HK1181305B - Pharmaceutical compositions including carbamoyloxy arylalkanoyl arylpiperazine compound - Google Patents

Description

Pharmaceutical composition containing carbamoyloxy arylalkanoyl arylpiperazine compound

Technical Field

This application claims priority from korean patent application No.10-2010-0065950, filed on 8.2010 to the korean intellectual property office, the disclosure of which is hereby incorporated by reference in its entirety.

The present invention relates to pharmaceutical compositions comprising carbamoyloxy arylalkanoyl arylpiperazine compounds and methods of treating diseases selected from the group consisting of diabetic nephropathy, diabetic neuropathy, diabetic vascular complications, hyperlipidemia, coronary artery disease, and inflammation by using the pharmaceutical compositions.

Background

Lipoxygenases are non-heme iron-containing enzymes present in plants and animals that catalyze the oxygenation of specific polyunsaturated fatty acids (e.g., lipids and lipoproteins). A number of different lipoxygenases are known to have specific oxidation reactions. Mammalian lipoxygenases are named according to their position in arachidonate to be oxygenated. Three types of lipoxygenase are known in the human body, which catalyse the addition of oxygen molecules to carbon positions 5, 12 and 15 in arachidonate. Thus, the enzyme is named 5-, 12-or 15-lipoxygenase depending on the position of the carbon site to which the oxygen molecule is added. 5-lipoxygenase converts arachidonic acid ester to 5-hydroperoxicosatetraenoic acid (5-HPETE). This is the first stage of the metabolic pathway to produce 5-hydroxyeicosatetraenoic acid (5-HETE) and Leukotrienes (LT). Likewise, 12-and 15-lipoxygenase converted arachidonic acid ester to 12-HPETE and 15-HPETE, respectively. The biochemical reduction of 12-HPETE produces 15-HETE, which is a precursor of the compound 15-HETE lipoxin. Various biological effects are associated with products having lipoxygenase activity, and many of these products are known to be regulators of various diseases. 15-lipoxygenase is normally expressed in endothelial cells, smooth muscle cells, monocytes/macrophages, mesangial cells, tubular epithelial cells and podocytes and catalyzes the formation of 15-S-hydroxyeicosatetraenoic acid (15-S-HETE) from arachidonic acid (Natarajan and Nadler, Front. biosci. (2003)8: S783-795; Reilly et al., J.biol. chem. (2004)279(10): 9440-9450).

The chronic complications of diabetes comprise macrovascular complications, diabetic nephropathy, diabetic neuropathy and the like. If diabetes persists for years, the microvasculature and macrovascular disease slowly changes pathologically. The occurrence of microvascular complications is directly caused by hyperglycemia. If sorbitol is formed promoted by hyperglycemia, the thickness of the blood vessel wall increases and blood flow is affected, thereby promoting blood coagulation. Chronic complications of diabetes such as diabetic nephropathy, diabetic neuropathy may occur if persistent hyperglycemia-induced microvascular damage occurs in the kidneys, neurons, etc. Meanwhile, hyperglycemia is an indirect cause of macrovascular complications, and an increase in cholesterol caused by abnormal lipid metabolism in a hyperglycemic state is a direct cause of hyperglycemia.

The prior art discloses that 15-lipoxygenase is involved in the development of arteriosclerosis, glomerulonephritis, hyperlipidemia or inflammation. Furthermore, KR2008-67364 discloses triazole compounds as 15-lipoxygenase inhibitors. However, there is no commercially available agent for preventing or treating these diseases by inhibiting 15-lipoxygenase. Therefore, there is a need to develop a pharmaceutical composition that can prevent or treat these diseases.

Disclosure of Invention

Technical problem

The present invention provides a pharmaceutical composition for preventing or treating a disease selected from the group consisting of diabetic nephropathy, diabetic neuropathy, diabetic vascular complications, hyperlipidemia, coronary artery disease, and inflammation.

The present invention also provides a method for treating a disease selected from the group consisting of diabetic nephropathy, diabetic neuropathy, diabetic vascular complications, hyperlipidemia, coronary artery disease, and inflammation by using the pharmaceutical composition.

Technical scheme

In one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating a disease selected from the group consisting of diabetic nephropathy, diabetic neuropathy, diabetic vascular complications, hyperlipidemia, coronary artery disease and inflammation, wherein the pharmaceutical composition comprises a therapeutically effective amount of a compound selected from the group consisting of a compound represented by the following formula I, a pharmaceutically acceptable salt, isomer, solvate or hydrate thereof and a combination thereof; and a pharmaceutically acceptable carrier:

formula I

Wherein, X₁Comprising at least one member selected from the group consisting of hydrogen, linear or branched C1-C6 alkyl, halogen including F, Cl or Br, linear or branched C1-C6 alkoxy, nitro and trifluoromethyl;

X₂comprising at least one member selected from the group consisting of hydrogen, linear or branched C1-C6 alkyl, halogen, linear or branched C1-C6 alkoxy, nitro and trifluoromethyl if X₂2 or more thereof, which are the same as or different from each other and form a ring with an adjacent carbon atom; and is

Y is hydrogen or methyl, or forms a carbonyl group with an adjacent carbon atom.

The pharmaceutical composition of one embodiment of the present invention may include a therapeutically effective amount of a compound selected from the group consisting of compounds represented by formula I above, pharmaceutically acceptable salts, isomers, solvates or hydrates thereof, and combinations thereof.

In another embodiment, there is provided a method of treating a disease associated with 15-lipoxygenase comprising administering to a subject in need thereof a therapeutically effective amount of a compound selected from the group consisting of a compound represented by formula I, a pharmaceutically acceptable salt, isomer, solvate or hydrate thereof and combinations thereof. The disease associated with 15-lipoxygenase is selected from the group consisting of diabetic complications, hyperlipidemia, coronary artery disease, and inflammation. The diabetic complications include diabetic nephropathy, diabetic neuropathy, diabetic vascular complications, diabetic hyperlipidemia, and diabetic inflammation.

The term "treating" should be interpreted as arresting the development of a disease or condition in an animal; inhibiting a disease or condition, i.e., inhibiting the development of a disease or condition; and alleviating, i.e., causing regression of, a disease or condition, said animal having a disease or condition selected from the group consisting of diabetic nephropathy, diabetic neuropathy, diabetic vascular complications, hyperlipidemia, coronary artery disease, and inflammation, or having not been diagnosed as having said disease or condition but is predisposed to having said disease or condition. Thus, the term "therapeutically effective amount" refers to an amount sufficient to obtain a pharmaceutical, i.e., therapeutic, effect.

The compound represented by formula I in one embodiment of the present invention may be prepared by one of ordinary skill in the art of compound synthesis using known compounds or using compounds that can be easily prepared from known compounds. In particular, the preparation process of said compounds is disclosed in detail in KR2008-40393, which was filed by the same inventors as the present invention, said patent being incorporated herein by reference. The compounds of the present invention can be chemically synthesized using methods disclosed in the references cited above. However, the method is for illustration purposes only. Accordingly, the order of operations used herein may be optionally changed if necessary, without limiting the scope of the present invention.

In addition to the compounds represented by formula I, the compounds may also include pharmaceutically acceptable salts thereof, i.e., acid addition salts or base addition salts thereof; and stereochemically isomeric forms of the compounds represented by formula I herein, which are pharmaceutically acceptable salts, may be any of the various salts which allow the parent compound to retain its activity in a subject to which the compound is administered and which do not cause adverse effects. The pharmaceutically acceptable salt may be an inorganic salt or an organic salt. Examples of acids are acetic acid, hydrochloric acid, nitric acid, aspartic acid, sulfonic acid, sulfuric acid, maleic acid, glutamic acid, formic acid, succinic acid, phosphoric acid, phthalic acid, tannic acid, tartaric acid, hydrobromic acid, propionic acid, benzenesulfonic acid, benzoic acid, stearic acid, ethanesulfonic acid, butyric acid, dicarbonic acid, disulfuric acid, ditartaric acid, oxalic acid, butyric acid, calcium ethylenediaminetetraacetate, camphorsulfonic acid, carbonic acid, chlorobenzoic acid, citric acid, ethylenediaminetetraacetic acid (ionic acid), toluenesulfonic acid, ethanedisulfonic acid (ethylenic acid), ethanesulfonic acid (ethylenic acid), fumaric acid, glucoheptonic acid, pamoic acid, gluconic acid, glycoloylarsonic acid (glycolic acid), methyl nitric acid, polygalacturonic acid (galacturonic acid), hexylresorcinol (hydroxyethylonic acid), ursolic acid, hydrobromic acid, hydrochloric acid (hydrochloric acid), hydroiodic acid, hydroxynaphthoic acid (hydroxynaphthoic acid), hydroxynaphthoic acid (glycolic acid), glycolic acid (glycolic acid), and glycolic acid (glycolic acid) Lactobionic acid, mandelic acid, propanoic acid dodecylsulphate (esterinic acid), mucic acid, naphthalenesulphonic acid (naphcylic acid), muconic acid, p-nitromethanesulphonic acid, cyclohexanesulphonic acid, pantothenic acid, monohydrogen phosphate, dihydrogen phosphate, salicylic acid, sulphamic acid, methanesulphonic acid and theachloric acid (theolic acid) for example, the acid used for the addition salt may be hydrochloric acid or methanesulphonic acid And (4) melting the mixture.

Meanwhile, the stereochemically isomeric form of the compound represented by formula I according to one embodiment of the present invention means any compound obtainable from the compound represented by formula I. If not otherwise defined, the chemical name of a compound indicates a mixture of any possible stereochemical isomeric types, examples of which are diastereomers and enantiomers each having a basic molecular structure. Specifically, the stereocenter may have an R-or S-coordination, and the substituent of the 2-valent cyclic (partially) saturated group may have a cis-or trans-coordination. The compound having a double bond may have an E or Z-stereochemistry at the double bond. Stereochemical isomers of the compounds represented by formula I are included in the scope of the present invention.

Non-limiting examples of compounds represented by formula I are carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -3-oxo-1-phenyl-propyl ester, carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -1- (4-fluoro-phenyl) -3-oxo-propyl ester, carbamic acid 3- (4-benzo [1,3] dioxol-5-yl-piperazin-1-yl) -3-oxo-1-phenyl-propyl ester, carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -1- (4-trifluoro-phenyl) -piperazin-1-yl Methyl-phenyl) -3-oxo-propyl ester; hydrochloride, carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -1- (4-nitro-phenyl) -3-oxo-propyl ester, (R) -carbamic acid 3- [4- (4-chloro-phenyl) -piperazin-1-yl ] -3-oxo-1-phenyl-propyl ester, (S) -carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -3-oxo-1-phenyl-propyl ester, (R) -carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -3-oxo-1-benzene -propyl ester, carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -1-phenyl-butyl ester and carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -1- (4-chloro-phenyl) -3-oxo-propyl ester. In one embodiment of the present invention, the compound represented by formula I may be (R) -carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -3-oxo-1-phenyl-propyl ester.

The pharmaceutical composition of one embodiment of the present invention may include a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier of the pharmaceutical composition may be any of various materials conventionally used in formulations, and non-limiting examples of the pharmaceutically acceptable carrier are lactose, glucose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. The pharmaceutical composition may further include lubricants, wetting agents, flavoring agents, emulsifiers, suspending agents, preservatives and the like. Suitable pharmaceutically acceptable carriers and formulations are disclosed in detail in Remington's Pharmaceutical Sciences (19 th edition, 1995).

In one embodiment of the present invention, the pharmaceutical composition may be administered orally or parenterally. Parenteral administration may be intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intravaginal administration, intrapulmonary administration or intrarectal administration. For oral administration, the active material may be coated or formulated to prevent decomposition in the stomach. At the same time, the pharmaceutical composition may also be administered by any means that allows the active material to reach the target cell. The route of administration may vary depending on the overall condition and age of the subject to be treated, the nature of the condition being treated and the active material used.

The suitable dosage amount of the pharmaceutical composition may depend on the formulation method; a method of administration; the age, weight, sex and disease state of the patient; a food; the time of administration; the route of administration; the rate of excretion; and reaction sensitivity. In general, the skilled pharmacist can determine and prescribe an effective therapeutically or prophylactically administered amount without any difficulty. The amount of the pharmaceutical composition to be administered may be administered all at once, or may be administered in several portions, for example, the pharmaceutical composition may be administered 1 to 4 times per day.

The pharmaceutical compositions may be presented in unit dosage form using procedures well known to those of ordinary skill in the art, as well as pharmaceutically acceptable carriers and/or excipients, or may be prepared using multi-dose containers. In this case, the formulation may be a solution, suspension or emulsion in an oil medium or aqueous medium, an extract, a powder, granules, a tablet or a capsule, and a dispersing agent or a stabilizing agent may also be included in the pharmaceutical composition. Meanwhile, the pharmaceutical composition may be administered in the form of suppository, spray, ointment, cream, gel, inhalant or skin patch.

The pharmaceutical composition can be used for preventing or treating diabetic nephropathy, diabetic neuropathy or diabetic vascular complications.

The term "diabetic" means that the cause of the corresponding disease is diabetes. Meanwhile, the term "diabetes" refers to a group of diseases characterized by chronic hyperglycemia induced by a defect in insulin metabolism and having various abnormal metabolisms due to the chronic hyperglycemia. Thus, diabetic nephropathy, diabetic neuropathy or diabetic vascular complications may be understood as diabetes-induced nephropathy, diabetes-induced neuropathy and diabetes-induced vascular complications, respectively.

Diabetic nephropathy refers to a disease in which the microvasculature of the kidney is damaged and thus proteins are directly discharged without filtration. The development of diabetic nephropathy is due to glomerular hyperfiltration (hyperfunction), abnormal thickening of glomerular basement membrane, proliferation of mesangial cells, thickening of glomeruli, and synthesis and increase of extracellular matrix, and causes gradually increasing glomerulosclerosis and renal failure. Diabetic neuropathy is a neurological complication of diabetes in which abnormalities in neuronal function and structure occur as a result of diabetes, usually in the peripheral nervous system. Meanwhile, diabetic vascular complications refer to diseases in which arteriosclerosis is generated due to blood flow disorders caused by diabetes-induced hyperglycemia and metabolic disorders (e.g., insulin resistance).

Also, the pharmaceutical composition is used for preventing or treating hyperlipidemia, coronary artery disease or inflammation.

Hyperlipidemia refers to a state in which metabolism of fat (e.g., triglyceride or cholesterol) proceeds abnormally, and thus, a large amount of fat is contained in blood, and when fatty substances appear in blood, it may accumulate on the blood vessel wall to cause inflammation and cause cardiovascular diseases. Meanwhile, coronary artery disease refers to a state in which fat deposits and fibrous tissues are accumulated in an artery (coronary artery or cardiac artery) through which blood supplied to the heart passes, and thus the amount of blood supplied to the myocardium is reduced so that angina pectoris, myocardial necrosis and myocardial infarction occur, and thus the function of the heart is seriously impaired. According to embodiments of the present invention, the cause of hyperlipidemia or coronary artery disease may or may not be diabetes.

Inflammation is understood by those of ordinary skill in the art to include symptoms characterized by local or global defense responses, which may be caused by chemical and/or physiological reactions (e.g., portions of allergic reactions) to external injuries, infections, the above-mentioned chronic diseases, and/or external stimuli to the body. The reaction causes damage, illusion or isolation of the injured tissue. For example, inflammation may occur due to heat, swelling, pain, excessive internal heat, vasodilation and/or increased blood flow, white blood cell invasion into the infected site or loss of function, and/or inflammatory conditions associated with other symptoms. Thus, inflammation is understood to include inflammatory diseases, disorders or conditions, particularly acute, chronic, ulcerative, specific, allergic and necrotic inflammation, as well as other types of inflammation known to those of ordinary skill in the art. Thus, the pharmaceutical composition may be used to treat asthma, Chronic Obstructive Pulmonary Disease (COPD), pulmonary fibrosis, allergic diseases, rhinitis, inflammatory bowel disease, ulcers, inflammatory pain, fever, arteriosclerosis, coronary artery disease, vasculitis, pancreatitis, arthritis, osteoarthritis, rheumatoid arthritis, conjunctivitis, iritis, scleritis, uveitis, wound healing, dermatitis, eczema, psoriasis, stroke, diabetes, autoimmune diseases, alzheimer's disease, multiple sclerosis, sarcoidosis, hodgkin's disease, other malignant diseases, or other types of diseases with inflammatory factors.

In one embodiment of the present invention, the compound may have an inhibitory activity of 15-lipoxygenase.

15-lipoxygenase is known to be involved in the etiology of a variety of diseases including arteriosclerosis, asthma, glomerulonephritis, chronic complications of diabetes etc. (Harats et al, Enterioscope, Thromb, Val. biol., (2000)20(9) 2100. about 2105; Natarajan et al, Frontbiosci., (2003)8: s783-795; Hatley et al, J. biol. chem., (2003)278(28) 25369. about 25375; Shannon et al, am. Rev. Respir. Dis., (1993)147(4) 1024. about 1028; Montero and Badr, exp. Nephe., (2000)8(1) 14-19; Ukon, J. biol. 2003, Thr. about 24. about 11, Val. about 24, Val. about # 23, 11. about # 12, Ha et al, J. about # 23, J. about # 11, Ha et al, J. about # 24, Val. about # 24, 11, No. (24, D., (24) 23, D., (9) 24, D., (11, D. about # 12, D., (11, D., (III) 3, D., (11, D., (III, D., (11) 3, D., (11, D., J.Vasc.Res., (2008)45(2): 132-. The enhanced expression of 15-lipoxygenase affects a variety of metabolism, signaling networks, transcriptional control, and gene expression, thereby increasing the formation of free radicals, lipid peroxidation, mitogen-activated protein (MAP) kinase, and inflammatory responses. These results contribute to the development of diabetic complications and diabetic vascular complications, diabetic nephropathy and diabetic neuropathy in animal models of Diabetes are preventable and treatable by artificially inhibiting the expression or activity of 15-lipoxygenase (Natarajan and dNadler, Front.Biosci. (2003)8: s783-795; Reilly et al, J. biol. Chem. (2004)279(10):9440-9450; Obrosova et al, Diabetes55 (Supl.1) (2006) A188; Xu et al, Nephrol Dial Transplant. (2009)24(6):1744-1752; Yuan et al, Am J Physiol Renal Physiol 2008 (295-617) 2). Furthermore, inhibition of the expression or activity of 15-lipoxygenase is suggested as a method for the prevention or treatment of diabetes-induced or non-diabetes-induced coronary artery disease (Siu, J cardiovascular Med (Hagerstown), (2010)11(1):1-6; Nagelin et al, J.biol.Chem. (2009)284(45):31303 31314; Natarajan et al, Frontbiosci. (2003)8: s783-95; Hatley et al, J.biol.Chem. (2003)278(28): 25369-25375), in addition, since 15-lipoxygenase plays an important role in the biosynthesis of asthma, allergy, psoriasis and inflammation regulators and inhibitors of said enzymes inhibit biochemical pathways associated with said disease state, so that 15-lipoxygenase can be used for the treatment of said inflammation-related diseases (Yang et al, J.1203, III.) (III), Cell et al, III, IV, III, clin Exp Allergy, (2009)39(6): 908-.

Therefore, a compound showing an inhibitory activity against 15-lipoxygenase is useful as an agent for preventing or treating the above-mentioned diabetic nephropathy, diabetic neuropathy, diabetic vascular complications, hyperlipidemia, coronary artery disease and inflammation.

In another aspect, the present invention provides a method for treating a disease selected from the group consisting of diabetic nephropathy, diabetic neuropathy, diabetic vascular complications, hyperlipidemia, coronary artery disease, and inflammation, wherein the method comprises contacting the pharmaceutical composition with a subject.

The contacting may be performed in vitro or in vivo. If the contacting is in vivo, the method may further comprise administering the pharmaceutical composition to the subject.

The subject may be a cell, tissue, organ, or individual. Meanwhile, the administration may include direct contact of a solution of the pharmaceutical composition dissolved in a suitable buffer with cells, tissues or organs, or may be parenteral administration. The pharmaceutical compositions and methods of administration for treatment are described above and therefore will not be described in detail herein

Meanwhile, the subject to which the pharmaceutical composition can be administered may be any type of animal. For example, the subject may be a human or non-human animal such as a dog, cat or mouse.

Drawings

The above and other features and advantages of the present invention will become more apparent by describing in detail exemplary embodiments thereof with reference to the attached drawings in which:

FIG. 1 shows the measurement results of the total cholesterol amount in the serum obtained after the compound according to one embodiment of the present invention is administered to a mouse in which diabetes is induced;

FIG. 2 shows the measurement results of the amount of triglycerides in the serum obtained after the compound according to one embodiment of the present invention was administered to mice in which diabetes was induced;

FIG. 3 shows the measurement of fibronectin expression levels in renal cortical tissue after administration of a compound of one embodiment of the present invention to a mouse in which diabetes is induced;

FIG. 4 is a graph showing the measurement results of the amount of mRNA transcribed from the PAI-1 gene in renal cortical tissue after administration of the compound according to one embodiment of the present invention to a mouse in which diabetes is induced;

FIG. 5 shows the measurement results of the amount of mRNA transcribed from the MCP-1 gene in renal cortical tissue after administration of a compound according to one embodiment of the present invention to a mouse in which diabetes is induced; and

FIG. 6 shows the measurement results of the amount of mRNA transcribed from TGF-. beta.1 gene in renal cortical tissue after administration of a compound of one embodiment of the present invention to a mouse in which diabetes is induced. Wherein in figures 1 to 6 indicates statistical significance between the control group and the test group to which no compound was administered, + indicates statistical significance between the test group to which no compound was administered and the test group to which compound was administered.

Detailed Description

The present invention will be described in more detail with reference to the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: synthesis of 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -3-oxo-1-phenyl-propyl carbamate

554mg (2.887mmole) of ethyl benzoylacetate and 641mg (2.887mmole) of 3, 4-dimethoxyphenylpiperazine were dissolved in toluene and refluxed for 24 hours. 736mg (1.99mmole) of the compound obtained after concentration under reduced pressure are dissolved in methanol and cooled to 0 ℃ and 109mg (2.887mmole) of sodium borohydride are slowly added. The resulting solution was stirred at room temperature for 2 hours, then the solvent was concentrated under reduced pressure and dissolved with water, extracted several times with ethyl acetate. Then, the resulting organic layer was dried with magnesium sulfate and filtered, and concentrated under reduced pressure. The obtained residue was purified by column chromatography (hexane: ethyl acetate =1: 1), thereby obtaining 1.592mmole (589 mg) of the compound. The resulting compound was dissolved in tetrahydrofuran (10 mL), followed by addition of 820mg (5 mmole) of 1,1 '-carbodiimidazole (1, 1' -carbomidiazol) and stirring at room temperature for 1 hour, addition of excess aqueous ammonia and stirring at room temperature for 1 hour. The reaction mixture was diluted with water and extracted several times with ethyl acetate, and the resulting organic layer was dried over magnesium sulfate and filtered, and concentrated under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate) to give the objective compound (yield: 329 mg, yield: 28%)

¹HNMR(200MHz，CDCl3)d：2.82(dd，lH)，3.04(m，5H)，3.61(m，2H)，3.77(m，2H)，3.88(d，6H)，4.77(br，2H)，6.15(t，1H)，6.42(d，1H)，6.57(s，1H)，6·82(d，1H)，7.41(m，5H)

Example 2: synthesis of 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -1- (4-fluoro-phenyl) -3-oxo-propyl carbamate

The target compound was obtained in the same manner as in example 1, except that 4-fluoro-benzoylacetic acid and 3, 4-dimethoxyphenylpiperazine were used as starting materials (yield: 542 mg, yield: 37%).

¹HNMR(2OOMHZ，CDCl3)d：2.82(dd，1H)，3.01(m，5H)，3.60(m，2H)，3.75(m，2H)，3.86(d，6H)，4.92(br，2H)，6.15(t，lH)，6.42(d，lH)，6.56(d，lH)，6.80(d，lH)，7.04(t，2H)，7.38(t，2H)

Example 3: synthesis of 3- (4-benzo [1,3] dioxol-5-yl-piperazin-1-yl) -3-oxo-1-phenyl-propyl carbamate

The objective compound was obtained in the same manner as in example 1, except for using benzoylacetic acid ethyl ester and 3, 4-methylenedioxyphenylpiperazine as starting materials (yield: 190 mg, yield: 48%).

¹HNMR(2OOMHz，CDC13)d：2.98(m，6H)，3.59(m，2H)，3.76(m，2H)，4.71(br，2H)，5.94(S，2H)6.15(t，1H)，6.36(dd，1H)，6.55(s，1H)，6.74(d，1H)，3.40(m，5H)

EXAMPLE 4 carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -1- (4-trifluoromethyl-phenyl) -3-oxo-propyl ester; synthesis of hydrochloride

The objective compound was obtained in the same manner as in example 1, except for using ethyl 4-trifluoromethyl-benzoylacetate and 3, 4-dimethoxyphenylpiperazine as starting materials (yield: 250mg, yield: 52%). The product was dissolved in dichloromethane and saturated HCl/ether solution was added to produce its hydrochloride salt.

¹HNMR(2OOMHz，DMSO)d：2.9O(dd，lH)，3·l2(dd，1H)，3.34(m，4H)，3.75(s，3H)，3.78(s，3M)，3.85(m，4H)，6.00(m，1H)，6.60(br，2H)，7·01(m，2H)，7.20(m，lH)，7·60(d，2H)，7·75(d，2M)

EXAMPLE 5 Synthesis of 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -1- (4-nitro-phenyl) -3-oxo-propyl carbamate

The objective compound was obtained in the same manner as in example 1, except for using ethyl 4-nitro-benzoylacetate and 3, 4-dimethoxyphenylpiperazine as starting materials (yield: 261mg, yield: 57%).

¹HNMR(200MHz，DMSO)d：2.96(dd，lH)，3.16(dd，lH)，3.42(m，4H)，3.76(s，3H)，3.78(s，3H)，392(m，4H)，6.05(m，1H)，6.64(br，2H)，7.02(m，1H)，7.24(m，2H)，7.65(d，2H)，8·24(d，2H)

Example 6: synthesis of (R) -carbamic acid 3- [4- (4-chloro-phenyl) -piperazin-1-yl ] -3-oxo-1-phenyl-propyl ester

1.0g (6.0mmole) of (R) -3-hydroxy-3-phenylpropionic acid and 1.18g (6.0mmole) of 4-chlorophenylpiperazine were dissolved in 50mL of tetrahydrofuran as a solvent at room temperature, and 1.24g (6.0mmole) of EDC and 0.81g (6mmole) of HOBt were added dropwise thereto, followed by stirring at 25 ℃ for 5 hours. The excess solvent was removed by distillation under reduced pressure, the resulting product was neutralized with 20mL of 1N aqueous sodium chloride solution, 25mL of ethyl acetate was added, and the resulting organic layer was separated and extracted 2 times with 15mL of ethyl acetate. The resulting organic layer was dried over 2g of anhydrous magnesium sulfate and filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (hexane: ethyl acetate =1:1 to 1: 10). 0.345g (1mmole) of the product obtained is dissolved in 15mL of tetrahydrofuran, then 0.325g (2mmole) of 1,1' -carbodiimidazole is added and stirred at room temperature for 1 hour, then excess aqueous ammonia is added and stirred at room temperature for 2 hours. The reaction mixture was diluted with water and extracted several times with ethyl acetate, and the resulting organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The obtained residue was purified by column chromatography (hexane: ethyl acetate =1: 1), thereby yielding the objective compound (yield: 1.2g, yield: 52.5%).

¹HNMR(200MHz，CDC13)d：2.82(dd，1H)，3.07(m，5H)，3.58(m，2H)，3.74(m，2H)，4.81(br，2H)，6.13(t，1H)，6.84(d，2H)，7.38(m，7H)

Example 7: synthesis of (S) -carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -3-oxo-1-phenyl-propyl ester

The objective compound was obtained in the same manner as in example 6 except for using (S) -3-hydroxy-3-phenylpropionic acid (6mmole) and 3, 4-dimethoxyphenylpiperazine (6mmole) as starting materials (yield: 1.38g, yield: 56%).

¹HNMR(20OMHZ，CDC13)d:2.82(dd，1H)，3.04(m，5H)，3.61(m，2H)，3.77(m，2H)，3·88(d，6H)，4·77(br，2H)，6·15(t，1H)，6.42(d，lH)，6.57(s，lH)，6.82(d，lH)，7.41(m，5H)

Example 8: synthesis of (R) -carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -3-oxo-1-phenyl-propyl ester

The objective compound was obtained in the same manner as in example 6, except that (R) -3-hydroxy-3-phenylpropionic acid and 3, 4-dimethoxyphenylpiperazine were used as starting materials (yield: 1.040g, yield: 42%).

¹HNMR(200MHz，CDCl3)d:2.82(dd，1H)，3.04(m，5H)，3.61(m，2H)，3.77(m，2H)，3.87(d，6H)，4.77(br，2H)，6.15(t，IH)，

Example 9: synthesis of carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -1-phenyl-butyl ester

Phenyl-1-propenyl (prophenyl) -one (4.1mmole) and 3, 4-dimethoxyphenylpiperazine (4.9mmole) were dissolved in 30mL of ethanol (as solvent) and stirred at 72 ℃ for 48 hours. The solvent was distilled under reduced pressure, and the resulting mixture was diluted with water, extracted twice with water, and extracted 2 times with ethyl acetate. The organic layer was distilled under reduced pressure, then dried over magnesium sulfate and filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (hexane: ethyl acetate =4: 1), thereby yielding a compound. The compound (2.9 mmole) was dissolved in 20mL of methanol and NaBH was added slowly₄(3.8 mmole). The resulting product was stirred at room temperature for 2 hours, the solvent was concentrated under reduced pressure, and the yellow residue was purified by column chromatography (hexane: ethyl acetate =1: 1). The purified compound (2mmole) was dissolved in 15ml of tetrahydrofuranThen, 1' -carbodiimidazole (4mmole) was added and stirred at room temperature for 1 hour, and an excess of aqueous ammonia was added and stirred at room temperature for 2 hours. The reaction mixture was diluted with water and extracted several times with ethyl acetate, and the resulting organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The obtained residue was purified by column chromatography (hexane: ethyl acetate =1: 1), thereby yielding a final product (yield: 90.9 mg, yield: 22%).

¹HNMR(20OMHz，CDCl3)d1.81(m，1H)，2.32(m，1H)，2.5(m，3H)，2.8(m，2H)，3.14(m，4H)，3.80.(s，6H)，4.80(br，2H)，6.02(t，1H)，6.92(m，4H)，7.36(m，5H)

Example 10: synthesis of 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -1- (4-chloro-phenyl-3-oxo-propyl carbamate

The objective compound was obtained in the same manner as in example 1, except for using ethyl 4-chloro-benzoylacetate and 3, 4-dimethoxyphenylpiperazine as starting materials (yield: 543mg, yield: 42%).

¹HNMR(200MHZ，CDCl3)d:2.82(dd，1H)，3.01(m，5H)，3，64(m，2H)，3.77(m，2H)，3·86(d，6H)，4.84(br，2H),6.15(t，lH)，6.42(d，lH)，6.57(s，IH)，6·82(d，lH)，7.35(s，4H)

Example 11: in vitro test for the inhibitory Effect of 15-lipoxygenase

To confirm that the compounds prepared in examples 1 to 10 specifically inhibit only 15-lipoxygenase, human platelet-induced 12-lipoxygenase was used as a negative control, and rabbit reticulocyte-induced 15-lipoxygenase was used as a test group to identify the inhibitory effect of the compounds on 12-lipoxygenase.

The 12-lipoxygenase is reacted with arachidonic acid ester as a substrate to produce 12-hydroxyeicosatetraenoic acid (12-HETE) as a product. Thus, the activity of 12-lipoxygenase was evaluated by measuring the amount of 12-HETE produced by spectroscopy. Meanwhile, 15-lipoxygenase reacts with linolenic acid as a substrate to produce 13-hydroperoxy-9, 11-octadecadienoic acid (13-HPODE) as a product. Therefore, the activity of 15-lipoxygenase was evaluated by measuring the amount of 13-HPODE produced by spectroscopy.

The activity of 12-lipoxygenase was measured at a concentration of 10. mu.M for each compound. Each sample was pretreated at 25 ℃ for 15 minutes by using a buffer (50 mM Tris-HCl,0.1% Triton X-100, pH 7.4), then 30. mu.M arachidonate was added (the concentration thereof was controlled to 30. mu.M during the reaction), reacted at 25 ℃ for 15 minutes, and the amount of 12-HETE as a produced product was measured by absorbance at a wavelength of 570 nm.

The activity of 15-lipoxygenase was measured at a concentration of 10. mu.M for each compound. Each sample was pretreated at 4 ℃ for 15 minutes by using a buffer (phosphate-buffered saline buffer, pH 7.4), then 260. mu.M linolenic acid (the concentration thereof was controlled at 260. mu.M during the reaction) was added, and reacted at 4 ℃ for 10 minutes, and the amount of 13-HPODE as the produced product was measured by absorbance at a wavelength of 660 nm.

The results obtained by measuring the inhibitory effect of the compounds on the activities of 12-lipoxygenase and 15-lipoxygenase are shown in table 1.

[ Table 1]

Example 12: drug effect test of diabetic hyperlipidemia on diabetic animal model

Sprague-Dawley rats (n =21, 4 weeks old, male) were used as a laboratory model (Central lab. Animal Inc.). Rats (n = 14) having diabetes induced by intraperitoneal administration of 50 mg/kg streptozotocin were used as a test group, and rats (n = 7) to which only a solvent (100 mM citrate buffer, ph 4.5) was administered intraperitoneally were used as a control. In general, streptozotocin destroys beta cells of the pancreas to induce diabetes, so that blood glucose is increased and the amounts of cholesterol and triglyceride in serum are increased. The test group was induced to diabetes 2 days after streptozotocin administration. The test components were divided into 2 groups, one group of rats orally administered 200mg/kg (r) -carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -3-oxo-1-phenyl-propyl ester daily for 4 weeks. Another group orally administered 30% polyethylene glycol (PEG) instead of the compound.

The amount of total cholesterol and triglycerides in the serum of each group was determined after administration of the compound and PEG4 weeks, and the results are shown in fig. 1 and 2. To measure the amount of total cholesterol and triglycerides, serum was isolated from the rats using a Hitachi 7600 autoanalyzer. As a result, it was confirmed that the increase in the concentration of total cholesterol caused by streptozotocin (120. + -.13 mg/dl) was reduced to 66. + -.9 mg/dl, which is similar to that of the control 62. + -.5 mg/dl, while the increase in the concentration of triglyceride caused by streptozotocin (760. + -.162 mg/dl) was greatly reduced to 126. + -.44 mg/dl, due to the administration of (R) -carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -3-oxo-1-phenyl-propyl ester. That is, the increase in cholesterol and triglycerides in the serum is significantly reduced as a result of the administration of the compound. From this result, it was confirmed that the compound is useful for preventing or treating hyperlipidemia.

Example 13: drug efficacy test for diabetic nephropathy

Sprague-Dawley rats (n =21, 4 weeks old, male) were used as a laboratory model (Central lab. Animal Inc.). Rats (n = 14) having diabetes induced by intraperitoneal administration of 50 mg/kg streptozotocin were used as a test group, and rats (n = 7) to which only a solvent (100 mM citrate buffer, ph 4.5) was administered intraperitoneally were used as a control. In general, in rats with streptozotocin-induced diabetes, diabetic nephropathy is induced by hyperglycemia, one of the symptoms is renal fibrosis, and gene expression of extracellular matrix proteins such as fibronectin is increased. The test group was induced to diabetes 2 days after streptozotocin administration. The test components were divided into 2 groups, one group of rats orally administered 200mg/kg (r) -carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -3-oxo-1-phenyl-propyl ester daily for 4 weeks. Another group orally administered 30% polyethylene glycol (PEG) instead of the compound.

After administration of the compound and PEG4 weeks, renal cortical tissue was extracted from each group, and the expression level of fibronectin (i.e., the amount of mRNA transcribed from fibronectin) was measured by real-time reverse transcriptase polymerase chain reaction (see fig. 3). The expression level of fibronectin was measured using a forward primer (5'-GCCACACCTACAACCAGTAT-3'; SEQ IDMO:1) and a reverse primer (5'-ATCACCACTCAGAAATGGAG-3'; SEQ ID NO: 2). In the RT-PCR, annealing was performed at a temperature of 60 ℃ for 1 minute, SYBRgreen (applied biosystem) was used as a fluorescent material, and a 7300 Real-time PCR instrument manufactured by applied biosystem was used. Reactions other than the annealing are performed using methods known in the art according to the manufacturer's protocol. For the control, mRNA transcribed from the β actin gene was used, and the assay results were expressed as the amount of fibronectin mRNA quantified from the results of the RT-PCR divided by the amount of β actin mRNA. As a result, the expression level of fibronectin in renal cortex was 1.4 ± 0.1 times that of the control due to the administration of streptozotocin, and was reduced to 1.2 ± 0.1 times that of the control due to the administration of (R) -carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -3-oxo-1-phenyl-propyl ester. From this result, it was confirmed that the compound is effective for inhibiting diabetes-induced renal fibrosis.

Example 14: pharmacodynamic testing of inflammatory response in animal models with induced diabetic nephropathy

When diabetic nephropathy is induced by streptozotocin, the inflammatory response in the kidney is increased, and thus the expression of various inflammation-related genes (e.g., PAI-1, MCP-1, and TGF-. beta.1) is increased. Therefore, to confirm the efficacy of the compounds on inflammatory responses, the amounts of mRNA transcribed from the PAI-1, MCP-1 and TGF-. beta.1 genes from the renal cortical tissues of each group used in example 13 were measured by RT-PCR used in example 13 (see FIGS. 4, 5 and 6). The gene was extended using the following primer sequences: the forward primer (5'-TCCGCCATCACCATTTT-3'; SEQ ID NO:3) and the reverse primer (5'-GTCAGTCATGCCCAGCTTCTC-3'; SEQ ID NO:4) were used to amplify PAI-1; a forward primer (5'-CCTCCACCACTATGCAGGTCTCC-3'; SEQ ID NO: 5) and a reverse primer (5'-GCACGTGGATGCTACAGGC-3'; SEQ ID NO:6) for the amplification of MCP-1; the forward primer (5'-CCAACTACTGCTTCAGCTCCA-3'; SEQ ID NO: 7) and the reverse primer (5' -GTCTCCAGGCTCCAAATGT-3; SEQ ID NO: 8) were used to amplify TGF-. beta.1.

As a result, the expression of the inflammation-associated genes PAI-1, MCP-1 and TGF-. beta.1 in the renal cortex was 1.8. + -. 0.1, 3.4. + -. 1.0 and 1.6. + -. 0.2 times that of the control, respectively, and the expression of the PAI-1, MCP-1 and TGF-. beta.1 genes in the renal cortex was reduced to 1.1. + -. 0.2, 1.6. + -. 0.3 and 1.3. + -. 0.2 times that of the control due to the administration of (R) -carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -3-oxo-1-phenyl-propyl ester, respectively. From this result, it was confirmed that the compound is effective for inhibiting the expression of diabetes-induced inflammation-associated genes.

From the results of examples 13 and 14, it was confirmed that the compound is useful for preventing or treating diabetic nephropathy since the compound can inhibit the expression of fibrosis and inflammation-associated genes causing diabetic nephropathy.

Example 15: administration of (R) -carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -3-oxo-1-phenyl-propyl ester and preparation of tablets containing the same (prospective)

The compounds of the present invention are useful for preventing or treating diabetic nephropathy, diabetic neuropathy, diabetic vascular complications, hyperlipidemia, coronary artery disease, or inflammation. A clinically suitable dose (oral) is 300mg per adult.

Based on the administration amounts, tablets containing the components shown in table 2 below can be prepared by using a conventional method. Avicel102 (microcrystalline cellulose) was used as excipient.

[ Table 2]

The compound is suitably administered in an amount of 60kg per adult, which corresponds to 1 or 2 tablets containing the compound.

When the pharmaceutical composition of the present invention is used, diabetic nephropathy, diabetic neuropathy, diabetic vascular complications, hyperlipidemia, coronary artery disease or inflammation can be effectively prevented or treated.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the following claims.

The nucleotide sequences or polypeptide sequences of SEQ ID NO 1 to SEQ ID NO 8 are filed as sequence Listing text and the contents of the sequence Listing are incorporated herein in their entirety.

Claims (4)

1. Use of a pharmaceutical composition for the manufacture of a medicament for the prevention or treatment of a disease selected from the group consisting of diabetic neuropathy, diabetic vascular complications, hyperlipidemia, coronary artery disease, and inflammation, wherein the pharmaceutical composition comprises a therapeutically effective amount of a compound selected from the group consisting of compounds of formula I, pharmaceutically acceptable salts thereof, and combinations thereof, and a pharmaceutically acceptable carrier

Formula I

Wherein, X₁Is at least one selected from hydrogen, linear or branched C1-C6 alkyl, F, Cl, Br, linear or branched C1-C6 alkoxy, nitro and trifluoromethyl; x₂Is at least one member selected from the group consisting of hydrogen, linear or branched C1-C6 alkyl, halogen, linear or branched C1-C6 alkoxy, nitro and trifluoromethyl if X₂2 or more thereof, which are the same as or different from each other and form a ring with an adjacent carbon atom; and is

Y is hydrogen or methyl, or forms a carbonyl group with an adjacent carbon atom.

2. The use of claim 1, wherein the compound is selected from the group consisting of: 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -3-oxo-1-phenyl-propyl carbamate, 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -1- (4-fluoro-phenyl) -3-oxo-propyl carbamate, 3- (4-benzo [1,3] dioxol-5-yl-piperazin-1-yl) -3-oxo-1-phenyl-propyl carbamate, 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -1- (4-trifluoromethyl-phenyl) -3-oxo-propyl carbamate -propyl ester; hydrochloride, carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -1- (4-nitro-phenyl) -3-oxo-propyl ester, (R) -carbamic acid 3- [4- (4-chloro-phenyl) -piperazin-1-yl ] -3-oxo-1-phenyl-propyl ester, (S) -carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -3-oxo-1-phenyl-propyl ester, (R) -carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -3-oxo-1-benzene -propyl ester, carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -1-phenyl-butyl ester and carbamic acid 3- [4- (3, 4-dimethoxy-phenyl) -piperazin-1-yl ] -1- (4-chloro-phenyl) -3-oxo-propyl ester.

3. The use of claim 1, wherein the compound has 15-lipoxygenase inhibitory activity.

4. The use of claim 1, wherein the diabetic vascular complication is diabetic nephropathy.