WO2008069518A1 - Pharmaceutical composition for controlling the function of spihngosylphosphorylcholine - Google Patents

Abstract

The present invention relates to a new use of alkylcarbamoyl naphthalenyloxyprophenylhydroxybenzamide derivatives for controlling the function of sphingosylphosphorylcholine.

Description

PHARMACEUTICAL COMPOSITION FOR CONTROLLING THE FUNCTION OF SPfflNGOSYLPHOSPHORYLCHOLINE

FIELD OF THE INVENTION

The present invention relates to a new use of alkylcarbamoyl naphthalenyloxyprophenylhydroxybenzamide derivatives for controlling the function of sphingosylphosphorylcholine.

BACKGROUND OF THE INVENTION

Sphingosylphosphorylcholine (SPC), represented by the following structural formula, is a member of the lysosphingolipid family, and it is the N- deacylated form of sphingomyelin (SM) in which choline is bound to a phosphate group. SPC is produced by the action of sphingomyelin N- deacylase on sphingomyelin (Higuchi K, et al., Biochem J. (2000), 350, 747-56).

SPC is known to function as a mitogen for various types of cells and to stimulate the secretion of some cytokines (Meyer et al., Biochim Biophys Acta. (2002), 1582: 178-89). SPC is also known to be closely associated with the proliferation of various cells (Desai et al., Biochem . Biophys. Res. Commun. (1991), 181, 361-366), angiogenesis (Boguslawski et al., Biochem. Biophys. Res. Commun. (2000), 272, 603-609), apoptosis (Jeon ES et al., Biochim Biophys Acta. (2005), 1 734(1); 25-33) and the like. In particular, SPC is responsible for wound healing by stimulating the proliferation of keratinocytes and fibroblasts (Wakita et al., /. Invest. Dermatol. (1998), 110, 253-258; and Berger et al., Proc. Natl. Acad. ScL U. S. A. (1995), 92, 5885-5889). In addition, SPC is involved in Ca²⁺ release from endoplasmic reticulum (Desai et al., J. Cell. Biol. (1993), 121, 1385-1395; and Okajima et al., J. Biol. Chem. (1995), 270, 26332-26340), and cell reconstitution or migration in various cells (Seufferlein et al., J. Biol. Chem. (1995), 270, 24343-24351; and Rumenapp et al., Naunyn- Schmiedeberg's Arch. Pharmacol. (2000), 361, 1-11).

The major diseases or disorders associated with SPC include Niemann- Pick disease (NPD), a hereditary metabolic disease induced by accumulation of considerable amount of lipids in cells, which induces a destruction of cells and a dysfunction in major organs due to deficiency of acidic sphingomyelinase (ASM).

SPC also causes various diseases such as atopic dermatitis induced by reduced antimicrobial activity and loss of barrier function due to a reduced amount of lipids in the stratum corneum, which weakens resistance to external stimuli thereby triggering inflammatory responses. Such inflammatory responses cause itching, which, in turn, tends to lead to secondary infections, resulting in hyperimmune responses. The most important fact associated with the pathology of atopic dermatitis is that the activity of sphingomyelin deacylase increases at the affected sites, or the proximity thereof and the expression level of SPC in the skin of patients with atopic dermatitis is more than 1000-fold higher than that in the skin of a healthy subject (Higuchi K et al., Biochem. J. (2000), 350, 747- 756; and Reiko Okamoto et al., Journal of Lipid Research (2003), 44, 93-102). This has been postulated to be caused by the lack of ceramide in the stratum corneum of patients suffering from atopic dermatitis (Junko Hara et al., J. invest. Dermatol. (2000), 115, 406-413), which has been the basis for the proposal that SPC plays a critical role in abnormal keratinization which manifests itself in atopic diseases (Higuchi et al., J. Lipid Res. (2001), 42, 1562-1570). These reports suggest that SPC may be directly responsible for the skin barrier dysfunction among others and for the inflammatory responses accompanying such dysfunction.

According to a corroborative report associated with the function of SPC in inflammatory response, SPC, when treated to keratinocyte, has been induced to increase markedly expression of such inflammatory response-mediating factors as TNF-α, ICAM-I, and the like (Imokawa et al., J. Invest. Dermatol.(l999), 112, 91-96; and Ito et al., J. Biol. Chem.(l999), 270, 24370- 24374). Once inflammatory response occurs, the neutrophils which is collected to the proximity of the affected sites by chemotactic migration and antagonized against the pathogens, wherein the important controlling factor of chemotactic migration is rho kinase (Verena Niggli, The International Journal of Biochemistry & Cell Biology. (2003), 35, 1619-1638) which also mediates to control the change in the concentration of Ca²⁺ induced by SPC (see, Zhu et al., Exp Dermatol. (2005), 14, 509-514; and Todoroki-Ikeda et al., FEBS Lett. (2000), 482, 85-90). Thus, the mechanism in cell response induced by SPC is established to share the essential factor thereof with the mechanism in inflammatory response.

Meanwhile, it is previously known that the eosinophil as a inflammatory cell leads to inflammation in respiratory tract, intestinal canal and other tissues associated with asthma, allergic rhinitis, eosinophilic pneumonia, eosinophilic enteritis, hypereosinophilic syndrome, and the like which induce proinflammatory cytokine in process of pathology such as allergic disease, asthma, etc. Except for digestive system, the eosinophil is normally not found in most organs. However, the hypereosinophilic syndrome is occurred in the tissue of patients with acute and chronic atopic dermatitis and the skin hypertrophy of epidermis and dermis in mouse model having atopic dermatitis is caused by healing process after cytotoxicity reaction of eosinophil major basic protein or eosinophil cationic protein.

The migration of eosinophil from bone marrow to other tissues is closely associated with the sphingolipid family, and, for example, it is recently reported that the chemotaxis and recruitment of human eosinophil have been postulated to be caused by Sphingosine-1 -phosphate (SlP) (Fiorentina Roviezzo et al., Proc Natl Acad Sci. (2004), 101, 11170-11175). In experiment applying SlP to rat paw tissue topically, the increase of the eosinophilia was founded in paw tissue and in its cell experiment, SlP induced cell migration of eosinophil and increased SlP acceptor mRNA, chemokine CCR3 and cytokine RANTES (Regulated on Activation Normal T cell- Expressed and Secreted) (Fiorentina Roviezzo et al., Proc Natl Acad Sci.(2004), 101, 11170-11175). In accordance with the result of above studies, based on structural similarity between SPC and SlP, the controlling factor of function of SPC in vivo is expected to be effective in various diseases caused by eosinophil as etiology.

However, a report is established that lysophosphatic acid (LPA) which is structurally comparable to SPC causes the itching which causes pain to a patient suffering therefrom and decrease the patient's quality of life (Hashimoto et al., Pharmacology.(2004), 72, 51-56). Thus, it is inferred that SPC also causes the itching in vivo and the present inventors established the fact that the administration of SPC through epidermic enjection causes itching directly (WO 2006/049451).

The present inventors have therefore endeavored to find that the negative effects of SPC as mentioned above can be reduced by efficiently controlling the function of SPC which led to the present invention.

SUMMARY OF THE INVENTION

Accordingly, it is a primary object of the present invention to provide a pharmaceutical composition for controlling the function of SPC.

In accordance with an aspect of the present invention, there is provided a pharmaceutical composition for controlling the function of SPC, comprising the alkylcarbamoyl naphthalenyloxyprophenylhydroxybenzamide derivatives of formula I or pharmaceutically acceptable salts thereof as an active ingredient:

wherein,

B and Z are independently H or halogen atom; X is CH₂, CH₂CH₂ or carbonyl group;

Y is H, C_1-I6 alkyl, C₂.₁₆ alkenyl, C_3-8 cycloalkenyl, aryl, aryl C_1-6 alkyl, tricyclo[4.3.1.13.8]undecanyl unsubstituted or substituted with C_1-4 alkyl, C_3-8 cycloalkyl unsubstituted or substituted with 2-(naphthalene-2-yloxy)-acetamide, adamantly or carboxyl unsubstituted or substituted with C_1-4 alkyl, C_1-4 alkoxy, benzoyl, morpholine, tetrahydrofuran, C_1-6 alkyl substituted with 2- (naphthalene-2-yloxy)-acetamide, or (3-methyl-l-oxo-l-((E)-2-((E)-3- phenylallylidene)hydrazinyl)butan-2-yl.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, which respectively show:

FIG. 1 shows a graph which represents the inhibitory effect of the compound of Example 1 on cell proliferation induced by SPC.

FIG. 2 shows a graph which represents inhibitory effect of the compound of Example 1 on cell chemotactic migration induced by SPC.

FIG. 3 shows a graph which represents inhibitory effect on inflammatory response of ear edema of mouse induced by TPA.

FIG. 4 shows a graph which represents inhibitory effect on eosinophilic inflammatory response of ear edema of mouse induced by oxazolone. FIG. 5 shows a graph which compares the changing aspects of the heart rate after the compound of Example 1 and FTY720-P administered by phleboclysis.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be described in more detail. The term "alkyl" as referred herein represents saturated hydrocarbon of straight or branched chain, and preferably includes methyl, ethyl, isopropyl, n- butyl, tert-butyl, neopentyl and hexyl.

The term "aryl" as referred herein represents monocyclic aromatic group or bicyclic group which has at least one aromatic ring, the term

"arylalkyl" as referred herein represents alkyl which has 1 to 6 carbon atoms, and preferably includes benzyl, trityl and phenylethyl, which is not limited thereto.

Examples of preferred alkylcarbamoyl napthalenyloxyprophenylhydroxybenzamide derivatives of formula I according to the present invention are: N-(2-(naphthalene-2-yloxy)ethyl)prop-2-en- 1 -amine;

N-(2-naphthalene-2-yloxy)ethyl)butan- 1 -amine;

N-(2-naphthalene-2-yloxy)ethyl)butan-2-amine;

3-(naphthalene-2-yloxy)-N-propylpropane- 1 -amine;

N-(3 -(naphthalene-2-yloxy)propyl)butan- 1 -amine; N-(2-( 1 -bromonaphthalene-2-yloxy)ethyl)butan-2-amine;

N-(2-( 1 -bromonaphthalene-2-yloxy)ethyl)butan- 1 -amine;

N-(2-( 1 -bromonaphthalene-2-yloxy)ethyl)cyclopentanamine;

N-(2-( 1 -bromonaphthalene-2-yloxy)ethyl)-3 -methoxypropane- 1 -amine;

2-(naphthalene-2-yloxy)-N-proρylacetamide; N-sec-butyl-2-(naphthalene-2-yloxy)acetamide; N-(3-methoxypropyl)-2-(naphthalene-2-yloxy)acetamide;

N-cyclopropyl-2-(naphtalane-2-yloxy)acetamide;

N-cyclohexyl-2-(naphtalane-2-yloxy)acetamide;

N-cyclooctyl-2-(naphtalane-2-yloxy)acetamide; N-(2-cyclohexenylethyl)-2-(naphthalene-2-yloxy)acetamide;

2-(naphthalene-2-yloxy)-N-phenethylacetamide;

2-(naphthalene-2-yloxy)-N-(4-phenylbutyl)acetamide;

2-(naphthalene-2-yloxy)-N-(4-phenylbutan-2-yl)acetamide;

N-(3 ,3 -diphenylpropyl)-2-(naphthalene-2-yloxy)acetamide; 2-(naphthalene-2-yloxy)-N-((tetrahydrofuran-2-yl)methyl)acetamide;

N-admantane-2-yl-2-(naphthalene-2-yloxy)-acetamide;

N-admantane- 1 -yl-2-(naphthalene-2-yloxy)-acetamide;

2-(naphthalene-2-yloxy)-N-tricyclo[4.3.1.13, 8]unde- 1 - cylmethylacetamide; N-(2-admantane- 1 -yl-ethyl)-2-(naphthalene-2-yloxy)-acetamide;

N-(2-methyl-admantane-2-ylmethyl)-2-(naphthalene-2-yloxy)- acetamide;

6-(2-(naphthalene-2-yloxy)acetamido)hexanoic acid;

N-butyl-2-(naphthalene-2-yloxy)acetamide; 2-(naphthalene-2-yloxy)-Ν-(pentane-3 -yl)acetamide;

N-isopropyl-2-(naphthalene-2-yloxy)acetamide;

N-(3 ,3 -diphenylpropyl)-2- (naphthalene-2-yloxy)acetamide;

2-(naphthalene-2-yloxy)-N-((tetrahydrofuran-2-yl)methyl)acetamide;

N-admantane-2-yl-2-(naphthalene-2-yloxy)-acetamide; N-admantane- 1 -yl-2-(naphthalene-2-yloxy)-acetamide;

2-(naphthalene-2-yloxy)-N-tricyclo[4.3.1.13, 8]unde- 1 -cylmethylacetamide;

N-(2-admantane- 1 -yl-ethyl)-2-(naphthalene-2-yloxy)-acetamide;

N-(2-mehtyl-admantane-2-ylmethyl)-2-(naρhthalene-2-yloxy)- acetamide;

6-(2-(naphthalene-2-yloxy)acetamido)hexanoic acid; N-butyl-2-(naρhthalene-2-yloxy)acetamide;

2-(naphthalene-2-yloxy)-Ν-(pentane-3-yl)acetamide;

N-isopropyl-2-(naphthalene-2-yloxy)acetamide;

N-tert-butyl-2-(naphthalene-2-yloxy)acetamide; N-benzyl-2-(naphthalene-2-yloxy)acetamide;

2-(naphthalene-2-yloxy)-N-phenylacetamide;

N-cyclopentyl-2-(naphthalene-2-yloxy)acetamide;

2-( 1 -bromonaphthalene-2-yloxy)-N-cyclohexy lacetamide;

2-(l-bromonaphthalene-2-yloxy)-N-(2-cyclohexenylethyl)acetamide; 2-( 1 -bromonaphthalene-2-yloxy)-N-((tetrahydrofuran-2- yl)methyl)acetamide;

2-( 1 -bromonaphthalene-2-yloxy)-N-hepthylacetamide;

N₅N' -(butan- 1 ,4-diyl)bis(2-(naphthalene-2-yloxy)acetamide);

N,N'-(hexan- 1 ,6-diyl)bis(2-(naphthalene-2-yloxy)acetamide); N₅N' -(cyclohexan- 1 ,2-diyl)bis(2-(naphthalene-2-yloxy)acetamide);

N-(3 -methyl- 1 -oxo- 1 -((E)-2-((E)-3 -phenylally lidene)hydrazynyl)butan- 2-yl)-2-(naphthalene-2-yloxy)acetamide;

2-(6-bromonaphthalene-2-yloxy)-N-cyclooctylacetamide;

3-methyl-2-(2-(naphthalene-2-yloxy)acetamido)methylbutanoate; and 3-methyl-2-(2-(naphthalene-2-yloxy)acetamido)butanoic acid.

The subject compound of formula I wherein X is CH₂ may be prepared, as shown in Reaction Scheme A:

Reaction Scheme A

n I(a) wherein, n is 1 or 2, and

B, Z and Y have the same meaning as defined above.

In reaction scheme A, the compound of formula I(a) was prepared by heating at a temperature ranging from 100 to 110°C for 20 to 25 hours following the addition of amine to a solvent in which the bromide of formula II was dissolved. The suitable solvents is preferably toluene, or DMSO and the amine may be used in an amount of ranging from 130 to 150 parts by weight, based on 100 parts weight of the compound of formula II.

And the subject compound of formula I wherein X is carbonyl may be prepared, as shown in Reaction Scheme B:

Reaction Scheme B

wherein,

B, Z and Y have the same meaning as defined above.

In reaction scheme B, the compound of formula I(b) was prepared by heating at a temperature ranging from 20 to 25 °C for 13 to 15 hours before adding amine in the presence of catalyst to a solvent in which the carboxyl compound of formula III was dissolved.

The solvent suitable for use therein is preferably dichloromethane, toluene, or DMSO and the catalyst for use therein is EDCl (l-(3- dimethylaminopropyl)-3-ethylcarbodiimide), DMAP (4- dimethylaminopyridine).

And, the amine may be used in an amount of ranging from 130 to 150 parts by weight, based on 100 parts by weight of the carboxyl compound of formula III.

The present invention provides a pharmaceutical composition for controlling the function of SPC in vivo and in vitro, which comprises said compound of formula I or a pharmaceutically acceptable salt thereof.

The compound of formula I of the present invention can also be used in the form of a pharmaceutically acceptable salt. The salt is formed by reacting the compound with a pharmaceutically acceptable inorganic acid, organic acid or base to yield additional salt thereof. The base which may be used in this reaction are alkali and alkaline earth metal such as Na, K, Mg, Ca; the inorganic acid which may be used in this reaction includes, not limited to, hydrochloric acid, sulfuric acid; the organic acid which may be used in this reaction includes, not limited to, acetic acid, maleic acid.

Further, the subject pharmaceutical composition may be formulated by any of the conventional methods. For the preparation of the formulation, it is preferred that the active ingredient is mixed, diluted or employed into injectable composition the active ingredient with a carrier. In case the carrier is used as diluent, it may be carrier, exipient or solid, semisolid, or liquid substance which serves as medium for an active ingredient. Thus, its formulation is in the form of tablet, pill, granule, powder, sachet, elixir, suspension, emulsion, solution, syrup, aerosol, hard and soft gelatin capsule, sterile injection, sterile powder, et al.

The carrier, excipient, and dilulent suitable for use therein include, not limited to lactose, dextrose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and minerals, et al. The formulation further includes filler, anticoagulant, lubricant, humectant, flavoring agent, emulsifier, antiseptics, et al. The subject pharmaceutical composition may be formulated by any of methods well known in the relevant art so as to allow instant sustained, or delayed- release of the active substance after administered to mammal.

The subject pharmaceutical composition may be administered via various routes including oral, transcutaneous, subcutaneous, intravenous, and intra-muscular route to a subject in a dose ranging from 0.001 to 1000 mg/kg, preferably 0.1 to 10 mg/kg weight per day with one portion or divided portions. However, it is appreciated that an actual dose of active ingredients is determined by various factor associated disease to be treated, route of administration, age, sexuality and weights of a patient, severity of disease and thus the dose is not limited to the present invention in any manners.

As described above, the subject pharmaceutical composition which effectively controls the proliferation of cells, inflammatory response, and eosinopilia conditions induced by in vitro and in vivo action of SPC, such as without side effects such as the decreased heart rate and thus may be useful in prevention and treatment of atopic dermatitis, or other diseases accompanied with inflammation, itch and skin infection.

Hereinafter, the present invention will be described more specifically by Examples. However, the following Examples are provided only for illustrations and thus the present invention is not limited to or by them.

Preparation of the subject compounds

Preparation Example 1: Synthesis of 2-(2-bromoethoxy)naphthalene

300 mg of 2-naphtol (2.08 mmol) was dissolved in 4 mL of 95% ethanol, and added 118 mg of potassium hydroxide (10 mmol), and the mixture was stirred for 30 minutes. Then, 0.8 mL of 1,2-dibromoethan (2.10 mmol) was added thereto and heated for 24 hours while refluxing. The reaction solution was cooled to room temperature, concentrated and extracted with 10% sodium hydroxide and dichloromethan. The organic layers thus obtained were combined, dried and concentrated over MgSO₄, purified by silica gel column chromatography (hexane/EtOAc, 30:1), to obtain 2-(2- bromoethoxy)naphthalene (yield: 35%)

Preparation Example 2: Synthesis of l-bromo-2-(2- bromoethoxy)naphthalene

The procedure similar to those shown in Preparation Example 1 was performed except that l-bromo-2-naphtol was used instead of 2-naphtol.

Preparation Example 3: Synthesis of 3-methyl-2-(2-(naphthalene-2- oxy)acetamido)butanoic acid

132 mg of cinnamaldehyde (1 mmol) was dissolved in 5 mL of EtOH, and 75 mg of 80% hydrazine hydrate (1.3 mmol) was added thereto for 5 minutes at room temperature. The solution was filtered, washed several times with diethylether and dried under reduced pressure to yield 3-methyl-2-(2- (naphthalene-2-oxy)acetamido)butanoic acid as yellow precipitates (yield: 66%).

Example 1: Synthesis of N-(2-(naphthaIene-2-yIoxy)ethyl)prop-2-en-l- aniiiie

500 mg of 2-(2-bromoethoxy)naρhthalene (2 mmol) obtained in Preparation Example 1 was dissolved in 20 mL of DMSO, and subsequently 1.4 mL of triethylamine (10 mmol) and 0.5 mL of alrylamine (6 mmol) was added, and heated at 60 ^°C for 12 hours. Then, the mixture was cooled to room temperature, diluted several times with water, and extracted with dichloromethane. The organic layer formed was washed several times with water, dried and concentrated over MgSO₄, and purified by silica gel column chromatography (dichloromethane/MeOH, 50:1), to obtain the title compound

(yield: 79%).

¹H NMR (CDCl₃, 300 MHz) δ 1.80 (s, IH), 3.08 (t, J = 5.1 Hz, 2H), 3.37 (td, J = 1.2, 6.0 Hz, 2H), 4.21 (t, J = 5.1 Hz, 2H), 5.15 (dd, J = 1.2, 10.2 Hz, IH), 5.25 (dd, J = 1.5, 17.4 Hz, IH), 5.96 (dddd, J = 5.7, 6.0, 10.2, 17.1 Hz, IH), 7.18 (m, 2H), 7.34 (t, J = 8.1, IH), 7.45 (t, J = 7.2, IH), 7.75 (m, 3H).

Examples 1 to 9

The procedure similar to those shown in Example 1 was performed with the respective starting material and amine to obtain the respective title compounds as listed in Table 1.

<Table 1>

Example 10: Synthesis of 2-(naphthalene-2-yloxy)-N-propyIacetamide

100 mg of 2-naphthoxyacetic acid (0.50 mmol) was dissolved in 4 mL of dichloromethane, and 250 mg of EDCl (1.30 mmol) and 31 mg of DMAP (0.25 mmol) were added. Propylamine (0.75 mmol) was added, and was stirred at room temperature for 4 hours. Then, water was added to the reaction mixture, extracted with dichloromethane. The organic layer was dried and concentrated over MgSO₄, purified by silica gel column chromatography (hexane/EtOAc, 3:1), to obtain the title compound.

¹U NMR (CDCl₃, 300 MHz) δ 0.93 (t, J = 7.2 Hz, 3H),1.52-1.64 (m, 2H), 3.33 (q, 2H), 4.61 (s, 2H), 7.17 (dt, J = 2.4, 8.7 Hz, 2H), 7.38 (t, J = 6.9 Hz, IH), 7.47 (t, J = 7.2 Hz, IH), 7.76 (m, 3H).

Examples 11 to 43

The procedure similar to those shown in Example 10 was performed with the respective starting material and amine to obtain the respective title compounds as listed in Table 2.

<Table 2>

Example 44: Synthesis of 3-methyl-2-(2-(naphthaIene-2- yloxy)acetamido)methylbutanoate

DL-valine acetate hydrochloride (1 mmol) was dissolved in 2 niL of acetonitrile at -5 ^°C, 0.1 mL of triethylamine, 135 mg of hydroxybenzotriazol(HOBT)(l mmol), 206 mg of DCC (1 mmol), and 222 mg of 2-naphthoxyacetic acid (1.1 mmol) were added thereto. The reaction mixture was stirred at 0^°C for 2 hours, and then was stirred at room temperature for 16 hours. Dicyclohexylurea(DCU) formed was filtered, concentrated and diluted with EtOAc, washed with salt solution, 5% sodium hydrocarbon, and IN hydrochloric acid. The organic layer formed was dried over MgSO₄ and concentrated, recrystallizated (EtOAc/Petroleum ether), to obtain the title compound (yield: 87%).

¹R NMR (CDCl₃, 300 MHz) δ 1.01 (d, J = 7.2 Hz, 6H), 3.09 (dd, J = 6.1, 7.2 Hz, IH), 3.67 (s, 3H), 4.41 (d, J = 6.1 Hz, H), 4.93 (s, 2H), 7.18 (m. 2H), 7.34 (t, J = 8.1, IH), 7.45 (t, J = 7.2, IH), 7.75 (m, 3H), 8.32 (br s IH)

Example 45: Synthesis of 3-methyl-2-(2-(naphthalene-2- yloxy)acetamido)butanoic acid

3-methyl-2-(2-(naphthalene-2-yloxy)acetamido)methylbutanoate obtained in Example 44 was dissolved in aqueous solution of ethylene glycol dimethylether(DME) and IM LiOH and was stirred at room temperature for 2 hours. 10% citric acid solution was added thereto to make it acidic to pH 4, and extracted with EtOAc. The organic layer was dried over MgSO₄ and concentrated, purified by silica gel column chromatography (EtO Ac/Petroleum ether, 10:1), to obtain the title compound (yield: 56%). 1B. NMR (CDCl₃, 300 MHz) δ 1.01 (d, J = 7.2 Hz, 6H), 3.09 (dd, J =

6.1, 7.2 Hz, IH), 4.45 (d, J = 6.1 Hz, H), 4.93 (s, 2H), 7.18 (m, 2H), 7.34 (t, J = 8.1, IH), 7.45 (t, J = 7.2, IH), 7.75 (m, 3H), 8.32 (br s, IH), 12.34 (br s, IH)

Pharmacological Activity

The results of Test Examples below were indicated by average values including their standard deviations. Specifically, the obtained results were evaluated using the Student's t-test used for determining significance of difference among average values of samples. Only when the obtained p- values of test and control groups were less than 0.05, the difference between the test group and the control group was determined to be significant.

Test Example 1: Control of the proliferation of cells by the subject compounds.

SPC induces excessive proliferation of cells when provided to cells, which may cause pathological symptoms (Desai et al., Biochem. Biophy. Res. Commun. (1991), 181, 361-366). Hence, the inventors tested the effects of the subject compounds on the proliferation of cells induced by SPC. NIH 3T3 cells (American Type Culture Collection, Manassas, VA,

USA) were seeded in a culture plate at a density of about I X lO⁵ cells (generally, 1 X 10⁴to 10⁶ cells), and cultured in RPMI medium unsupplemented with fetal bovine serum (FBS) for 24 hours, thereby resulting in serum starvation. Then, the cells were treated with 1 ppm of the compounds of Examples 1 to 48 and FTY 720 as a control compound, which is an agonist of Sphingosine-1 -phosphate ("SPl") (Seoul National University, Lipidomics NRL), and cultured for 30 minutes, SPC (Biomol, Plymouth Meeting, PA, USA) was added in the concentration of 7 μ M, and cultured for 24 hours at 37 ^°C . The amount of proliferation of cells was measured using a method of MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Beales IL, Life ScI (2004), 75:29 83-95), and the results of proliferation rate (%) of cells were determined by the following Equation I, as shown in Table 3.

Equation I

Proliferation rate (%) = 100 x (cell number of a group treated with subject compound and SPC — cell number of SPC-untreated group)/(cell number of a group treated with SPC only— cell number of SPC-untreated group)

<Table 3>

As shown in Table 3, the each compound acted as either an agonist or an antagonist on the proliferation of cells. The excessive proliferation of cells induced by an inflammatory response occurring in the process of wound healing after trauma results in scar formation. Thus a substance inhibiting the excessive proliferation of cells may be used as an agent for preventing the formation of undesirable scars. On the other hand, those augmenting the excessive proliferation of cells may be used to accelerate the wound healing process after trauma. Particularly, the above procedure was performed with regard to 0, 0.1, and 1 ppm of the compound of Example 1 and FTY 720. The results are shown in FIG. 1.

As can be seen in FIG. 1, the compound of Example 1 showed dose- dependent suppression of cell proliferation induced by SPC. To the contrary, FTY 720 having a chemical structure similar to SPC as a SlP agonist did not exhibit such suppression. Thus, the inhibition of cell proliferation is induced by the characteristic structure of the present compound and it can inhibit scar formation resulting from excessive proliferation of cells due to an inflammatory response induced by the process of wound healing after trauma.

Test Example 2: Inhibitory effect to cell chemotactic migration induced by SPC

It has been reported that SPC plays a critical role in cell chemotactic migration similar to vascular endothelial growth factor (VEGF) (Boguslawski et al., Biochem Biophys Res Commun. (2000), 272: 603-609). The migration of cell induced by certain cytokine or chemokine in vivo is an essential step in the process of immune cell recruitment to an inflammatory region, or angiogenesis induced by endothelial cell migration. Hence, the present inventors tested the effect of the subject compound of Example 1 on cell chemotactic migration induced by SPC using a Boy den chamber analysis.

First, a polycarbonate membrane of 25 x 80 mm (Neuro Probe, Inc.) which has a pore of 8 μm was put it in 0.01% gelatin and 0.1% acetic acid solution overnight and then dryied at room temperature.

Meanwhile, human umbilical vein endothelial cells (HUVECs) cultured in complete EBM-2 medium (Cambrex, Catalog No. CC-3121) supplemented with 2% FBS were cultured in EBM-2 medium unsupplemented with FBS for 4 hours, thereby resulting in serum starvation, and then harvested with trypsin/EDTA solution. The HUVECs were suspended in EBM-2 medium supplemented with 0.1% FBS albumin and seeded in silicon-coated eppendorf tubes and treated with 0 to 10 μg/ml of the compound of Example 1 as a test substance at 37^°C for 30 minutes. 27 μJL of EBS-2 medium with or without 10 μ M SPC was loaded in each well of the lower chamber of a Boy den chamber, and the gelatin-coated membrane was placed over the lower chamber with its shinning surface facing polycarbonate downward, and a gasket was placed thereon and the upper chamber was assembled. HUVECs treated with the test substance were seeded in the upper chamber to have 5 x 10⁴ cells (56 μi), and then incubated in a CO₂ incubator at 37 ^°C for 8 hours. Thereafter, the membrane separated therefrom was dyed with Diff-Quik dye (Sysmex Corporation), washed with deionized water and attached to a slide glass while the shinning surface of the membrane was placed downward. The cells attached on the membrane were collected using kimwipers or cotton swabs, 5 fields per each well were photographed (magnifying power: 200-fold) and analyzed for the cell count. The inhibition rate (%) was determined by the following Equation II and the results are shown in FIG. 2.

Equation II

Inhibition rate (%) = 100 x (cell number of a group treated with SPC only— cell number of a group treated with test substance and SPC)/(cell number of a group treated with SPC only— cell number of SPC-untreated group)

As shown in FIG. 2, the subject compound of Example 1 inhibited the cell chemotactic migration induced by SPC. Therefore, these results suggest that the process of angiogenesis in a tumor or the process of amplification of immunological reaction against a foreign antigen can be controlled by the subject compound.

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

To evaluate the inhibiting effect of the subject compounds on inflammatory response, the following experiment was performed using tetradecanoyl phorbol acetate (TPA)-induced inflammation model. TPA- induced inflammation model is a method commonly used for evaluating the mechanism of inflammatory response and the effect of inhibiting agents (De Young LM et al., Agents and Actions (1989), 26, 335-341). TPA is an inflammation-inducing agent, which induces erythema and edema when applied to the skin of ear of a subject. The induced inflammation can be measured by increased activity of meloperosidase (MPO) which is an essential material for a leukocyte's attack against bacteria.

40 male ICR mice of 6 weeks old were subjected to TPA treatment. Specially, their left ear skin was coated with 20 μi of TPA (Sigma Aldrich Korea) dissolved in acetone (125 μg/mi). After 1 hour, each 20 μi of acetone, 1% compound of Example 1 dissolved in acetone, or 1% hydrocortisone (Sigma Aldrich Korea) dissolved in acetone was applied to TPA-coated region, and after 6 hours, additional 20 μi was applied again. At a point of 24 hours after TPA coating, the mice were euthanized using a method of ruptured cervical disk, and the weight, and the activity of MPO of tissue samples collected from their left ears were determined. The inhibition rate (%) was calculated by Equation III, and the results are shown in Table 4 and FIG. 3.

Equation III

Inhibition rate (%) = 100 x (weight/MPO activity of a group treated with TPA only— weight/MPO activity of a group treated with test substance and TPA)/(weight/MPO activity of a group treated with TPA only -weight/MPO activity of TPA-untreated group)

<Table 4>

Inhibition rate (%) of the subject compounds at 1 ppm

As shown in Table 4, the compounds of Example 1 and Example 1-1 significantly inhibited the inflammatory response induced by TPA in both ear edema and MPO activity. Further as shown in FIG. 3, the effect of the compound of Example 1 was comparable to that of hydrocortisone commonly used as an anti-inflammatory agent. These results indicate that the anti- inflammatory effect of the subject compounds is achieved by inhibiting infiltration of neutrophils around ear region. Test Example 4: Control of inflammatory response in oxazolone-induced mouse eosinophilic model

4-ethoxymethylene-2-phenyl-2-oxazolin-5-one was administered to 6

BALB/c mice to induce prolonged hypersensitivity reaction and eosinophilia, and these mice were treated with the compounds of Example 1 and Example 1-1 by topical administration. The inhibition of tissue edema and the activity of eosinophil peroxidase (EPO) was observed. 6 male BALB/c mice weighing about 20-25g were depilated in their abdominal skin areas, and the depilated areas were coated with 50 μi of 2% oxazolone solution (solvent: 96% alcohol) per day for 2 continuous days. At a point of 6 days following application of oxazolone, 20 μJL of 2% oxazolone solution was treated to their skin of the both ears. The topical administration of test substances was performed at 4 hours prior to oxazolone treatment and 3 hours after oxazolone treatment. At 24 hours after oxazolone treatment, 6 mm of tissue of the both ears of mice was collected using biopsy punch method to measure its weight, determine the activity of EPO and carry out its histologic examination. EPO activity is determined by a method for evaluating the eosinophil count present in tissues of identical size by determining the activity of peroxidase, based on the fact that peroxidase is contained in the eosinophil.

The tissue of ear collected was added to 0.05 mol/L Tris-HCl buffer solution (pH 8.0) containing Triton X-IOO (i.e., EPO buffer solution) and well grinded and centrifuged under 1500 g, at 4^°C for 10 minutes. 50 μJt of the supernatant was seeded in a 96-well plate and was reacted with 100/^ of a substrate solution containing a mixture of 10 mmol/L of o-phenylenediamine, 0.05 mmol/L of Tris-HCl buffer solution (pH 8.0) and 4 mmol/L of hydrogen peroxidase at room temperature for 1 hour. After the reaction, the optical density was determined at a wavelength of 490 nm and the inhibition rate (%) was calculated by Equation IV, and the results are shown in FIG. 4. Equation IV

Inhibition rate (%) = [(weight/EPO activity of right ear of a group treated with test substance and oxazolone— weight/EPO activity of right ear of untreated group)/(weight/EPO activity of right ear of a group treated with oxazolone only —weight/EPO activity of right ear of untreated group)] x 100

<Table 5>

Inhibition rate of inflammatory response in oxazolone-induced mouse eosinophilia model

As shown in Table 5, the compounds of Example 1 and Example 1-1 exhibited significant inhibition of eosinophilic inflammatory response and the inhibition rate of the compound of Example 1 was comparable to that of hydrocortisol commonly used as an anti-inflammatory agent. Thus, it is expected that the subject compounds are effective in inhibiting allergic diseases, asthma, atopic dermatitis, etc. which are known to be primarily caused by eosinophils such as allergic rhinitis, eosinophilic pneumonia, hemic and tissue hypereosinophilia.

Test Example 5: Effect of the subject compound on heart rate

SPC, an active phospholipid in vivo, has a structure similar to SlP, and it has been reported that FTY 720 (SlP agonist) shows a side effect of decreasing heart rate (Dragun et al., Transplantation Proceedings (2004), 36 (Suppl 2S), 544S-548S). Therefore, as described in Test Examples above, it may be possible that the subject compound inhibiting the function of SPC in vitro and in vivo may affect the heart rate like FTY 720. Hence, the present inventors tested the effect of the subject compound on heart rate. 15 male Sprague-Dawley (SD) rats weighing 200 to 30Og were subjected to anesthesia by administering phentobarbital (Sigma Aldrich Korea) by intraperitional injection. The rats stabilized for 20 minutes were fixed on dissection tables and a cannula was inserted to their femoral vein after dissection of their inguinal area. Acusectors connected to a biosignal physiograph were sequentially connected to right anterior cms (Gl), left anterior cms (Comp), left hind limb (G2), and then the data of electrocardiogram were measured for 15 minutes until the heart rate per minute became stable. lmg/kg of a solution containing 10% hydroxyprophy- β -cyclodextin (HP- β -CD, Sigma Aldrich Korea) dissolved in physiological saline was applied to the cannula for 1 minute, and the data of electrocardiogram was measured for 20 minutes until the heart rates per minute became stable. The average value of the heart rate per minute between 15 and 20 minutes after administering the above solution was selected as a standard value of heart rate. At a point of 20 minutes after application of the above solution, 1 mg/kg of the solution containing compound of Example 1 or 0.1 mg/kg of the solution containing FTY 720-P (an active in vivo form of FTY-720, Seoul National University) was applied to the cannula for 1 minute, and the data of electrocardiogram were obtained for 20 minutes. The heart rate per minute was determined, the effect on the heart rate was calculated by Equation V, and the results are shown in FIG. 5.

Equation V

Effect on the heart rate (%) = 100 x heart rate per minute after application of test substance/(average heart rate for 5 minutes immediately before application of test substance) As shown in FIG. 5, the compound of Example 1 was demonstrated to be safe even at a 10-fold higher concentration than FTY 720-P, without showing any side effects.

Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical composition for controlling the function of sphingosylphosphorylcholine comprising a compound of formula I or a pharmaceutically acceptable salt thereof as an active ingredient:

wherein,

B and Z are independently H or halogen atom; X is CH₂, CH₂CH₂ or carbonyl group;

Y is H, C_1-16 alkyl, C₂.₁₆ alkenyl, C_3-8 cycloalkenyl, aryl, aryl C_1-6 alkyl, tricyclo[4.3.1.13.8]undecanyl unsubstituted or substituted with C_1-4 alkyl, C_3-8 cycloalkyl unsubstituted or substituted with 2-(naphthalene-2-yloxy)-acetamide, adamantly or carboxyl unsubstituted or substituted with C_1-4 alkyl, C_1-4 alkoxy, benzoyl, morpholine, tetrahydrofuran, C_1-6 alkyl substituted with 2- (naphthalene-2-yloxy)-acetamide, or (3 -methyl- 1 -oxo- 1 -((E)-2-((E)-3 - phenylallylidene)hydrazinyl)butan-2-yl.

2. The composition of claim 1, wherein the compound of formula I is selected from the group consisting of:

N-(2-(naphthalene-2-yloxy)ethyl)prop-2-en- 1 -amine; N-(2-naphthalene-2-yloxy)ethyl)butan- 1 -amine; N-(2-naphthalene-2-yloxy)ethyl)butan-2-amine; 3-(naphthalene-2-yloxy)-N-propylpropane-l-amine; N-(3-(naphthalene-2-yloxy)propyl)butan-l -amine;

N-(2-( 1 -bromonaphthalene-2-yloxy)ethyl)butan-2-amine;

N-(2-( 1 -bromonaphthalene-2-yloxy)ethyl)butan- 1 -amine;

N-(2-( 1 -bromonaphthalene-2-y loxy)ethyl)cyclopentanamine;

N-(2-( 1 -bromonaphthalene-2-yloxy)ethyl)-3 -methoxypropane- 1 -amine; 2-(naphthalene-2-yloxy)-N-propylacetamide;

N-sec-butyl-2-(naphthalene-2-yloxy)acetamide;

N-(3-methoxypropyl)-2-(naphthalene-2-yloxy)acetamide;

N-cyclopropyl-2-(naphtalane-2-yloxy)acetamide; N-cyclohexyl-2-(naphtalane-2-yloxy)acetamide;

N-cyclooctyl-2-(naphtalane-2-yloxy)acetamide;

N-(2-cyclohexenylethyl)-2-(naphthalene-2-yloxy)acetamide;

2-(naphthalene-2-yloxy)-N-phenethylacetamide;

2-(naphthalene-2-yloxy)-N-(4-phenylbutyl)acetamide; 2-(naphthalene-2-yloxy)-N-(4-phenylbutan-2-yl)acetamide;

N-(3 ,3 -diphenylpropyl)-2-(naphthalene-2-yloxy)acetamide;

2-(naphthalene-2-yloxy)-N-((tetrahydrofuran-2-yl)methyl)acetamide;

N-admantane-2-yl-2-(naphthalene-2-yloxy)-acetamide;

N-admantane- 1 -yl-2-(naphthalene-2-yloxy)-acetamide; 2-(naphthalene-2-yloxy)-N-tricyclo[4.3.1.13,8]unde- 1 -cylmethylacetamide;

N-(2-admantane- 1 -yl-ethyl)-2-(naphthalene-2-yloxy)-acetamide;

N-(2-methyl-admantane-2-ylmethyl)-2-(naphthalene-2-yloxy)-acetamide;

6-(2-(naphthalene-2-yloxy)acetamido)hexanoic acid;

N-butyl-2-(naphthalene-2-yloxy)acetamide; 2-(naphthalene-2-yloxy)-Ν-(pentane-3 -yl)acetamide;

N-isopropyl-2-(naphthalene-2-yloxy)acetamide;

N-(3 ,3 -diphenylpropyl)-2- (naphthalene-2-yloxy)acetamide;

2-(naphthalene-2-yloxy)-N-((tetrahydrofuran-2-yl)methyl)acetamide;

N-admantane-2-yl-2-(naphthalene-2-yloxy)-acetamide; N-admantane- 1 -yl-2-(naphthalene-2-yloxy)-acetamide;

2-(naphthalene-2-yloxy)-N-tricyclo[4.3.1.13,8]unde- 1 -cylmethyl-acetamide;

N-(2-admantane- 1 -yl-ethyl)-2-(naphthalene-2-yloxy)-acetamide;

N-(2-mehtyl-admantane-2-ylmethyl)-2-(naphthalene-2-yloxy)-acetamide;

6-(2-(naphthalene-2-yloxy)acetamido)hexanoic acid; N-butyl-2-(naphthalene-2-yloxy)acetamide;

2-(naphthalene-2-yloxy)-Ν-(pentane-3-yl)acetamide; N-isopropyl-2-(naphthalene-2-yloxy)acetamide;

N-tert-butyl-2-(naphthalene-2-yloxy)acetamide;

N-benzyl-2-(naphthalene-2-yloxy)acetamide;

2-(naphthalene-2-yloxy)-N-phenylacetamide; N-cyclopentyl-2-(naphthalene-2-yloxy)acetamide;

2-( 1 -bromonaρhthalene-2-yloxy)-N-cyclohexylacetamide;

2-(l-bromonaphthalene-2-yloxy)-N-(2-cyclohexenylethyl)acetamide;

2-(l-bromonaphthalene-2-yloxy)-N-((tetrahydrofuran-2-yl)methyl)acetamide;

2-( 1 -bromonaphthalene-2-yloxy)-N-hepthylacetamide; N,N'-(butan- 1 ,4-diyl)bis(2-(naphthalene-2-yloxy)acetamide);

N₅N' -(hexan- 1 ,6-diyl)bis(2-(naphthalene-2-yloxy)acetamide);

N,N'-(cyclohexan-l,2-diyl)bis(2-(naphthalene-2-yloxy)acetamide);

N-(3-methyl- 1 -oxo- 1 -((E)-2-((E)-3-phenylallylidene)hydrazynyl)butan-2-yl)-2-

(naphthalene-2-yloxy)acetamide; 2-(6-bromonaphthalene-2-yloxy)-N-cyclooctylacetamide;

3-methyl-2-(2-(naphthalene-2-yloxy)acetamido)methylbutanoate; and

3 -methyl-2-(2-(naphthalene-2-yloxy)acetamido)butanoic acid.

3. The pharmaceutical composition of claim 1, wherein the composition inhibits scar formation after trauma.

4. The pharmaceutical composition of claim 1, wherein the composition improves wound healing after trauma.

5. The pharmaceutical composition of claim 1, wherein the composition inhibits an inflammatory response.

6. The pharmaceutical composition of claim 5, wherein the inflammatory response is induced by increased eosinophils.

7. The pharmaceutical composition of claim 1, wherein the composition controls a symptom mediated by cell chemotactic migration.