WO2009041789A2

WO2009041789A2 - Novel 3-chloro-5-substituted-quinoxaline-2-amine derivatives and pharmaceutically acceptable salt thereof, method for preparation, therapeutic agent for inflammatory disease induced by spc activity containing 3-chloro-5-substituted-quinoxaline-2-amine derivatives as an effective ingredient

Info

Publication number: WO2009041789A2
Application number: PCT/KR2008/005725
Authority: WO
Inventors: Young-Dae Gong; Heeyeong Cho; Moon-Kook Jeon; Taeho Lee; Gildon Choi; Jae-Yang Kong; Woo-Kyu Park; Soon-Hee Hwang; Jung Ju Kim; Chang-Hoon Lee; Jaeyoung Ko; Minsoo Noh; Eun Sil Han; Hyuk Kim; Jun-Won Yun; Joo Hyun Moh; Do Hoon Kim
Original assignee: Korea Research Institute Of Chemical Technology; Amorepacific Corporation
Priority date: 2007-09-27
Filing date: 2008-09-26
Publication date: 2009-04-02
Also published as: KR20090032373A; WO2009041789A3; KR100916716B1

Abstract

Provided are a 3-chloro-5-substiuted-quinoxalin-2-amine derivative represented by the following Chemical Formula 1, a pharmaceutically acceptable salt thereof, a method for preparation thereof, and a use thereof as an effective ingredient in a therapeutic agent for inflammatory disease induced by activity of sphingosyl phosphorylcholine (SPC) receptor. The 3-chloro-5-substiuted-quinoxalin-2-amine derivative of the present invention has been confirmed to have superior inhibition activity against SPC receptor in an animal experiment using human-derived endothelial cells and mice. Thus, a therapeutic agent for inflammatory disease containing the 3- chloro-5-substiuted-quinoxalin-2-amine derivative or a pharmaceutically acceptable salt thereof as an effective ingredient may be useful for treating inflammation, itching or skin infection associated with atopic dermatitis or other disease induced by activity of SPC receptor.

Description

[DESCRIPTION]

[Invention Title]

NOVEL 3-CHL0R0-5-SUBSTITUTED-QUIN0XALINE-2-AMINE DERIVATIVES AND PHARMACEUTICALLY ACCEPTABLE SALT THEREOF, METHOD FOR PREPARATION THEREOF, AND THERAPEUTIC AGENT FOR INFLAMMATORY DISEASE INDUCED BY SPC ACTIVITY CONTAINING 3-CHL0R0-5-SUBSTITUTED-QUIN0XALINE-2-AMINE DERIVATIVES AS AN EFFECTIVE INGREDIENT

[Technical Field]

Example embodiments of the present invention relate to a 3-chloro^~5- substiuted-quinoxalin-2-amine derivative, a pharmaceutically acceptable salt thereof, a method for preparation thereof, and a therapeutic agent for inflammatory disease induced by activity of sphingosylphosphorylcholine (SPC) receptor containing the same as an effective ingredient, more particularly, to a 3-chloro-5-substiuted-quinoxalin-2-amine derivative as a novel compound exhibiting inhibition activity against SPC receptor, a pharmaceutically acceptable salt thereof, and pharmaceutical composition for treating inflammatory disease containing the derivative or the pharmaceutically acceptable salt thereof as an effective ingredient.

[Background Art]

Sphingosylphosphorylcholine (SPC) is the member of the Lysophospholipid family along with structurally similar sphingosine-1-phosphate (SlP) and lysophosphatidic acid (LPA). These materials act as important signaling mediators in immune actions, including cell proliferation, migration, inflammation, and the like.

SPC is produced from sphingomyelin, a component of the cell membrane, by the action of the enzyme sphingomyelin deacylase [Higuchi K, Biochem. J., 2000, 350, 747-56]. SPC is known to be deeply associated with growth and proliferation of various types of cells [Desai , Biochem. Biophys. Res. Commun. , 1991, 181, 361-366], angiogenesis [Bogus1awski , Biochem. Biophys. Res. Commun., 2000, 272, 603-609], apoptosis [Jeon ES, Biochem. Biophys. Acta., 2005, 1734(1); 25-33], and the like. A typical example of SPOrelated diseases is atopic dermatitis. Atopic dermatitis results in reduced antibacterial activity due to decreased lipid content in the stratum corneum and reduced resistance to external stimulants because of reduced barrier capability. As a result, it causes inflammatory reactions and itching. Since the itching may lead to secondary infections, the hyperimmune response may result in a vicious cycle.

While SPC exists hardly or at very low concentrations in healthy people, its concentration in the skin of atopic dermatitis patients increases thousands of times [Higuchi K, Biochem. J., 2000, 350, 747-756; Reiko Okamoto, Journal of Lipid Research, 2003, 44, 93-102]. It is the main cause of deficiency of intracellular lipids (ceramides) in the stratum corneum of atopic dermatitis patients [Junko Hara, /. invest. Dermatol., 2000, 115, 406- 413]. Further, SPC plays an important role in the abnormal cornificat ion associated with the atopic disease [Higuchi, /. Lipid Res. , 2001, 42, 1562- 1570]. These researches suggest that SPC may be not only the direct cause of skin barrier function disorder characteristic of the atopic dermatitis, but also the cause of secondary inflammatory responses. Thus, the control of the production of SPC may lead to the development of a new therapeutic agent for skin inflammatory disease.

With regard to itching, which is a symptom of the atopic dermatitis afflicting the patient with pain and decreased quality of life, it was reported that LPA, which is structurally similar to SPC, induces itching [Hashimoto, Pharmacology, 2004, 72, 51-56]. Accordingly, it can be inferred that SPC may cause itching, too. Recently, it was shown that an intradermal injection of SPC may directly cause itching [WO 06/049451].

The inventors of the present invention have researched to develop novel compounds that can be used as pharmaceutical composition for treating inflammatory disease. They designed and synthesized a 3-chloro-5-substiuted- quinoxalin-2-amine derivative, which has not yet been reported to exhibit inhibition activity against SPC receptor. Through experiments using human- derived endothelial cells and mice, they confirmed that the 3-chloro-5- substiuted-quinoxalin-2-amine derivative has superior ant iinflamatory effect and completed the present invention. [Disclosure] [Technical Problem]

In an aspect, the present invention provides a 3-chloro-5-substiuted- quinoxal in-2-amine derivative prepared through an organic synthesis technique, a pharmaceutically acceptable salt thereof and a method for preparation thereof.

In another aspect, the present invention provides a use of the 3- ch1oro-5-substiuted-quinoxal in-2-amine derivative or the pharmaceutically acceptable salt thereof as an effective ingredient of a pharmaceutical composition for treating inflammatory disease induced by activity of sphingosylphosphorylcholine (SPC) receptor. [Technical Solution]

The present invention provides a 3-chloro-5-substiuted-quinoxalin-2- amine derivative represented by the following Chemical Formula 1 and a pharmaceutically acceptable salt thereof: [Chemical Formula 1]

wherein R i s hydrogen, Ci-C₁₀ l inear or branched alkyl , C₁-Ci₀ alkoxy or

2 halogen; R is one to four Ci-Cio linear or branched alkyl, C₁-Ci₀ alkoxy,

hydroxy, halogen, amine, substituted amine, amide, carbamate or urea, the substituent being phenyl substituted by 1-4 substituents selected from the group consisting of Ci-C₁₀ linear, branched or cyclic alkyl, halogen, nitro,

Ci-Cio alkyl, Ci-Ci₀ alkoxy and Ci-Ci₀ haloalkyl.

In the 3-chloro-5-substiuted-quinoxal in-2-amine derivative, R may be 2

C₁-Cs linear or branched alkyl or alkoxy, or fluoro; and R may be selected

from the group consisting of one or two CrCs linear or branched alkyl, C₁-Cs alkoxy, nitro and halogen.

The present invention further provides a method for preparation of a 3- chloro-5-substiuted-quinoxalin-2-amine derivative using an organic synthesis technique.

The present invention further provides a pharmaceutical composition for treating inflammatory disease induced by activity of sphingosylphosphorylcholine (SPC) receptor containing the 3-chloro-5^~ substiuted-quinoxalin-2-amine derivative represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an effective ingredient.

The present invention further provides a pharmaceutical composition containing the 3-chloro-5-substiuted-quinoxalin-2-amine derivative represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an effective ingredient for preventing scarring after injury and promoting wound healing.

The present invention further provides a modulator of chemotaxis- mediated symptoms containing the 3-chloro-5-substiuted-quinoxalin-2-amine derivative represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an effective ingredient.

[Advantageous Effects]

The present invention provides a 3-chloro-5-substiuted-quinoxalin-2- amine derivative and a pharmaceutically acceptable salt thereof, which exhibits superior inhibition activity against sphingosylphosphorylcholine (SPC) receptor in an animal model experiment using human-derived endothelial cells and mice. The present invention further provides a pharmaceutical composition for treating inflammatory disease induced by activity of SPC receptor. [Mode for Invention]

Hereinafter, the present invention will be described in more detail.

The present invention provides a 3-chloro-5-substiuted-quinoxalin-2- amine derivative represented by the following Chemical Formula 1 and a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

1 2

(In Chemical Formula 1, R and R are the same as defined above.) i In the 3-chloro-5-substiuted-quinoxalin-2-amine derivative, R may be

2

C₁-C₃ linear or branched alkyl or alkoxy, or fluoro! and R may be selected

from the group consisting of one or two C₁-Cs linear or branched alkyl, C₁-Cs alkoxy, nitro and halogen.

The present invention further provides a method for the preparation of the 3-chloro-5-substiuted-quinoxal in-2-amine derivative represented by Chemical Formula 1, and it may be prepared by the following Scheme 1: [Scheme 1]

1 2

(wherein R and R are the same as defined above.)

In detail, the method for the preparation of the 3-chloro-5-substiuted- quinoxalin-2-amine derivative represented by Chemical Formula 1 according to the present invention comprises: reacting an R -substituted 1,2-diamino compound represented by Chemical

Formula 2 with oxalic acid to synthesize quinoxaline-2,3-dione represented by i Chemical Formula 3 in which the substituent R is introduced; reacting the compound represented by Chemical Formula 3 with thionyl chloride to synthesize 2,3-dichloroquinoxaline represented by Chemical Formula 4; and reacting the 2-chloro substituent of the compound represented by

2

Chemical Formula 4 with R-substitued 4-amino-l-arylmethylpiperidine represented by Chemical Formula 5 to synthesize the 3-chloro-5-substiuted- quinoxalin-2-amine derivative represented by Chemical Formula 1.

The reaction process, composition of the solvent system and reaction condition in accordance with the present invention will be described in detai 1.

In the first step, water (H₂O), dioxane or tetrahydrofuran (THF) is used as solvent. Preferably, water may be used. In this step, a 1,2-diamine compound having the substituent R may be used. Oxalic acid may be used in an amount of about 1.5 equivalents. Preferably, it may be used in an amount of about 1 equivalent, considering economy. An acid such as hydrochloric acid (HCl) or sulfuric acid (H₂SO₄) may be used. Use of 3 N HCl may be effective.

In the second step, chloroform (CHCI3) or 1,2-dichloroethane (DCE) is used as solvent. Preferably, chloroform may be used. In this step, thionyl chloride may be used in an amount of about 5 equivalents. Preferably, it may be used in an amount of about 3 equivalents, considering economy. Addition of a catalytic amount of dimethylformamide (DMF) may be effective.

In the third step, dimethyl sulfoxide (DMSO), DMF, chloroform or dichloromethane (CH₂Cl₂) is used as solvent. Preferably, DMSO may be used.

2 In this step, an R -substituted 4-amino-l-arylmethylpiperidine represented by

Chemical Formula 5 may be used in an amount of about 1.2 equivalents.

Preferably, it may be used in an amount of about 1.0 equivalent, considering

2 economy. The R -substituted 4-amino-l-arylmethylpiperidine is the same as defined above.

In the preparation of the 3-chloro-5-substiuted-quinoxalin-2-amine derivative represented by Chemical Formula 1 according to the present invention, the progress of reaction of each step may be confirmed by TLC. Structural analysis of the reaction intermediates represented by Chemical Formulas 3 and 4 may be carried out by MR or mass spectroscopy after separation and purification.

The present invention further provides a pharmaceutical composition for treating inflammatory disease induced by activity of sphingosylphosphorylcholine (SPC) receptor containing the 3-chloro-5- substiuted-quinoxalin-2-amine derivative represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an effective ingredient.

The pharmaceutical composition for treating inflammatory disease may comprise ^(l-benzylpiperidin-4-yl)-3-chloro-5-methoxyquinoxalin-2-amine (Compound No. 1-1, see Table 1 below) or 3-chloro-τV-[l-(3,5- diphenoxybenzyl)ρiperidin-4-yl]-5-methoxyquinoxalin-2-amine (Compound No. 1- 13, see Table 1) of the 3-chloro-5-substiuted-quinoxalin-2-amine derivative represented by Chemical Formula 1, as an effective ingredient.

The 3-chloro^~5-subst iuted-quinoxal in-2-amine derivative of the present invention was confirmed to have an antagonistic effect in selective cell proliferation induced by SPC (see Table 2). Therefore, it may be effective for atopic dermatitis or other skin disease caused by excessive cell division and proliferation induced by SPC. Further, because excessive cell division and proliferation during wound healing may result in scars through inflammatory response, the 3-chloro-5-subst iuted-quinoxal in-2-amine derivative of the present invention, which inhibits the excessive cell division and proliferation, may be used to prevent unwanted scarring. In addition, it may be used to facilitate wound healing after injury.

Further, in tetradecanoylphorbol acetate (TPA)-induced inflammatory response test, the 3-chloro-5^~subst iuted-quinoxal in-2-amine derivative of the present invention reduced ear edema and inhibited MPO activity, comparable to hydrocortisone which is commonly used to treat inflammation (see Table 4).

Accordingly, the 3-chloro^~5-subst iuted-quinoxal in-2-amine derivative of the present invention may be effective in treating inflammation, itching, skin infections, etc. associated with atopic dermatitis or other disease, and may be useful as a pharmaceutical composition for preventing scarring after injury and promoting wound healing.

Further, the present invention provides a modulator of chemotaxis- mediated symptoms containing the 3-chloro-5-subst iuted-quinoxal in-2-amine derivative represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an effective ingredient.

Chemotaxis is the phenomenon in which endothelial cells or immune cells are attracted by specific materials such as cytokines or chemokines. By this, immune cells move to inflamed area or endothelial cells migrate to result in angiogenesis.

The 3-chloro-5-subst iuted-quinoxal in-2-amine derivative represented by Chemical Formula 1 according to the present invention was confirmed to be able to strongly inhibit the migration of endothelial cells or immune cells induced by SPC (see Table 3). Accordingly, a modulator of chemotaxis- mediated symptoms comprising the 3-chloro-5-substiuted-quinoxalin-2-amine derivative represented by Chemical Formula 1 according to the present invention or a pharmaceutically acceptable salt thereof as an effective ingredient may inhibit angiogenesis caused by the migration of endothelial cells and may control the amplification of immune response to antigens from outside.

Preferably, the modulator of chemotaxis-mediated symptoms may comprise yV^L(l-benzylpiperidin-4-yl)-3-chloro-5-methoxyquinoxalin-2-amine of the 3- chloro-5-substiuted-quinoxalin-2-amine derivative represented by Chemical Formula 1 as an effective ingredient.

Specific examples of the chemotaxis-mediated symptoms that can be controlled by the modulator of chemotaxis-mediated symptoms according to the present invention may include inflammation, itching and skin infection associated with atopic dermatitis or other disease.

The pharmaceutically acceptable salt according to the present invention may be one that can be prepared by a method commonly used in the related art. For example, a pharmaceutically acceptable acid salt may be prepared using an inorganic acid such as hydrochloric acid, hydrogen bromide, sulfuric acid, sodium bisulfate, phosphoric acid, carbonic acid, etc. or an organic acid such as formic acid, acetic acid, oxalic acid, benzoic acid, citric acid, tartaric acid, gluconic acid, gentisic acid, fumaric acid, lactobionic acid, salicylic acid, acetylsalicylic acid (aspirin), etc., a metal salt may be prepared using an alkali metal ion such as sodium, potassium, etc., or other pharmaceutically acceptable salt may be prepared using an ammonium ion.

Further, a commonly used non-toxic pharmaceutically acceptable carrier, modifier or excipient may be added to the 3-chloro-5-substiuted-quinoxalin-2- amine derivative of the present invention or a pharmaceutically acceptable salt thereof to prepare a pharmaceutical composition in oral or parenteral preparation forms common in the pharmaceutical field, e.g. tablet, capsule, troche, liquid, suspension, etc.

The administration dose of the compound of the present invention may vary depending on the age, body weight and sex of the patient, administration route, physical conditions and severity of disease. For an adult patient weighing 70 kg, a usual dosage may be 0.01-1,000 mg/day. Depending on the physician's or pharmacist's decision, it may be administered once or several times a day at predetermined intervals.

Hereinafter, the present invention will be described in detail through examples.

However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by them.

Example 1: Synthesis of 3-chloro-5-substiuted-quinoxalin-2-amine derivative (Chemical Formula 1-1)

Step 1: Reaction of 1,2-diamino compound (Chemical Formula 2) with oxalic acid

A 3-methoxybenzene-l,2-diamine compound (1.86 g, 13.5 mmol) represented by Chemical Formula 2-1 and oxalic acid (1.23 g, 13.6 mmol) were added to 10 mL of 3 N HCl solution. After stirring at room temperature for 10 minutes, reaction was carried out for 3 days under reflux. Then, after cooling to room temperature, the reaction mixture was filtered. The filtered compound was repeatedly washed with cold water to obtain a quinoxaline-2,3- dione compound (2.26 g, 87%) represented by Chemical Formula 3-1.

¹H NMR (500 MHz, DMS(Hi₆) 3.86 (s, 3H), 6.75 (d, J = 8.1 Hz₁ IH), 6.80 (d, J

= 8.0 Hz, IH), 7.05 (t, J = 8.2 Hz, IH), 11.24 (br s, IH), 11.91 (br s, IH); n/z ( [M+l] ) 139.

Step 2: Conversion of quinoxaline-2,3-dione (Chemical Formula 3-1) to 2,3-dichloroquinoxal ine

The 5-methoxyquinoxaline-2,3(L//,4/0-dione compound (2.26 g, 11.7 mmol) represented by Chemical Formula 3-1 was dissolved in 10 mL of chloroform and a catalytic amount (0.3 mL) of DMF, and thionyl chloride (SOCl₂; 2.60 mL, 35.1 mmol) was added at room temperature. After 3 days of reaction under reflux, the reaction mixture was cooled to room temperature, distilled under reduced pressure, and separated and purified by silica gel column chromatography to obtain a 2,3-dichloroquinoxal ine compound (1.74 g, 65%) represented by Chemical Formula 4-1.

¹H NMR (500 MHz, CDCl₃) 4.13 (s, 3H), 7.19 (d, J = 7.9 Hz, IH), 7.64 (d, J =

8.1 Hz, IH), 7.77 (t, J = 8.2 Hz, IH); m/z ([M+l]⁺) 228.

Step 3: Addition of amine (Chemical Formula 5-1) to 2,3- dichloroquinoxal ine (Chemical Formula 4-1)

The 2,3-dichloro-5-methoxyquinoxaline compound (60 mg, 0.26 mmol) represented by Chemical Formula 4-1 and a l-benzylpiperidine-4-amine compound (54 mg, 0.28 mmol) represented by Chemical Formula 5-1 were added to 3 mL of DMSO solution. After stirring for 10 minutes at room temperature, reaction was carried out at 60 ^°C for 2 days. Then, the reaction mixture was dissolved in 20 mL of EtOAc and washed with 20 mL of brine. After drying the organic layer using Na₂SO₄, the reaction mixture was filtered and concentrated. The concentrated reaction mixture was purified by silica gel column chromatography (hexane:Et0Ac = 1:1) to obtain the target compound 3- chloro-5-methoxyquinolin-2-amine (Chemical Formula 1-1, 41 mg, 41%).

¹H NMR (500 MHz, CDCl₃) 1.60-1.67 (m, 2H), 2.12-2.17 (m, 2H), 2.25-2.31 (m,

4H), 2.87-2.89 (m, 2H), 3.56 (s, 2H), 4.01 (s, 3H), 4.12-4.16 (m, IH), 5.43- 5.45 (m, IH), 6.77-6.78 (m, IH), 7.26-7.30 (m, IH), 7.31-7.34 (m, 5H), 7.46-

7.50 (m, IH); m/z ([M+l]⁺) 383.

Examples 2-40: Synthesis of 3-chloro-5-substiuted-quinoxalin-2-amine derivatives

3-Chloro-5-substiuted-quinoxalin-2-amine derivatives represented by Chemical Formula 1 were synthesized in the same manner as in Example 1, with

1 2 the substituents R and R being listed in Table 1.

Analysis result for the synthesized 3-chloro-5-substiuted-quinoxalin-2- amine derivatives is also given in the table.

[Chemical Formula 1]

[Table 1]

Experimental Example l: Control of cell division and proliferation Treatment of cells with SPC results in excessive cell division and proliferation, which may lead to pathological symptoms such as atopic dermatitis or other skin disease [Desai , Biochem. Biophys. Res. Commun., 1991, 181, 361-366]. The effects of the compounds prepared in Examples on the cell division and proliferation induced by SPC was tested as follows.

IxIO⁵ (normally lxl0⁴~10⁶) NIH 3T3 cells (American Type Culture Collection, Manassas, VA, USA) were cultured on a culture plate. Then, they were cultured in an RPMI medium free of bovine serum for 24 hours until serum _starvation. After treating with the compounds prepared in Examples or with FTY720 (fingolimod), an agonist of sphingosine-1-phosphate (SlP), as control compound at concentrations of 0.001 μM, 0.01 μM, 0.1 μM and 1 μM, the cells were cultured for 30 minutes. Then, after adding SPC (Biomol, Plymouth Meeting, PA, USA) at a concentration of 7 μM, the cells were cultured for 24 hours at 37°C. Cell proliferation was measured by [3H]-thymidine incorporated into DNA strands during cell division [Beales IL, Life Sci. , 2004, 75, 83-95]. Proliferation rate (%) was calculated by the following Equation 1, and the result is given in the following Table 2. [Equation 1]

p_r.._ifor,_ti._{n rat}. ₍o_/o) - (Test compound treated group) - (SPC non ^ treated group) ^ _{1 QQ}

(SPC treated group) - (SPC non - treated group)

[Table 2]

As seen from Table 2, the compounds of the present invention prepared in Examples exhibited antagonistic effect against selective cell proliferation induced by SPC. Because inflammatory response due to excessive cell division and proliferation during wound healing after injury results in scars, the material which inhibits cell division and proliferation may be used to prevent unwanted scarring. And, the material which enhances cell division and proliferation may be used to promote wound healing after injury.

Especially, the compounds of Examples 1-1 and 1-16 inhibited the cell division and proliferation induced by SPC in a dose-dependent manner. It is to be noted that FTY720, an agonist of SlP, which has a chemical structure similar to that of SPC and shares some of membrane receptors, did not inhibit the cell division and proliferation induced by SPC. Accordingly, it is conjectured that the inhibition of cell division and proliferation is due to the inherent structural activity of the compound of the present invention, and the compound of the present invention may be used to prevent scarring caused by inflammatory response due to excessive cell division and proliferation during wound healing after injury.

Experimental Example 2: Inhibition of chemotactic cell migration induced by SPC

Recently, it was reported that SPC plays an important role in chemotactic cell migration similarly to vascular endothelial growth factor (VEGF) [Boguslawski et al., Biochem. Biophys. Res. Com/nun., 2000, 272, 603- 609]. Chemotaxis, the phenomenon in which cells are attracted by specific materials such as cytokines or chemokines, is critical to the migration of immune cells or endothelial cells. The effect of the compound prepared in Examples on the chemotactic migration of cells induced by SPC was tested by the Boyden chamber technique.

A 25 x 80 mm polycarbonate membrane (Neuro Probe, Inc.) having 8 ? pores was immersed in 0.01% gelatin, 0.1% acetic acid solution. After coating overnight, the membrane was allowed to be dried at room temperature.

Human umbilical vein endothelial cells (HUVECs) cultured in a complete EBM-2 medium containing 2% fetal bovine serum (FBS) were cultivated for 4 hours in a EBM-2 medium (Cambrex, Catalog No. CC-3121) without containing bovine serum until serum starvation, and harvested with trypsin/EDTA solution. The HUVECs were suspended in a EBM-2 medium containing 0.1% bovine serum albumin (BSA), transferred to a si Iicone-coated Eppendorf tube, and treated with the test compound of Example 1-1 at concentrations of 0, 0.1, 1 and 10 βg/mL, at 37°C for 30 minutes. 27 μl of EBM-2 medium with or without containing 10 μM SPC was added to each well of the lower compartment of a Boyden chamber. The gelatin-coated membrane was placed so that a glossy surface faced downward. A gasket was placed thereon and the upper compartment was assembled. The HUVEC cells treated with the compound were

4 transferred to the upper compartment, 5x10 (56 μi) each, and cultured for 8 hours at 37°C in a CO2 incubator. The membrane was separated, stained with a

Diff-Quik stain (Sysmex Corporation), washed with deionized water, and attached on a slide glass so that a glossy surface faced upward. The cells attached on the upper portion of the membrane were cautiously wiped out using KimWipes or a swab. Photographs were taken arbitrarily, 5 fields per each well (x 200), in order to count the cells. Inhibition rate (%) was calculated by the following Equation 2, and the result is given in the following Table 3. [Equation 2]

Inhibition rπtc (^0/c) - ^{(SPC treated groυp) " (Test com}P°^{υnd treated} 9^"P) _^100

(SPC treated group) - (SPC non - treated group)

[Table 3]

As seen from Table 3, the compound of Example 1-1 strongly inhibited the chemotactic cell migration induced by SPC. This suggests that, by inhibiting the migration of endothelial cells or immune cells, the compound may control the process of angiogenesis in tumors or amplification of immune response to antigens from outside.

Experimental Example 3: Control of inflammatory response in mouse TPA- induced ear inflammation model

In order to confirm the inhibition effect against inflammatory response, experiment was carried out as follows using a Tetradecanoyl phorbol acetate(TPA)-induced inflammation model, as follows. The TPA-induced inflammation model is widely used to test the mechanism of inflammatory response and the efficiency of inhibiting substance [De Young LM et al., Agents and Actions, 1989, 26, 335-341]. TPA is a potent tumor promoter resulting in inflammatory response. When applied on the ear of a subject, it results in erythema and edema. This inflammatory response can be measured by the increased activity of myeloperoxidase (MPO), which is essential when white blood cells attack bacteria.

Forty 6-week-old male ICR mice were prepared. TPA (Sigma Aldrich Korea) dissolved in acetone at 125 μg/mL was applied on the left ears of the mice, 20 μJt per each. One hour later, acetone, 0.3% test compounds dissolved in acetone, or 0.3% hydrocortisone (Sigma Aldrich Korea) dissolved in acetone was applied on the TPA-applied area, 20 μJt per each. 24 hours later, TPA was applied again on the same area, 20 μi per each. 24 hours later, the mice were euthanized by cervical dislocation, and the left ears were taken to measure weight and MPO activity. Inhibition rate (%) was calculated by the following Equation 3, and the result is given in the following Table 4. [Equation 3]

Inhibition rate (%) = (TPA treated group) - (Test compound treated group) „ _{1 QQ}

(TPA treated group) - (TPA non - treated group)

[Table 4]

( ): MPO inhibition rate

As seen from Table 4, the compound of Example 1-1 was superior in inhibiting ear edema caused by TPA-induced inflammatory response and MPO activity, comparable to hydrocortisone, which is commonly used as antiinflammatory drug. This result signifies that the compound of Example 1- 1 inhibited the infiltration of neutrophils at the inflammation area.

Preparation Example 1: Preparation of tablet (compression) 5.0 mg of the compound represented by Chemical Formula 1, as an effective ingredient, was sieved, mixed with 14.1 mg of lactose, 0.8 mg of crospovidone USNF and 0.1 mg of magnesium stearate, and compressed into a tablet.

Preparation Example 2: Preparation of tablet (wet granulation)

5.0 mg of the compound represented by Chemical Formula 1, as an effective ingredient, was sieved, and mixed with 16.0 mg of lactose and 4.0 mg of starch. 0.3 mg of Polysorbate 80 dissolved in pure water was added in an adequate amount and subjected to granulation. After drying and sieving, the granule was mixed with 2.7 mg of colloidal silicon dioxide and 2.0 mg of magnesium stearate. The granule was compressed into a tablet.

Preparation Example 3: Preparation of powder and capsule 5.0 mg of the compound represented by Chemical Formula 1, as an effective ingredient, was sieved, and mixed with 14.8 mg of lactose, 10.0 mg of polyvinylpyrrolidone and 0.2 mg of magnesium stearate. The mixture was filled in a hard No. 5 gelatin capsule using an appropriate apparatus.

Preparation Example 4: Preparation of injection

100 mg of the compound represented by Chemical Formula 1, as an effective ingredient, was mixed with 180 mg of mannitol, 26 mg of Na2HPU4 ^•

12H₂O and 2,974 mg of distilled water to prepare an injection.

The invention has been described in detail with reference to example embodiments thereof. However, it will be appreciated by those skilled in the art that changes may be made in these embodiments without departing from the principles and spirit of the present invention, the scope of which is defined in the accompanying claims and their equivalents. [Industrial Applicability]

As described above, the present invention

1) provides a 3-chloro-5-substiuted-quinoxalin-2-amine derivative and a pharmaceutically acceptable salt thereof through a solid-phase chemical synthesis technique,

2) elucidates superior inhibition activity of the 3-chloro-5- substiuted-quinoxalin-2-amine derivative against sphingosylphosphorylcholine (SPC) receptor through an animal experiment using human-derived endothelial cells and mice, and

3) provides an inhibitor of SPC receptor and a pharmaceutical composition for treating inflammatory disease induced by SPC containing the -chloro-5-substiuted-quinoxalin-2-amine derivative as an effective ingredient, and a use thereof.

Claims

[CLAIMS] [Claim 1]A 3-chloro-5-substiuted-quinoxalin-2-amine derivative represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof:

[Chemical Formula 1]

1 wherein R i s hydrogen, C₁-CiO l inear or branched alkyl , C₁-CiO alkoxy or

2 halogen; R is one to four Ci-C₁O linear or branched alkyl, C₁-C₁O alkoxy,

hydroxy, halogen, amine, substituted amine, amide, carbamate or urea, the substituent being phenyl substituted by 1-4 substituents selected from the group consisting of Ci-Cio linear, branched or cyclic alkyl, halogen, nitro,

Ci-Cio alkyl, C₁-CiO alkoxy and Ci-Ci₀ haloalkyl.

[Claim 2]

The 3-chloro-5-substiuted-quinoxalin-2-amine derivative or the pharmaceutically acceptable salt thereof as set forth in claim 1, wherein, in

Chemical Formula 1, R is C₁-Cs linear or branched alkyl or alkoxy, or fluoro;

2 and R is selected from the group consisting of one or two C1-C5 linear or

branched alkyl, C1-C5 alkoxy, nitro or halogen.

[Claim 3]

A method for the preparation of the 3-chloro-5-substiuted-quinoxalin-2- amine derivative represented by Chemical Formula 1 as set forth in claim 1, comprising: reacting an R -substituted 1,2-diamino compound represented by Chemical Formula 2 with oxalic acid to synthesize a quinoxaline-2,3-dione represented i by Chemical Formula 3 in which the substituent R is introduced; reacting the compound represented by Chemical Formula 3 with thionyl chloride to synthesize a 2,3-dichloroquinoxaline represented by Chemical Formula 4; and reacting the 2-chloro substituent of the compound represented by

2

Chemical Formula 4 with an R -substitued 4-am i no- 1-ar yl met hylpi peri dine represented by Chemical Formula 5 to synthesize the 3-chloro-δ-substiιιted- quinoxalin-2-amine derivative represented by Chemical Formula 1: [Scheme 1]

rreefflluuxx _C|,

- ' -

1 2 wherein R and R are the same as defined in claim 1.

[Claim 4]

A pharmaceutical composition for treating inflammatory disease induced by activity of sphingosylphosphorylcholine (SPC) receptor containing the 3- chloro-5-substiuted-quinoxalin-2-amine derivative represented by Chemical Formula 1 or the pharmaceutically acceptable salt thereof as set forth in claim 1 as an effective ingredient.

[Claim 5]

The pharmaceutical composition for treating inflammatory disease induced by activity of SPC receptor as set forth in claim 4, wherein the 3- chloro-5-subst iuted-quinoxal in-2-amine derivative represented by Chemical Formula 1 is jV-(l-benzylpiperidin-4-yl)-3-chloro-5-methoxyquinoxal in-2-amine or S-chloro-jV-tl-CS.δ-diphenoxybenzyDpiperidin^-yπ-δ-methoxyquinoxalin^- amine.

[Claim 6]

The pharmaceutical composition for treating inflammatory disease induced by activity of SPC receptor as set forth in claim 4, wherein the inflammatory disease is selected from the group consisting of inflammation, itching and skin infection associated with atopic dermatitis or other disease.

[Claim 7]

A pharmaceutical composition for preventing scarring after injury and promoting wound healing containing the 3-chloro-5-substiuted-quinoxalin-2- amine derivative represented by Chemical Formula 1 or the pharmaceutically acceptable salt thereof as set forth in claim 1 as an effective ingredient.