Hyperfilm ECL (mersham Pharmacia Biotech, KOREA).

2. siRNA ( small interfering RNA ) Transformation

For transformation, VSMCs were cultured in 6-well plates in serum-free medium and the compounds were treated at different concentrations and siRNA (ERK) was transformed with Lipofectamine 2000 (Invitrogen). Transformed cells were subjected to Western blotting to confirm that the level of the desired protein in the cells was decreased.

Experiment result

Colmeside

In the colchicine-treated VSMC, the cell proliferation was significantly inhibited by concentration, and the BrDU assay, which observed the amount of DNA labeled with BrDU, also inhibited DNA synthesis in a concentration dependent manner. (Fig. 1).

Since cell proliferation was confirmed to be inhibited by the callide, cell cycle progression analysis was conducted to determine whether the cell cycle regulates the cell cycle. As a result, G ₀ / G ₁ corresponding to the resting period of the cell cycle (Fig. 2). These results suggest that colismamide suppresses the proliferation of VSMC, and that proliferation inhibition is due to the G ₀ / G ₁ arrest in the cell cycle.

In order to investigate the mechanism of action to regulate the cell cycle and inhibit cell proliferation, we measured the degree of phosphorylation of ERK and Akt, which play a key role in the signal transduction pathway of PDGF-BB used to induce VSMC proliferation, I investigated whether it was influential.

As shown in FIG. 3, phosphorylation of Akt, ERK1 / 2 and PDGFR beta was observed by the concentration of each of the callides, which inhibited the phosphorylation of ERK1 / 2 (FIG. 3). In addition, since the calliemide affects the cell cycle, the expression of CDK2 / cyclin E and CDK4 / cyclin D1, which are checkpoints of G ₀ / G ₁ , and the expression of p21 and p27, the CDK inhibitor (CKI) Respectively. As a result, it was observed that callide suppressed the phosphorylation of pRb and the expression of Cyclin E and CDK2, and the expression of CKI p27 was increased (FIG. 4). These results suggest that the use of p27 and ERK1 / 2 immunoprecipitation (IP) immunotherapy is based on the assumption that the interaction between the two proteins is due to the inhibition of phosphorylation of ERK1 / 2 and the increased expression of p27 ) Were performed. In addition, in order to determine whether the inhibition of phosphorylation of ERK1 / 2 is the key to suppress the proliferation of VSMC, the inhibition of cell proliferation by the callide was reduced when the ERK1 / 2 was rusted down in the cells. RNA transformation was performed.

IP was confirmed to interact with p27 and ERK1 / 2, and the inhibition of cell proliferation by the callide was reduced by si-ERK transformation and cell proliferation was further observed (Fig. 5). These results demonstrate that callame suppresses the phosphorylation of ERK, which is the main pathway in PDGF-BB signaling pathway, increases the expression of p27, inhibits the cell cycle and inhibits cell proliferation.

No Somaiyin

In the VSMCs treated with anismaicin, cell proliferation was significantly inhibited by concentration, and in BrDU assays, which observed the amount of DNA labeled with BrDU, anismaicin also induced DNA synthesis (Fig. 6).

Based on the above results, it was observed that the effect of anismaicin on the PDGF-BB-mediated signal was not affected by the observed phosphorylation of Akt and ERK1 / 2 at each concentration of an isomycin (Fig. 7).

Cell cycle progression analysis was performed to determine if an isomycin regulates the cell cycle since it was observed that it was inhibited by somaicin, not cell proliferation. No, and observed that the soma who G ₀ / G ₁ corresponding to the resting stage of the cell cycle by which eoreseuteu (Fig. 8), that this is not the soma, who by inhibiting the growth of VSMC, growth inhibition of the cell cycle G ₀ / It is understood that it is due to the G ₁ arrest. In order to investigate whether the suppression effect of an isomicin on cell cycle is due to the effect of anismaicin on the cell cycle, the expression of CDK2 / cyclin E and CDK4 / cyclin D1, which are checkpoints of G ₀ / G ₁ , The inhibitors p21, p27, and p53 were also observed. An isomicin suppressed the phosphorylation of pRb and the expression of CDK2, and the expression of p21, a CKI, was observed to increase (Fig. 9). Since the expression of p21 was activated by p53, the expression of p53 and p-p53 was observed, and p-p53 and p53 were activated (FIG. 9). These results indicate that an isomycin activates p53, and that it will affect DNA damage-related signals, thus determining whether it affects the expression of ATM / ATR and Chk1 / Chk2 associated with DNA damage Respectively. In addition, since DNA damage is activated by UV irradiation, oxidative stress, and so on, we observed the formation of reactive oxygen species (ROS) in order to investigate the mechanism of DNA damage by an isomycin.

Cells were stained with DCFH-DA, which detects ROS. As a result of observing whether anismaicin produced ROS in cells, we observed that ROS was produced at the same level as the positive control LPS group. ATM / ATR, Chk1 / Chk2, and increased the phosphorylation of p-MDM2 (Fig. 10). These results suggest that anismaicin induces intracellular production of ROS, resulting in DNA damage, increased p53 and p21 activation, and ultimately inhibits cell proliferation.

Emertin

Emertin-treated VSMCs significantly inhibited cell proliferation by concentration. In the BrDU assay for monitoring the amount of DNA labeled with BrDU, emertin also inhibited DNA synthesis in a concentration-dependent manner (Fig. 11).

Based on the above results, it was observed that phosphorylation of Akt and ERK1 / 2 at each concentration of the ermatine did not have any effect in order to determine the effect of the ermatine on PDGF-BB-mediated signal. 12).

Since we observed that cell proliferation was inhibited by emetic, cell cycle progression analysis was performed to determine whether emeticin regulates the cell cycle. G ₀ / G ₁ corresponding to the rest period of the cell cycle was arrested by emetine (FIG. 13). These results demonstrate that emertin inhibits the proliferation of VSMCs and that proliferation inhibition is due to the G ₀ / G ₁ arrest in the cell cycle. In order to investigate whether the effect of emetine on cell cycle proliferation is due to the effect of emetin on cell proliferation, we examined the expression of CDK2 / Cyclin E, CDK4 / Cyclin D1, the checkpoint of G ₀ / G ₁ , (CKI), p21, p27, and p53 were also examined. As a result, it was observed that emetin inhibited the phosphorylation of pRb and the expression of CDK2 and increased the expression of p21, a CKI. Since the expression of p21 is activated by p53, expression of p53 and p-p53 was examined, and p-p53 and p53 were activated (FIG. 14). These results suggest that emetin activates p53, and therefore, it affects the expression of ATM / ATR in relation to DNA damage. In addition, since DNA damage is activated by UV irradiation and oxidative stress, we investigated the production of reactive oxygen species to investigate the mechanism of DNA damage by emetin.

Cells were stained with DCFH-DA, which detects ROS. As a result, ROS was produced at the same level as the positive control LPS group, and phosphorylation of ATM / ATR, which is a typical DNA damage signal, was increased. (Fig. 15). These results suggest that emertin induces DNA damage by producing ROS in the cells, increases the activation of p53 and p21, and ultimately inhibits cell proliferation.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

A composition for preventing or treating hyperproliferative vascular disorders comprising the compound represented by Formula 1, Formula 2, or Formula 3 as an active ingredient:
Formula 1

In the above formula, R ₁ To R ₆ are each independently hydrogen or C ₁ -C ₃ alkoxy; R ₇ and R ₈ are each independently hydrogen or C ₁ -C ₅ Alkyl;
(2)

In the above formula, A ₁ is C ₁ -C ₅ Alkyl; A ₂ to A ₆ are each independently hydrogen or C ₁ -C ₃ alkoxy; A ₇ is hydrogen or C ₁ -C ₅ Alkyl; n is an integer of 0-2;
(3)

In the above formulas, B ₁ to B ₄ and B ₉ to B ₁₂ are each independently hydrogen or C ₁ -C ₃ alkoxy; B ₅ to B ₇ are each independently hydrogen or C ₁ -C ₅ Alkyl.
According to claim 1, wherein in Formula 1 R ₁ , R ₅ And R ₇ is hydrogen; R ₂ To R ₄ and R ₆ are C ₁ -C ₃ alkoxy; R ₈ is C ₁ -C ₅ Composition comprising alkyl.
According to claim 1, A ₁ of Formula 2 is C ₁ -C ₅ Alkyl; A ₂ , A ₃ , A ₅ and A ₆ are hydrogen; A ₄ is C ₁ -C ₃ alkoxy; A ₇ is hydrogen, characterized in that the composition.
According to claim 1, wherein in Formula 3 B ₁ , B ₄ , B ₅ , B ₇ , B ₉ and B ₁₂ are hydrogen, and B ₂ , B ₃ , B ₁₀ and B ₁₁ are C ₁ -C ₃ alkoxy and B ₆ is C ₁ -C ₅ Composition comprising alkyl.
The composition according to claim 1, wherein the compound represented by Formula 1 is a compound represented by Formula 4 below:
Formula 4

The composition of claim 1, wherein the compound represented by Formula 2 is a compound represented by Formula 5 below:
Formula 5

The composition of claim 1, wherein the compound represented by Chemical Formula 3 is a compound represented by Chemical Formula 6 below:
6

The composition according to claim 1, wherein the abnormal proliferative vascular disease is arteriosclerosis or vascular stenosis.
2. The composition of claim 1, wherein the composition inhibits the proliferation of smooth muscle cells.

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