KR20030060286A - Chroman derivatives and a pharmaceutical composition comprising same - Google Patents

Abstract

PURPOSE: Chroman derivatives and a pharmaceutical composition comprising the same as an effective component are provided, which compounds are useful for prevention and treatment of apoplexy, Alzheimer's diseases and Parkinson's disease. CONSTITUTION: Chroman derivatives represented by the formula 1 and pharmaceutically acceptable salt thereof are provided, wherein R is H, C1-C4 lower alkyl, benzyl or phenyl; and X is C=O or CH2. A pharmaceutical composition for inhibiting the activation of microglia comprises the chroman derivatives or pharmaceutically acceptable salts thereof, and carriers.

Description

CHROMAN DERIVATIVES AND A PHARMACEUTICAL COMPOSITION COMPRISING SAME}

Degenerative neurological diseases such as stroke (stroke), Alzheimer's disease (dementia), and Parkinson's disease reduce the quality of life by impairing motor and sensory functions and inhibiting higher-order functions such as memory, learning, arithmetic, and reasoning. It causes a lot of mental and physical pain for you and your family until you get there. In addition, in light of the recent trend of the rapid increase of the elderly population due to the aging trend of modern society, as the incidence rate of neurodegenerative diseases increases and the survival time after the occurrence increases, it becomes a big social problem. According to a 1997 report by the National Statistical Office of Korea, stroke is a single disease and occupies the 1st-2nd place of death in Korea. Currently, the mortality rate from stroke is higher in Korea than in the United States, Canada and Australia. Therefore, stroke is now an important social problem that cannot be seen in our country.

Degenerative neuropathy is characterized by the death of nerve cells by rapidly or slowly progressing necrosis or apoptosis. Therefore, understanding the neuronal death mechanism must be made for the development of prevention, control and treatment of various central diseases such as trauma, stroke, Alzheimer's disease, Parkinson's disease, multiple sclerosis. For these reasons, many efforts to treat brain diseases have been devoted to studying the mechanism of neuronal death and finding ways to suppress it.

Glial cell activation and neuronal death

The central nervous system is composed of about 10% neurons and 90% glial cells, which in turn are composed of 90% astrocytes and the remaining microglia and oligodendrocytes. Glial cell activation has been observed in neurodegenerative diseases. In stroke, the activation of glial cells occurs very quickly. In ischemic stroke, activation of microglia is observed within several hours after onset, and activation of astrocytes is observed after several tens of hours. It is well known that microglia play an important role in the formation of senile plague even in Alzheimer's disease (Wegiel, J. et al., Acta Neuropathol (Berl), 100 (4): 356-64 (2000) ). An important example is that anti-inflammatory agents such as ibprofen can inhibit the formation of lytic plaques and prevent the development of dementia (Weggen, S. et al . , Nature, 414 : 212-216 (2001); Wyss-Coray, T. and Mucke). , L., Nat. Med., 6 : 973-974 (2000); Lim, GP et al ., J. Neurosci., 20 : 5709-5714 (2000)). Recently, cytokines such as those produced in peripheral blood or activated glia have been found to be factors that induce neuronal toxicity by excitatory neurotransmitters, epilepsy and degenerative neurological diseases.

Heewette et al. Found that activated glioblastoma cells can affect neuronal death (Hewett, S.J. et al.,Neuron, 13 :487-494(1994); Hewett, S.J. Etc,Stroke, 27 :1586-1591 (1996). The present inventors have studied the interrelationship between the death of activated glioblastoma cells and neurons, and the neuronal toxicity caused by N-methyl-D-aspartate (NMDA) or glucose deficiency is increased by activated microglial cells. Increased death has been attributed to increased expression of inducible NO synthase (iNOS) (Choi, JJ and Kim, WK,J. Neurosci. Res., 54: 870-875 (1998); Kim, W. K. et al.,Neurosci. Res., 33: 281-289 (1999). Therefore, in order to suppress neuronal cell death and deterioration of function, inhibiting the activation of glial cells, particularly microglia, is very important for the development of treatment for degenerative brain diseases such as stroke, Alzheimer's disease, head trauma, and the like.

Increased death of activated glia

In general, the survival rate of glial cells in brain disease is known to be higher than that of neurons. Recently, however, there are reports that glioblastoma cells die first compared to neurons in cerebral ischemia (Borlongan, CV et al., FASEB J. , 14 : 1307-1317 (2000)). The death of glial cells after ischemia seems to be due to overproduction of reactive oxygen species, decreased supply of energy sources, and decreased oxygen supply, as in neurons. Recently, however, the present inventors have revealed that glial cell death is not easy under normal conditions, but is very vulnerable to cytotoxicity after glial cells are activated (Choi and Kim, see above). The present inventors found that activating primary cultured glial cells with IFN-γ and IL-1β or IFN-γ and lipopolysaccharide (LPS) caused these glial cells to react very sensitive to energy source deficiency or oxygen deficiency, causing them to die quickly. It was. The present inventors found that the survival rate of non-activated normal glial cells is higher than that of neurons, which is due to the antioxidant system of glial cells (reducible glutathione is the main antioxidant system). It has been shown that the rapid loss of glutathione makes it susceptible to oxidative damage of peroxynitrite (Ju, C. et al ., J. Neurochem., 74 : 1989-1998 (2000)). The present inventors found that activation of microglia plays a very important role even in the death of glial cells.

Effect of Immune Inhibitors on Central Immune Activation by Ischemia

Ischemia causes activation of microglia and astrocytes. Glioblastoma cell activity has been reported to be inhibited by the administration of immunosuppressive cyclosporin A (Li, PA et al . , Exp. Neurol., 165 : 153-163 (2000); Wakita, H., et al.). , Stroke, 26 (8): 1415-1422 (1995); Kondo, Y. et al . , Neurosci.Res., 22 (1): 123-127 (1995); Pakzaban, P. et al., Neuroscience, 65 (4) : 983-996 (1995)). It has also been reported that the activity of these glia is also inhibited by cyclophosphamide or cytarabine-A (Cyt-A) (Gould, DJ and Goshgarian, HG.Exp . Neurol. 158 (2). ): 394-402 (1999)). Inhibition of glial activity by cyclophosphamide or Cyt-A appears to be due to cell division inhibitory action. Inhibition of glial activity by cyclosporin A has been reported to be enhanced by the combination of prednisolone and azathioprine (Pedersen, EB et al., Neuroscience, 78 (3): 685-701 (1997). )). In general, astrocytes are known to protect neurons. However, when microglia are activated by cytokines, they have a function similar to that of macrophages of peripheral tissues having lysosomal activity or phagocytic activity. Excessive activity of microglia is believed to be toxic to surrounding cells. Therefore, in order to suppress neuronal cell death in brain diseases such as stroke, Alzheimer's disease and Parkinson's disease, it is considered that it is very important to suppress the activation of microglia.

Development of microglia activation inhibitor

Recently, methyl dopamine derivatives have been reported to inhibit the expression of several cytokines and NOs following activation of microglial cells and to reduce neuronal damage in stroke mice (Cho, S. et al. , J. Cereb. Blood Flow Metab). , 21 : 550-556 (2001); Cho, S. et al., J. Neurosci ., 19 : 878-889 (1999). The present inventors synthesized several Chroma-based derivatives in the process of developing similar derivatives and confirmed the effect on the activation inhibition of microglia.

It is an object of the present invention to provide novel Chromamann derivative compounds useful as microglia activation inhibitors.

Another object of the present invention is to provide a method for preparing the compound.

Another object of the present invention to provide a pharmaceutical composition for inhibiting microglial activation containing the compound.

1A to 1C: Reduced nitrite production by the present compounds in BV2 microglia. 1A: compound KL1037; 1B: compound KL1044; 1C: Western analysis using iNOS antibody.

2: Reduced TNF-α production by the present compounds.

3: Amount of LDH released from BV2 / SK-N-BE (2) C coculture.

In order to achieve the above object, the present invention provides a compound of formula 1 and pharmaceutically acceptable salts thereof useful as microglia activation inhibitor.

Formula 1

Where

R is H, a C ₁ -C ₄ lower alkyl, benzyl or phenyl group and X is C═O or CH ₂ .

"C ₁ -C ₄ lower alkyl" in the present invention includes, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl.

Preferred compounds of the present invention are 6-hydroxy-7-methoxy-4-oxochroman-2-carboxylic acid propylamide, 6-hydroxy-7-methoxy-4-oxochroman-2-carboxylic acid butylamide, 6-hydroxy-7-methoxy-4-oxochroman-2-carboxylic acid phenylamide, 6-hydroxy-7-methoxy-4-oxochroman-2-carboxylic acid benzylamide, 6-hydroxy-7 -Methoxychroman-2-carboxylic acid butylamide, 6-hydroxy-7-methoxychroman-2-carboxylic acid propylamide, 6-hydroxy-7-methoxychroman-2-carboxylic acid phenylamide and 6- Hydroxy-7-methoxychroman-2-carboxylic acid benzylamide.

Most preferred compounds of the invention are 6-hydroxy-7-methoxy-4-oxochroman-2-carboxylic acid propylamide and 6-hydroxy-7-methoxy-4-oxochroman-2-carboxylic acid butylamide Includes,

The present invention is prepared according to conventional methods, for example, inorganic metal salts such as alkali metal salts (eg, sodium salts, potassium salts, etc.), alkaline earth metal salts (magnesium salts, calcium salts, etc.), ammonium salts, organic base salts ( (Eg, trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, etc.), and the like in the form of various pharmaceutically acceptable salts known in the art.

The present invention also provides a method for preparing the compound of Formula 1.

The compounds of the present invention may be prepared by the method shown in the following schemes, but are not limited to these examples. The following schemes represent the preparation steps of representative compounds of the present invention, and other compounds may be prepared by appropriate changes in reagents and starting materials known to those skilled in the art.

Derivatives of the invention are obtained using the Friedel-Crafts acylation reaction as the main step.

The process for preparing the compound of Formula 1 is illustrated by the following Scheme 1.

Methoxyhydroquinone and maleic anhydride were subjected to Friedel-Crafts acylation under an aluminum chloride catalyst to obtain an intermediate, which was then cyclized with sodium acetate to prepare intermediate KL-1055 , which was converted to oxalylchloride or dicyclohexane. After treatment with silica carbamide (DCC), the corresponding ammonia, propylamine, butylamine, aniline, benzylamine, and the like are used to treat derivatives KL-1090 , KL-1037 , KL-1044 , KL-1134 , and KL-1135 . Can be prepared.

Preparation of benzyl rigs ketone is removed carboxamide derivative compounds, intermediates KL-1055 ethanol - preparing a reduced to an intermediate KL-1073 contact under 10% pd / c catalyst in acetic acid solvent, and the intermediate keto the KL-1073 Derivatives KL-1154 , KL-1155 , KL-1056 and KL-1157 may be prepared by treating butylamine, propylamine, aniline, benzylamine and the like in the same manner as the synthesis of amide derivatives.

The present invention also provides a pharmaceutical composition for inhibiting microglial activation comprising the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. Since activated microglial cells induce the death of neuronal cells, the pharmaceutical composition of the present invention which inhibits the activation of microglial cells can be used to prevent brain diseases such as stroke, Alzheimer's disease and Parkinson's disease, which can be affected by activated glioblastoma cells. It can be used for treatment.

Chromman derivative of Formula 1 may be mixed with a suitable carrier or excipient or diluted with a diluent according to conventional methods to prepare a pharmaceutical composition for treating the disease. Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like. Compositions of the present invention may be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders and the like.

The pharmaceutical compositions of the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular. Typical daily dosages of croman derivatives range from 0.1 to 500 mg / kg, preferably 0.5 to 100 mg / kg body weight, and may be administered once or in several doses. However, it is to be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the route of administration, the age, sex and weight of the patient, and the severity of the patient, and therefore the dosage may be determined in any aspect of the invention. It does not limit the scope of.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Preparation Example 1 Preparation of 4- (2,5-dihydroxy-4-methoxyphenyl) -4-oxobut-2-inoic acid

Methoxyhydroquinone (5 g, 35.68 mmol) was added to a 500 ml round flask, refluxed under nitrogen stream, and dissolved in dichloromethane (250 ml), followed by addition of maleic anhydride (5.948 g, 60.66 mmol). Then, aluminum chloride (16.651 g 128.87 mmol) was added little by little in an ice bath, followed by stirring under reflux for 24 hours. After cooling with stirring, 15 ml of distilled water was added slowly over 10 minutes in an ice bath and 35 mL of distilled water was added again over 10 minutes (Note: Adding a large amount of distilled water at once releases a lot of heat and gas). After the heat was released, 5 ml of concentrated hydrochloric acid was added thereto, and the mixture was left to stir for 40 minutes. Concentrated under reduced pressure to remove dichloromethane, dissolved in 100 ml of 60 ° C water, transferred to a beaker, cooled for 2 hours to crystallize to obtain the product.

Yield: 28.8%, 2.45 g

m. p. 177 to 179 ° C

IR (KBr): 3346, 1634 cm ^-1

¹ H-NMR (CD ₃ OD, TMS) δ: 7.05 (s, 1H, Ar-H), 6.90 (d, J = 12Hz, 1H, = C- H ), 6.47 (s, 1H, Ar- H ) , 6.28 (d, J = 12 Hz, = C- H ), 3,93 (s, 3H, O Me )

Preparation Example 2 Preparation of 6-hydroxy-7-methoxy-4-oxochroman-2-carboxylic acid (KL-1055)

Dissolve 4- (2,5-dihydroxy-4-methoxyphenyl) -4-oxobut-2-inoic acid (2 g, 8.4 mmol) in 200 ml of ethanol, dissolve sodium acetate in 50 ml of distilled water, and put it in a 500 ml round flask. The mixture was stirred at reflux for 6 hours. 50 ml of 1N hydrochloric acid was added and acidified, and only ethanol was concentrated under reduced pressure. The remaining water layer was extracted with ethyl acetate and concentrated under reduced pressure to crystallize.

Yield: 67%, 1.34 g

m. p .: 200 to 204 ° C

IR (KBr): 3441, 1680, 1624 cm ^-1

¹ H-NMR (CD ₃ OD, TMS) δ: 6.91 (s, 1H, Ar- H ), 6.72 (s, 1H, Ar- H ), 5.48 (s, 1H, OC H (CH ₂ ) COOH), 3.94 (s, 3H, O Me ), 2.96 (d, J = 16.7 Hz, 1H, COC H ₂ ), 2.68 (dd, J = 16.7 Hz, J = 7.6 Hz, 1H, COC H ₂ )

Preparation Example 3 Preparation of 6-hydroxy-7-methoxychroman-2-carboxylic Acid (KL-1073)

6-hydroxy-7-methoxy-4-oxochroman-2-carboxylic acid (KL-1055) (1 g, 4.20 mmol) was dissolved in ethanol and placed in a 250 ml flask, where 10% pd / c (500 mg) and AcOH (0.3 ml) were added, followed by stirring under hydrogen stream for one day. The platinum catalyst was filtered through a celite pad, concentrated and then 3% MeOH / CH₂Cl₂Purified by flash column chromatography (silica gel 230-400 mesh, Merck 9385) using (500 ml) + AcOH (2 ml) as the developing solvent.KL-1073Got.

Yield: 55.8%, 525 mg

m. p .: 129-131.1 ° C

IR (KBr): 3426, 1707 cm ^-1

¹ H-NMR (CD ₃ OD, TMS) δ: 6.37 (s, 1H, Ar-H), 6.13 (s, 1H, Ar-H), 4.78 (t, 1H, J = 7.2 Hz, 1H, OC H (CH ₂ ) CO), 3.51 (s, 3H, OMe), 3.00-2.95 (m, 1H, C H ₂ Ph), 2.57-2.50 (m, 1H, C H ₂ Ph), 2.47-2.33 (m, 2H, CH ₂ ).

Example 1 Preparation of 6-hydroxy-7-methoxy-4-oxochroman-2-carboxylic acid amide (KL-1090)

6-hydroxy-7-methoxy-4-oxochroman-2-carboxylic acid (KL-1055) (300 mg, 1.26 mmol) was placed in a 100 ml flask and dissolved with 40 ml of dichloromethane. 0.45 ml of oxalyl chloride was added, followed by adding two drops of dimethylformamide and stirring for 18 hours under nitrogen. The reaction solution was concentrated under reduced pressure and dissolved in 6 ml of dichloromethane.2ml)Was dissolved in 12 ml of dichloromethane and stirred for 18 hours. Extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated and purified by flash column chromatography (silica gel 230-400 mesh, Merck 9385) using 5% methanol / dichloromethane as a developing solvent.L-1090Got.

Yield: 65.6%, 196 mg

m. p .: 219 ° C (dec)

IR (KBr): 3518, 3386, 1729 cm ^-1

¹ H-NMR (CD ₃ OD, TMS) δ: 7.11 (s, 1H, Ar-H), 6.78 (s, 1H, Ar-H), 3.80 (s, 3H, OMe), 3.62 (s, 2H, COC H ₂ ), 3.15 (s, 2H, N H ₂ )

Example 2 Preparation of 6-hydroxy-7-methoxy-4-oxochroman-2-carboxylic acid propylamide (KL-1037)

(Manufacturing method 1)

Dissolve 6-hydroxy-7-methoxy-4 -oxochroman- 2-carboxylic acid ( KL-1055 ) (200mg, 0.84mmol) in 10ml of dichloromethane, add 0.3ml of oxalylchloride and 18 hours under nitrogen. Was stirred. The reaction solution was concentrated and dissolved in 4 ml of dichloromethane, and a solution of 0.26 ml (3.17 mmol) of propylamine in dichloromethane (8 ml) was added little by little and stirred for 18 hours. The reaction solution was concentrated and purified by flash column chromatography (silica gel 230-400 mesh, Merck 9385) using EtOAc: hexane (1: 2) as a developing solvent to obtain the derivative KL-1037 .

Yield: 15.2%, 36 mg

m. p .: 115.9 ~ 157.6 ℃

IR (KBr): 3436, 3285, 1646 cm ^-1

¹ H-NMR (CD ₃ OD, TMS) δ: 7.09 (s, 1H, Ar-H), 6.79 (s, 1H, Ar-H), 3.88 (s, 3H, OMe), 3.30 (s, 2H, COCH ₂ ), 3.30 (s, 1H, NH), 3.16 (t, J = 7 Hz, 2H, NHC H ₂ CH ₂ CH ₃ ), 1.57-1.47 (m, 2H, NHCH ₂ C H ₂ CH ₃ ), 0.92 (t, J = 7.4 Hz, 3H, NHCH ₂ CH ₂ C H ₃ )

(Manufacturing Method 2)

6-hydroxy-7-methoxy-4-oxochroman-2-carboxylic acid (KL-1055) (238 mg, 1 mmol) was dissolved in 10 ml of anhydrous tetrahydrofuran (THF) and nitrogen-substituted. Cool to 0 ° C., add dicyclohexylcarbodiamide (DCC) (206 mg, 1 mmol) and stir for 5 minutes, add hydroxybenzotriazole (HOBT) (163 mg, 1 mmol) and stir at 0 ° C. for 10 minutes. It was. After reacting at room temperature for 3 hours, the mixture was concentrated under reduced pressure at 40 ° C. The concentrate was dissolved in 10 ml of dichloromethane, and 0.7 ml of 8.23 mmol propylamine was added in excess to react with nitrogen for 3 hours. The resulting dicyclohexyl urea (DCU) was filtered off and concentrated, then EtOAc: CH₂Cl₂(1: 1) was purified by flash column chromatography (silica gel 230-400 mesh, Merck 9385) using a developing solvent.KL-1037Got.

Yield: 63%, 176 mg

Example 3 : Preparation of 6-hydroxy-7-methoxy-4-oxochroman-2-carboxylic acid butylamide (KL-1044)

( Manufacturing method 1) : The derivative KL-1044 was obtained like the manufacturing method of Example 2 except having used 0.33 ml of 3.17 mmol butyl amines instead of propylamine.

Yield: 18.1%, 45 mg

m. p .: 67-69.3 degreeC

IR (KBr): 3371, 2928, 1697 cm ^-1

¹ H-NMR (CD ₃ OD, TMS) δ: 6.86 (s, 1H, Ar-H), 6.66 (s, 1H, Ar-H), 3.89 (s, 3H, OMe), 3.11 (t, J = 6.8 Hz, 2H, NHC H ₂ CH ₂ CH ₂ CH ₃ ), 2.75 (dd, J = 15.2 Hz, J = 3.6 Hz, 1H, COC H ₂ ), 2.48 (dd, J = 15.2 Hz, J = 8.4 Hz , 1H, COC H ₂ ), 1.45-1.36 (m, 2H, NHC H ₂ C H ₂ CH ₂ CH ₃ ), 1.32-1.22 (m, 2H, NHC H ₂ CH ₂ C H ₂ CH ₃ ), 0.87 ( t, J = 7.2 Hz, 3H, NHC H ₂ CH ₂ CH ₂ C H ₃ )

Production method 2 : Derivative KL-1044 was obtained by the same method as Production method 2 of Example 2, except that 0.7 ml of 6.72 mmol butyl amine was used instead of propylamine.

Yield: 75%, 220 mg

Example 4 : Preparation of 6-hydroxy-7-methoxy-4-oxochroman-2-carboxylic acid phenylamide (KL-1134)

It was prepared in the same manner as in Preparation 2 of Example 2, except that 1 ml of 10.9 mmol aniline was used instead of propylamine.

Yield: 89%, 313 mg

m. p .: 145-165 degreeC

IR (KBr): 3325, 2928, 1690 cm ^-1

¹ H-NMR (CD ₃ OD, TMS) δ: 7.70 (d, J = 7.8 Hz, 2H, Ar-H), 7.46 (t, J = 7.7 Hz, 2H, Ar-H), 7.26 (t, J = 7.3 Hz, 1H, Ar-H) 7.11 (s, 1H, Ar-H), 6.91 (s, 1H, Ar-H), 5.18-5.14 (m, 1H, OC H (CH ₂ ) CO), 4.11 (s, 3H, OMe), 3.20 (dd, J = 15.5 Hz, J = 3.3 Hz, 1H, COC H ₂ ), 2.91 (dd, J = 15.5 Hz, J = 8.6 Hz, 1H, COC H ₂ )

Example 5 Preparation of 6-hydroxy-7-methoxy-4-oxochroman-2-carboxylic acid benzylamide (KL-1135)

It was prepared in the same manner as in Preparation 2 of Example 2, except that 1 ml of 9.1 mmol benzyl amine was used instead of propylamine.

Yield: 61.5%, 200 mg

m. p .: 97.2-99.9 ° C

IR (KBr): 3289, 3096, 1682 cm ^-1

¹ H-NMR (CD ₃ OD, TMS) δ: 6.89-6.82 (m, 5H, Ar-H), 6.47 (s, 1H, Ar-H), 6.26 (s, 1H, Ar-H), 3.91 ( s, 2H, NHC H ₂ ), 3.50 (s, 3H, OMe), 2.44 (d, J = 15.2 Hz , 1H, COC H ₂ ), 2.17 (d, J = 15.2 Hz , 1H, COC H ₂ )

Example 6 : Preparation of 6-hydroxy-7-methoxychroman-2-carboxylic acid butylamide (KL-1154)

6-hydroxy-7-methoxychroman-2-carboxylic acid ( KL-1073 ) (100 mg, 0.45 mmol) was prepared in the same manner as in Production Example 2 of Example 3, using the starting material.

Yield: 89%, 113.4 mg

m. p .: 90-160 degreeC

IR (KBr): 3325, 2929, 1627 cm ^-1

¹ H-NMR (CD ₃ OD, TMS) δ: 6.55 (s, 1H, Ar-H), 6.31 (s, 1H, Ar-H), 4.96 (s, 1H, OC H (CH ₂ ) CO), 3.68 (s, 3H, OMe), 3.35 (s, 1H, C H ₂ Ph), 3.18-3.10 (m, 2H, NHC H ₂ CH ₂ CH ₂ CH ₃ ), 2.75-2.68 (m, 1H, C H ₂ Ph), 2.56 (m, 2H, CH ₂ ), 1.77-1.73 (m, 2H, NHC H ₂ C H ₂ CH ₂ CH ₃ ), 1.38-1.36 (m, 2H, NHC H ₂ CH ₂ C H ₂ CH ₃ ), 0.86-0.84 (m, 3H, NHC H ₂ CH ₂ CH ₂ C H ₃ ).

Example 7 : Preparation of 6-hydroxy-7-methoxychroman-2-carboxylic acid propylamide (KL-1155)

6-hydroxy-7-methoxychroman-2-carboxylic acid ( KL-1073 ) (100 mg, 0.45 mmol) was prepared in the same manner as in Preparation Example 2 of Example 2, using the starting material.

Yield: 77.7%, 92 mg

m. p .: 120.9-124.5 ° C

IR (KBr): 3308, 2931, 1643 cm ^-1

¹ H-NMR (CD ₃ OD, TMS) δ: 6.80 (s, 1H, Ar-H), 6.55 (s, 1H, Ar-H), 5.20 (t, 1H, J = 7.3 Hz, 1H, OC H (CH ₂ ) CO), 3.92 (s, 3H, OMe), 3.60-3.46 (m, 2H, NHC H ₂ CH ₂ CH ₃ ), 3.33-3.25 (m, 1H, C H ₂ Ph), 2.98-2.93 (m, 1H, C H ₂ Ph), 2.76-2.66 (m, 2H, CH ₂ ), 1.68-1.48 (m, 2H, NHC H ₂ C H ₂ CH ₃ ), 1.08 (t, J = 7.4 Hz, 3H, NHCH ₂ CH ₂ C H ₃ )

Example 8: Preparation of 6-hydroxy-7-methoxychroman-2-carboxylic acid phenylamide (KL-1156)

6-hydroxy-7-methoxychroman-2-carboxylic acid ( KL-1073 ) (100 mg, 0.45 mmol) was prepared in the same manner as in Example 4, using the starting material.

Yield: 70.4%, 94 mgg

m. p .: 115.4 ~ 116.9 ° C

IR (KBr): 3346, 1634 cm ^-1

¹ H-NMR (CD ₃ OD, TMS) δ: 7.68 (d, J = 7.2 Hz, 2H, Ar-H), 7.43 (t, J = 7.2 Hz, 2H, Ar-H), 7.22 (t, J = 7.2 Hz, 1H, Ar-H), 6.80 (s, 1H, Ar-H), 6.57 (s, 1H, Ar-H), 5.29 (t, J = 7.2 Hz, 1H, OC H (CH ₂ ) CO), 3.90 (s, 3H, OMe), 3.32 (s, 1H, C H ₂ Ph), 2.83-2.79 (m, 1H, C H ₂ Ph), 2.76-2.62 (m, 2H, CH ₂ ).

Example 9 : 6-hydroxy-7-methoxychroman-2-carboxylic acid benzylamide (KL-1157)

6-hydroxy-7-methoxychroman-2-carboxylic acid ( KL-1073 ) (100 mg, 0.45 mmol) was prepared in the same manner as in Example 5 using starting material.

Yield: 61.6%, 111 mg

m. p .: 159.8-161.5 ° C

IR (KBr): 3323, 2926, 1625 cm ^-1

¹ H-NMR (CD ₃ OD, TMS) δ: 7.46 (s, 5H, Ar-H), 6.81 (s, 1H, Ar-H), 6.56 (s, 1H, Ar-H), 5.26 (t, J = 7.3 Hz, 1H, OC H (CH ₂ ) CO), 4.56 (s, 2H, NHC H ₂ ), 3.94 (s, 3H, OMe), 3.42-3.37 (m, 1H, C H ₂ Ph), 3.04-2.96 (m, 1H, C H ₂ Ph), 2.89-2.70 (m, 2H, CH ₂ ).

As described below, the biological efficacy of the target compound obtained from the above-described methods was measured. The glial cell activation inhibitory effect was measured through nitrite production, iNOS expression inhibition, and TNF-α production inhibition, and activated glial cells through LDH analysis. The effect of inhibiting neuronal cell death by was measured.

Test Example 1: Confirmation of inhibition of microglial activation of Chromamann compounds

Inhibition of iNOS Expression and Production of Nitrite by Activated Microglial Cells

Dissolve 100,000 microglial cell line BV2 cells (Weill Medical College of Cornell University at Burke Medical Research Institute, White Plains, NY, USA) in a 24-well plate at 100,000 wells per well and incubate overnight in medium, then 100ng / ml LPS And test drugs (see FIG. 1 for dose) were added to the culture medium, and further incubated for 24 hours, and then the amount of nitrite released into the medium was measured using grease reagent (Choi, JJ and Kim, WK, J. Neurosci.Res., 54 : 870-875 (1998). Cells were treated with RIPA buffer (1x phosphate buffered saline (PBS), 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 0.4 mM PMSF, 30 ug / ml aprotinin, 1 nM sodiumorthovanadate) Proteins were isolated and electrophoresed on 10% SDS polyacrylamide gels and then analyzed by Western analysis using iNOS antibody (BD Biosciences, USA) to compare the amount of iNOS protein expressed (Cho, S. et al . , Glia. 33). : 324-33 (2001).

As shown in FIG. 1A, KL1037 reduced the amount of NO released by LPS (100 ng / ml) to about 2/3 at a concentration of 40 μM and to about 1/12 at a concentration of 400 μM.

As shown in FIG. 1B, KL1044 reduced the amount of NO liberated by LPS (100 ng / ml) to about 2/3 at a concentration of 20 μM and to about 1/4 at a concentration of 200 μM.

As shown in FIG. 1C, it was confirmed that the production of iNOS protein was significantly reduced by KL1037 (400 μM) and KL1044 (200 μM) in Western analysis using iNOS antibody.

The inhibitory effect of KL1037 and KL1044 on NO and iNOS expression were obtained in small neuroglial cells of primary culture rats.

Inhibition of TNF-α Production by Activated Microglial Cells

The BV2 mouse microglia were treated with LPS and test drugs in the same manner as described above, and TNF-α released into the medium after 6 or 24 hours of incubation was measured using a TNF-α ELISA kit (DIACLONE Research, USA). .

As shown in FIG. 2, 0.4 mM KL1037 reduced TNF-α liberated by 100 ng / ml LPS from about 1/20 to 1/10 (reduced to nearly basal levels), while 0.2 mM KL1044 was about Reduction from 1/3 to 1/4 fold. The degree of reduction was the same at 6 hours and 24 hours.

TNF-α free inhibitory effects of KL1037 and KL1044 were obtained in primary cultured rat microglial cells.

IC on NO and TNF-α Free Inhibitory Activity of Activated Microglial Cells ₅₀ value

IC ₅₀ values for the NO and TNF-α free inhibitory action produced in activated microglia of the compounds of the present invention, including KL1037 and KL1044, are shown in Tables 1 and 2.

compound NHR IC ₅₀ (μM) NO glass TNF-α glass KL-1055 -OH > 500 > 500 KL-1037 NHCH ₂ CH ₂ CH ₃ 94 88 KL-1044 -NHCH ₂ CH ₂ CH ₂ CH ₃ 42 43 KL-1134 -NHC ₆ H ₅ 22 32 KL-1135 -NHCH ₂ C ₆ H ₅ 29 33

compound NHR IC ₅₀ (μM) NO glass TNF-α glass KL-1073 -OH > 500 > 500 KL-1154 -NHCH ₂ CH ₂ CH ₂ CH ₃ 32 41 KL-1155 NHCH ₂ CH ₂ CH ₃ 33 37 KL-1156 -NHC ₆ H ₅ 39 40 KL-1157 -NHCH ₂ C ₆ H ₅ 40 42

Test Example 2 Confirmation of Inhibition of Neuronal Apoptosis of Chromann Compound

Inhibition of neuronal cell death by activated microglia

Mouse microglial BV2 cells were grown in microporous membranes of Transwell cell culture inserts (NUNC, USA) and then treated with 100 ng / ml LPS for 6 hours to activate microglial cells. Inserts containing activated small neuroglial cells were co-cultured in wells containing SK-N-BE (2) C neuroblastoma cells (ATCC #: CRL-2271, USA). At this time, KL1037 and KL1044 were added to the coculture medium at 0.4 mM and 0.2 mM, respectively. After 36 h of incubation, the morphology of SK-N-BE (2) C neuroblastoma cells was observed and the degree of cell death was compared by lactate dehydrogenase (LDH) free assay, an indicator of cell death ( Choi, JJ and Kim, WK, J. Neurosci.Res., 54 : 870-875 (1998)).

As shown in FIG. 3, 0.4 mM KL1037 reduced neuronal cell death by activated small neuroglial cells by about 70% and 0.2 mM KL1044 by about 77%.

The neuronal apoptosis inhibitory effects of KL1037 and KL1044 were obtained in the primary microcultured rat microglia cells.

The novel Chromann-based compound of the present invention and the pharmaceutical composition containing the same are useful as therapeutic agents for brain diseases such as stroke, Alzheimer's disease and Parkinson's disease by acting as inhibitors of activation of microglia and inhibiting neuronal cell death.

Claims (4)

Chroman derivatives of the formula (1) or pharmaceutically acceptable salts thereof: Formula 1 Where R is H, a C ₁ -C ₄ lower alkyl, benzyl or phenyl group and X is C═O or CH ₂ . The method of claim 1, Chromane derivatives selected from the group consisting of: 6-hydroxy-7-methoxy-4-oxochroman-2-carboxylic acid propylamide, 6-hydroxy-7-methoxy-4-oxochroman-2-carboxylic acid butylamide, 6-hydroxy-7 -Methoxy-4-oxochroman-2-carboxylic acid phenylamide, 6-hydroxy-7-methoxy-4-oxochroman-2-carboxylic acid benzylamide, 6-hydroxy-7-methoxychroman- 2-carboxylic acid butylamide, 6-hydroxy-7-methoxychroman-2-carboxylic acid propylamide, 6-hydroxy-7-methoxychroman-2-carboxylic acid phenylamide and 6-hydroxy-7-meth Oxychroman-2-carboxylic acid benzylamide. Claim 1 Chromman derivative or a pharmaceutically acceptable salt thereof as an active ingredient comprising a pharmaceutically acceptable carrier, pharmaceutical composition for inhibiting microglial activation. The method of claim 3, wherein A pharmaceutical composition, characterized in that it is used for the prevention and treatment of stroke, selected from Alzheimer's disease and Parkinson's disease.