KR20050108234A - Anti-atherosclerosis composition containing synthetic flavone derivatives - Google Patents

Abstract

The present invention relates to anti-atherosclerosis compositions of synthetic flavone derivatives. In particular, the present invention provides an anti-atherosclerosis composition comprising a flavone derivative and / or a chalcone-based compound as an active ingredient, which can prevent the adhesion of monocytes to vascular endothelial cells and block the development of atherosclerosis at an early stage.

Description

ANTI-ATHEROSCLEROSIS COMPOSITION CONTAINING SYNTHETIC FLAVONE DERIVATIVES}

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to an anti-atherosclerosis composition, and more particularly, to a flavone derivative and / or a chalcone derivative that can prevent the adhesion of monocytes to vascular endothelial cells and thereby block the development of atherosclerosis at an early stage. .

[Private Technology]

High cholesterol in blood has been considered a key factor in the development of atherosclerosis (Ross R. 1993. Nature 362: 801-809; Krauss RM. 1987. Am Heart J 113: 578-582; Steinberg D, Parthasarathy S, Carew TE , Khoo JC, Witztum J L. 1989. N Eng J Med 320: 915-924). The early stages of atherosclerosis appear as lymphocytes, polymorphonuclear leukocytes, and monocytes aggregate into damaged vascular endothelial tissue, such as cell adhesion molecules (CAMs), for example VCAM-1. adhesion molecule-1), ICAM-1 (intracellular cell adhesion molecule-1), E-selectin, and the like (Scalia R, Appel JZ III, Lefer AM. 1998. Arterioscler Thromb Vasc Biol 18: 1093 -1100; Schonbeck U, Mach F, Libby P. 2001. Circ Res 89: 1092-1103; Osborn L, Hessian C, Tizard R, Vassalio C, Luhowskyj S, Chi-Rosso S, Lobb R. 1989. Cell 59: 1203-1211; Cybulsky ML, Gimbrone MA Jr. 1991. Science 251: 788-791).

The CAMs have increased expression by inflammatory cytokines such as interleukin-I and tumor necrosis factor-α (TNF-α) (Dustin ML, Rothlein R, Bhan AK, Dinarello CA, Springer TA). 1986. J Immunol 137: 245-254. In addition, the expression of CAMs is various, and it is assumed that complex regulatory mechanisms by growth factors, platelet activators and chemotactic factors are involved. CAMs are found in atherosclerotic lesions of human coronary and abdominal aorta as well as in rabbits and mice, and have been reported to be involved in atherosclerotic plaque formation in neovascular and inflammatory invasive sites (Iiyama K, Hajra L, Iiyama). M, Li H, Di Chiara M, Medoff BD, Cybulsky MI. 1999. Circ Res 85: 199-207; Lessner SM, Prado HL, Waller EK, Galis ZS. 2002. Am J Pathol 160: 2145-2155; Van der Wal AC, Das PK, Tigges AJ, Becker AE. 1992. Am J Pathol 141: 161-168; O'Brien KD, Allen MD, McDonald TO, Chait A, Harlan JM, Fishbein D, McCarty J, Furgerson M, Hudkins K , Benjamin CD, Lobb R, Alpers CE. 1993. J Clin Invest 92: 945-951).

Flavonoids, on the other hand, are water-soluble pigments present in plants such as green tea, legumes, grapes, garlic, and onions, and plants, such as green tea, beans, grapes, garlic, and onions. (Levy A, Fuhrman B, Markel A, Dankner G, Ben-Amotz A, Presser D, Aviram M. 1994. Ann Nutr Metab 38: 287-294). Flavonoids exist in various forms and are classified into subgroups of flavonols, flavones, isoflavones, flavonones, flavan-3-ols, and anthocyanidins. However, not all flavonoids have the same physiological activity. Flavonoids are reported to have the role of natural antioxidants in the removal of free radicals from cells and tissues (Kris-Etherton PM, Keen CL. 2002. Curr Opin Lipidol 13: 41-49: Nijveldt RJ, van Nood E , van Hoorn DE, Boelens PG, van Norren K, van Leeuwen PA. 2001. Am J Clin Nutr 74: 418-425; Braca A, Sortino C, Politi M, Morelli I, Mendez J. 2002. J Ethnopharmacol 79: 379 -381), similar to proanthocyanidins and phenolic acids, are polyphenolic compounds, and are currently being suggested for use in the prevention or treatment of circulatory diseases (Cotelle N. 2001. Curr Top Med Chem 1: 569-). 590; Hirano R, Sasamoto W, Matsumoto A, Itakura H, Igarashi O, Kondo K. 2001. J Nutr Sci Vitaminol ( Tokyo ) 47: 357-362). However, it has not been confirmed that the physiological effects of flavonoids are effective in vivo, and no report on the mechanism of action has been made.

In order to solve the problems of the prior art, the present invention provides an anti-atherosclerosis composition of flavone derivatives that can inhibit the progression to atherosclerosis.

It is another object of the present invention to provide a substance capable of inhibiting cell adhesion of monocytes to vascular endothelial cells.

In order to achieve the above object, the present invention provides an anti-atherosclerosis composition comprising a compound represented by Formula 1 or a compound represented by Formula 2.

(Formula 1)

In Chemical Formula 1,

R ₁ is OCH ₃ ,

R ₂ is H or OCH ₃ ,

R ₃ is H or OCH ₃ ,

R ₄ is H or CH ₃ , and

R ₅ is H, OH or OCH ₃ ;

(Formula 2)

In Chemical Formula 2,

R ₆ is OCH ₃ ,

R ₇ to R ₁₀ are each independently H or OCH ₃ .

In another aspect, the present invention provides a composition for inhibiting the expression of cell adhesion protein induced by tumor necrosis factor-α comprising the compound represented by Formula 1 or the compound represented by Formula 2 as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present inventors, while screening for a compound having anti-arteriosclerosis activity, confirmed that the compound represented by Formula 1 and the compound represented by Formula 2 inhibit the expression of cell adhesion molecules induced by tumor necrosis factor. Completed.

Examples of the flavone-based compound represented by Formula 1 include the compounds of Table 1 below, and examples of the calon compound represented by Formula 2 include the compounds of Table 2 below, but are not limited thereto.

Table 1

R ₁ R ₂ R ₃ R ₄ R ₅ Compound 1 OCH ₃ H H H OCH ₃ Compound 2 OCH ₃ H OCH ₃ H H Compound 3 OCH ₃ OCH ₃ H H OCH ₃ Compound 4 OCH ₃ OCH ₃ OCH ₃ H H Compound 5 OCH ₃ OCH ₃ H CH ₃ CH ₃ Compound 6 OCH ₃ OCH ₃ H CH ₃ H Compound 7 OCH ₃ OCH ₃ H H OH

Table 2

R ₆ R ₇ R ₈ R ₉ R ₁₀ Compound 8 OCH ₃ H H H OCH ₃ Compound 9 OCH ₃ H OCH ₃ H H Compound 10 OCH ₃ OCH ₃ H H OCH ₃ Compound 11 OCH ₃ OCH ₃ OCH ₃ H H

The flavone derivatives and the chalcone-based compounds of the present invention inhibit the expression of cytomegaloproteins induced by the tumor necrosis factor (TNF) -α, an inflammatory tumor necrosis factor, thereby preventing progression to atherosclerosis. Representative cell adhesion proteins include E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (IMA-1).

In one embodiment of the present invention, TNF-α treated vascular endothelial cells significantly increased THP-1 monocyte adhesion compared to the non-treated group, but when additionally treated with the flavone derivative represented by the formula (1) It was confirmed that the adhesion of monocytes to endothelial tissue was suppressed. This cytostatic inhibitory activity is not due to the cytotoxicity of the flavone derivatives, but by inhibiting the expression of TNF-α-induced cell adhesion protein (FIGS. 2A and 2B).

Therefore, the flavone derivatives or chalcone-based compounds of the present invention can be used for the prevention and treatment of atherosclerosis, especially atherosclerosis, by significantly reducing the expression of TNF-α-induced cell adhesion proteins.

Accordingly, the present invention provides an anti-atherosclerosis composition comprising a flavone derivative represented by Formula 1 and / or a chalcone-based compound represented by Formula 2 as an active ingredient.

The anti-atherosclerosis composition of the present invention may include only the active ingredient alone, and may further include a pharmacologically acceptable carrier. The carriers include, but are not limited to, saline, buffered saline, water, glycerol, ethanol, and the like, and any suitable agent known in the art (Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA)) can be used.

The anti-atherosclerosis composition of the present invention may include only one or two or more active ingredients, and the content is preferably adjusted appropriately, but preferably, 10 to 70% by weight of the atherosclerosis composition. If the content is less than 10% by weight, the anti-atherosclerosis effect may be insignificant, and if the content is more than 70% by weight, the effect on the dose may be somewhat reduced, which is uneconomical.

The anti-atherosclerosis composition of the present invention can be used as a pharmaceutical or food additive. The formulation of the anti-atherosclerosis composition may be a warning, granule, powder, syrup, liquid, liquid extract, emulsion, suspension, acupuncture, tablet, injection, capsule, cream, troche or pasta, and oral or parenteral Can be used as a sphere. Preferably it is oral.

The dosage of the anti-atherosclerosis composition of the present invention is a conventional dosage of a medicament, for example, 300 to 1,000 mg of flavone derivatives or quercetin may be used per day. The dosage is not limited to this, and is preferably applied differently according to the age, sex, condition of the patient and the drug used in combination.

In addition, the flavone derivative represented by the formula (1) and / or the chalcone-based compound represented by the formula (2) can be used as a composition for inhibiting the expression of cell adhesion protein induced expression by tumor necrosis factor-α.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

Example

The derivatives of Table 3 represented by Formula 1 and the compounds of Table 4 represented by Formula 2 were prepared, respectively.

(Formula 1)

(Formula 2)

Table 3

R ₁ R ₂ R ₃ R ₄ R ₅ denomination Example 1 OCH ₃ H H H OCH ₃ 5G Example 2 OCH ₃ H OCH ₃ H H 5H Example 3 OCH ₃ OCH ₃ H H OCH ₃ 11G Example 4 OCH ₃ OCH ₃ OCH ₃ H H 11H Example 5 OCH ₃ OCH ₃ H CH ₃ CH ₃ 11I Example 6 OCH ₃ OCH ₃ H CH ₃ H 11D Example 7 OCH ₃ OCH ₃ H H OH 11N

Table 4

R ₆ R ₇ R ₈ R ₉ R ₁₀ Example 8 OCH ₃ H H H OCH ₃ Example 9 OCH ₃ H OCH ₃ H H Example 10 OCH ₃ OCH ₃ H H OCH ₃ Example 11 OCH ₃ OCH ₃ OCH ₃ H H

Example 1: 4 ', 7'-dimethoxyflavone

Yield: 58.6%. Pale brown needles. ^{_{IR (KBr) ν max cm -1}} : 1604, 1257. 1 H-NMR (acetone-d 6, 300MHz) δ: 6.68 (s, 1H), 7.99 (d, J = 8.7, 1H), 7.15 (dd, J = 2.4, 8.7,1H), 7.01 (d, J = 8.7, 1H), 7.21 (dd, J = 2.4, 8.7, 2H), 7.04 (d, J = 8.4, 2H), 3.92 (s, 3H) ; Tlc, R f 0.48 (5: 3 Hexane / EtOAc); mp 128-130 ℃

Example 2: 5,4'-Dimethoxyflavone

Yield: 78.6%. Pale brown needles. IR (KBr) ν _max cm ^-1 : 1602, 1259. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 6.75 (s, 1H), 7.10 (d, J = 8.7, 1H), 7.26 (d, J = 8.7, 1H), 6.96 (d, J = 8.7, 1H), 7.65 (d, J = 8.7, 2H), 7.08 (d, J = 8.7, 2H), 3.84 (s, 3H), 3.72 (s , 3H); Tlc, R f 0.52 (5: 3 Hexane / EtOAc); mp 136-138 ℃

Example 3: 7, 7 ', 4'-trimethoxyflavone

Yield: 65.4%. Pale brown needles. IR (KBr) ν _max cm ^-1 : 1603, 1260. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 6.71 (s, 1H), 6.70 (d, J = 8.7, 1H), 6.75 (d, J = 8.7, 1H), 6.61 (d, J = 8.7, 1H), 6.43 (d, J = 8.7, 1H), 6.52 (d, J = 8.7, 1H), 7.53 (d, J = 8.7, 1H) , 3.84 (s, 6 H), 3.72 (s, 3 H); Tlc, R f 0.63 (5: 3 Hexane / EtOAc); mp 129-131 ℃

Example 4: 5,3 ', 4'-dimethoxyflavone

Yield: 85.2%. Pale brown needles. IR (KBr) ν _max cm ^-1 : 1601, 1258. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 6.71 (s, 1H), 6.70 (d, J = 8.7, 1H), 6.75 (d, J = 8.7, 1H), 6.61 (d, J = 8.7, 1H), 6.81 (d, J = 8.7, 1H), 6.88 (d, J = 8.7, 1H), 7.15 (d, J = 8.7, 1H) , 3.84 (s, 6 H), 3.72 (s, 3 H); Tlc, R f 0.60 (5: 3 Hexane / EtOAc); mp 123-124 ℃

Example 5: 3 ', 4'-dimethoxy-6,7-dimethylflavone

Yield: 77.3%. Pale brown needles. IR (KBr) ν _max cm ^-1 : 1603, 1259. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 6.71 (s, 1H), 6.70 (d, J = 8.7, 1H), 6.75 (d, J = 8.7, 1H), 6.61 (d, J = 8.7, 1H), 6.60 (d, J = 8.7, 1H), 7.32 (d, J = 8.7, 1H), 3.84 (s, 6H), 2.35 (s , 6H); Tlc, R f 0.69 (5: 3 Hexane / EtOAc); mp118-120 ℃

Example 6: 3'4'-dimethoxy-6-methylflavone

Yield: 83.5%. Pale brown needles. IR (KBr) ν _max cm ^-1 : 1603, 1260. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 6.71 (s, 1H), 6.70 (d, J = 8.7, 1H), 6.75 (d, J = 8.7, 1H), 6.61 (d, J = 8.7, 1H), 6.80 (d, J = 8.7, 1H), 7.17 (d, J = 8.7, 1H), 7.44 (d, J = 8.7, 1H) , 3.84 (s, 6 H), 2.35 (s, 3 H); Tlc, R f 0.71 (5: 3 Hexane / EtOAc); mp 160-162 ℃

Example 7: 7-Hydroxy-3 ', 4'-Dimethoxyflavone

Yield: 34.6%. Pale brown needles. IR (KBr) ν _max cm ^-1 : 1603, 1260. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 6.71 (s, 1H), 6.70 (d, J = 8.7, 1H), 6.75 (d, J = 8.7, 1H), 6.61 (d, J = 8.7, 1H), 6.39 (d, J = 8.7, 1H), 6.48 (d, J = 8.7, 1H), 7.47 (d, J = 8.7, 1H) , 3.84 (s, 6 H); Tlc, R f 0.39 (5: 3 Hexane / EtOAc); mp 168-171 ℃

Example 8: 2'-Hydroxy-4,3'-dimethoxychalcone

Yield: 39.8%. Yellow needles. IR (KBr) v _max cm ^-1 : 3404, 1603. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 8.00 (d, J = 15.3, 1H), 7.67 (d, J = 8.7, 2H), 7.54 (d, J = 15.3, 1H), 7.25 (d, J = 8.7, 2H), 7.53 (d, J = 8.4, 1H), 6.43 (d, J = 8.4, 1H), 6.52 (d, J = 8.4, 1H), 3.83 (s, 6H). Tlc, R f 0.50 (5: 3 Hexane / EtOAc); mp 169-172 ℃

Example 9: 2'-Hydroxy-4,6'-dimethoxychalcone

Yield: 86.5%. Yellow needles. IR (KBr) ν _max cm ^-1 : 3404, 1603. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 8.00 (d, J = 15.3, 1H), 7.67 (d, J = 8.7, 2H), 7.54 (d, J = 15.3, 1H), 7.25 (d, J = 8.7, 2H), 7.09 (d, J = 8.4, 1H), 6.48 (d, J = 8.4, 1H), 6.52 (d, J = 8.4, 1H), 3.83 (s, 6H). Tlc, R f 0.54 (5: 3 Hexane / EtOAc); mp 101-105 ℃

Example 10 2'-Hydroxy-3,3 ', 4-trimethoxychalcone

Yield: 42.1%. Yellow needles. IR (KBr) ν _max cm ^-1 : 3404, 1603. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 8.00 (d, J = 15.3, 1H), 6.71 (d, J = 8.7, 1H), 6.70 (d, J = 8.7, 1H), 7.54 (d, J = 15.3, 1H), 6.75 (d, J = 8.7, 1H), 7.53 (d, J = 8.4, 1H), 6.43 (d, J = 8.4, 1H), 6.52 (d, J = 8.4, 1H), 3.83 (s, 9H). Tlc, R f 0.46 (5: 3 Hexane / EtOAc); mp 203-205 ℃

Example 11: 2'-Hydroxy-3,4,6'-trimethoxychalcone

Yield: 33.1%. Yellow needles. IR (KBr) ν _max cm ^-1 : 3404, 1603. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 8.00 (d, J = 15.3, 1H), 6.48 (d, J = 8.7, 1H), 652 (d, J = 8.7, 1H), 7.54 (d, J = 15.3, 1H), 7.26 (d, J = 8.7, 1H), 7.53 (d, J = 8.4, 1H), 6.43 (d, J = 8.4, 1H), 6.52 (d, J = 8.4, 1H), 3.83 (s, 9H). Tlc, R f 0.53 (5: 3 Hexane / EtOAc); mp 155-158 ℃

Comparative example

A total of seven compounds of Table 5 represented by Formula 1 were synthesized, respectively.

Table 5

R ₁ R ₂ R ₃ R ₄ R ₅ denomination Comparative Example 1 OCH ₃ OCH ₃ H OCH ₃ H 11B Comparative Example 2 H H H H OCH ₃ 1G Comparative Example 3 OCH ₃ H H Cl H 5E Comparative Example 4 OCH ₃ OCH ₃ H Cl H 11E Comparative Example 5 N (CH ₃ ) ₂ H H Cl H 20E Comparative Example 6 CH ₃ H H Cl H 27E Comparative Example 7 CH ₃ H H CH ₃ H 27D Comparative Example 8 OCH ₃ H H H H 5A Comparative Example 9 OCH ₃ H H Br H 5F Comparative Example 10 OCH ₃ H H CH ₃ CH ₃ 5I Comparative Example 11 OCH ₃ OCH ₃ H H H 11A Comparative Example 12 OCH ₃ OCH ₃ H Br H 11F Comparative Example 13 2-OH H H CH ₃ CH ₃ 2I Comparative Example 14 OH H H H OH 4N Comparative Example 15 2-OH H H H OH 2N Comparative Example 16 OCH ₃ H H OH 5C Comparative Example 17 OCH ₃ H H H OH 5N Comparative Example 18 OH 2'-OH H H OH 21N

Comparative Example 1: 6,3 ', 4'-Trimethoxyflavone (6,3', 4'-Trimethoxyflavone)

Yield: 24.3%. Pale brown needles. IR (KBr) ν _max cm ^-1 : 1616, 1238. 1 H-NMR (acetone-d ₆ , 300 MHz) δ: 7.68 (d, J = 8.4, 1H), 7.13 (d, J = 8.4, 1H), 6.78 (d, J = 8.4, 1H), 7.65 (s, 1H), 7.69 (d, J = 8.7, 1H), 7.48 (d, J = 8.7, 1H), 7.36 (d, J = 8.7, 1H) 3.91 (s, 3H), 3.85 (s, 3H); Tlc, R f 0.37 (1: 3 Hexane / EtOAc); mp 186-188 ℃

Comparative Example 2: 7-Methoxyflavone

Yield: 39.8%. Pale brown needles. IR (KBr) ν _max cm ^-1 : 1616, 1238. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 6.43 (d, J = 8.4, 1H), 6.52 (d, J = 8.4, 1H), 7.53 (d, J = 8.4, 1H), 7.65 (s, 1H), 7.14 (d, J = 8.7, 1H), 7.21 (d, J = 8.7, 2H), 7.30 (d, J = 8.7, 2H) 3.85 (s, 3 H); Tlc, R f 0.47 (1: 3 Hexane / EtOAc); mp 168-171 ℃

Comparative Example 3: 6-Chloro-4'-methoxyflavone

Yield: 48.0%. Pale brown needles. IR (KBr) ν _max cm ^-1 : 1604, 1269. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 6.80 (s, 1H), 8.02 (s, 1H), 7.38 (dd, J = 2.4, 8.4, 1H), 6.86 (dd, J = 2.4, 8.4, 2H), 7.91 (dd, J = 2.4, 8.7, 2H), 3.91 (s, 3H); Tlc, R f 0.44 (1: 3 Hexane / EtOAc); mp 160-162 ℃

Comparative Example 4: 6-Chloro-3 ', 4'-dimethoxyflavone

Yield: 36.8%. Yellow needles. IR (KBr) ν _max cm ^-1 : 1599, 1228. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 7.85 (d, J = 8.7, 1H), 7.14 (d, J = 8.7, 1H), 7.01 (d, J = 8.7, 1H), 7.71 (s, 1H), 7.59 (d, J = 8.7, 1H), 7.51 (d, J = 8.7, 1H), 7.39 (d, J = 8.7, 1H) 3.91 (s, 3 H), 3.87 (s, 3 H); Tlc, R f 0.45 (1: 3 Hexane / EtOAc); mp 116-118 ℃

Comparative Example 5: 6-Chloro-4 '-(N, N-dimethylamineflavone) (6-Chloro-4'-(N, N-dimethylamineflavone))

Yield: 52.6%. Pale brown needles. IR (KBr) ν _max cm ^-1 : 1605, 1270. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 6.86 (d, J = 8.4, 1H), 7.38 (d, J = 8.4, 1H), 7.65 (d, J = 8.4, 1H), 6.86 (s, 1H), 6.54 (d, J = 8.7, 2H), 7.12 (d, J = 8.7, 2H), 2.85 (s, 6H); Tlc, R f 0.39 (1: 3 Hexane / EtOAc); mp 220-222 ℃

Comparative Example 6: 6-Chloro-4'-methylflavone

Yield: 35.2%. Pale brown needles. IR (KBr) ν _max cm ^-1 : 1605, 1269. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 6.86 (d, J = 8.4, 1H), 7.38 (d, J = 8.4, 1H), 7.65 (d, J = 8.4, 1H), 6.86 (s, 1H), 7.01 (d, J = 8.7, 2H), 7.18 (d, J = 8.7, 2H), 2.35 (s, 3H); Tlc, R f 0.39 (1: 3 Hexane / EtOAc); mp 156-158 ℃

Comparative Example 7: 6,4'-Dimethoxyflavone

Yield: 43.5%. Pale brown needles. IR (KBr) ν _max cm ^-1 : 1605, 1269. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 6.80 (d, J = 8.4, 1H), 7.17 (d, J = 8.4, 1H), 7.44 (d, J = 8.4, 1H), 6.86 (s, 1H), 7.01 (d, J = 8.7, 2H), 7.18 (d, J = 8.7, 2H), 2.35 (s, 6H); Tlc, R f 0.35 (1: 3 Hexane / EtOAc); mp 132-135 ℃

Comparative Example 8: 4'-Methoxyflavone

Yield: 33.8%. Pale brown needles. IR (KBr) ν _max cm ^-1 : 1605, 1269. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 6.92 (d, J = 8.4, 1H), 7.01 (d, J = 8.4, 1H), 7.37 (d, J = 8.4, 1H), 7.64 (d, J = 8.4, 1H), 6.86 (s, 1H), 6.72 (d, J = 8.7, 2H), 7.19 (d, J = 8.7, 2H) , 3.73 (s, 3 H); Tlc, R f 0.37 (1: 3 Hexane / EtOAc); mp 152-155 ℃

Comparative Example 9: 6-Bromo-4'-methoxyflavone

Yield: 77.8%. Pale brown needles. IR (KBr) ν _max cm ^-1 : 1605, 1269. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 6.81 (d, J = 8.4, 1H), 7.54 (d, J = 8.4, 1H), 7.81 (d, J = 8.4, 1H), 6.86 (s, 1H), 6.72 (d, J = 8.7, 2H), 7.19 (d, J = 8.7, 2H),, 3.73 (s, 3H); Tlc, R f 0.32 (1: 3 Hexane / EtOAc); mp 189-191 ℃

Comparative Example 10: 4'-methoxy-6,7-dimethylflavone

Yield: 71.0%. Orange needles. IR (KBr) ν _max cm ^-1 : 1604, 1259. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 6.68 (s, 1H), 7.32 (d, J = 8.4, 1H), 6.69 (d, J = 8.7, 1H), 7.22 (dd, J = 2.4, 8.7, 2H), 6.74 (dd, J = 2.7, 8.4, 2H), 3.73 (s, 3H), 2.41 (s, 3H), 2.34 (s , 3H); Tlc, R f 0.45 (1: 3 Hexane / EtOAc); mp 174-176 ° C

Comparative Example 11: 3 ', 4'-Dimethoxyflavone

Yield: 32.1%. Pale brown needles. IR (KBr) ν _max cm ^-1 : 1601, 1242. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 7.81 (dd, J = 2.1, 8.4, 1H), 7.14 (dd, J = 2.1, 8.4 , 1H), 7.02 (dd, J = 2.1, 8.4, 1H), 6.81 (d, J = 8.4, 1H), 7.61 (s, 1H), 7.62 (d, J = 8.7,1H), 7.45 (dd, J = 2.4, 8.7, 1H), 7.36 (dd, J = 2.4, 8.7, 1H), 3.95 (s, 3H), 3.87 (s, 3H); Tlc, R f 0.42 (1: 3 Hexane / EtOAc); mp 112-114 ℃

Comparative Example 12: 6-Bromo-3 ', 4'-dimethoxyflavone

Yield: 23.5%. Orange needles. ^{_{IR (KBr) ν max cm -1}} : 1599, 1267. 1 H-NMR (acetone-d 6, 300MHz) δ: 7.97 (s, 1H), 7.14 (d, J = 8.7, 1H), 7.02 (d, J = 8.7, 1H), 7.72 (s, 1H), 7.62 (d, J = 8.4, 1H), 7.52 (d, J = 8.4, 1H), 7.41 (d, J = 8.4, 1H), 3.87 (s , 3H), 3.84 (s, 3H); Tlc, R f 0.44 (1: 3 Hexane / EtOAc); mp 184-186 ° C

Comparative Example 13: 4'-Hydroxy-6,7-dimethylflavone

Yield: 23.9%. Pale brown needles. IR (KBr) ν _max cm ^-1 : 1605, 1269. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 6.60 (d, J = 8.4, 1H), 7.32 (d, J = 8.4, 1H), 6.86 (s, 1 H), 6.68 (d, J = 8.7, 2H), 7.13 (d, J = 8.7, 2H), 2.35 (s, 6H); Tlc, R f 0.36 (1: 3 Hexane / EtOAc); mp 199-201 ℃

Comparative Example 14 7,4'-Dihydroxyflavone

Yield: 18.7%. Yellow needles. IR (KBr) ν _max cm ^-1 : 1601, 1209. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 7.97 (dd, J = 2.1, 8.4, 1H), 6.52 (dd, J = 2.1, 8.4 , 1H), 6.63 (d, J = 8.7, 1H), 6.67 (s, 1H), 7.88 (d, J = 8.4, 2H), 6.90 (d, J = 8.4 2H); Tlc, R f 0.35 (1: 3 Hexane / EtOAc); mp 190-193 ℃

Comparative Example 15: 7,2'-Dihydroxyflavone

Yield: 21.6%. Pale brown needles. IR (KBr) ν _max cm ^-1 : 1602, 1219. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 7.98 (d, J = 8.4, 1H), 7.14 (s, 1H), 6.51 (d, J = 8.4, 1H), 7.61 (d, J = 8.4, 1H), 6.9-7.0 (m, 1H), 7.13 (dd, J = 2.1, 8.4, 1H), 7.38 (d, J = 8.4, 1H) ; Tlc, R f 0.44 (1: 3 Hexane / EtOAc); mp 221-226 ° C

Comparative Example 16: 6-Hydroxy-4'-methoxyflavone

Yield: 22.9%. Pale brown needles. IR (KBr) ν _max cm ^-1 : 1605, 1269. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 6.75 (d, J = 8.4, 1H), 6.84 (d, J = 8.4, 1H), 7.11 (d, J = 8.4, 1H), 6.86 (s, 1H), 6.72 (d, J = 8.7, 2H), 7.19 (d, J = 8.7, 2H), 3.73 (s, 3H); Tlc, R f 0.27 (1: 3 Hexane / EtOAc); mp 142-145 ℃

Comparative Example 17: 7-hydroxy-4'methoxyflavone

Yield: 32.8%. Pale brown needles. IR (KBr) ν _max cm ^-1 : 1605, 1269. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 6.39 (d, J = 8.4, 1H), 6.48 (d, J = 8.4, 1H), 7.47 (d, J = 8.4, 1H), 6.86 (s, 1H), 6.72 (d, J = 8.7, 2H), 7.19 (d, J = 8.7, 2H), 3.73 (s, 3H); Tlc, R f 0.33 (1: 3 Hexane / EtOAc); mp 201-205 ℃

Comparative Example 18: 6,3 ', 4'-Trihydroxyflavone (6,3', 4'-Trihydroxyflavone)

Yield: 15.9%. Pale brown needles. IR (KBr) ν _max cm ^-1 : 1605, 1269. ¹ H-NMR (acetone-d ₆ , 300 MHz) δ: 6.39 (d, J = 8.4, 1H), 6.48 (d, J = 8.4, 1H), 7.47 (d, J = 8.4, 1H), 6.86 (s, 1H), 6.51 (d, J = 8.7, 1H), 6.60 (d, J = 8.7, 1H), 6.69 (d, J = 8.7, 1H) ; Tlc, R f 0.17 (1: 3 Hexane / EtOAc); mp 229-233 ℃

Experimental Example 1: Verification of monocyte cell adhesion inhibition effect by flavone derivatives

1-1. Experiment method

Vascular endothelial cells

Human umbilical vein endothelial cells were isolated using collagenase enzymes (collagenase type III, Worthington Biocemicals Co., Lakewood, NJ, USA) and cultured at 37 ° C, 95% O ₂ + 5% CO ₂ Primary culture under conditions (Jaffe EA, Nachman RL, Becker CG, Minick CR. 1973. Clin Invest 52: 2745-2756). The authenticity of vascular endothelial cells was determined by the accumulation of acetylated low density lipoproteins labeled with a fluorescent substance of MoI (1.1'-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate) (Molecular Probes Co., Eugene, OR, USA). Experimental (Voyta JC, Via DP, Butterfield CE, Zetter BR. 1984. J Cell Biol 99: 2034-2040).

Cell culture experiment

Microvascular endothelial cells prepared in primary culture were 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 0.9 mg / mL bovine brain extract, 0.75 mg / mL human cortical growth Culture was carried out in 25 mM HEPES-M199 medium (Sigma Co. St. Louis, MO, USA) to which factor and 0.075 mg / mL hydrocortisone were added. Human monocyte leukemia cells, THP-1 monocyte cell line, were cultured in RPMI-1640 medium.

compound

Each compound of Examples 1 to 11 and Comparative Examples 1 to 7 was previously dissolved in dimethyl sulfoxide (DMSO) for cell culture experiments (Anderson JJ, Garner SC. 1998. Baillieres Clin Endocrinol Metab 12: 543-557 ), The final concentration of DMSO used for cell culture was 0.5%, and the concentration for the culture experiment was 50 μM.

In vitro adhesion experiment

Vascular endothelial cells were pretreated for 20 minutes in M199 medium to which the compounds of Examples 1 to 11 and Comparative Examples 1 to 7 were added at 50 μM, and then activated for 6 hours at 10 ng / mL TNF-α. THP-1 monocytes cultured in RPMI 1640 medium were stained with 5 μM calcein-AM (Molecular Probes Co.) for 30 minutes, washed incubated with vascular endothelial cells and 485 on a fluorescence microscope attached with a SPOT II digital camera. The degree of adhesion to vascular endothelial cells of THP-1 monocytes was measured by nm excitation and emission wavelength of 538 nm.

Statistical processing

Experimental results were expressed as mean ± SEM, and the difference between groups (p <0.05) was compared using a SAS PC program (SAS Institute Inc., Cary, NC, USA). Two-way ANOVA was used and the group with significant difference was tested by Tukey correction.

1-2. result

The effects of the compounds of Examples 1 to 11 and Comparative Examples 1 to 7 on the adhesion of vascular endothelial cells and monocytes activated with TNF-α were investigated. According to the results of Choi YJ, Choi JS, Lee SH, Lee YJ, Kang JS, Kang YH. 2002. J Soc Food Sci Nutr 31: 672-678, 50 μM flavonoids were used for cell culture. The concentration did not show cytotoxicity during the proliferation of vascular endothelial cells.

As a result, vascular endothelial cells treated with TNF-α for 6 hours showed significantly increased THP-1 monocyte adhesion compared with vascular endothelial cells not treated with TNF-α. However, it was confirmed that monocyte adhesion by TNF-α was inhibited when TNF-α-treated vascular endothelial cells were treated with the compounds of Examples 1-11, whereas the compounds of Comparative Examples 1-7 were treated. In one case, monocyte adhesion was observed, which did not effectively inhibit monocyte adhesion.

Figures 1a and 1b confirms the monocyte adhesion inhibitory effect of 7-hydroxy-3 ', 4'-dimethoxyflavone (Example 7) in TNF-α-activated vascular endothelial cells, 2a is a vascular endothelial cells Pretreatment with 50 μM of 3'4'-dimethoxy-7-hydroxyflavone for 20 minutes followed by 6 hours with 10 ng / mL TNF-α followed by 24 hours with calcaine-AM-labeled THP-1 monocytes After culturing it was observed with a fluorescence microscope (x200), 2b is a result of quantitative analysis of the excitation wavelength 485 nm and emission wavelength 538 nm with a Fluoroscan ELISA plate reader. That is, it can be seen that 7-hydroxy-3 ', 4'-dimethoxyflavone (Example 7) significantly inhibits monocyte adhesion by tumor necrosis factor.

In another experiment, to determine whether the inhibition of monocyte adhesion by the compounds of Examples 1 to 11 induced cytotoxicity to TNF-α-activated vascular endothelial cells resulting from a decrease in the number of vascular endothelial cells, Example 1 Cytotoxicity of each of the compounds of 11 to 11 was tested. After treatment of each compound to TNF-α treated cells, cell viability was confirmed by MTT method after 5 hours. As a result, the survival rate of the vascular endothelial cells treated with the compounds of Examples 1 to 11 was confirmed to be almost similar to the cell survival rate of the untreated vascular endothelial cell control group, and 50 μM of the compounds of Examples 1 to 11 were expressed as TNF-α. Treated vascular endothelial cells did not show any cytotoxicity. Thus, monocyte adhesion of vascular endothelial cells activated with TNF-α could be seen to be inhibited due to physiological activity and not cytotoxicity of the compounds of Examples 1-11.

Experimental Example 2: Verification of the inhibitory effect of TNF-α-activated CAMs protein expression by flavone derivatives

2-1. Experiment method

Protein Isolation and Western Blot Analysis

Western blots were performed to measure VCAM-1 protein expression in vascular endothelial cells.

Endovascular endothelial cells were treated with 1.0 M Tris-HCl (pH 6.8) cell blood buffer containing 100% glycerol, 10% SDS, 1% β-glycerophosphate, 0.1 M Na ₃ VO ₄ , 0.5 M NaF and a protease inhibition cocktail. The cell extracts were prepared by treatment. Protein of the cell extract was subjected to 8% SDS-PAGE. Electrophoretic protein bands were transferred to nitrocellulose membranes, treated with 5% scheme milk to prevent nonspecific binding, and then subjected to the monoclonal rabbit anti-human antibody, Santa Cruz Biotech. , Santa Cruz, CA, USA) was diluted 1,000 times to react with stirring, and as a secondary antibody of chlorine anti-rabbit horseradish peroxidase (1: 7,500, Jackson Immuno Research Lab., West Grove, PA, USA) The reaction was time. The CAMs protein in the nitrocellulose membrane was detected by a Supersignal West pico chemiluminescence (Pierce Biotech. Inc., Rockford, IL, USA) and photographed on a Konica X-ray film. The amount of VCAM-1 protein expression was quantified by an optical densitometer.

2-2. result

CAMs are mainly responsible for the aggregation of monocytes in vascular endothelial tissue, causing atherosclerotic vascular damage and being involved in the generation of atherosclerotic plugs (Dustin ML, Springer TA. 1988. J Cell Biol 107: 321-331). . We investigated whether the inhibitory effect of flavonoids on monocyte adhesion of vascular endothelial cells by TNF-α was due to the inhibition of CAMs expression in vascular endothelial cells. To this end, Western blot analysis was attempted using antibodies specific for CAMs protein.

2a and 2b confirm the changes in VCAM-1 protein expression induced by TNF-α by the compounds of Examples 1 to 7 and the compounds of Comparative Examples 1 to 18, respectively. As a result, the vascular endothelial cell control group expressed little VCAM-1 protein when TNF-α was not treated, but expressed more than 10-fold VCAM-1 protein when TNF-α was treated. On the other hand, when endothelial cells treated with TNF-α were treated with 50 μM of the compound of Example 1-7, VCAM-1 expression was inhibited. On the other hand, the compounds of Comparative Examples 1-18 showed no significant inhibitory effect on VCAM-1 expression.

Figure 3 shows the effect of inhibiting the expression of VACM-1 according to the throughput of 7-hydroxy-3 ', 4'-dimethoxyflavone (Example 7), which is 1-50 μM and 10 ng / mL vascular endothelial cells After treatment with TNF-α, the cell extracts were developed with 12% SDS-PAGE followed by Western blot with VACM-1 primary antibody. In Figure 3 it can be seen that 7-hydroxy-3 ', 4'-dimethoxyflavone inhibits VCAM-1 expression even below 50 μM concentration.

Therefore, it was confirmed that the compounds of Examples 1 to 7 inhibit the adhesion of monocytes to vascular endothelial tissue by inhibiting the expression of CAMs protein of vascular endothelial cells. Thus, the compounds of Examples 1 to 7 can be provided for the purpose of preventing and treating atherosclerosis and coronary artery disease while blocking the initial process of atherosclerosis.

As described above, the flavone derivatives and the chalcone-based compounds of the present invention can prevent the adhesion of monocytes to vascular endothelial cells at an early stage to prevent the occurrence of arteriosclerosis, and can be used as an anti-atherosclerosis composition.

1a and 1b confirm the monocyte adhesion inhibition effect of 7-hydroxy-3 ', 4'-dimethoxyflavone in TNF-α-activated vascular endothelial cells.

2A and 2B confirm the effects of the flavone derivatives of Examples 1 to 7 on VCAM-1 protein expression induced by TNF-α.

Figure 3 shows the effect of inhibiting the expression of VACM-1 according to the throughput of 7-hydroxy-3 ', 4'-dimethoxyflavones.

Claims (6)

An atherosclerosis composition comprising a compound represented by Formula 1 or a compound represented by Formula 2: (Formula 1) In Chemical Formula 1, R ₁ is OCH ₃ , R ₂ is H or OCH ₃ , R ₃ is H or OCH ₃ , R ₄ is H or CH ₃ , and R ₅ is H, OH or OCH ₃ ; (Formula 2) In Chemical Formula 2, R ₆ is OCH ₃ , R ₇ to R ₁₀ are each independently H or OCH ₃ . The composition of claim 1, wherein the compound represented by Formula 1 is selected from the group consisting of compounds 1 to 7 shown in Table 1 below. Table 1 R ₁ R ₂ R ₃ R ₄ R ₅ Compound 1 OCH ₃ H H H OCH ₃ Compound 2 OCH ₃ H OCH ₃ H H Compound 3 OCH ₃ OCH ₃ H H OCH ₃ Compound 4 OCH ₃ OCH ₃ OCH ₃ H H Compound 5 OCH ₃ OCH ₃ H CH ₃ CH ₃ Compound 6 OCH ₃ OCH ₃ H CH ₃ H Compound 7 OCH ₃ OCH ₃ H H OH The composition of claim 1, wherein the compound represented by Formula 2 is selected from the group consisting of compounds 8 to 11 shown in Table 2 below. Table 2 R ₆ R ₇ R ₈ R ₉ R ₁₀ Compound 8 OCH ₃ H H H OCH ₃ Compound 9 OCH ₃ H OCH ₃ H H Compound 10 OCH ₃ OCH ₃ H H OCH ₃ Compound 11 OCH ₃ OCH ₃ OCH ₃ H H The composition of claim 1, wherein the composition is used for preventing and treating atherosclerosis. A composition for inhibiting expression of a cell adhesion protein, the expression of which is induced by tumor necrosis factor-α comprising a compound represented by Formula 1 or a compound represented by Formula 2: (Formula 1) In Chemical Formula 1, R ₁ is OCH ₃ , R ₂ is H or OCH ₃ , R ₃ is H or OCH ₃ , R ₄ is H or CH ₃ , and R ₅ is H, OH or OCH ₃ ; (Formula 2) In Chemical Formula 2, R ₆ is OCH ₃ , R ₇ to R ₁₀ are each independently H or OCH ₃ . The composition of claim 5, wherein the cell adhesion protein is vascular cell adhesion molecule-1 (VCAM-1).