JP2009143908A - Treatment of restenosis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of inhibiting the expression or activity of an adhesion molecule associated with an endothelial cell by bringing the adhesion molecule or the endothelial cell into contact with one or more isoflavone compounds or derivatives thereof. <P>SOLUTION: Disclosed are a method of preventing or reducing a risk of restenosis after angioplasty by bringing an adhesion molecule or an endothelial cell into contact with one or more isoflavone compounds or derivatives thereof to thereby inhibit expression or activity of the adhesion molecule associated with the endothelial cell, and a method of the treatment or prophylaxis of atherosclerosis, coronary artery diseases, other cardiovascular diseases and inflammatory diseases mediated by the adhesion molecules. The invention further discloses pharmaceutical compositions useful in these methods, and methods for producing such medicaments. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates generally to a method of inhibiting the expression or activity of an adhesion molecule associated with an endothelial cell by contacting the adhesion molecule or endothelial cell with one or more isoflavone compounds or derivatives thereof. The present invention generally relates to methods for preventing or reducing the risk of restenosis after angioplasty, and adhesion molecule mediated atherosclerosis, coronary artery disease, and other cardiovascular diseases. And also a method of treating or preventing inflammatory diseases. The present invention further relates generally to pharmaceutical compositions useful in these methods, and methods for producing such agents. Further aspects of the present invention will become apparent from the following description.

Note: Bibliographic details of the publications referred to by author in this specification are collected at the end of the specification.

Atherosclerosis is a chronic inflammatory disease of the arterial intima characterized by focal accumulation of leukocytes, smooth muscle cells, lipids and extracellular matrix. Plaque formation is caused by the deposition of lipids and other blood derivatives, particularly in the vessel walls of large arteries. Continual thickening of the vessel wall is observed as lipid and leukocyte attachment continues to the vessel wall. Increased plaque size results in stenotic properties that cause vascular occlusion due to atheroma, thrombosis or embolism. Serious vascular problems such as infarction, heart failure, stroke and sudden death can occur.

An important predisposition to the development of atherosclerosis is blood cholesterol levels.
Atheromatous plaques consist primarily of cells that contain cholesterol. Consequently, high blood cholesterol levels are associated with an increased risk of atherosclerosis. A high absolute level of cholesterol is an important risk factor, while that risk is more specifically related to the type of lipoprotein present in the blood. According to a wide range of medical aspects, low density lipoprotein (LDL) and very low density lipoprotein (VLDL) are detrimental to the risk of developing atherosclerosis, but high density lipoprotein (HDL) Has been shown to be a beneficial factor. In short, HDL in the bloodstream
The ratio to LDL is an important factor. The higher the ratio of HDL to LDL, the more patients will be protected against the development of atherosclerosis, even if their total cholesterol levels are slightly elevated. This view is supported by clinical trials conducted in many centers and countries.

Numerous clinical and epidemiological studies have shown that high levels of total cholesterol in the blood, especially high levels of low-density cholesterol in the blood, especially high LDL: HDL cholesterol ratios It was determined as the main risk factor for the development of sclerosis. This has resulted in a variety of therapeutic strategies intended to reduce this risk. There are two general strategies that show varying degrees of success. The first is the use of drugs that interfere with cholesterol synthesis. The second strategy is to reduce cholesterol absorption from the intestine through the use of resins, thereby reducing the pool of cholesterol in the body.
In general, however, neither of these two strategies is optimal due to the resulting side effects and limited efficacy.

The development of atherosclerosis proceeds by the oxidation of LDL cholesterol in the arterial wall, resulting in an inflammatory lesion. If left unchecked, the inflammatory process proceeds until the lesion causes vascular construction, occlusion and infarction.

Antioxidants are molecules or compounds that can inhibit oxidation (ie, damage) by chemically inactivating free radicals produced during normal physiological function. If the body lacks the supply of antioxidants, some free radicals remain active and attack healthy cells or convert safe compounds into damaging compounds. Antioxidants play a number of important roles in cardiovascular diseases. They are of particular interest as potential inhibitors of atherosclerosis by inhibiting the oxidative modification of low density lipoprotein (LDL). Lipoprotein is the main carrier of cholesterol in the body. The majority of cholesterol associates with LDL and with LDL that is distributed throughout the body from the liver. High density lipoprotein (HDL) “wipes” excess cholesterol present in the blood and returns it to the liver. Lipoproteins are highly oxidizable and, once oxidized, can accumulate in healthy arteries and blood vessel walls without being metabolized. Such accumulation of lipoprotein greatly increases the risk of atherosclerosis. In particular, the sub-endothelial space
) Oxidation of LDL in the middle represents an early causative process in atherogenesis (Non-patent Document 1). Thus, inhibition of oxidized LDL formation by antioxidants is generally considered to delay disease progression. This is another area of protection in cardiovascular disease where cardioprotective agents are effective, in which case even better agents are sought.

Another area in the development of vascular disease and plaque formation is the role played by cell adhesion molecules, as outlined in [2]. Cell adhesion molecules are involved in the adhesion of leukocytes and monocytes to tissues including the vascular endothelium. Known cell adhesion molecules include intercellular adhesion molecules (ICAM1, 2 and 3), vascular cell adhesion molecule-1 (VCAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1). The role of adhesion molecules in the pathogenesis of cardiovascular disease is supported by the observation that cell adhesion molecules are expressed in atherosclerotic lesions. In addition, cell adhesion molecules are upregulated by several coronary heart disease risk factors, and antibodies against cell adhesion molecules are believed to prevent reperfusion injury in animal models. Thus, modulation of the expression or activity of adhesion molecules associated with endothelial cells may be useful for the treatment or prevention of cardiovascular pathology by limiting the development of atherosclerotic plaques.

Other molecules such as E-selectin, P-selectin and L-selectin exhibit adhesion function. In particular, E-selectin is a cell surface protein that is inducibly expressed in cytokine-activated endothelial cells in response to inflammatory factors such as those caused by tissue damage. By endothelial cells
E-selectin expression can also be induced by inflammatory factors including interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α) and various endotoxins. E-selectin expression is associated with the binding of endothelial cells and platelets to leukocytes and lipids. Leukocyte binding to endothelial cells is observed at an early stage after tissue injury and is associated with various acute and chronic inflammations. Suppression or inhibition of the expression or activity of adhesion molecules associated with endothelial cells limits the focal accumulation and adhesion of leukocytes to the vessel wall, particularly in the area of injury, damage or infection.

In the treatment of atherosclerosis, angioplasty has been developed to enable non-surgical intervention of atherosclerotic plaque. During angioplasty treatment, a balloon catheter is used to fully recoil and dilate the blood vessel beyond its ability to widen the blood vessel lumen, thereby increasing blood flow. However, this technique can cause mechanical damage to the arterial wall and then restenosis
Usually happens. Indeed, restenosis is a major problem after traumatic injury caused to blood vessels during vascular surgery and treatment.

A variety of other treatments have been proposed to address the development of restenosis, including surgical treatments and drugs and gene therapy. Such therapies are useful for cell division and hyperproliferative disorders (hy
administration of compounds that inhibit perproliferative disorders), necrotize vascular smooth muscle cells, and block the expression of endothelial cell adhesion molecules. Additional compounds useful for combating restenosis include hyperlipidemia drugs, antiplatelet drugs, antithrombotic drugs, calcium channel blockers,
Angiotensin converting enzyme (ACE) inhibitors and β-blockers are included. In general, however, therapeutic agents are not selective and often have side effects that need to be monitored or countered. In this regard, reference is generally made to drugs in use, such as Ticlid (ticlopidine), Plavix (clopidrogel) and Cardiprin (aspirin). Typically, gastrointestinal disorders and skin rashes are commonly reported side effects with their use. Other agents, such as heparin, are reportedly in
Inhibits smooth muscle cell proliferation in vitro but has the adverse side effect of inhibiting clotting when used in vivo.

Given that vascular disease is generally the leading cause of death in today's society, and there is a strong need to identify new, improved and better alternative methods and pharmaceuticals for its treatment and prevention .

Therefore,
It is a preferred object of the present invention to provide a method for the treatment, amelioration or prevention of vascular and inflammatory diseases, particularly restenosis associated with vascular treatment. It is a further preferred object of the present invention to provide a pharmaceutical composition for the treatment, amelioration or prevention of vascular and inflammatory diseases, in particular cardiovascular diseases. It is yet another preferred object of the present invention to provide methods and compositions that inhibit the expression or activity of adhesion molecules in endothelial cells.
Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL "Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity". N Engl J Med 1989 Apr 6; 320 (14): 915-24 Hillis GS and Flapan AD "Cell adhesion molecules in cardiovascular disease: a clinical perspective". Heart 1998 May; 79 (5): 429-31

Surprisingly, the inventors have found that isoflavone compounds, metabolites and derivatives thereof are particularly useful for inhibiting or downregulating adhesion molecule expression or activity in endothelial cells. . Isoflavones and their derivatives have also been found to be particularly useful for inhibiting endothelial cell surface adhesion molecules, particularly E-selectin and VCAM-1.

The inventors have found that isoflavones and derivatives thereof are angioplasty treatments of vascular treatments such as atherosclerosis and the resulting mechanical damage at the site of angioplasty during the treatment of atherosclerotic lesions. It has also been surprisingly found that it finds use in preventing or reducing the risk of restenosis associated with.

Isoflavone compounds, metabolites, derivatives and analogs useful in the methods of the present invention are represented by the general formula I as presented below.

Thus, according to the first aspect of the present invention, there is provided a method of inhibiting the expression or activity of an adhesion molecule associated with endothelial cells, the method inhibiting the expression or activity of the adhesion molecule or endothelial cell. Contacting with one or more compounds of formula I in an amount sufficient to do so.

Preferably, the adhesion molecule is E-selectin or vascular cell surface adhesion molecule (VCAM-1).

According to a second aspect of the invention, there is provided a method of inhibiting the expression or activity of an adhesion molecule associated with an endothelial cell of a subject, said method comprising a therapeutically effective amount of one or more formulas Administering to the subject a compound of I.

According to a third aspect of the invention, there is provided a method of treating a disease mediated by expression or activity of an adhesion molecule associated with an endothelial cell of a subject, the method comprising an adhesion molecule associated with the endothelial cell. Administering to the subject one or more compounds of formula I in an amount sufficient to inhibit said expression or activity of

Preferably, the disease is a vascular disease such as restenosis, inflammatory disease, coronary artery disease, angina or small vessel disease, more preferably restenosis after angioplasty.

According to a fourth aspect of the invention, there is provided a method of treating, ameliorating, preventing or reducing the risk of restenosis in a subject, said method comprising a therapeutically effective amount of one or more of formula I Administering the compound to the subject.

Typically, restenosis is associated with vascular treatment such as coronary intervention. Preferably, the vascular coronary artery treatment is percutaneous transluminal coronary artery angioplasty.
luminal coronary angioplasty), direction coronary atherotomy
therectomy) or stent, more preferably angioplasty.

According to a fifth aspect of the invention, a procedural vascular trauma in a subject.
trauma) is provided, wherein the method comprises a therapeutically effective amount of one or more Formula I.
Administering a compound of the above to a subject.

Preferably, the procedural vascular trauma is angioplasty, vascular surgery, graft or transplant procedure.

According to a sixth aspect of the invention, there is provided a method of treating or preventing a vascular disease in a subject comprising administering to the subject a therapeutically effective amount of one or more compounds of formula I. The process of carrying out is included.

Preferably, the vascular disease is restenosis, inflammatory disease, coronary artery disease, angina or small vessel disease, more preferably restenosis after angioplasty.

According to a seventh aspect of the present invention there is provided a pharmaceutical composition in a dosage form suitable for use in the treatment of a disease mediated by the expression or activity of adhesion molecules associated with endothelial cells in a subject, The composition comprises one or more compounds of formula I together with a pharmaceutically acceptable carrier.

According to an eighth aspect of the invention, there is provided a pharmaceutical composition in a dosage form suitable for use in preventing or reducing the risk of vascular disease in a subject, said composition being pharmaceutically acceptable One or more compounds of formula I are included with the carrier.

According to a ninth aspect of the invention, the use of one or more compounds of formula I in the manufacture of a medicament for use in the treatment of a disease mediated by the expression or activity of adhesion molecules associated with endothelial cells. Provided.

According to a tenth aspect of the present invention there is provided the use of one or more compounds of formula I in the manufacture of a medicament for the treatment of vascular diseases and / or procedural vascular trauma.

These and other aspects of the present invention will become apparent from the following description and claims, taken in conjunction with the accompanying drawings.

Throughout this specification and the appended claims, unless otherwise specified, “including”
se) "and its variants" comprises "or" contains "(c
“omprising” is meant to include the described whole body or step or groups of multiple whole bodies or steps, but any other whole body or process, or multiple whole bodies or steps. It is understood that it does not mean to exclude a group.

The accompanying FIGS. 1-21 illustrate embodiments of the present invention, but are not limited thereto. A description of each of the figures is provided through the following examples.

The term “stenosis” is taken in its broadest sense and means in particular the narrowing or tightening of the diameter of a body passage or orifice such as a blood vessel or artery, generally reducing blood flow As well as the attendant problem of vascular occlusion. Typically, stenosis occurs as a result of focal accumulation and deposition of lipids and other blood derivatives.

The term “restenosis” or restenosis or secondary stenosis is interpreted in its broadest sense and typically means recurrence of stenosis after vascular treatment, injury or surgery, eg after balloon catheter treatment . Stenosis and restenosis can occur in blood vessels throughout the body, but of particular medical importance is the life-threatening and often fatal effect of stenosis in the coronary arteries.

Isoflavones and their derivatives respond to numerous signals known to be active in atherosclerosis, restenosis, inflammatory responses and other diseases mediated by the expression of cell adhesion molecules It was surprisingly discovered that it blocks the inducible expression of adhesion molecules, particularly E-selectin and VCAM-1. This result shows that isoflavone compounds and their derivatives are useful for the treatment and prevention of restenosis, coronary artery disease, angina and other vascular and cardiovascular diseases, inflammatory diseases mediated by adhesion molecules E-selectin and VCAM-1 It shows that it is. The specific molecular mechanism by which isoflavones and derivatives function in inhibiting cell adhesion molecule expression is not well understood.

It was further shown that isoflavones and their derivatives inhibit cell proliferation of human vascular smooth muscle cells and also inhibit PDGF-induced Erk activation in human vascular smooth muscle cells. This activity indicates that isoflavones and their derivatives may prevent the onset and progression of atherosclerotic lesions and provide potential benefits in vascular protection.

Isoflavones and their derivatives have been shown to inhibit endothelial cell proliferation and inhibit endothelial cell migration, and thus can be used as cardioprotective therapies such as the treatment, amelioration or prevention of restenosis after vascular treatment Have sex.

It has also been shown that isoflavones and their derivatives have potent vascular regulatory capabilities. Thus, isoflavones and their derivatives can counteract contractile activity, directly counteract vasodilation, and protect against endothelial damage by oxidized low density lipoprotein. Their activity is comparable to the ovarian steroid 17β-estradiol but also appears to be unique to their mechanism of action. Thus, isoflavones and derivatives also show potential for use as cardioprotective therapies such as treatment, amelioration or prevention of restenosis after vascular treatment.

Isoflavone compounds and derivatives are used to treat patients with small vascular diseases that cannot be treated by surgery or angioplasty, or other vascular diseases for which surgery is not an option, and before and after revascularization therapy. Can be administered to stabilize. The active compound is a period immediately before and after vascular treatment such as coronary or angioplasty as a means to reduce or eliminate the hyperproliferative and inflammatory responses that generally result in clinically significant restenosis Can also be administered. In addition, isoflavones can also be used to treat heart transplant rejection and vascular transplantation and transplantation procedures.

The methods of the present invention represent a significant advance in the treatment of vascular conditions and diseases in that they simply go beyond known therapies intended to inhibit disease progression. Experimental data provided herein has unexpectedly shown that restenosis after vascular treatment or angioplasty is inhibited or at least significantly reduced in various mammalian animal models . Thus, when used appropriately, the methods of the invention medically treat restenosis,
And possibly demonstrates the potential of isoflavone compounds and their derivatives to cure atherosclerosis by preventing the development of new lesions and stabilizing or regressing established lesions.

Isoflavones encompass a class of phytoestrogens found primarily in legumes that have estrogenic activity related to their structural similarity to steroidal estrogens. Only recently has the importance of isoflavones been recognized due to the reduced risk of developing osteoporosis in mammals, some forms of cancer and its obvious link to cholesterol levels and their ability to lower blood pressure. It has also been suggested that many of the biological effects of isoflavones can be explained by their conversion in vivo to various other active metabolites. Given the importance of isoflavone metabolites, synthetic derivatives of isoflavones and metabolites are also considered important therapeutic agents. In addition, isoflavones have been ingested for many years as a staple component that is very resistant to few or no known side effects, unlike many known symptomatic treatments in vascular disease and related surgery. .

Isoflavone compounds, metabolites, derivatives and analogs useful in the methods of the present invention are represented by the following general formula, including pharmaceutically acceptable salts thereof:

Where
R1, R2 and Z are independently hydrogen, hydroxy, OR9, OC (O) R10, OS
(O) R10, CHO, C (O) R10, COOH, CO2R10, CONR3R4, alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, alkylaryl, alkoxyaryl, thio, alkylthio, amino, alkylamino, Dialkylamino, nitro or halo, or R2 is as defined above, and R1 and Z together with the carbon atom to which they are attached, form a five-membered ring selected from Forming,

Alternatively, R1 is as defined above, and R2 and Z together with the carbon atom to which they are attached form a 5-membered ring selected from:

And W is R1, A is hydrogen, hydroxy, NR3R4 or thio, and B is selected from the following formula:

Alternatively, W is R1 and A and B together with the carbon atom to which they are attached form a 6-membered ring selected from:

Alternatively, W, A and B, together with the group with which they are associated, are selected from the following formula:

Alternatively, W and A, together with the group with which they are associated, are selected from the following formula:

  And B is selected from the following chemical formula:

in this case,
R3 is hydrogen, alkyl, arylalkyl, alkenyl, aryl, amino acid, C (
O) R11 (where R11 is hydrogen, alkyl, aryl, arylalkyl or amino acid), or CO2R12 (where R12 is hydrogen, alkyl, haloalkyl,
Aryl or arylalkyl),
R4 is hydrogen, alkyl or aryl, or R3 and R4 together with the nitrogen to which they are attached constitute pyrrolidinyl or piperidinyl;
R5 is hydrogen, C (O) R11 (where R11 is as defined above) or C
O2R12 (where R12 is as defined above);
R6 is hydrogen, hydroxy, alkyl, aryl, amino, thio, NR3R4, C (O)
R11 (where R11 is as defined above), CO2R12 (where R12
Is as defined above) or CONR3R4,
R7 is hydrogen, C (O) R11 (where R11 is as defined above), alkyl, haloalkyl, alkenyl, aryl, arylalkyl or Si (R13) 3 (
Wherein each R13 is independently hydrogen, alkyl or aryl),
R8 is hydrogen, hydroxy, alkoxy or alkyl;
R9 is alkyl, haloalkyl, aryl, arylalkyl, C (O) R11 (where R11 is as defined above) or Si (R13) 3 (where R13 is as defined above). ) And
R10 is hydrogen, alkyl, haloalkyl, amino, aryl, arylalkyl, amino acid, alkylamino or dialkylamino;
The drawing "--- (dashed and solid double lines)" represents a single or double bond,
T is independently hydrogen, alkyl or aryl,
X is O, NR4 or S, and Y is

Wherein R14, R15 and R16 are independently hydrogen, hydroxy, OR9, OC (O)
R10, OS (O) R10, CHO, C (O) R10, COOH, CO2R10, CONR3
R4, alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, thio, alkylthio, amino, alkylamino, dialkylamino,
Nitro or halo.

  The invention particularly relates to the use of compounds of the following general formulas II to VIII:

Where
R1, R2, R5, R6, R14, R15, W and Z are as defined above;
More preferably R1, R2, R14, R15, W and Z are independently hydrogen, hydroxy, OR9,
OC (O) R10, C (O) R10, COOH, CO2R10, alkyl, haloalkyl, arylalkyl, aryl, thio, alkylthio, amino, alkylamino, dialkylamino, nitro or halo,
R5 is hydrogen, C (O) R11 (where R11 is hydrogen, alkyl, aryl or an amino acid), or CO2R12 (where R12 is hydrogen, alkyl or aryl);
R6 is hydrogen, hydroxy, alkyl, aryl, C (O) R11 (where R11 is as defined above), or CO2R12 (where R12 is as defined above);
R9 is alkyl, haloalkyl, arylalkyl or C (O) R11 (where R
11 is as defined above), and R10 is hydrogen, alkyl, amino, aryl, amino acid, alkylamino or dialkylamino;

More preferably,
R1 and R14 are independently hydroxy, OR9, OC (O) R10 or halo;
R2, R15, W and Z are independently hydrogen, hydroxy, OR9, OC (O) R10,
C (O) R10, COOH, CO2R10, alkyl, haloalkyl or halo,
R5 is hydrogen, C (O) R11 (where R11 is hydrogen or alkyl) or C
O2R12 (R12 is hydrogen or alkyl);
R6 is hydrogen or hydroxy;
R9 is alkyl, arylalkyl or C (O) R11 where R11 is as defined above, and R10 is hydrogen or alkyl;

More preferably,
R1 and R14 are independently hydroxy, methoxy, benzyloxy, acetyloxy or chloro;
R2, R15, W and Z are independently hydrogen, hydroxy, methoxy, benzyloxy, acetyloxy, methyl, trifluoromethyl or chloro;
R5 is hydrogen or CO2R12 (where R12 is hydrogen or methyl) and R6 is hydrogen.

  Particularly preferred compounds of the invention are selected from the following chemical formula:

Preferred compounds of the invention are derived from the isoflavone or derivative molecule to which it is attached in v
Also included are all derivatives having a physiologically cleavable leaving group that can be cleaved in ivo. Leaving groups include acyl, phosphate, sulfate, sulfonate, preferably mono-, di- and per-acyloxy substituted compounds, in which one or more pendant hydroxy groups are acyl groups, preferably Protected by an acetyl group.
Typically, acyloxy substituted isoflavones and derivatives thereof are readily cleavable to the corresponding hydroxy substituted compound. Furthermore, protection of the functional groups on the isoflavone compounds and derivatives of the present invention can be carried out by methods well established in the art, for example as described in TW Greene (1981).

Most preferred isoflavone compounds contemplated for use in accordance with the present invention include formonenetin (formononetin), biochanin, genistein
), Daidzein and equol, and functional derivatives, equivalents or analogs thereof. Equally important compounds are isoflavone metabolites such as dihydrodaidzein, cis and trans tetrahydrodaidzein and dehydroequol and their derivatives and prodrugs.

The chemical and functional equivalents of a particular isoflavone are to be understood as molecules that exhibit any one or more functional activities of isoflavones and are either chemically synthesized or of natural product screening. From any source as identified by such screening methods.

The term “alkyl” refers to straight-chain, branched and cyclic (for 5 or more carbon atoms) saturated alkyl groups of 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, such as methyl, ethyl Propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, cyclopentyl and the like. The alkyl group is more preferably methyl,
Ethyl, propyl or isopropyl. The alkyl group is optionally one or more of fluorine, chlorine, bromine, iodine, carboxyl, C1-C4-alkoxycarbonyl, C1-C4-alkylaminocarbonyl, di- (C1-C4-alkyl) -amino-
It can be substituted by carbonyl, hydroxyl, C1-C4-alkoxy, formyloxy, C1-C4-alkyl-carbonyloxy, C1-C4-alkylthio, C3-C6-cycloalkyl or phenyl.

The term “alkenyl” refers to straight, branched and cyclic (for 5 or more carbon atoms) having 2 to 10 carbon atoms, preferably 2 to 6 carbon atoms and having at least one double bond. Of hydrocarbons such as ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl and the like. The alkenyl group is more preferably ethenyl, 1-propenyl or 2-propenyl. An alkenyl group optionally contains one or more fluorine, chlorine, bromine, iodine, carboxyl, C
1-C4-alkoxycarbonyl, C1-C4-alkylamino-carbonyl, di- (C
1-C4-alkyl) -amino-carbonyl, hydroxyl, C1-C4-alkoxy,
It can be substituted by formyloxy, C1-C4 alkyl-carbonyloxy, C1-C4-alkylthio, C3-C6-cycloalkyl or phenyl.

The term “alkynyl” refers to 2 to 10 carbon atoms, preferably 2 to 6 carbon atoms,
It is understood to include straight and branched chain hydrocarbons having at least one triple bond, such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl and the like. The alkynyl group is more preferably ethynyl, 1-propynyl or 2-propynyl. An alkynyl group optionally contains one or more fluorine, chlorine, bromine, iodine, carboxyl, C,
1-4-alkoxycarbonyl, C1-C4-alkylamino-carbonyl, di- (C1
-C4-alkyl) -amino-carbonyl, hydroxyl, C1-C4-alkoxy, formyloxy, C1-C4-alkyl-carbonyloxy, C1-C4-alkylthio,
It can be substituted by C3-C6-cycloalkyl or phenyl.

The term “aryl” is taken to include phenyl, biphenyl and naphthyl;
It can be optionally substituted by one or more C1-C4-alkyl, hydroxy, C1-C4-alkoxy, carbonyl, C1-C4-alkoxycarbonyl, C1-C4-alkylcarbonyloxy or halo.

The term “heteroaryl” is taken to include 5- and 6-membered rings containing at least one oxygen, sulfur or nitrogen in the ring, which ring is optionally furyl, pyridyl, pyrimidyl, thienyl, imidazolyl, Can be fused to other aryl or heteroaryl rings such as tetrazolyl, pyrazinyl, benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl, purinyl, morpholinyl, oxazolyl, thiazolyl, pyrrolyl, xanthinyl, purine, thymine, cytosine, uracil and isoxazolyl It is not limited to these. The heteroaromatic group optionally has one or more fluorine, chlorine, bromine,
Iodine, carboxyl, C1-C4-alkoxycarbonyl, C1-C4-alkylamino-carbonyl, di- (C1-C4-alkyl) -amino-carbonyl, hydroxyl,
C1-C4-alkoxy, formyloxy, C1-C4-alkyl-carbonyloxy,
It can be substituted by C1-C4-alkylthio, C3-C6-cycloalkyl or phenyl. The heteroaromatic can be partially or fully hydrogenated as desired.

The term “halo” is taken to include fluoro, chloro, bromo and iodo, preferably fluoro and chloro, more preferably fluoro. For example, “haloalkyl”
Reference to includes monohalogenated alkyl groups, dihalogenated alkyl groups and perhalogenated alkyl groups. Preferred haloalkyl groups are trifluoromethyl and pentafluoroethyl.

The term “pharmaceutically acceptable salt” refers to an organic or inorganic moiety that carries charge and can be administered with a pharmaceutical agent, for example, as a counter cation or counter anion in the salt. Pharmaceutically acceptable cations are known to those skilled in the art and include sodium,
Examples include but are not limited to potassium, calcium, zinc and quaternary amines. Pharmaceutically acceptable anions are known to those skilled in the art and include, but are not limited to, chloride, acetate, citrate, bicarbonate and carbonate.

The term “pharmaceutically acceptable derivative” or “prodrug” refers to an activity that can directly or indirectly provide a parent compound or metabolite upon administration to a recipient, or that itself exhibits activity. Refers to a derivative of a compound.

As used herein, “treatment”, “prophylaxis” or “
The terms “prevention”, “amelioration” and the like are to be considered in their broadest context. In particular, the term “treatment” does not necessarily mean that the animal is treated until complete recovery. Thus, “treatment” includes the improvement of the symptoms or severity of a particular disease, or the prevention or otherwise reduction of the risk of developing a particular disease.

The amount of one or more compounds of formula I required for therapeutic treatment of the present invention depends on a number of factors such as the particular application, the nature of the particular compound used, the condition being treated, the mode of administration and Depends on patient condition. The compound of formula I can be administered in the manner and amounts as conventionally practiced. See, for example, Goodman and Gilman, et al. (1995).
The specific dosage utilized will depend on the condition being treated, the condition of the subject, the route of administration and other known factors as described above. In general, the daily dose per patient ranges from 0.1 mg to 5
g, typically in the range of 0.5 mg to 1 g, preferably 50 mg to 200 mg. The length of administration depends on the severity of the condition to be treated or alleviated, from a single dose administered once a day or 2 days to 1 week, if necessary months to 2 days a day, administered for up to several years
It can be in the range of single or triple doses. For any individual patient, the specific dosing regimen is
It is further understood that it should be adjusted over time according to the individual needs and according to the professional judgment of the person administering or managing the composition. A relatively short-term treatment with the active compound can be used to stabilize or reduce coronary artery disease lesions that cannot be treated by either angioplasty or surgery. Longer term treatment can be employed to prevent the development of advanced lesions in high-risk patients.

The manufacture of pharmaceutical compositions for the treatment of therapeutic indications described herein typically involves the compounds of the invention (for convenience, hereinafter referred to as “active compounds”).
And one or more pharmaceutically or veterinarily acceptable carriers and / or excipients known in the art.

The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and not deleterious to the subject. The carrier or excipient may be a solid or liquid or both and preferably comprises a unit dose which may contain up to 100% by weight of active compound, preferably 0.5% to 59% by weight of active compound, for example Formulated with the compound as a tablet. One or more active compounds may be formulated into the formulations of the present invention, which essentially consists of combining the components, optionally containing one or more accessory ingredients. It can be prepared by any known technique of formulation. The preferred concentration of the active compound in the drug composition will depend on absorption, distribution, inactivation and excretion rates of the drug, as well as other factors known to those of skill in the art.

Formulations of the present invention include those suitable for oral, rectal, ocular, buccal (eg, sublingual), parenteral (eg, subcutaneous, intramuscular, intradermal or intravenous) as well as transdermal administration. The most suitable route in a given case will depend on the nature and severity of the condition being treated and the nature of the particular active compound being used.

Formulations suitable for oral administration contain a predetermined amount of the active compound as a powder or granules, as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil emulsion, respectively. It can be provided in discrete units such as capsules, sachets, lozenges or tablets. Such formulations may be prepared by any suitable method of formulation including the step of bringing into association the active compound and a suitable carrier, which may contain one or more accessory ingredients as described above. In general, the formulations of the present invention uniformly mix the active compound with a liquid or finely divided solid carrier or both, and then, if necessary, form the resulting mixture to form a unit dosage. It is prepared by molding. For example, a tablet may be prepared by compressing or molding a powder or granules containing the active compound, optionally with one or more accessory ingredients. Compressed tablets can be made by suitable machines, optionally with binders, lubricants, inert diluents, and / or surfactant / dispersant (s).
It can be prepared by compressing a free-flowing compound such as a powder or granule mixed with. Molded tablets may be made by molding, in a suitable machine, the powdered compound moistened with an inert liquid binder.

Formulations suitable for buccal (sublingual) administration include flavor treatment bases, usually lozenges containing the active compound in sucrose and acacia or tragacanth, and inert bases such as gelatin and glycerin or sucrose and gum arabic Examples include pastilles containing the compound therein.

Compositions of the present invention suitable for parenteral administration comprise, for convenience, sterile aqueous formulations of the active compound,
This formulation is preferably isotonic with the blood of the intended recipient. These formulations are preferably administered intravenously, although administration can also be accomplished by subcutaneous, intramuscular or intradermal injection. Such formulations may be prepared for convenience by combining the compound with water or glycerin buffer and sterilizing the resulting solution to make it isotonic with blood. Injectable formulations according to the invention generally contain from 0.1 to 60% (w / v) of active compound and are administered at a rate of 0.1 ml / min / kg.

Formulations suitable for rectal administration are preferably provided as unit dose suppositories. These combine the active compound with one or more conventional solid carriers such as cocoa butter,
The resulting mixture can then be prepared by molding.

Formulations or compositions suitable for topical administration to the skin preferably take the form of ointments, creams, lotions, pastes, gels, sprays, aerosols or oils. Carriers that can be used include petrolatum, lanolin, polyethylene glycols, alcohols, and combinations of two or more thereof. The active compound is generally from 0.1 to 5
% (W / w), especially 0.5-2% (w / w). Examples of such compositions include:
Examples include beauty skin creams.

Formulations suitable for transdermal administration may be provided as individual patches that are intended to remain in intimate contact with the recipient's epidermis for an extended period of time. Such patches suitably contain the active compound, for example as an optionally buffered aqueous solution with a concentration of 0.1 to 0.2 M with respect to the active compound. See, for example, Brown, L., et al. (1998).

Formulations suitable for transdermal administration are
It can also be delivered by iontophoresis (see, for example, Panchagnula R, et al., 2000) and typically takes the form of an aqueous solution of the active compound, optionally buffered. Suitable formulations include citrate or bis / Tris buffer (pH 6) or ethanol / water, 0.1
Contains ~ 0.2M active ingredient.

Formulations suitable for inhalation may be delivered as a spray composition in the form of a solution, suspension or emulsion. The inhalation spray composition can further comprise a pharmaceutically acceptable propellant such as carbon dioxide or nitrous oxide.

The active compound may be provided in the form of a food product, for example in the form of being added, mixed, coated, integrated, or otherwise added to the food product. The term groceries is used in its broadest possible meaning, and is a liquid formulation such as a beverage containing dairy products,
And other foods such as health food bars and desserts. Food formulations containing the compounds of the invention can be readily prepared according to standard techniques.

Treatment methods, uses and compositions include, for example, mammals such as companion animals and domestic animals (eg, dogs and cats) and livestock animals (eg, cows, sheep, pigs and goats), birds (eg, chickens, turkeys, ducks) For human or animal administration.

The active compound or a pharmaceutically acceptable derivative prodrug thereof or a salt thereof also includes other active substances that do not impair the initial action, or substances that supplement the initial action,
For example, it can be co-administered with antibiotics, antifungal agents, anti-inflammatory agents or antiviral compounds. The active agent may be a combination or synergy of two or more isoflavones or derivatives thereof (
synergistic) may be included in the mixture. Active compounds also include lipid lowering agents such as probucol and nicotinic acid, platelet aggregation inhibitors such as aspirin, antithrombotic agents such as coumadin, calcium channel blockers such as verapamil, diltiazem and difedipine, captopril and enalapril Angiotensin converting enzyme (ACE) inhibitors, as well as “-blockers such as propanolol, terbutalol and labetalol. The compounds may be administered as non-steroidal anti-inflammatory agents such as ibuprofen, indomethacin, aspirin, fenopro It can also be administered in combination with phen, mefenamic acid, flufenamic acid and sulindac The compound can also be administered with corticosteroids.

Co-administration may be simultaneous or sequential. Simultaneous administration can be accomplished with compounds present in the same unit dose administered at the same or similar time points, or in individual and individual unit doses. Sequential administration may be in any order as needed, typically
When a second or subsequent active agent is administered, especially if an accumulation or synergistic effect is desired, the ongoing physiological action of the first or first active agent must be persisted There is.

Isoflavones for use in the present invention are derived from any number of sources that are readily identifiable to those skilled in the art. Preferably they are obtained in the form of concentrates or extracts from plant sources. Moreover, although those skilled in the art can easily identify an appropriate plant species, for example, leguminous plants can be mentioned as plants of a specific use in the present invention. More preferably, the isoflavone extract is obtained from chickpea, lentil, pea, purple clover or subthalenian clover species.

The isoflavone extract can be prepared by any number of techniques known in the art.
For example, suitable isoflavone extracts can be prepared by water / organic solvent extraction from plant sources. It is understood that an isoflavone extract can be prepared from any single tissue of a single species of plant, or a combination of two or more different tissues thereof. Similarly, extracts can be prepared from starting materials containing a heterogeneous mixture of tissues from two or more different species of plants.

Generally, when an isoflavone extract is prepared from plant material, the material is crushed or chopped into smaller pieces, or partially pulverized or chopped into water and organic It can be contacted with a solvent, such as a water-miscible organic solvent. Or
The plant material is contacted with water and organic solvents without any pretreatment. The ratio of water to organic solvent is generally in the range of 1:10 to 10: 1, for example, an equivalent ratio of water and solvent or 1 to 30
% (V / v) organic solvent. Any organic solvent or mixture of such solvents can be used. The organic solvent is preferably C2-C10, more preferably C
1 to C4 organic solvents (eg, methanol, chloroform, ethanol, propanol,
Propylene glycol, erythritol, butanol, butanediol, acetonitrile,
Ethylene glycol, ethyl acetate, glycidol, glycerol dihydroxyacetone or acetone). If desired, the water / organic solvent mixture can include an enzyme that cleaves the isoflavone glycoside into an aglycone form. The mixture can be vigorously stirred to form an emulsion. The mixing temperature can range, for example, from ambient temperature to boiling temperature. The exposure time can be between 1 hour and several weeks. One convenient extraction time is
24 hours at 90 ° C. The extract can be separated from undissolved plant material and the organic solvent is removed, for example, by distillation, rotary evaporation, or other standard techniques for solvent removal. The resulting extract containing water-soluble and water-insoluble components can be dried to obtain an isoflavone-containing extract, which according to the present invention comprises one or more pharmaceutically acceptable carriers, It can be formulated with excipients and / or adjuvants.

Extracts made in accordance with the instructions set forth in the previous paragraph may contain a small amount of oil containing isoflavones in their aglycone form (referred to herein as isoflavones). This isoflavone concentrated oil can be HPLC processed to adjust the isoflavone ratio, or if it is at the desired isoflavone ratio, it is dried, for example, in the presence of silica, one or more carriers, loaded. It can be formulated with a form and / or adjuvant to obtain an isoflavone-containing extract. Alternatively, the isoflavone contained in the small amount of oil is hexane,
It can be further concentrated by adding to an oil of a water-insoluble organic solvent such as heptane, octane, acetone or a mixture of one or more of such solvents. One example is 80% hexane, 20% (w / w), which has a high solubility for oil but a low solubility for isoflavones.
w) Acetone. The oil is easily distributed in the organic solvent and the concentrated isoflavone-containing extract separates out of solution. The recovered extract is dried, for example, in an oven at 50 ° C. to about 120 ° C. and formulated with one or more pharmaceutically acceptable carriers, excipients and / or adjuvants.

It is understood that the present invention also contemplates the generation of suitable isoflavones, functional derivatives, equivalents or analogs thereof by established synthetic techniques known in the art. See, for example, Chang et al. (1994), which discloses a suitable method for the synthesis of various isoflavones.

Other suitable methods can be found, for example, in International Patent Application Publication Nos. WO98 / 08503 and WO00 / 49009 and references cited therein. These descriptions are incorporated herein by reference in their entirety.

  The invention is further illustrated by the following non-limiting examples and the accompanying drawings.

Anti-vasoconstriction and protection from oxidative LDL damage Vascular elasticity and vascular responses are important factors that promote cardiovascular risk. Normal physiological conditions indicate a healthy circulatory system, whereas arteries are differential stimuli of stress (di
It is responsive to fferential stimuli. Cholesterol accumulation and age are involved in poorly responsive blood vessels. Experiments were therefore conducted to test the ability of isoflavones to produce vasodilation and protect blood vessels from oxidized LDL damage. Furthermore, the importance of endothelium in these actions was investigated.

Methods Isolation of Rat Thoracic Aorta Male Sprague-Dawley rats (250 ± 50 g) were gassed to death with 80% CO 2 and 20% O 2. The thoracic aorta was removed and 199 mM NaCl, 4.7 mM HCl, 1.17 mM MgSO4 · 7H2O, 25
mM NaHCO3, 1.18 mM KH2PO4, 2.5 mM CaCl2, 11 mM glucose and 0.03 mM EDTA
Was quickly placed in an ice-cold Krebs modified solution consisting of next,
Fat and connective tissue were removed and cut into 2-3 mm wide rings. Hold at 37 ° C, 95% O2 + 5
Each ring was mounted on two parallel stainless steel hooks in a 3 mL organ bath with a single water jacket containing a modified Krebs solution aerated with% CO2. The lower hook is attached to a movable support foot and the upper hook is FT03 for isometric recordings
It was attached to a force transducer (Grass Scientific Instruments, Mitutoyo, Japan). Amplifying the force change (Quadbridge amplifier, Scientific Concepts Inc., Victor
ia, Australia), and MacLab 8E data collection system (Adinstuments Pty Ltd., NSW, Australia) linked to Apple Computer (Apple Computer Inc., Cupertina, CA). The rings were set to 2 g initial tension and allowed to equilibrate for 30 minutes, after which they were reset to 2 g tension before the start of each experimental protocol.

Protocol 1: Effect of isoflavone metabolites on the contraction curve for noradrenaline β-estradiol (1 μg / ml), Cpd. 5 (0.1 and 1 μg / ml), Cpd. 7 (0.1 and 1
μg / ml), Cpd. 8 (0.1 and 1 μg / ml) and Cpd. 12 (1 μg / ml), and noradrenaline (0.1 nM to 10 mM) in the presence and absence of vehicle equivalent volume DMSO A concentration-contractile curves were obtained. Only one compound at any one concentration was tested on any one ring from any one animal.

Protocol 2a Vasodilatory ability of isoflavone metabolite derivatives A full concentration response to noradrenaline was obtained. From now on, the submaximal of about 80%
) The concentration causing contraction was selected (0.03-0.3 μM). The ring was then constricted at this sub-maximal concentration and allowed to stabilize before total concentration − against β-estradiol and Cpd.5, 7, 8 and 12 and vehicle DMSO (in equal volume) — A relaxation curve was obtained. Only one compound was tested using any one ring from any one animal.

Protocol 2b. Vasodilatory ability of isoflavone metabolite derivatives: mechanism of action endothelium (the endothelium was peeled off by gently rotating the lumen against the rough surface), nitric oxide synthase inhibitor nitro-L-arginine (NOLA 10 μM ; Except Cpd.8 for limited supply of compounds), 40 mM KCl (to inhibit endothelium-derived hyperpolarizing factor), cyclo-oxygenase inhibitor indomethacin (10 μM) and soluble guanylate cyclase inhibitor 1H-
For Cpd. 5, 7, 8, and 12 and β-estradiol in the absence and presence of [1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ; 10 μM), A total relaxation curve was obtained. Only one compound with the only treatment was tested on any one ring from any one animal.

Protocol 3 Protective effect of isoflavone metabolite derivatives against endothelial damage induced by oxidized low density lipoprotein. A total concentration response curve to phenylephrine was constructed from which the concentration showing submaximal contraction of approximately 80% was selected (usually 0.3 μM ). Then tighten the tissue with phenylephrine at the selected concentration,
Allowed to stabilize. A total concentration expansion curve was then constructed for acetylcholine. After this,
Preliminary experiments demonstrated no effect on response to acetylcholine
0.1% antifoam B (SIGMA) was added to the bath. Next, β-estradiol (10 pg / ml), C
pd. 5 (300 ng / ml), Cpd. 7 (1 μg / ml), Cpd. 8 (3 μg / ml) or Cpd. 12 (3 μg / ml)
The total concentration response curve to acetylcholine was repeated after 1 hour incubation with oxidized low density lipoprotein (bovine LDL: 0.3 mg protein / ml) in the absence and addition of. Concentrations were selected based on neg log EC45-EC50 from experiments performed in protocol 2a.

Preparation of low density lipoprotein Human plasma was obtained from a blood bank. Beckman vertical rotor using a Beckman centrifuge (70
Individual lipoprotein fractions were obtained using discontinuous density gradient ultracentrifugation at Ti). That is, add 0.018 g of KBr to 1 ml of plasma, transfer it to a fast sealed tube, 4 ° C at 65,000 rpm
And rotated overnight. The top (VLDL) fraction was then removed and 0.064 g of KBr was added to the bottom of 1 ml. This was transferred to quick-seal tubes and rotated overnight at 45,000C at 65,000 rpm. The upper (LDL) fraction was transferred to a rapidly sealed tube, filled with dl.063 solution and rotated. The upper LDL fraction was collected after each rotation and dialyzed against 0.05 M NH4HCO3 pH 8.0, changing the solution daily for 4 days. The protein content of the final LDL fraction obtained was determined by Lowry's simplified protein assay. LDL was oxidized by 2 hours incubation with 5 μM CuSO4.

Data presentation and statistical analysis Contractile responses are expressed in g tension (mean + standard error of the mean). The diastolic response is expressed as a percentage of the contractile force produced by the pre-constricting agent used. Individual concentration-response curves for treatment with noradrenaline and acetylcholine ± were fitted to the following logistic equation:

E = MAp / (ApXKp)

Where E is the response, M is the maximum response, and K is the concentration that elicits 50% of the maximum response (ie neg log EC50).

Sigmastat statistical software (Jandel Scientific, San Rafael, CA) that analyzes results by two-way repeated measures analysis of variance and, where appropriate, then essentially tests the data for normality before performing parameter analysis ) Was used to perform a post-hoc t test with appropriate correction.

Drugs and solutions
1,3,5 [10] -estraditrien-3,17β-diol (17β-estradiol, SIGMA), Cp
d.5, Cpd. 7, Cpd. 8 and Cpd. 12 (Novogen Ltd) were dissolved in DMSO and diluted to the desired concentration in Krebs. Nω-nitro-L-arginine (SIGMA), indomethacin (SIGMA)
), Norepinephrine bitartrate (SIGMA), acetylcholine chloride (SIGMA) and 1H
-(1,2,4) oxadiazolo (4,3-a) quinoxalin-1-one (SIGMA) was dissolved according to the manufacturer's instructions.

Results Protocol 1: Effect of isoflavone metabolites on the contraction curve for noradrenaline Table 1 shows β-estradiol, a synthetic isoflavone derivative (Cpd. 5, 7, 8 and 12).
) And the maximum force obtained for noradrenaline in the presence and absence of the used.

FIG. 1 graphically illustrates the effect on response to noradrenaline for various compounds when used at 1 microg / ml. FIG. 2 shows the absence after 30 minutes incubation (
The total concentration-contraction response to noradrenaline in the pre-compound) and in the presence (post-compound) is shown.

All active compounds tested had significant antagonism on noradrenaline-induced contraction. In order to obtain an indication of the order of potency of the compounds tested, the difference in maximal response to noradrenaline (maximum response in the absence of compound-maximum response in the presence of compound) was calculated as 1 μg / m
Calculated for each compound in l (Table 1). From this it was clear that Cpd.12 was comparable to β-estradiol in offsetting the contractile response to noradrenaline, while Cpd.5, Cpd.7 and Cpd.8 were active while β-estradiol It was less effective.

Protocol 2a: Vasodilatory capacity of isoflavone metabolites: mechanism of action FIG. 3 shows the concentration-expansion effect of the compounds tested. Concentration-dilation curves obtained for 17β-estradiol and Cpd. 5, 7, 8 and 12 using rat isolated aortic rings pre-constricted with submaximal concentrations of noradrenaline. All values are mean ± standard error of the mean. All four compounds were at least as potent as β-estradiol in their vasodilatory capacity. The mechanism by which these compounds exert an expanding effect was further investigated using specific antagonists.

β-estradiol: FIG. 4 was pre-constricted with submaximal concentrations of noradrenaline (pre-con
stricted) concentrations obtained for 17β-estradiol using isolated rat aortic rings-
An extended curve is shown.
these,
(a) intact endothelium,
(b) Nitric oxide synthase inhibitor N “-nitro-L-arginine (NOLA 10 μM),
(c) 40 mM KCl,
(d) a soluble guanylate cyclase inhibitor 1H- [1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ 10 μM),
(e) performed in the absence and presence of the cyclo-oxygenase inhibitor indomethacin (10 μM);
(f) shows time (30 minutes) control.
n = number of experiments.

The vasodilatory response to β-estradiol consists of endothelium removal (FIG. 3a), incubation with nitro-L-arginine (FIG. 4b), high levels of KCl (FIG. 4c) and ODQ (
Inhibited by FIG. 4d). Indomethacin had no effect on the diastolic action of this steroid (Fig. 4e) and no time-dependent changes were observed (Fig. 4f).

Cpd. 5: FIG. 5 shows the concentration-dilation curve obtained for Cpd. 5 using rat isolated aortic rings pre-constricted with submaximal concentrations of noradrenaline.
these,
(a) intact endothelium,
(b) Nitric oxide synthase inhibitor N ″ -nitro-L-arginine (NOLA 10 μM),
(c) 40 mM KCl,
(d) a soluble guanylate cyclase inhibitor 1H- [1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ 10 μM),
(e) performed in the absence and presence of the cyclo-oxygenase inhibitor indomethacin (10 μM);
(f) shows time (30 minutes) control.
n = number of experiments.

The vasodilator response to Cpd.5 is inhibited by endothelium removal (FIG. 5a), incubation with nitro-L-arginine (FIG. 5b), high levels of KCl (FIG. 5c) and ODQ (FIG. 5d). It was. Indomethacin has no effect on the diastolic action of this steroid (Figure 5e).
), No time-dependent changes were observed (FIG. 5f).

Cpd. 7: FIG. 6 shows the concentration-expansion curve obtained for Cpd. 7 using rat isolated aortic rings pre-constricted with submaximal concentrations of noradrenaline.
these,
(a) intact endothelium,
(b) Nitric oxide synthase inhibitor N ″ -nitro-L-arginine (NOLA 10 μM),
(c) 40 mM KCl,
(d) a soluble guanylate cyclase inhibitor 1H- [1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ 10 μM),
(e) performed in the absence and presence of the cyclo-oxygenase inhibitor indomethacin (10 μM);
(f) shows time (30 minutes) control.
n = number of experiments.

The vasodilatory response to Cpd.7 is the removal of endothelium (Fig. 6a), high levels of KCl (Fig. 6c)
And was inhibited by ODQ (FIG. 6c). There was a trend toward inhibition of these responses by NOLA, which was not statistically significant. Indomethacin did not affect the diastolic action of this steroid (Fig. 6d) and no time-dependent changes were observed (Fig. 6e).

Cpd. 8: FIG. 7 shows the concentration-dilation curve obtained for Cpd. 8 using aortic rings isolated from rats pre-constricted with submaximal concentrations of noradrenaline.
these,
(a) intact endothelium,
(b) 40 mM KCl,
(c) a soluble guanylate cyclase inhibitor 1H- [1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ 10 μM),
(d) carried out in the absence and presence of the cyclo-oxygenase inhibitor indomethacin (10 μM);
(e) shows time (30 minutes) control.
n = number of experiments.

The vasodilatory response to Cpd.8 was inhibited by endothelium removal (FIG. 7a), high KCl levels (FIG. 7b), but not by ODQ (FIG. 7c). Indomethacin is Cpd
The inhibitory action of .8 was enhanced (FIG. 7d). No time-dependent change was observed (Figure 7e).

Cpd. 12: FIG. 8 shows the concentration-expansion curve obtained for Cpd. 12 using aortic rings isolated from rats pre-constricted with submaximal concentrations of noradrenaline. these,
(a) intact endothelium,
(b) Nitric oxide synthase inhibitor N “-nitro-L-arginine (NOLA 10 μM),
(c) 40 mM KCl,
(d) a soluble guanylate cyclase inhibitor 1H- [1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ 10 μM),
(e) performed in the absence and presence of the cyclo-oxygenase inhibitor indomethacin (10 μM);
(f) shows time (30 minutes) control. n = number of experiments.

The vasodilatory effect on Cpd.12 was inhibited by endothelium removal (FIG. 8a) and high levels of KCl (FIG. 8c), but either NOLA (FIG. 8b) or ODQ (FIG. 8d) or indomethacin (FIG. It was not inhibited even in 8e). No time dependent change was observed (Figure 8e).

Protocol 3: Protective effect of isoflavone metabolites against endothelial damage induced by oxidized low density lipoprotein FIG.
(A) Oxidized low density lipoprotein (Bull LDL, 0.3 mg protein / ml),
(B) Bull LDL + Cpd.8 (3 μg / ml)
2 shows total concentration response curves for acetylcholine obtained using an aortic ring isolated from rats pre-constricted with submaximal phenylephrine with and without 30 min incubation. FIG. 10 shows submaximal concentrations with and without bull LDL alone and bull LDL + 17β-estradiol or Cpd. 5, 7, 8 or 12 co-incubation. 2 represents a histogram showing the maximum dilation obtained for acetylcholine using rat isolated aortic rings pre-constricted with phenylephrine. n = number of experiments.

Incubation with bull LDL significantly reduced the response to acetylcholine (FIG. 9a). Co-incubation of bull LDL with Cpd. 8 reduced the inhibitory effect of bull LDL (FIG. 9b).

Furthermore, the maximum response obtained for acetylcholine when co-incubated with this compound was significantly lower than in the case of single incubation with bull LDL (FIG. 10). Co-incubation of bull LDL with β-estradiol and other compounds reduced the effect of bull LDL so that the significant difference between maximal expansion obtained before and after bull LDL incubation was no longer apparent. However, the maximum expansion for acetylcholine obtained with co-incubation is the bull LD
It was not different from that obtained in the presence of L alone (FIG. 10).

Discussion These tests
Isoflavone compounds and derivatives are
(a) inhibits the vasoconstrictive response to noradrenaline;
(b) be vasodilator, and
(c) Demonstrate that Cpd. 8 significantly protects against endothelial damage due to oxidized LDL. The in vitro vascular profile of some of these compounds is comparable to and in some cases more effective than the ovarian steroid 17β-estradiol. This suggests that these compounds can play a cardioprotective effect attributed to high isoflavone diets, but more importantly, especially from isoflavones taken in the normal diet It suggests that these compounds may be useful as potential cardioprotective therapeutics when administered at higher doses than are obtained.

Cpd. 5, 7, 8 and 12 were all able to antagonize the contractile action of noradrenaline, but to a different extent. Cpd. 12 is the most effective and comparable to 17β-estradiol. Cpd
.5 and Cpd. 7 seemed to be comparable but less potent than Cpd. Cpd
.8 was the least effective. The antagonism of Cpd.5, 7 and 8 was dose dependent, but the dose dependency of Cpd.12 and β-estradiol was not examined. At equal volumes, vehicle (DMSO) was not effective in inhibiting the response to noradrenaline.

The vasodilatory effect of isoflavone metabolite synthetic derivatives was at least as strong, if not more potent than 17β-estradiol, but the mechanism of action was that the dilatory effect of all four compounds was reduced upon removal of the endothelium. However, the mechanism of action of 17β-estradiol was different.

None of the compounds were affected by indomethacin, suggesting that prostacyclins are unlikely to play a substantial role in their vasodilatory capacity. However, indomethacin has enhanced the action of Cpd.8 in which an increase in vasodilatory capacity is observed. Cpd.8 is a vasoconstrictive prostanoid (va
increase the release of soconstrictory prostanoid), suggesting that an expansion was observed upon its removal. Both Cpd.5 and Cpd.7 extended responses are associated with high levels of KCl, ODQ and NOLA.
It was inhibited by. Thus, the dilating action of both of these compounds is endothelium-derived relaxing factor,
It may possibly be associated with the release of nitric oxide that activates soluble guanylate cyclase activity.

Inhibition with KCl suggests that endothelium-derived hyperpolarizing factor (EDHF) is also released. It is recognized that there are limitations to the use of KCl in isolating EDHF responses, which warrants further investigation through the use of specific K + channel inhibitors such as apamin and charybdotoxin. Assuming limitations, Cpd.5 and Cpd.
There is no evidence to suggest that the hyperpolarizing factor released by 7 is not nitric oxide.

On the other hand, the expansion effect of Cpd. 8 and Cpd. 12 can contribute to EDHF as a whole,
This is clearly not nitric oxide and is not activated by soluble guanylate cyclase.
Thus, it is clear that the mechanism of action by which these four compounds cause vasodilation is not only different from ovarian steroid β-estradiol and its parent compound, but also from each other.

In the final series of experiments, the protective effect of these compounds against endothelial damage induced by oxidized LDL was examined. Oxidized LDL inhibited the vasodilatory ability of the endothelium-dependent vasodilator acetylcholine. All compounds tested reduced the response to acetylcholine caused by bull LDL, but Cpd. 8 has a significant effect on the response to acetylcholine in direct comparison with single incubation with bull LDL. It was the only compound that could demonstrate It has already been demonstrated that the 17β-estradiol can protect against endothelial damage by bull LDL. From the current data it is clear that in this connection the protective action of Cpd.8 is at least 10 times stronger than 17β-estradiol. Since Cpd. 8 is not the most potent compound in other protocols, ie, in antagonism of noradrenaline or in its direct vasodilatory capacity, the mechanism of cardioprotective action of this compound is And may be present in its antioxidant capacity.

These results indicate that Cpd. 5, 7, 8, 12 has a strong vascular regulatory ability. Therefore, isoflavones and their derivatives can offset contractile activity (Cpd. 12>
Cpd.5 = Cpd.7> Cpd.8), directly offsets vasodilation and protects against endothelial damage by oxidized low density lipoprotein (Cpd.8 is at least more than 17β-estradiol in this context) 10 times more powerful). While comparable to the ovarian steroid 17β-estradiol, their activity also appears to be unique in their mechanism of action. Thus, isoflavones and derivatives are used as cardioprotective therapies, especially the treatment of restenosis and other types of accelerated intimal thickening
The potential for use in the treatment of

Vascular smooth muscle cell action Vascular smooth muscle cell proliferation is an important step in the atherosclerotic process,
Inhibited by estrogen. Atheroprotection of isoflavone compounds and derivatives (at
To investigate the eroprotective properties, experiments were performed to select simple derivatives of DNA synthesis and cell proliferation, namely selected synthetic derivatives of isoflavone metabolites on [3H] -thymidine incorporation and cell number, Cpd. The effects of 8 and 12 were examined. In view of the promising results obtained for Cpd. 7, mitogen-activated protein (MAP) kinase (Erk, JUNK, p38)
The signal pathways mediating these activities were examined by determining the effect of Cpd.

Method Cell Preparation Vascular smooth muscle cells (VSMC) from female internal thoracic artery (IMA) were cultured in 10% FBS-DMEM. Cells are grown to confluence in LG (glucose 5 mM) medium, then trypsinized, and 10% FBS-LG
Dilute to approximately 10,000 cells / ml with media and aliquot the 1 ml suspension into four 24-well plates. Cells were then grown for 2 days to visually confluence. LG serum-free medium was changed for 48 hours to synchronize most cells to G0 / G1.

Cell processing:
After 24 hours in serum-free medium, the cells are treated with compound (Cpd.5, Cpd.7, Cpd.8 or Cpd.12).
Treated for 4 hours and added FBS overnight (18-20 hours) (final concentration 2.5%). Four wells were assigned to each treatment:
(1) Control: 2.5% FBS
(2) FBS + isoflavone treatment (0.01 mM)
(3) FBS + isoflavone treatment (0.1 mM)
(4) FBS + isoflavone treatment (1 mM)
(5) FBS + isoflavone treatment (10 mM)
(6) FBS + isoflavone treatment (100 mM)

Cell growth was induced by various growth factors such as serum and platelet derived growth factor (PDGF).

Thymidine incorporation assay:
The medium was changed to LG serum-free DMEM and cells were treated with [3H] -thymidine at 1 μCi / well for 3 hours. Cells were washed twice with ice-cold Dulbecco's PBS. Then ice-cooled to 0.
2M HClO4 (1 ml / well) was added and the cells were incubated on ice for 30 minutes. The cells were then washed 3 times with ice-cold 0.2M HClO4 (0.5 ml / well). Medium (0.5 ml / well 0.2 M NaO
H) was changed and the cells were incubated at 37 ° C. After 1 hour, the cells were acetic acid (6%, 0.2 ml /
Well) and transferred to scintillation vials with 3 ml scintillation fluid (Instagel) and counted for 2 minutes per vial.

Results In preliminary experiments, Cpd.7 at a concentration as low as 0.1 mM significantly inhibits the growth of IMA-derived VSMC cells (n = 3) compared to control (0) (# cell damage) (* P <0.05)
(See FIG. 11). Interestingly, the effect is low (up to 1 mM)
Only the observed increase in proliferation was measured at 10 mM and 100 mM (FIG. 11). C
pd.12 did not appear to inhibit cell proliferation, but caused significant cell damage at high concentrations (10 mM and 100 mM) (FIG. 11). Tests are currently underway to examine this effect in more detail and determine whether it is due to apoptosis. Interestingly, it was found that Cpd.8 caused a significant increase in proliferation at high concentrations (10 mM and 100 mM) (FIG. 11). Cpd. 5 was found to be relatively inactive.

The effects of Cpd. 5 and Cpd. 7 were further tested in IMA-derived human VSMCs sensitized with PDGF BB instead of serum. In addition, Cpd. 7 inhibited the growth of IMA cells at a concentration as low as 0.1 nM (
(N = 1) (* P <0.005) vs. control (0); #P <0.01 vs. control (0))
(FIG. 12). This effect was equivalent to that observed with estradiol. Cpd. 5 had no effect on cell proliferation.

In further experiments, Cpd. 7 inhibits platelet-derived growth factor (PDGF) BB-induced DNA synthesis and is assessed by [3H] -thymidine incorporation in a dose-dependent manner with 10% inhibition at 1 nmol / L, 10 and 100 nmol. It was 17% inhibition at / L. The inhibition was statistically significant but was low compared to the effect of 17β-estradiol (27% at 1 or 10 nmol / L, 33% at 100 nmol / L). ICI 182780, a non-selective estrogen receptor (ER) antagonist (100 nmol / L)
It completely eliminates the inhibitory action of Cpd.7 at 10 nmol / L, partially eliminates the inhibitory action of Cpd.7 at 100 nmol / L, and shows ER mediated. Consistent with our results using thymidine incorporation, cell count PD
The GF BB induced increase was partially but significantly reduced by Cpd.7 (1-100 nmol / L).

To further investigate the signaling pathway that mediates this phytoestrogens action in VSMC, the effect of Cpd. On mitogen-activated protein (MAP) kinase (Erk, JUNK, p38).
The effect of 7 was measured by immunoprecipitation analysis, and the effect on the early growth response gene (Egr-1, Sp-1) was measured by Western blotting. PDGF BB activates Erk, which is
It was significantly inhibited by Cpd. 7 in a dose-dependent manner, and the activity of JUNK and p38 was observed in PDGF BB or Cpd.
Although not affected by any of 7, the cells strongly expressed both proteins as well as Erk. Egr-1 or Sp-1 protein was not altered by either PDGF BB or Cpd.

These results indicate that isoflavones and their derivatives, and in particular Cpd. 7, inhibit cell proliferation of human vascular smooth muscle cells and also inhibit PDGF-induced Erk activation in human vascular smooth muscle cells. This activity demonstrates the potential for the development and progression of atherosclerotic lesions, particularly isoflavones and their derivatives that prevent accelerated stenosis and provide a potential benefit in vascular protection.

Antioxidant activity Oxidation is a process that plays a role in many major disease processes. One of the main contributors to the development of atherosclerosis in the context of cardiovascular disease is in the arterial wall
Oxidation of LDL cholesterol, which leads to inflammatory lesions. If left unexamined, the inflammatory process proceeds until the lesion causes vascular occlusion and infarction and presents a particularly important problem in the accelerated intimal thickening of restenosis. In view of potential cardiovascular benefits of antioxidant activity, selected isoflavone compounds of the present invention were assayed to determine their antioxidant activity.

LDL antioxidant test
The LDL antioxidant test measures the ability of a compound to scavenge free radicals directly or chelate transition metals. Therefore, in this series of experiments, isoflavones and their derivatives were tested for their ability to act as free radical scavengers.

Method
Fresh whole blood was collected by venipuncture from healthy human volunteers aged 25 years or younger and EDTA (91
mg / ml) and BHT (4.4 mg / ml) were added immediately. LDL was prepared by step-wise ultracentrifugation within a density gradient of 1.02 to 1.05 g / cm3. EDTA and
BHT was present throughout all stages of isolation. EDTA / BHT-containing stock solution (15-30 mg LDL / ml
) Was stored in the dark in a nitrogen atmosphere until use, but for a period of up to 2 weeks.

Before the oxidation experiment, 100 times the volume of 0.00 by vacuum degassing and then purged with nitrogen to make oxygen free.
The LDL stock solution was dialyzed in 1 M phosphate buffer pH 7.4, 0.16 M NaCl, 0.1 mg / ml chloroamphenicol. The buffer was changed 4 times. This EDTA and BHT free LDL stock solution was used for all oxidation tests. The stock solution was stored at 4 ° C. for up to 24 hours. To perform oxidation experiments, EDTA and BHT-free LDL stock solutions were diluted with oxygen-saturated 0.01 M phosphate buffer pH 7.4, 0.16 M NaCl and oxidized by addition of freshly prepared CuCl 2 aqueous solution. Started. Final conditions are in all experiments: room temperature, 0.25 mg LDL / ml and 1.66
mM CuCl2 (see Esterbauer et al., 1989).

The following compounds were tested for antioxidant activity: daidzein (10 mM), genistein (10 mM)
Glycitein (10 mM), biocanin (10 mM), formonenetin (10 mM), Promensil (Novogen) (2.5 mg / ml, 5 mg / ml), Cpd. 5 (10 mM), Cpd. 7
(10 mM), Cpd. 8 (10 mM), Cpd. 12 (10 mM), Cpd. 4 (10 mM) and Cpd. 6 (10 mM).
In each experiment, controls were used to compare the relative potency of Novogen isoflavone derivatives with common and known antioxidants such as ascorbate (2.5 mM; vitamin C).

Results At the concentration tested (10 mM), it was found that the parent isoflavone did not show any ability to scavenge free radicals. On the other hand, promencil was a moderate scavenger and showed an increase in lag time to 100% (at 5 mg / ml) when compared to the positive control (see Table 2).

When isoflavone derivatives are tested, Cpd. 12 (10 mM) and Cpd. 8 (10 mM) are very active scavengers and can increase lag time by over 600% when compared to positive controls. (See Table 3). Cpd. 7 was found to be significantly less active than Cpd. 8 and Cpd. 12, but was still nearly as active as ascorbate.

Prevention of tocopherol-mediated peroxidation (anti-TMP test)
A test was performed to determine if the isoflavone derivative could prevent LDL oxidation in the presence of vitamin E. This study is physiologically due to the presence of vitamin E (a-tocopherol) in the bloodstream with LDL and because LDL oxidation is considered to be one of the major factors in the development of atherosclerosis. is important.

Methods The anti-TMP test is described in detail by Bowry et al. (1995). The test indirectly evaluates the ability of compounds to synergize with a-tocopherol in human LDL undergoing mild chemically controlled oxidation. Oxidation is measured by the accumulation of cholesterol ester hydroperoxide at a time point corresponding to 20% consumption of endogenous a-tocopherol.

In short, ascorbate and ubiquinol-10 free LDL (1 mM in Apo B)
An aliquot of a stock solution of the compound to be tested was added. The mixture was incubated at 37 ° C. for 10 minutes, then with low flux water soluble ROO − produced from AAPH (4 mM)
Oxidized at 37 ° C. The time-dependent consumption of LDL a-tocopherol and the accumulation of cholesteryl linoleate hydroperoxide (Ch18: 2-OOH) were monitored by HPLC using electrochemical and post-column chemiluminescence detection, respectively. Antioxidant efficacy, identified as an anti-TMP index (redox index), is compared to that of endogenous a-tocopherol in a control sample (ie, in the absence of antioxidant).
Measured after% consumption, Ch18 produced with and without antioxidant
: Defined as the relative amount of 2-OOH. The active compound produces a low redox index. High redox activity suggests that the compound can interact with a-tocopherol in LDL, possibly by reducing a-tocopheroxyl radicals. Butylated hydroxytoluene (BHT, 10 mM) was used as a positive control.

result
Cpd. 12 (10 mM) was found to have a strong LDL protective action (low redox index) and prevent LDL oxidation in the presence of vitamin E. In addition, Cpd. 7 and Cpd. 8 showed low levels of activity. The results are summarized in Table 4.

In addition, a number of parent isoflavones were tested and found to have no antioxidant action. Promencil showed a low level of activity (see Table 5).

Synergistic action with a-tocopherol (TRAA test)
The purpose of this test was to cetyltrimethyl ammonium chloride
To evaluate the ability of isoflavone derivatives to reduce a-tocopheroxyl radicals in chloride and sodium dodecyl sulfate micelles.

Methods a-Tocopheroxyl radical attenuating ability (TRAA) assay is described by Witting et al. (1997).
Are described in detail. That is, 100 mM stock solution of cetyltrimethylammonium chloride (HTAC) and sodium dodecyl sulfate (SDS) was prepared in phosphate buffer. A micelle dispersion of a-tocopherol was prepared by diluting an ethanol solution of a-tocopherol (0.2M) into micelles at a final vitamin concentration of 500 mM. The resulting solution was sonicated for 15 seconds, at which point it became completely homogeneous.

LDL was isolated by ultracentrifugation of freshly collected heparinized plasma from a non-fasting healthy male donor (27 years old). LDL was obtained by direct aspiration and stored at 4 ° C. for 16 hours before use. Immediately before use, excess KBr and residual low molecular weight water-soluble antioxidants can be removed from LDL to PD-10 columns (Pharm
acia, Uppsala, Sweden) was removed by gel filtration chromatography and the concentration of LDL was measured as described by Lowry et al. (1951).

An aliquot of micelles containing a-tocopherol is placed in the neck of an ESR flat cell (100 mL) and 0.5 m from a 125 W Osram HQL-Mercury fluorescent bulb used as a UV light source.
Arranged. In order to increase the light intensity, the frosted casing of the bulb was removed. Sample 3
Irradiate for 1 minute, then mix well, then transfer the flat cell to the corresponding temperature-controlled Dewar insert in the ESR cavity where the sample is 37 ° C
Equilibrated to This approach is similar to that estimated for the 10 mM TPDI nitroxide standard.
It resulted in a-tocopheroxyl radical (a-TO) between ˜2 mM. Using a Bruker ESP 300 ESR spectrometer equipped with an X-band cavity, an ESR spectrum was obtained at 9.41 GHz with a modulation amplitude of 1.0 G, a microwave power of 20 nW and a modulation frequency of 12.5 kHz.

After accumulation of the T = 0 min spectrum, the flat cell array containing a-TO was removed from the ESR cavity, and the solution was gently introduced into the neck of the flat cell under positive pressure. The selected compound was then added to bring the final concentration to 10 mM and the treated sample was re-entered into the cavity to equilibrate with standard conditions and sampling was resumed. Time-dependent decay of ESR signal intensity for a-TO, with and without co-antioxidant (10 mM), using a 20.5 second sweep time Measured and averaged the output from three consecutive cleanings at each time point and averaged the results of three separate experiments.

Results Results are expressed as the relative rate constant of decay of the a-tocopheroxyl radical in the presence of the test sample divided by the relative rate constant of decay of the a-tocopheroxyl radical in the absence of the test sample. . Although the TRAA approaching unity is considered to have weak synergistic activity, the active compounds show large values because they immediately eliminate a-tocopheroxyl radicals when mixed. Ascorbate was used as a positive control.

Cpd. 12 induced a high rate of decay and was found to be active when the a-tocopheroxyl radical was eliminated. The results are summarized in Table 6.

However, oxidation reactions other than those catalyzed by tocopheroxyl radicals may also be important. Therefore, it was investigated whether the derivatives possess additional antioxidant activity.

4.5 Scavenging of peroxyl radicals
The efficacy of isoflavone derivatives to inhibit linoleate oxidation induced by peroxyl radicals (in the absence of vitamin E) was tested. This test provided information on whether the derivatives had radical scavenging activity independent of vitamin E.

Methods Peroxyl radicals are natural byproducts of many enzymes, such as lipoxygenase and cyclo-oxygenase. Peroxyl radicals are thermolabile azo initiators (thermo
-labile azo-initiator) produced by AAPH. AAPH-induced oxidation of linoleate was performed as described above (Pryor et al., 1993) except that lower linoleate concentrations were used and SDS was omitted. An aliquot of linoleate stock solution was added to PBS at 37 ° C. to a final concentration of 500 mM. AAPH (final concentration 87.5) in the absence and presence of either 10 mM Cpd. 7, Cpd. 8 or Cpd. 12, or 500 mM ascorbate (positive control).
Oxidation was initiated by the addition of mM). Formation of linoleate hydroperoxide, Perkin
Monitoring by UV234nm using an Elmer UV / Vis Lambda 40 spectrophotometer. The test was performed twice as a single analysis. Appropriate concentrations of alcohol (ethanol and isopropanol; 0.13-0.2% (v / v)) were included in each control sample.

Dulbecco's PBS was purchased from Sigma, St Louis, MO. Water-soluble azoperoxyl radical generator 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) is a Polyscie
Purchased from nces, Warrington, PA. Ascorbate (sodium salt) was purchased from Merck. AAPH (350 mM) stock solution was prepared by serial dilution with Dulbecco's PBS. A stock solution of test compound (10 mM) was prepared as an ethanol solution, while a stock solution of linoleate (300 mM) was made by dissolving in isopropanol.

result
Inclusion of 10 mM Cpd. 12 was found to significantly inhibit the time-dependent increase in 234 nm absorbance induced by exposure of linoleic acid to AAPH, which indicates that Cpd. It shows that it is an effective agent for ruradicals. Similar results were obtained in two separate experiments. Ascorbate (used as a positive control) is linoleic acid AAPH
Prevents induced oxidation, which is consistent with existing literature.

4.6 Prevention of Peroxidase-Induced Oxidation of Linoleate The purpose of this test was to test whether isoflavone derivatives could affect the heme-catalyzed oxidation reaction. Such oxidation reactions are believed to be related in vivo and can occur using lipids, proteins and / or DNA as substrate (s).

Method The test is similar to the peroxyl scavenging described in Section 4.5 above, except that horseradish peroxidase (HRP) + hydrogen peroxide (H2O2) was used instead of the peroxyl radical generator.

Increased heme levels are considered a potential risk factor for atherosclerosis,
Heme-containing proteins can be involved in oxidative modification of LDL. In the presence of H2O2, heme proteins are potent oxidants (referred to as compound I) that can oxidatively modify lipids, such as lipids in LDL.
Produce. Therefore, the ability of isoflavones to inhibit such lipid oxidation was tested using horseradish peroxidase as a model heme protein. The HRP / H2O2-induced oxidation of linoleate is mainly due to the replacement of LDL with linoleate,
It was similar to that described by ng et al (1997). Add linoleate stock at 37 ° C.
Add to BS to a final concentration of 300 mM, 10 mM Cpd. 7, Cpd. 8 or Cpd. 12 or 2
HRP (40 U / mL) / H in the absence and presence of any of 5 mM BHT (positive control)
Oxidized with 2O2 (2 mM). The formation of linoleate hydroperoxide, Perkin Elmer
Was monitored by UV 234 nm using a UV / visible lambda 40 spectrophotometer. The above test was performed twice as a single analysis. Appropriate concentrations of alcohol (ethanol and isopropanol; 0.13-0.2% (v / v)) were included in each control sample.

Dulbecco's PBS, butylated hydroxytoluene (BHT) and acid free linoleate were purchased from Sigma, St. Louis, MO. Horseradish peroxidase (HRP; Gra
d II; 100,000 U / 502.5 mg lyop. Powder) was purchased from Boehringer Mannheim (Germany). Hydrogen peroxide (H2O2; 30% (w / v)) was purchased from Merck. HRP (6000 U / mL) stock solution and H2O2 (1 M) stock solution were prepared by serial dilution with Dulbecco's PBS. BHT (25 mM) and test compound (10 mM) were prepared as ethanol solutions, while a stock solution of linoleate (300 mM) was made by dissolving in isopropanol.

Results A test was carried out to see if isoflavones could react with the strong oxidized compound I.
Therefore, horseradish peroxidase + H2O2 was converted to an isoflavone derivative (Cp
d.7, Cpd. 8 or Cpd. 12). Both Cpd.7 and Cpd.8 are responsible for linoleic acid oxidation.
It has been found that it can be effectively inhibited by Compound I as well as 25 mM BHT (used as a positive control). Surprisingly, the inhibitory activity of Cpd. 7 and Cpd. 8 is equivalent to equimolar amounts of Cpd.
Although greater than 12, this suggests that their mechanism of action can be complex.

4.7 Inhibition of lipoxygenase Lipoxygenase is a potentially pro-inflammatory enzyme that requires oxidative activation and releases peroxyl radicals upon catalysis. Isoflavone derivatives were tested for their ability to affect lipoxygenase activity in vitro using soybean and rabbit reticulocyte 15-lipoxygenase and linoleic acid as a substrate.

Methods 15-lipoxygenase has been suggested to be involved in the early stages of LDL oxidation in vivo and has been suggested to contribute to atherogenesis (Kuhn et al., 1994; Fol
cik et al., 1995). Soybean 15-lipoxygenase (SLO) is an appropriate model for 15-lipoxygenase and was therefore used to assess the ability of Cpd. 7, Cpd. 8 and Cpd. 12 to inhibit this enzyme. As above, SLO-induced oxidation of linoleate was performed (U
pston et al., 1996). In short, linoleate (100 mM) in PBS was replaced with 10 mM Cpd. 7, Cp.
d.8 or Cpd.12 or 100 mM eicosatetraenoic acid (ETYA, positive control)
Oxidized with SLO (60 U / mL) in the absence and presence of. The formation of linoleate hydroperoxide was monitored by UV234nm using a Perkin Elmer UV / visible lambda 40 spectrophotometer. The above test was performed twice as a single analysis. Appropriate concentrations of alcohol (ethanol and isopropanol; 0.13-0.2% (v / v)) were included in each control sample.

Dulbecco's PBS, acid-free linoleate, soybean 15-lipoxygenase (SLO; 630,000)
U / mg protein) was purchased from Sigma (St. Louis, MO). Ascorbate (sodium salt) and eicosatetraenoic acid (ETYA) are Aldrich and Cayman Chemic, respectively.
Purchased from als. Stock solution of SLO (12,000 U / mL) and ascorbate (100 mM)
Stock solutions were prepared by serial dilution with Dulbecco's PBS. ETYA (33.3mM)
The stock solutions of and the test compound (10 mM) were prepared as ethanol solutions, while the linoleate (300 mM) stock solution was made by dissolving in isopropanol.

result
Cpd. 12 is an effective inhibitor of type 1 lipoxygenase, as shown by the strong decay of the increase in 234 nm absorbance. Indeed, even at 10 mM, Cpd. 12 showed an inhibitory effect comparable to 100 mM ETYA, a well-established inhibitor of lipoxygenase. As with the peroxyl radical, neither Cpd. 7 nor Cpd. 8 was able to inhibit SLO.

5.0 Lipid test 5.1 Distribution of isoflavones in lipoprotein subfractions The effect of edible soy protein on blood cholesterol levels in rabbits was determined by Meek
first reported by er and Kesten in the 1940s (see review by Kristchevsky, 1995)
. Later, evidence for the independent effects of isoflavones on blood cholesterol levels has been demonstrated in various models such as rats, hamsters, non-human primates and humans (Carroll, 1991; Anderson et al., 1995; Potter , 1996; Balmir et al., 1996; C
larkson et al., 1998). According to a report by Cassidy et al. (1995), in humans, 4
5 mg isoflavone results in a significant reduction in total and LDL cholesterol levels in young women, but not 23 mg isoflavone. Similar findings
er et al (1993) and Bakhit et al. (1994). In contrast, Nestel et
al. (1997) does not report a significant effect of 45 mg genistein (Cpd. 3) administered over a period of 5-10 weeks on blood lipid levels.

To confirm the relevance isoflavones of isoflavones in the regulation of blood cholesterol, experiments were performed to examine the affinity of isoflavone derivatives for serum LDL and HDL. In the preliminary test, the results are shown below, but one compound, namely Cpd.
Only (Daizein) was used.

Method
Cpd. 1 (10 mM and 50 mM) was incubated with whole plasma for 0, 4 and 8 hours. Next, the different subfractions LDL / VLDL, HDL and protein (no lipoprotein) were analyzed for their Cpd.1 concentration. Cpd. 1 was used because it has the polarity most similar to the isoflavone synthetic derivative.

Results It was found that a small but significant amount of Cpd.1 (daidzein) associates with lipoproteins. 2% of Cpd. 1 was found to be associated with LDL, 6% was found to be associated with HDL, and 92% was associated with the protein fraction. The amount associated with lipoproteins increased with time,
It was not accompanied by concentration. The results are shown in FIG. 13 (Cpd.1, 10 mM) and FIG. 14 (Cpd.1, 50 mM).

5.2 Modulation of LDL receptors A high risk factor for cardiovascular disease is elevated LDL cholesterol levels. Excessive cholesterol makes the chances of oxidative damage and thus atherosclerosis greater. Estrogens have a number of potentially beneficial effects on the development of atherosclerosis and the outcome of cardiovascular events. 17β-estradiol has been shown to increase LDL receptor activity. Although there is evidence that plant-derived estrogens (ie, phytoestrogens such as isoflavones) may have protective activity against the development of cardiovascular disease, the underlying mechanism for this remains unclear. Given the structural similarity between isoflavones and their derivatives and certain parts of estrogens, it is interesting to test whether isoflavones and related compounds can have similar molecular actions. Several studies have suggested that the mechanism by which isoflavones (and estrogens) improve blood lipid profiles may be due to changes in the amount or activity of LDL receptors (Baum et al., 1998; Kirk et al., 1998). It remains unclear whether isoflavone derivatives increase cholesterol turnover and increase its removal from the bloodstream by affecting LDL receptor activity. Therefore, by examining the specific binding of labeled LDL in control cells versus cells pretreated with test compounds, in vitro
A study was conducted to examine the ability of Cpd. 7, Cpd. 8 and Cpd. 12 to stimulate LDL receptor binding in human hepatocytes in tro.

Method
In complete DMEM medium containing 10% fetal bovine serum (FBS), test compounds (final concentration 50 nM, 5
Confluent human hepatoma (HepG2) cells were preincubated for 48 hours in the absence or presence of 00 nM and 10 mM Cpd. 7, Cpd. 8 and Cpd. 12). Serum deprivation (ie, 0.5% FBS) is used as a positive control and L
Induced DL receptor expression. After preincubation, after washing the cells, 50 mg [125
I] -LDL (specific activity 6.5-29.5 cpm / fmol) was added and the cells were added for an additional 4 hours at 4 ° C. in the absence (due to total binding) and presence (non-specific) of 500 mg unlabeled LDL In order to assess binding). The cells were then washed and lysed (0.1 M NaOH) to determine the lysate radioactivity and protein content (BCA assay). The number of LDL receptors was determined from specific binding (total binding-non-specific binding), but one molecule of LDL was assumed to bind per LDL receptor. Three independent experiments (each tested in triplicate) were performed using different batches of cells and [125I] -LDL.

Results To determine the concentration of LDL required to reach binding saturation, confluent HepG2 cells were titrated in [125I] -LDL concentration in the presence of excess (ie 500 mg) unlabeled LDL. Incubated in well plates. Saturation was reached with about 50 mg of [125I] -LDL. Using this concentration, the time required to reach saturation binding at 4 ° C. was determined to be about 4 hours. These established optimal conditions were used for all subsequent binding assays.

Overall,
A number of LDL receptors measured in different series of experiments were different from each other (Table 7).

As can be seen from the results expressed as an incremental increase in LDL receptor expression over control, Cpd. 12 did not show consistent effects, but Cpd. 7 and Cpd.
8 increased LDL binding. In the case of Cpd. 7 (but not Cpd. 8), LDL receptor expression increased in a concentration-dependent manner, but the magnitude of this increase was small when compared to that obtained by serum withdrawal. Cpd. 8 had no obvious concentration-dependent effect.

Although the observed effects were small, at least as compared to serum withdrawal, increased LDL binding was consistently observed in separate sets of experiments using different batches of both cells and LDL, which Suggests that the observations made reflect the true activity of Cpd.7 and Cpd.8. These results show the potential of isoflavones and their derivatives to prevent or delay the onset and progression of atherosclerotic plague restenosis.

Angiogenic activity Angiogenesis occurs through a number of distinct steps that can be defined as endothelial cell activation, migration, proliferation and reorganization, forming mature blood vessels characterized by the lumen. The effects of isoflavone derivatives on angiogenesis, particularly endothelial cell proliferation and endothelial cell migration, are evaluated by in vitro assays.

4.1 Method of action of isoflavone derivatives on endothelial cell proliferation:
Cell preparation Human umbilical vein endothelial cells between passages 1 and 3 were used. Cells were grown in gelatin-coated flasks in medium 199 containing Earle salt supplemented with 20% fetal calf serum, 25 mg / ml endothelial cell growth supplement and 25 mg / ml heparin. Cells from two different donors were used.

Proliferation assay
5 × 10 3 cells in 100 ml growth medium were plated into gelatin coated microtiter wells. Test compounds were added during plating. Next, the cells were incubated for 3 days, and the number of cells was measured by a colorimetric assay (CellTiter96 Aqueous assay, Promega). The results were compared with a standard curve and performed in each experiment. The assay was performed on two different days using two different endothelial cell isolates. The effect of isoflavone derivatives on human umbilical vein endothelial cell proliferation is shown for donor No. 1 (FIG. 15) and donor No. 2 (FIG. 16).

result:
Figures 15 and 16 show the effect of Cpd.5, Cpd.7, Cpd.8 and Cpd.12 on endothelial cell proliferation. Both Cpd. 8 (1-10 mg / ml) and Cpd. 12 (0.01-10 mg / ml) inhibited cell growth and produced no toxicity. Interestingly, considerable lineage variability was observed in cellular responsiveness to these compounds (FIGS. 15a and 16a). Cpd.5 (0.01-10
mg / ml) was found to have no apparent effect on endothelial cell proliferation in either of the two cell isolates tested (FIGS. 15b and 16b). Similarly, Cpd. 7 (0.
01-10 mg / ml) did not show any appreciable antiproliferative effect on the two cell isolates tested (FIGS. 15c and 16c).

4.2 Method of action of isoflavone derivatives on endothelial cell migration:
Cell preparation Human umbilical vein endothelial cells between passages 1 and 3 were used. Cells were grown in gelatin-coated flasks in medium 199 with Earle salt supplemented with 20% fetal bovine serum, 25 mg / ml endothelial cell growth supplement and 25 mg / ml heparin.

Migration assay
5 × 10 5 cells / well in growth media were plated on fibronectin-coated 6-well trays and grown to confluence. The use of a cell scraper (Costar) created a wound along the monolayer. The cells were then washed once and fresh medium containing the test compound was added. Ink capacitors (Olympus microscope) are defined points along the wound.
s) was used to visualize cell migration at fixed points for the next 48 hours. Photographs were taken at appropriate times to assess the extent of cell migration from the front of the wound.

result:
FIG. 17 shows the effect of Cpd. 12 on endothelial cell migration. The photograph shows the amount of cell migration at the baseline (0 hours) (Figure 17a) and 30 hours after wounding. Treatment with Cpd. 12 (final concentration 1 mg / ml) markedly inhibited endothelial cell migration towards the wound after 30 hours (Figure c).
). DMSO (solvent control) had no effect on proliferation, and after 30 hours of treatment, the cells migrated towards the wound to form a monolayer (FIG. 17b). Pictures were taken at various points along the wound, which remained constant throughout the course of the experiment. An initial wound front was marked.

The results of this example show that Cpd. 8 and Cpd. 12 inhibited endothelial cell proliferation and Cpd. 12 inhibited endothelial cell migration. Thus, isoflavones and derivatives thereof show potential for use as cardioprotective therapy, particularly after vascular treatment and surgery.

Inhibition of cell adhesion molecule expression
The ability of isoflavone compounds and derivatives to inhibit the expression of E-selectin has been demonstrated by Litwin et a
l. Measured according to the protocol described in (1997), which is incorporated herein by reference.

FIG. 18 presents comparative data regarding the ability of a number of isoflavone compounds to inhibit E-selectin expression performed according to the method of Litwin et al. The interaction of isoflavone metabolites Cpd. 8 and 12 in human umbilical vein endothelial cells activated by TNF- "was measured against compound concentration. Cells were either 2 or 18 hours prior to TNF stimulation. In comparison to DMSO control, isoflavone Cpd.8 showed very good inhibition of E-selectin expression, especially at 10 "g / ml. Isoflavone Cpd. 12 is especially 1 "g
It showed excellent inhibition of E-selectin expression at concentrations of / ml and 10 "g / ml.

This example demonstrates the ability of the claimed isoflavones and derivatives to respond to numerous signals known to be active in restenosis, inflammatory responses and other diseases mediated by cell adhesion molecule expression. It demonstrates that it specifically blocks the ability of E-selectin to be expressed.

Restenosis model-cell migration and proliferation An important feature of atherosclerosis in humans is the migration of vascular smooth muscle cells (VSMC) from the arterial media to the intima, followed by the intima It is the growth within.
The animal model of atherosclerosis used in this experiment is VS due to mechanical damage to the endothelium.
The aim was to induce MC migration and neointimal proliferation.
The probe was introduced into the femoral artery of the mouse and introduced into the aortic branch through the iliac artery. The probe was then withdrawn and the femoral artery was ligated to prevent bleeding. Collateral circulation (colat
eral circulation) prevents any limb-related ischemia. Neointimal proliferation occurred in the iliac arteries over the next 4 weeks.

Proliferation was enhanced by feeding the mice with cholesterol during this time (starting one week before surgery). At 4 weeks, mice were sacrificed and bilateral iliac blood vessels were collected (non-operative blood vessels were used as controls). Neointimal proliferation is expressed as the ratio of intima area to intima + media area.

This technique (mainly using gene knockout mice) was found to be very reproducible using a sample size of 5-10 mice. FIG. 19 shows a typical growth profile in normal mice. Minimal proliferation was observed at 3 weeks, and a substantial increase in proliferation was observed at 4 weeks, followed by further increase at 5 weeks. Minimal neointimal proliferation occurred in control non-surgical blood vessels (“non-vascular”) from the opposite side.

The ability of isoflavone derivatives to reduce and / or prevent “atherosclerotic” responses in a mouse model of neointimal hyperplasia was investigated. The compounds tested were Cpd. 5 and
Cpd.12.

Methods Atherosclerosis was induced in the femoral artery of each mouse by mechanically removing the endothelium using a probe. The ability of isoflavone compounds to inhibit atherosclerotic responses was assessed by comparing treated and placebo groups. C57 / B16 mice (8-
A 10-week-old male was anesthetized and the right femoral artery was deendothelialized using a dissecting microscope (de-endot
hilialized). These mice were also fed a 2% cholesterol diet to enhance disease progression. Mice were sacrificed at 4 weeks. Their treated arteries (right) and control arteries (
Left) were collected together and fixed in formalin for histological evaluation.

Mice were fed 2% cholesterol diet mixed with normal mouse diet for 1 week prior to surgery. Treatment groups were fed a diet containing Cpd.5 or Cpd.12 or Cpd.5 + 12. Mice were anesthetized with avertin and femoral artery angioplasty was performed. After the operation, the mice were moved onto a quiet dark warm mat and the recovery was closely monitored. Mice were housed in a sterile cage with a sterile bed to prevent infection. Mice continued on a cholesterol diet mixed with normal mouse diet for the next 4 weeks. Mice were euthanized 4 weeks after surgery. Those femoral arteries were collected for pathological examination. The sections were evaluated by image analysis to confirm the areas of the various sections of the arterial wall and the number of cells in each area. The main parameter evaluated was the increase in intima area expressed as the ratio of intima area to intima + media area. Sectional sections (every 50 "m or every 10th section) were sampled and stained with H & E. Endothelial, intima and medial cross-sectional areas were evaluated using image analysis.

Results FIG. 20 shows a transverse section through the iliac artery 4 weeks after surgery. FIG. 20a shows the absence of neointimal proliferation in the iliac artery from the non-operative side, where the intima is about 1 cell thick. FIG. 20b shows substantial neointimal proliferation in the iliac arteries in response to surgery, showing about 50% thickness of the vessel wall. Figures 20c and 20d are post-operative iliac arteries from mice treated with Cpd. 12 and Cpd. 5, respectively. Neointimal proliferation can be observed in each case, but proliferation is
In Cpd.5, it was significantly reduced.

FIG. 21 quantifies the effect of test compounds on neointimal proliferation, both individually and together. Untreated blood vessels (no surgery) show 5 ± 1% intimal thickness, whereas 50% of surgically treated blood vessels
Shows ± 5% inner film thickness. Cpd.5, both alone and in combination with Cpd.12, reduced neointimal proliferation to 30 ± 6% and 32 ± 10%, respectively, whereas Cpd.12 was the extent of neointimal proliferation. Had no detectable effect (50 ± 29%). Notably, these data are
Consistent with Cpd. 5 that significantly reduces neointimal proliferation, but has no appreciable effect Cpd.
Does not match.

It is noted that the area of neointimal proliferation corresponds well with the number of cells in the intima measured using image analysis.

These results indicate that isoflavones and their derivatives, particularly Cpd. 5, significantly inhibit VSMC proliferation in the neointima of a mouse model of atherosclerosis. The neointimal area was reduced by approximately 50% at the time tested (week 4). Cpd. 12 had no appreciable effect on neointimal proliferation in the mouse model at this point. Cpd.5 and Cp
The combination of d.12 did not appear to change the inhibitory effect observed with Cpd.5 alone. These results regulate VSMC migration and progression, and atherosclerotic plaques,
Therefore, it demonstrates the potential of isoflavones and their derivatives to prevent or delay the onset and progression of restenosis after mechanical injury, thereby providing an advantage in vascular protection.

The invention has been described herein with reference to certain preferred embodiments so that the reader may practice the invention without undue experimentation. However, one of ordinary skill in the art will readily recognize that numerous components and parameters may be changed or modified to the extent that they do not depart from the scope of the present invention. Further, titles, headings, etc. are presented to enhance the reader's understanding of the specification and should not be understood as limiting the scope of the invention.

The entire disclosure of all applications, patents and publications cited in this specification, if any,
Included herein by reference.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It should be understood that the invention encompasses all such changes and modifications. The invention includes all of the steps, features, compositions and compounds mentioned or shown herein individually or collectively, and any two or more of the steps or features. Includes any and all combinations.

Any reference to any prior art in the present specification will refer to the prior art as being subject to the research field (the
it is not a recognition or any form of suggestion that forms part of common general knowledge in the field of endeavor) and should not be so construed.

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Claims (27)

A method of inhibiting the expression or activity of an adhesion molecule associated with an endothelial cell, wherein said adhesion molecule or said endothelial cell comprises one or more compounds of formula I (including pharmaceutically acceptable salts thereof) ) With a sufficient amount to inhibit said expression or activity, wherein formula I is represented by:

Wherein R1, R2 and Z are independently hydrogen, hydroxy, OR9, OC (O) R10, O
S (O) R10, CHO, C (O) R10, COOH, CO2R10, CONR3R4, alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, alkylaryl, alkoxyaryl, thio, alkylthio, amino, alkylamino , Dialkylamino, nitro or halo, or R2 is as defined above, and R1 and Z, together with the carbon atom to which they are attached, are:

Or R1 is as defined above, and R2 and Z, together with the carbon atom to which they are attached, form the following:

And W is R1, A is hydrogen, hydroxy, NR3R4 or thio, and B is:

Or W is R1 and A and B together with the carbon atom to which they are attached are:

Or a W, A and B together with the group with which they are associated, the following:

Or W and A together with the group with which they are associated, the following:

And B is:

Selected from
in this case,
R3 is hydrogen, alkyl, arylalkyl, alkenyl, aryl, amino acid, C (
O) R11 (where R11 is hydrogen, alkyl, aryl, arylalkyl or amino acid), or CO2R12 (where R12 is hydrogen, alkyl, haloalkyl, aryl or arylalkyl);
R4 is hydrogen, alkyl or aryl, or R3 and R4 together with the nitrogen to which they are attached include pyrrolidinyl or piperidinyl,
R5 is hydrogen, C (O) R11 (where R11 is as defined above) or C
O2R12 (where R12 is as defined above);
R6 is hydrogen, hydroxy, alkyl, aryl, amino, thio, NR3R4, C (O)
R11 (where R11 is as defined above), CO2R12 (where R12
Is as defined above) or CONR3R4,
R7 is hydrogen, C (O) R11 (where R11 is as defined above), alkyl, haloalkyl, alkenyl, aryl, arylalkyl or Si (R13) 3 (
Wherein each R13 is independently hydrogen, alkyl or aryl),
R8 is hydrogen, hydroxy, alkoxy or alkyl;
R9 is alkyl, haloalkyl, aryl, arylalkyl, C (O) R11 (where R11 is as defined above) or Si (R13) 3 (where R13 is as defined above). ) And
R10 is hydrogen, alkyl, haloalkyl, amino, aryl, arylalkyl, amino acid, alkylamino or dialkylamino;
The drawing "---" represents either a single bond or a double bond,
T is independently hydrogen, alkyl or aryl;
X is O, NR4 or S, and Y is represented by the following formula:

Wherein R14, R15 and R16 are independently hydrogen, hydroxy, OR9, OC (O) R
10, OS (O) R10, CHO, C (O) R10, COOH, CO2R10, CONR3R
4, alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, aryl,
Heteroaryl, thio, alkylthio, amino, alkylamino, dialkylamino, nitro or halo. The method of claim 1, wherein the compound of formula I is represented by the following formulas II-VIII and comprises a pharmaceutically acceptable salt thereof:

(Wherein R1, R2, R5, R6, R14, R15, W and Z are as defined in claim 1 above). R1, R2, R14, R15, W and Z are independently hydrogen, hydroxy, OR9,
OC (O) R10, C (O) R10, COOH, CO2R10, alkyl, haloalkyl, arylalkyl, aryl, thio, alkylthio, amino, alkylamino, dialkylamino, nitro or halo,
R5 is hydrogen, C (O) R11 (where R11 is hydrogen, alkyl, aryl or amino acid) or CO2R12 (where R12 is hydrogen, alkyl or aryl);
R6 is hydrogen, hydroxy, alkyl, aryl, C (O) R11 (where R11 is as defined above), CO2R12 (where R12 is as defined above);
R9 is alkyl, haloalkyl, arylalkyl or C (O) R11 (where R
11 is as defined above), and R10 is hydrogen, alkyl, amino, aryl, amino acid, alkylamino or dialkylamino;
The method of claim 2 comprising a pharmaceutically acceptable salt thereof. R1 and R14 are independently hydroxy, OR9, OC (O) R10 or halo;
R2, R15, W and Z are independently hydrogen, hydroxy, OR9, OC (O) R10
, C (O) R10, COOH, CO2R10, alkyl, haloalkyl or halo,
R5 is hydrogen, C (O) R11 (where R11 is hydrogen or alkyl) or C
O2R12 where R12 is hydrogen or alkyl;
R6 is hydrogen or hydroxy;
R9 is alkyl, arylalkyl or C (O) R11 where R11 is as defined above, and R10 is hydrogen or alkyl;
The method of claim 2 comprising a pharmaceutically acceptable salt thereof. R1 and R14 are independently hydroxy, methoxy, benzyloxy, acetyloxy or chloro;
R2, R15, W and Z are independently hydrogen, hydroxy, methoxy, benzyloxy, acetyloxy, methyl, trifluoromethyl or chloro;
R5 is hydrogen or CO2R12 where R12 is hydrogen or methyl, and R6 is hydrogen,
The method of claim 2 comprising a pharmaceutically acceptable salt thereof. Compounds of formula I are represented by the following general formulas 1-30:

The method of claim 1 comprising a pharmaceutically acceptable salt thereof selected from. The method according to any one of claims 1 to 6, wherein the adhesion molecule is E-selectin or a vascular cell surface adhesion molecule (VCAM-1). A method for inhibiting the expression or activity of adhesion molecules associated with endothelial cells of a subject, comprising a therapeutically effective amount of one or more compounds of formula I as defined in any of claims 1-6. Administering to the subject. The method according to claim 8, wherein the adhesion molecule is E-selectin or vascular cell surface adhesion molecule (VCAM-1).   The method according to claim 8 or 9, wherein the subject is a human. In a method of treating a disease mediated by expression or activity of an adhesion molecule associated with an endothelial cell of a subject in an amount sufficient to inhibit the expression or activity of the adhesion molecule associated with the endothelial cell. One or more formulas I as defined in any of paragraphs 1-6
Administering a compound of the above to the subject.   The method according to claim 11, wherein the disease is a vascular disease. 13. The method of claim 12, wherein the vascular disease is selected from restenosis, inflammatory disease, coronary artery disease, angina and small vessel disease.   The method according to claim 13, wherein the vascular disease is restenosis after angioplasty. 7. A method of treating, ameliorating, preventing or reducing the risk of restenosis in a subject, comprising a therapeutically effective amount of one or more formulas as defined in any of claims 1-6. Administering a compound of I to said subject. 16. The method of claim 15, wherein restenosis is associated with a vascular treatment selected from percutaneous transluminal coronary angioplasty, directional coronary atherectomy and stents. A method of treating procedural vascular trauma in a subject comprising the step of administering to said subject a therapeutically effective amount of one or more compounds of formula I as defined in any of claims 1-6. Method. 18. The method of claim 17, wherein the procedural vascular trauma is selected from angioplasty, vascular surgery, transplantation and transplantation procedures. A method of treating or preventing a vascular disease in a subject comprising:
7. A method comprising administering to a subject a therapeutically effective amount of one or more compounds of formula I as defined in any of claims 1-6. 20. The method of claim 19, wherein the vascular disease is selected from atherosclerosis, restenosis, hypertension, inflammatory disease, coronary artery disease, angina and small vessel disease.   21. The method of claim 20, wherein the vascular disease is restenosis after angioplasty. A pharmaceutical composition in a dosage form suitable for use in the treatment of a disease mediated by expression or activity of adhesion molecules associated with endothelial cells in a subject, together with a pharmaceutically acceptable carrier. A composition comprising one or more compounds of formula I as defined in any of 6. A pharmaceutical composition in a dosage form suitable for use in preventing vascular disease or reducing the risk of vascular disease in a subject, as defined in any one of claims 1-6 together with a pharmaceutically acceptable carrier. A composition comprising one or more compounds of formula I Use of one or more compounds of formula I as defined in any of claims 1 to 6 in the manufacture of a medicament for inhibiting the expression or activity of adhesion molecules associated with endothelial cells of a subject. One or more formulas as defined in any of claims 1 to 6 in the manufacture of a medicament for the treatment of a disease mediated by the expression or activity of adhesion molecules associated with endothelial cells
Use of compounds of I. In the manufacture of a medicament for the treatment, amelioration, prevention or reduction of risk of restenosis.
Use of one or more compounds of formula I as defined in any of 6-6. Use of one or more compounds of formula I as defined in any of claims 1 to 6 in the manufacture of a medicament for the treatment of vascular diseases and / or procedural vascular trauma.

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