KR20040009047A - Novel sphingosine-1-phosphate derivatives and preparation thereof - Google Patents

Abstract

PURPOSE: Spingosin-1-phosphate derivatives and a preparation method thereof are provided. The compounds are useful as both agonist and antagonist of EDG(endothelial differentiation gene) receptor. CONSTITUTION: Spingosin-1-phosphate derivatives are represented by the formula 1, wherein R1 and R2 are independently hydrogen, C1-6 alkyl, sugars or inositols; R3 and R4 are independently hydrogen, C1-6 alkyl, (CH2)m-benzene-(R7)p or form 3 to 8-membered heterocyclic ring together with bound nitrogen; m is 0, 1, 2 or 3; p is 0, 1 or 2; R7 is hydroxy, amino, C1-6 alkyl, halogen, C1-6 alkyloxy or C1-6 alkylamino; R5 is hydrogen or OH or 0; L is O, S, CH2 or NH; R6 is -R8, X-benzene-R9, -NR10R11 or -OR12; R8 is hydrogen, C1-20 alkyl, 1 to 9 halogen-substituted C1-20 alkyl, 1 to 8-membered cyclic alkyl, 1 to 9 halogen-substituted 3 to 8-membered cyclic alkyl or (CH2)n-X-benzene-(R13)q; R9 is hydrogen, amino, hydroxy, halogen, C1-20 alkylamino, 3 to 8-membered cyclic alkylamino, C1-20 alkyloxy, 3 to 8-membered cyclic alkyloxy, 1 to 9 halogen substituted C1-20 alkyl or 1 to 9 halogen substituted 3 to 8-membered cyclic alkyl; R10, R11 and R12 are independently hydrogen, C1-20 alkyl, 3 to 8-membered cyclic alkyl or (CH2)n-X-(R13)q; R13 is the same as R9; X is N or CH; n is 0, 1, 2, 3, 4 or 5; and q is 0, 1 or 2.

Description

Novel sphingosine-1-phosphate derivatives and preparation method thereof {NOVEL SPHINGOSINE-1-PHOSPHATE DERIVATIVES AND PREPARATION THEREOF}

The present invention relates to novel sphingosine-1-phosphate derivatives, methods for their preparation and pharmaceutical compositions containing the same, which have an effective and selective efficacy and antagonism against EDG (endothelial differentiation gene) receptors.

Recently, sphingosine-1-phosphate (S1P), a type of sphingolipid metabolite in vivo, is an EDG (endothelium) which is a type of G-protein coupled receptor present in the cell membranes of various tissues in vivo. It has been shown to act as a ligand of endothelial differentiation gene receptors (EDG-1, 3, 5, 6, and 8) (M.-J. Lee et al., Science 1998 , 279 , 1552-1555 and N. Ancellin and T. Hla, J. Biol. Chem . 1999 , 274 , 18997-19002.

Inhibition or activation of EDG receptors causes cell migration, proliferation, differentiation and death, and thus plays a very important role in various physiological and pathological phenomena such as angiogenesis, inflammation and development (S. Spiegel et al., J. Cell Biol . 1998 , 142 , 229-240 and S. Pyne and NJ Pyne, Biochem. J. 2000 , 349 , 385-402). Inhibition or activation of EDG receptor action by S1P is a Although it may be useful for the treatment of various refractory diseases such as healing, proliferation and metastasis of cancer, angiogenesis-related diseases such as rheumatoid arthritis, inflammation-related diseases and circulatory system diseases, so far, S1P derivatives exhibiting these activities No synthetic efforts have been reported for.

Accordingly, it is an object of the present invention to provide novel sphingosine-1-phosphate derivatives and methods for their preparation which can be used as effective and selective agonists and antagonists for EDG (endothelial differentiation gene) receptors.

Another object of the present invention is to provide a pharmaceutical composition containing the sphingosine-1-phosphate derivative as an active ingredient.

In order to achieve the above object, the present invention provides a sphingosine-1-phosphate derivative of Formula 1 or a pharmaceutically acceptable salt thereof:

Formula 1

Where

Represents a single bond or a double bond;

R ₁ and R ₂ are each independently hydrogen, C _1-6 alkyl, sugars or inositols;

R ₃ and R ₄ are each independently hydrogen, C _1-6 alkyl, Or together with the nitrogen atom to which it is attached form a 3-8 membered heterocyclic ring, wherein m is 0, 1, 2 or 3, p is 0, 1 or 2 and R ₇ is hydroxy, Amino, C _1-6 alkyl, halogen, C _1-6 alkyloxy or C _1-6 alkylamino);

R ₅ is Is hydrogen or OH when is a single bond, Is O when is a double bond;

L is O, S, CH ₂ or NH;

R ₆ is -NR ₁₀ R ₁₁ or -OR ₁₂ { Represents a single bond or a double bond, R ₈ is hydrogen, C _1-20 alkyl, 1 to 9 halogen atoms substituted C _1-20 alkyl, 3 to 8-membered cyclic alkyl, one to nine halogen atoms 3-8-membered cyclic alkyl, or R ₉ is hydrogen, amino, hydroxy, halogen, C _1-20 alkylamino, 3 to 8 membered cyclic alkylamino, C _1-20 alkyloxy, 3 to 8 membered cyclic alkyloxy, 1 to C _1-20 alkyl substituted with 9 halogen atoms or 3-8 membered cyclic alkyl substituted with 1 to 9 halogen atoms, R ₁₀ , R ₁₁ and R ₁₂ are each independently hydrogen, C _1-20 alkyl, 3 to 8-membered cyclic alkyl or Wherein R ₁₃ is as defined above for R ₉ , X is N or CH, n is 0, 1, 2, 3, 4 or 5 and q is 0, 1 or 2).

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

The sphingosine-1-phosphate derivative of formula 1 according to the present invention includes all possible stereoisomers, and the term "3- to 8-membered heterocyclic ring" used in the definition of substituents R ₃ and R ₄ is 1 to It means a saturated or unsaturated heteromonocyclic ring substituted with four nitrogen atoms, specific examples include aziridinyl, pyrrolyl, pyrrolidyl and the like.

In the definition of the compound of Formula 1 according to the present invention, Preferably,

R ₁ and R ₂ are each independently hydrogen or C _1-4 alkyl;

R ₃ and R ₄ are each independently hydrogen, C _1-4 alkyl, Or together with the nitrogen atom to which it is attached form a 3-6 membered heterocyclic ring, wherein m is 0, 1, 2 or 3, p is 0, 1 or 2 and R ₇ is hydroxy, Amino, C _1-3 alkyl, halogen, C _1-3 alkyloxy or C _1-3 alkylamino);

L is O, S or CH ₂ .

Specific examples of the compound of formula 1 according to the present invention are as follows;

L-erythro-sphingosine-1-phosphate,

L-threo-sphingosine-1-phosphate,

D-stereo-sphingosine-1-phosphate,

D-erythro-sphingosine-1- (O, O'-dimethylphosphate) hydrochloride,

D-erythro-N-methyl-sphingosine-1-phosphate,

L-erythro-N-methyl-sphingosine-1-phosphate,

D-sreo-N-methyl-sphingosine-1-phosphate,

L-threo-N-methyl-sphingosine-1-phosphate,

D-erythro-N, N-dimethyl-sphingosine-1-phosphate,

L-erythro-N, N-dimethyl-sphingosine-1-phosphate,

D-sreo-N, N-dimethyl-sphingosine-1-phosphate,

L-threo-N, N-dimethyl-sphingosine-1-phosphate,

D-erythro-N-ethyl-sphingosine-1-phosphate,

L-erythro-N-ethyl-sphingosine-1-phosphate,

D-sreo-N-ethyl-sphingosine-1-phosphate,

L-threo-N-ethyl-sphingosine-1-phosphate,

D-erythro-N-propyl-sphingosine-1-phosphate,

L-erythro-N-propyl-sphingosine-1-phosphate,

D-threo-N-propyl-sphingosine-1-phosphate,

L-Threo-N-propyl-sphingosine-1-phosphate,

L-threo-dihydrosphingosine-1-phosphate,

D-sreo-dihydrosphingosine-1-phosphate,

L-erythro-dihydrosphingosine-1-phosphate,

( 2S , 3R , 4E ) -2-amino-5-phenyl-4-pentene-1,3-diol-1-phosphate,

(2 S , 3 S , 4 E ) -2-amino-5-phenyl-4-pentene-1,3-diol-1-phosphate,

( 2S , 3R , 4E ) -2-amino-5- (3-decanyl) -phenyl-4-pentene-1,3-diol-1-phosphate,

(2 S, 3 S, 4 E) -2- amino-5- (3-decanyl) -phenyl-4-pentene-1,3-diol-1-phosphate,

( 1R , 2S ) -2-amino-1-phenyl-propane-1,3-diol-3-phosphate,

( 1S , 2S ) -2-amino-1-phenyl-propane-1,3-diol-3-phosphate,

(1 R, 2 S) -2- amino-1- (3-dodecyl Canillo) -phenyl-propan-1,3-diol-3-phosphate,

(1 S, 2 S) -2- amino-1- (3-dodecyl Canillo) -phenyl-propan-1,3-diol-3-phosphate,

(1 R, 2 S) -2 -N, N- dimethylamino-1- (3-dodecyl Canillo) -phenyl-propan-1,3-diol-3-phosphate,

(2 S) -3- oxo-sphingosine-1-phosphate,

(2 R) -3- oxo-sphingosine-1-phosphate,

(1 R) -1-hydroxymethyl-1-hexahydro-1-phosphate to Dorsett acid,

(1 S) -1-hydroxymethyl-1-hexahydro-1-phosphate to Dorsett acid,

( 2S ) -2-amino-3-hydroxy-N-tetradecyl-propionamide-3-phosphate,

(2 R) -2- amino-3-hydroxy -N- tetradecyl-propionamide-3-phosphate, and

( 2S ) -2-Amino-3-hydroxy-propionic acid tetradecane ester-3-phosphate.

In the present invention, the compound of Formula 1 may be prepared by the methods (I) to (VII) shown in the following schemes 1-7.

Wherein R ₁ , R ₃ , R ₄ , R ₈ and L are as defined above,

P ₁ and P ₂ are each independently hydrogen or a hydroxy- or amine-protecting group,

R 'is hydrogen, C _1-6 alkyl or Where m, p and R ₇ are as defined above.

According to Method (I) of Scheme 1 according to the present invention, Compound (2) can be reacted with lithium dimethylmethylphosphonate to produce Compound (3). Any solvent that can be suitably used for this reaction can be used as long as it is an organic solvent that does not adversely affect the reaction. Preferably, solvents such as tetrahydrofuran, ethyl ether, dioxane and hexane are used. It is most suitable to carry out the reaction in tetrahydrofuran. The reaction is preferably carried out under anhydrous conditions. The reaction temperature is not particularly limited, and the reaction can generally be carried out at cooling to room temperature, preferably under cooling. The resulting compound (3) can be reacted with compound (4) in the presence of a base to give compound (5). Examples of bases that can be used include 1,8-diazabicyclo [5.4.0] -7-undecene (DBU), triethylamine, pyridine, sodium hydroxide, potassium hydroxide, sodium methoxide, sodium ethoxide and the like. , In particular using the DBU. The reaction is also generally carried out in the presence of a solvent which does not adversely affect the reaction, and examples of solvents which may be used for this purpose include tetrahydrofuran, ethyl ether, dioxane, hexane, methanol, ethanol, dimethylformamide (DMF) , Dimethyl sulfoxide (DMSO), and the like, in particular, tetrahydrofuran is used to carry out the reaction. The resulting compound (5) can be reduced to produce compound (6). In this case, a conventional reduction method may be used, and for example, sodium borohydride may be used as the reducing agent. Compound (7) can be produced by deprotection of the resulting compound (6). For deprotection, conventional deprotection methods such as hydrolysis, reduction and the like can be used, for example in the presence of acids or bases. The resulting compound (7) can be reacted with compound (8) in the presence of sodium borohydride and then reduced to form compound (9). This reaction is generally carried out in the presence of a solvent that does not adversely affect the reaction, and examples of solvents that can be used for this purpose include methanol, ethanol, tetrahydrofuran, dioxane, and the like, in particular using methanol. To perform. The resulting compound (9) can be reacted with compound (10) in the presence of a base and carbon tetrabromide and then hydrolyzed to give the desired compound (1a). Examples of bases that can be used include pyridine, triethylamine, DBU, etc., which can simultaneously serve as solvents, in particular pyridine. The reaction temperature is not particularly limited, and generally, the reaction is carried out under cooling or warming.

Alternatively, compound (7) may be protected through amine protection to give compound (11). At this time, conventional amine protecting groups such as benzyl, trityl, t-butyloxycarbonyl and the like can be used. The resulting compound (11) can be reacted with compound (10) in the presence of a base and carbon tetrabromide to give compound (12). Examples of bases that can be used include pyridine, triethylamine, DBU, etc., which can simultaneously serve as solvents, in particular pyridine. The reaction temperature is not particularly limited, and generally, the reaction is carried out under cooling or warming. The resulting compound (12) can be deprotected to give the desired compound (1b).

Alternatively, compound (12) may be reduced (hydrogenated) to produce compound (13). At this time, a conventional reduction method may be used, for example, hydrogenation may be carried out under a palladium / carbon catalyst. This reaction is generally carried out in the presence of a solvent that does not adversely affect the reaction, examples of the solvent that can be used for this purpose include ethyl acetate, tetrahydrofuran, ethyl ether, hexane, dioxane, methanol, ethanol, and the like. In particular, the reaction is carried out using ethyl acetate. The reaction temperature is not particularly limited and is preferably carried out at room temperature. The resulting compound (13) can be deprotected to give the desired compound (1c).

Wherein R ₁ , R ₃ , R ₄ , R ₈ , L, P ₁ , P ₂ and R ′ are as defined above.

According to Method (II) of Scheme 2 according to the present invention, Compound (5) can be deprotected to produce Compound (14). The resulting compound (14) can be reduced to produce compound (7). At this time, a conventional reduction method can be used, for example, zinc borohydride can be used as the reducing agent. This reaction is generally carried out in the presence of a solvent which does not adversely affect the reaction, and examples of the solvent that can be used for this purpose include tetrahydrofuran, ethyl ether, hexane, dioxane, and the like, in particular using tetrahydrofuran. To carry out the reaction. The reaction temperature is not particularly limited, and generally, the reaction is performed at cooling to room temperature. From the produced compound (7), the target compounds (1a), (1b) and (1c) can be produced in the same manner as in the above-mentioned method (I).

Alternatively, compound (14) may be protected through an amine protection reaction to produce compound (15). At this time, conventional amine protecting groups such as benzyl, trityl, t-butyloxycarbonyl and the like can be used. The resulting compound (15) can be reacted with compound (10) in the presence of a base and carbon tetrabromide and then deprotected to give the desired compound (1d). Examples of bases that can be used include pyridine, triethylamine, DBU, etc., which can simultaneously serve as solvents, in particular pyridine. The reaction temperature is not particularly limited, and generally, the reaction is carried out under cooling or warming.

Wherein R ₁ , R ₃ , R ₄ , R ₈ , P ₁ , P ₂ and R ′ are as defined above and R is the same as the definition of R ′.

According to the method (III) of Scheme 3 according to the present invention, compound (17) can be produced by esterifying compound (16) in the presence of acetyl chloride and methanol. The resulting compound (17) can be protected via hydroxy and amine protection to give compound (18). At this time, conventional protecting groups such as t-butyldimethylsilyl, methoxymethyl, benzyl, trityl, t-butyloxycarbonyl and the like can be used. The resulting compound (18) can be reacted under the same conditions as in the preparation of the compound (11) from the compound (2) of the above method (I) to produce the compound (22). The resulting compound (22) can be brominated by reaction with carbon tetrabromide and triphenylphosphine, followed by reaction with compound (10) and deprotection to prepare the desired compound (1e). This reaction is generally carried out in the presence of a solvent which does not adversely affect the reaction, and examples of the solvent that can be used for this purpose include chloroform, dichloromethane, tetrahydrofuran, ethyl ether, hexane, dioxane, and the like. The reaction is carried out using dichloromethane. The reaction temperature is not particularly limited, and in general, the reaction can be carried out under cooling to warming, preferably under heating.

Alternatively, the compound (20) may be reacted under the same conditions as in the preparation of the compound (7) from the compound (5) of the above method (II) to give the compound (24). The resulting compound (24) is reacted with compound (25) under the same conditions as in the preparation of compound (9) from compound (7) of method (I), followed by reaction with compound (8) in succession to compound (26). Can be generated. The resulting compound (26) can be subjected to the same reaction as in the preparation of the compound (1e) from the compound (22) to give the target compound (1f).

Wherein R ₁ , R ₃ , R ₄ , R ₉ , X, L, P ₁ , P ₂ , R and R 'are as defined above.

According to method (IV) of Scheme 4 according to the present invention, compound (29) can be produced by reacting compound (27) with compound (28) in the presence of a base. Examples of bases that can be used include n-butyllithium, lithium hexamethyldisilase, lithium diisopropylamide, and the like, in particular using n-butyllithium to carry out the reaction. This reaction is generally carried out in the presence of a solvent which does not adversely affect the reaction, and examples of the solvent that can be used for this purpose include tetrahydrofuran, ethyl ether, hexane, dioxane, and the like, in particular using tetrahydrofuran. To carry out the reaction. The reaction temperature is not particularly limited, and generally, the reaction is performed at cooling to room temperature. The resulting compound (29) can be deprotected to produce compound (30). The resulting compound (30) is reacted with compound (25) under the same conditions as in the preparation of compound (9) from compound (7) of method (I), followed by reaction with compound (8) in succession to compound (31). Can be generated. The resultant compound (31) can be subjected to the same reaction as in the preparation of the compound (1a) from the compound (9) of the above method (I) to give the target compound (1 g).

Wherein R ₁ , R ₃ , R ₄ , R ₆ , R ₇ , L, P ₁ , P ₂ , R and R 'are as defined above.

According to Method (V) of Scheme 5 according to the present invention, Compound (27) can be reacted with Compound (32) in the presence of a base and then reduced to form Compound (33). In the reaction with compound (32), examples of the base that can be used include n-butyllithium, lithium hexamethyldisilazein, lithium diisopropylamide, and the like, in particular, lithium hexamethyldisilazane is used to carry out the reaction. . The reaction with compound (32) is also generally carried out in the presence of a solvent which does not adversely affect the reaction, and examples of solvents which can be used for this purpose include tetrahydrofuran, ethyl ether, hexane, dioxane, and the like. The reaction is carried out using tetrahydrofuran. The reaction temperature is not particularly limited, and generally, the reaction is performed at cooling to room temperature. In addition, conventional reduction methods may be employed in the reduction, for example hydrogenation under a palladium / carbon catalyst. The reduction reaction is generally carried out in the presence of a solvent that does not adversely affect the reaction. Examples of the solvent that can be used for this purpose include ethyl acetate, tetrahydrofuran, ethyl ether, hexane, dioxane, methanol, ethanol, and the like. In particular, the reaction is carried out using ethyl acetate. The reaction temperature is not particularly limited and is preferably carried out at room temperature. The resulting compound (33) can be subjected to the same reaction as in the preparation of the compound (1 g) from the compound (29) of the above method (IV) to produce the target compound (1h).

Alternatively, the compound (34) may be subjected to the same reaction as the preparation of the compound (1b) from the compound (7) of the above method (II) to give the desired compound (1i).

Wherein R ₁ , R ₃ , R ₄ , R ₁₀ , R ₁₁ , L, P ₁ , P ₂ , R and R 'are as defined above.

According to the method (VI) of Scheme 6 according to the present invention, compound (39) can be produced by reacting compound (37) with compound (38) in the presence of a catalyst. Examples of catalysts that can be used include N, N'-dicyclohexylcarbodiimide, N, N'-carbonyldiimidazole, 1,3-diisopropylcarbodiimide, 1-hydroxybenzotriazole, and the like. Preferably the reaction is carried out using 1,3-diisopropylcarbodiimide and 1-hydroxybenzotriazole. This reaction is also generally carried out in the presence of a solvent which does not adversely affect the reaction, and examples of solvents which may be used for this purpose include dichloromethane, chloroform, DMF, DMSO, pyridine, tetrahydrofuran, ethyl ether, hexane, di Oxane and the like, in particular using dichloroform or DMF to carry out the reaction. The reaction temperature is not particularly limited, and generally, the reaction is carried out under cooling or warming. The resulting compound (39) can be subjected to the same reaction as in the preparation of compound (1h) from compound (33) of the above method (V) to give the desired compound (1j).

Wherein R ₁ , R ₃ , R ₄ , R ₁₂ , L, P ₁ , P ₂ , R and R 'are as defined above.

According to the method (VII) of Scheme 7 according to the present invention, compound (43) can be produced by reacting compound (37) with compound (42) in the presence of a catalyst. Examples of catalysts that can be used include N, N'-dicyclohexylcarbodiimide, N, N'-carbonyldiimidazole, 1,3-diisopropylcarbodiimide, chlorosulfonyl isocyanate, triphenylphosphine-carbon tetrachloride And the like, and preferably, the reaction is performed using N, N'-carbonyldiimidazole. This reaction is also generally carried out in the presence of a solvent which does not adversely affect the reaction, and examples of solvents which may be used for this purpose include dichloromethane, chloroform, DMF, DMSO, pyridine, tetrahydrofuran, ethyl ether, hexane, di Oxane and the like, in particular, the reaction is carried out using dichloroform or DMF. The reaction temperature is not particularly limited, and generally, the reaction is carried out under cooling or warming. The resulting compound (43) can be subjected to the same reaction as in the preparation of the compound (1h) from the compound (33) of the above method (V) to give the target compound (1k).

Starting materials used in the above methods (I) to (VII) can be easily prepared by conventional methods, and commercially available ones can be purchased and used. In addition, the resultant compounds of interest can be separated and purified using conventional methods such as column chromatography, recrystallization and the like.

Thus prepared, sphingosine-1-phosphate derivatives of the present invention and pharmaceutically acceptable salts thereof have structural characteristics and activity as effective and selective agonists and antagonists for EDG (endothelial differentiation gene) receptors. It can be usefully used as a therapeutic agent for various refractory diseases such as wound healing, cancer proliferation and metastasis, angiogenesis related diseases such as rheumatoid arthritis, inflammation related diseases and circulatory diseases.

Accordingly, the present invention provides an agonistic and antagonistic pharmaceutical composition for the EDG receptor, comprising an effective amount of a compound of Formula 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

The pharmaceutical compositions of the present invention are prepared by conventional preparations in the pharmaceutical field, for example, oral preparations such as tablets, capsules, troches, solutions, suspensions, etc., injectable solutions or suspensions, or distilled water for injection at the time of injection. It may be formulated into a variety of preparations, such as injectable preparations in the form of dry powder for immediate use for immediate use. Carriers that can be used in the compositions of the present invention are conventional in the pharmaceutical art, for example in the case of oral preparations, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments And fragrances. In the case of injectables, there are preservatives, analgesics, solubilizers, stabilizers and the like. The pharmaceutical preparations thus prepared can be administered orally or parenterally, eg, by intravenous, subcutaneous or intraperitoneal injection. In addition, in order to prevent the decomposition of the drug by gastric acid during oral administration, an antacid may be used in combination, or a solid dosage form for oral administration such as tablets may be formulated into a formulation coated with enteric skin.

The amount of the compound of formula (I) as an active ingredient is appropriately selected depending on the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age and condition of the patient, the severity of the disease to be treated, but generally 1 to 500 mg per day, preferably Preferably, the amount of 5 to 200 mg may be administered orally or parenterally via once or divided doses per day.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1 Preparation of L-Sreo-Sphingosine-1-Phosphate (Method (II))

(1) N- tert -Butyloxycarbonyl-L-sreo-sphingosine

L-Sreo-sphingosine (3.34 mmol) and di- t -butyl dicarbonate (10.0 mmol) were dissolved in methanol (40 ml), stirred at 40 ° C. for 9 hours and then cooled. Water was added to the reaction solution, and the organic substance obtained by extraction with ethyl acetate was dried over magnesium sulfate and concentrated under reduced pressure. The residue was separated by column chromatography (n-hexane: ethyl acetate = 3: 1) to give 1.15 g (yield: 87%) of the title compound as a colorless oil.

[a] _D ²⁵ = + 2.32 (c 0.58, CHC1 ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 5.75 (1H, dt), 5.51 (1H, dd), 5.16 (1H, br), 4.34 (1H, dd), 3.79 (2H, d), 3.62 (1H, dd), 2.37 (2H, br), 2.04 (2H, m), 1.45-1.26 (31H, m), 0.88 (3H, t).

(2) N- tert -Butyloxycarbonyl-L-sreo-sphingosine-1- (O, O'-dimethylphosphate)

N- tert -butyloxycarbonyl-L-sreo-sphingosine (1.68 mmol) and tetrabromomethane (2.11 mmol) were added to anhydrous pyridine (3 ml) under an argon atmosphere and cooled to 0 ° C. Trimethyl phosphite (2.52 mmol) was slowly added dropwise, followed by stirring at room temperature for 2 hours. Water was added to the reaction solution, and the organics obtained by extraction with ethyl acetate were washed sequentially with 2N hydrochloric acid, saturated sodium bicarbonate solution, and brine, and then dried over magnesium sulfate. The residue obtained by concentration under reduced pressure was separated by column chromatography (n-hexane: ethyl acetate = 1: 1) to give 630 mg (yield: 74%) of the title compound as a colorless oil.

[a] _D ²⁵ = -4.09 (c 1.7, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 5.77 (1H, dt), 5.48 (1H, dd), 5.50 (1H, d), 4.35 (1H, m), 4.10 (2H, pseudo t), 3.81-3.77 (7H, m), 2,03 ( 2H, m), 1.44-1.26 (31H, m), 0.88 (3H, t).

(3) L-Sreo-sphingosine-1-phosphate

N- tert -butyloxycarbonyl-L-sreo-sphingosine-1- (O, O'-dimethylphosphate) (0.2 mmol) is dissolved in anhydrous dichloromethane (3 ml) under argon atmosphere and cooled to 0 ° C. I was. Bromotrimethylsilane (20 mmol) was slowly added dropwise, followed by stirring at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, methanol (2 ml) and water (1 ml) were added thereto, and then concentrated under reduced pressure. The residue obtained was recrystallized from a tetrahydrofuran / water mixed solvent to give 47 mg (yield: 63%) of the title compound as a white solid.

[a] _D ²⁵ = -27.38 (c 0.22, AcOH).

¹ H-NMR (300 MHz, CD ₃ CO ₂ D); δ 5.91 (1H, dt), 5.48 (1H, dd), 4.31 (1H, pseudo t), 4.26-4.01 (2H, m), 3.43 (1H, m), 2,10 (2H, m), 1.41- 1.29 (22H, m), 0.89 (3H, t).

Example 2 Preparation of D-Sreo-Sphingosine-1-Phosphate (Method (II))

(1) N- tert -Butyloxycarbonyl-D-sreo-sphingosine

The title compound was obtained in the same manner as in Example 1 (1), using D-stereo-sphingosine as a starting material.

[α] _D ²⁵ = -1.58 (c 2.02, CHC1 ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 5.75 (1H, dt), 5.51 (1H, dd), 5.16 (1H, br), 4.34 (1H, dd), 3.79 (2H, d), 3.62 (1H, dd), 2.37 (2H, br), 2.04 (2H, m), 1.45-1.26 (31H, m), 0.88 (3H, t).

(2) N- tert -Butyloxycarbonyl-D-sreo-sphingosine-1- (O, O'-dimethylphosphate)

The title compound was obtained in the same manner as in Example 1 (2) using N- tert -butyloxycarbonyl-D-sreo-sphingosine as a starting material.

[a] _D ²⁵ = -4.83 (c 1.52, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 5.77 (1H, dt), 5.48 (1H, dd), 5.50 (1H, d), 4.35 (1H, m), 4.10 (2H, pseudo t), 3.81-3.77 (7H, m), 2,03 ( 2H, m), 1.44-1.26 (31H, m), 0.88 (3H, t).

(3) D-stereo-sphingosine-1-phosphate

The title compound was obtained in the same manner as in Example 3 (3), using N- tert -butyloxycarbonyl-D-sreo-sphingosine-1- (O, O'-dimethylphosphate) as a starting material.

[a] _D ²⁵ = + 29.04 (c 0.21, AcOH).

¹ H-NMR (300 MHz, CD ₃ CO ₂ D); δ 5.91 (1H, dt), 5.48 (1H, dd), 4.31 (1H, pseudo t), 4.26-4.01 (2H, m), 3.43 (1H, m), 2,10 (2H, m), 1.41- 1.29 (22H, m), 0.89 (3H, t).

Example 3 Preparation of L-erythro-sphingosine-1-phosphate (Method (I))

(1) N- tert -Butyloxycarbonyl-L-erythro-sphingosine

The title compound was obtained by the method of Example 1 (1) using L-erythro-sphingosine as starting material.

[a] _D ²⁵ = + 1.47 (c 1.15, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 5.78 (1H, dt), 5.53 (1H, dd), 5.29 (1H, br), 4.32 (1H, pseudo t), 3.94 (1H, dd), 3.71 (1H, dd), 3.61 (1H, m) , 2.05 (2H, m), 1.45-1.26 (31H, m), 0.88 (3H, t).

(2) N- tert -Butyloxycarbonyl-L-erythro-sphingosine-1- (O, O'-dimethylphosphate)

The title compound was obtained by the method in Example (2) using N- tert -butyloxycarbonyl-L-erythro-sphingosine as starting material.

[a] _D ²⁵ = -2.64 (c 2.26, CHC1 ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 5.76 (1H, dt), 5.32 (1H, dd), 5.01 (1H, d), 4.33 (1H, m), 4.13 (2H, m), 3.81-3.70 (7H, m), 2.79 (1H, br ), 2,03 (2H, m), 1.48-1.26 (31H, m), 0.88 (3H, t).

(3) L-erythro-sphingosine-1-phosphate

The title compound was obtained in the same manner as in (3) of Example 1, using N- tert -butyloxycarbonyl-L-erythro-sphingosine-1- (O, O'-dimethylphosphate) as a starting material.

[a] _D ²⁵ = + 4.84 (c 0.37, AcOH).

¹ H-NMR (300 MHz, CD ₃ CO ₂ D); δ 5.91 (1H, dt), 5.53 (1H, dd), 4.43 (1H, pseudo t), 4.27-4.09 (2H, m), 3.61 (1H, m), 2,09 (2H, m), 1.47- 1.29 (22H, m), 0.89 (3H, t).

Example 4 Preparation of D-erythro-sphingosine-1- (O, O'-dimethylphosphate) Hydrochloride (Method (I))

(1) N- tert -Butyloxycarbonyl-D-erythro-sphingosine

The title compound was obtained by the method of Example 1 (1) using D-erythro-sphingosine as starting material.

[a] _D ²⁵ = -1.27 (c 1.0, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 5.78 (1H, dt), 5.53 (1H, dd), 5.29 (1H, br), 4.32 (1H, pseudo t), 3.94 (1H, dd), 3.71 (1H, dd), 3.61 (1H, m) , 2.05 (2H, m), 1.45-1.26 (31H, m), 0.88 (3H, t).

(2) N- tert -Butyloxycarbonyl-D-erythro-sphingosine-1- (O, O'-dimethylphosphate)

The title compound was obtained in the same manner as in Example 2 (2) using N- tert -butyloxycarbonyl-D-erythro-sphingosine as a starting material.

[a] _D ²⁵ = + 4.78 (c 1.24, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 5.76 (1H, dt), 5.32 (1H, dd), 5.01 (1H, d), 4.33 (1H, m), 4.13 (2H, m), 3.81-3.70 (7H, m), 2.79 (1H, br ), 2,03 (2H, m), 1.48-1.26 (31H, m), 0.88 (3H, t).

(3) D-erythro-sphingosine-1- (O, O'-dimethylphosphate) hydrochloride

N- tert -butyloxycarbonyl-D-erythro-sphingosine-1- (O, O'-dimethylphosphate) (81 mg, 0.16 mmol) was dissolved in methanol (3 ml) under nitrogen atmosphere and cooled to 0 ° C. I was. Chlorotrimethylsilane (2 ml, 16 mmol) was slowly added dropwise, followed by stirring at room temperature for 6 hours. The reaction solution was concentrated under reduced pressure to give 62 mg (yield: 88%) of the title compound as a white solid.

[a] _D ²⁵ = -2.61 (c 0.34, CHC1 ₃ ).

¹ H-NMR (300 MHz, CD ₃ OD); δ 5.90 (1H, dt), 5.48 (1H, dd), 4.34-4.17 (3H, m), 3.85 (3H, s), 3.81 (3H, s), 3.46 (1H, m), 2,11 (2H , m), 1.43-1.29 (22H, m), 0.89 (3H, t).

Example 5 Preparation of L-Sreo-Dihydrosphingosine-1-Phosphate (Method (II))

(1) N- tert -Butyloxycarbonyl-L-sreo-dihydrosphingosine-1- (O, O'-dimethylphosphate)

N- tert -butyloxycarbonyl-L-sreo-sphingosine-1- (O, O'-dimethylphosphate) (188 mg, 0.37 mmol) was dissolved in ethyl acetate (6 ml) and 10% Pd / C 50 mg was added followed by stirring under hydrogen. After 18 hours, the reaction solution was filtered and concentrated, and then the residue was separated by column chromatography (n-hexane: ethyl acetate = 1: 1) to give 163 mg (yield: 87%) of the title compound as a colorless oil.

[a] _D ²⁵ = + 4.60 (c 1.22, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 5.01 (1H, d), 4.07 (2H, m), 3.84-3.76 (8H, m), 1.44-1.25 (37H, m), 0.88 (3H, t).

(2) L-Sreo-dihydrosphingosine-1-phosphate

The title compound was prepared in the same manner as in (3) of Example 1, using N- tert -butyloxycarbonyl-L-sreo-dihydrosphingosine-1- (O, O'-dimethylphosphate) as a starting material. Obtained.

[a] _D ²⁵ = -17.58 (c 0.24, AcOH).

¹ H-NMR (300 MHz, CD ₃ CO ₂ D); δ 4.34 (1H, m), 4.21 (1H, m), 3.95 (1H, m), 3.50 (1H, m), 1.61-1.29 (28H, m), 0.89 (3H, t).

Example 6 Preparation of D-Sreo-Dihydrosphingosine-1-Phosphate (Method (II))

(1) N- tert -Butyloxycarbonyl-D-sreo-dihydrosphingosine-1- (O, O'-dimethylphosphate)

The title compound was obtained in the same manner as in Example (1) of Example 5, using N- tert -butyloxycarbonyl-D-sreo-sphingosine-1- (O, O'-dimethylphosphate) as a starting material.

[a] _D ²⁵ = -4.96 (c 0.80, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 5.01 (1H, d), 4.07 (2H, m), 3.84-3.76 (8H, m), 1.44-1.25 (37H, m), 0.88 (3H, t).

(2) D-Sreo-dihydrosphingosine-1-phosphate

N- tert -butyloxycarbonyl-D-sreo-dihydrosphingosine-1- (O, O'-dimethylphosphate) as starting material to obtain the title compound in the same manner as in Example (2) It was.

[a] _D ²⁵ = + 18.34 (c 0.26, AcOH).

¹ H-NMR (300 MHz, CD ₃ CO ₂ D); δ 4.34 (1H, m), 4.21 (1H, m), 3.95 (1H, m), 3.50 (1H, m), 1.61-1.29 (28H, m), 0.89 (3H, t).

Example 7 Preparation of L-erythro-dihydrosphingosine-1-phosphate (Method (I))

(1) N- tert -Butyloxycarbonyl-L-erythro-dihydrosphingosine-1- (O, O'-dimethylphosphate)

The title compound was obtained in the same manner as in (1) of Example 5, using N- tert -butyloxycarbonyl-L-erythropo-sphingosine-1- (O, O'-dimethylphosphate) as a starting material. .

[a] _D ²⁵ = −19.46 (c 0.71, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 5.05 (1H, d), 4.42 (1H, m), 4.12 (1H, m), 3.82-3.61 (8H, m), 3.00 (1H, br), 1.57-1.25 (37H, m), 0.88 (3H , t).

(2) L-erythro-dihydrosphingosine-1-phosphate

Obtained the title compound in the same manner as in (2) of Example 5, using N- tert -butyloxycarbonyl-L-erythro-dihydrosphingosine-1- (O, O'-dimethylphosphate) as a starting material. It was.

[a] _D ²⁵ = + 6.90 (c 0.29, AcOH).

¹ H-NMR (300 MHz, CD ₃ CO ₂ D); δ 4.31 (2H, m), 3.99 (1H, dd), 3.68 (1H, dd), 1.58-1.29 (28H, m), 0.89 (3H, t).

Example 8 (2 S ) -3-oxo-sphingosine-1-phosphate (method (II))

(1) (2 S ) -N- tert -Butyloxycarbonyl-3-oxo-sphingosine

(2 S) -C3- Kane The title compound was a monohydrate ping high signal in the same manner as in (1) of Example 1 as a starting material was obtained.

[a] _D ²⁵ = + 0.09 (c 0.76, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 7.06 (1H, dt), 6.28 (1H, dd), 5.72 (1H, br), 4.61 (1H, br), 3.94-3.86 (2H, m), 2.84 (1H, dd), 2.25 (2H, m ), 1.46-1.26 (31 H, m), 0.88 (3 H, t).

(2) (2 S ) -N- tert -Butyloxycarbonyl-3-oxo-sphingosine-1- (O, O'-dimethylphosphate)

(2 S) -N- tert-butyloxycarbonyl-3-oxo-sphingosine was obtained in the same manner as the title compound and (2) in Example 1 as a starting material.

[a] _D ²⁵ = + 0.67 (c 1.15, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 7.05 (1H, dt), 6.29 (1H, d), 5.63 (1H, d), 4.71 (1H, br), 4.34 (2H, m), 3.77 (3H, d), 3.72 (3H, d), 2.25 (1 H, m), 1.52-1.26 (31 H, m), 0.88 (3 H, t).

(3) (2 S ) -3-oxo-sphingosine-1-phosphate

Title to sphingosine-1-same manner as in (3) of Example 1, the (O, O'- dimethyl phosphate) as a starting material - (2 S) -N- tert - butyloxycarbonyl-3-oxo The compound was obtained.

¹ H-NMR (300 MHz, CD ₃ CO ₂ D); δ 7.15 (1H, dt), 6.73 (1H, dd), 4.66 (1H, dd), 4.09 (1H, s), 3.65 (1H, t), 2.30 (2H, m), 1.50-1.29 (22H, m ), 0.88 (3H, t).

Example 9: (2 R ) -3-oxo-sphingosine-1-phosphate (method (II))

(1) (2 R ) -N- tert -Butyloxycarbonyl-3-oxo-sphingosine

(2 R) -C3- Kane The title compound was prepared in the same manner as in Example 8, the high signal to the Ping monohydrate starting material (1) was obtained.

[a] _D ²⁵ = + 0.37 (c 1.85, CHC1 ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 7.06 (1H, dt), 6.28 (1H, dd), 5.72 (1H, br), 4.61 (1H, br), 3.94-3.86 (2H, m), 2.84 (1H, dd), 2.25 (2H, m ), 1.46-1.26 (31 H, m), 0.88 (3 H, t).

(2) (2 R ) -N- tert -Butyloxycarbonyl-3-oxo-sphingosine-1- (O, O'-dimethylphosphate)

(2 R) -N- tert-butyloxycarbonyl-3-to a sphingosine as a starting material to give the title compound in the same manner as in (2) of Example 8.

[a] _D ²⁵ = -2.24 (c 1.09, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 7.05 (1H, dt), 6.29 (1H, d), 5.63 (1H, d), 4.71 (1H, br), 4.34 (2H, m), 3.77 (3H, d), 3.72 (3H, d), 2.25 (1 H, m), 1.52-1.26 (31 H, m), 0.88 (3 H, t).

(3) (2 R ) -3-oxo-sphingosine-1-phosphate

Title by sphingosine-1 the same way as in (O, O'- dimethyl phosphate) (3) of Example 8 as a starting material - (2 R) -N- tert - butyloxycarbonyl-3-oxo The compound was obtained.

1 H-NMR (300 MHz, CD ₃ CO ₂ D); δ 7.15 (1H, dt), 6.73 (1H, dd), 4.66 (1H, dd), 4.09 (1H, s), 3.65 (1H, t), 2.30 (2H, m), 1.50-1.29 (22H, m ), 0.88 (3H, t).

Example 10: (1 R ) -1-hydroxymethyl-1-hexadecylcarbamic acid-1-phosphate (method (V))

(1) (4 S ) -4- (hexadecanyl) -2,2-dimethyl-oxazolidine-3-carboxylic acid tert -Butyl ester

Dissolve 1,1,1,3,3,3-hexamethyldisilazane (0.6 ml, 2.85 mmol) in anhydrous tetrahydrofuran (7 ml) under nitrogen atmosphere and n-butyllithium (2.5M, 1.1 at room temperature). ml, 2.85 mmol) was slowly added dropwise and stirred for 30 minutes. Phosphonium (1.63 g, 3.1 mmol) was added thereto, dissolved in anhydrous tetrahydrofuran (7 ml), stirred for 30 minutes, and then cooled to 78 ° C. Oxazolidine-3-carboxylic acid tert - - butyl ester was dissolved in a solution (284 mg, 1.24 mmol) in anhydrous tetrahydrofuran (5 ml) slowly here (4 S) -4- formyl-2,2-dimethyl-a After dropwise addition, the mixture was stirred at room temperature for 20 hours. After cooling the reaction solution to 0 ℃ 1N hydrochloric acid solution was added and extracted with ethyl acetate. The obtained organics were washed with saturated sodium bicarbonate solution and brine, and dried over magnesium sulfate. The colorless oil obtained by concentration under reduced pressure was dissolved in ethyl acetate (20 ml), 10% Pd / C (220 mg) was added, followed by stirring under hydrogen. After 18 hours, the reaction solution was filtered and concentrated, and then the residue was separated by column chromatography (n-hexane: ethyl acetate = 20: 1) to give 471 mg (yield: 72%) of the title compound as a colorless oil.

[a] _D ²⁵ = -19.47 (c 0.60, CHCl ₃ ).

¹ H-NMR (300 MHz, CD ₃ CO ₂ D); δ 3.91 (1H, dd), 3.73 (2H, d), 1.59-1.26 (48H, m), 0.88 (3H, t).

(2) (1 R ) -1-hydroxymethyl-1-hexadecylcarbamic acid tert -Butyl ester

(4 S ) -4- (hexadecanyl) -2,2-dimethyl-oxazolidine-3-carboxylic acid tert -butyl ester (417 mg, 0.98 mmol) and lithium chloride (83 mg, 1.96 mmol) 90% It was dissolved in aqueous acetic acid solution (8 ml) and stirred at room temperature for 12 hours. Water was added to the reaction solution, and the organics obtained by extraction with ethyl acetate were washed with water and brine, and dried over magnesium sulfate. The residue obtained by concentration under reduced pressure was separated by column chromatography (n-hexane: ethyl acetate = 5: 1) to give 210 mg (yield: 82%) of the title compound as a white solid.

[a] _D ²⁵ = + 9.14 (c 0.78, CHC1 ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 4.57 (1H, br), 3.70-3.51 (3H, m), 2.37 (1H, br), 1.57-1.25 (39H, m), 0.88 (3H, t).

(3) (1 R ) -1-hydroxymethyl-1-hexadecylcarbamic acid tert -Butyl ester-1- (O, O'-dimethylphosphate)

Anhydrous pyridine (2 ml) under-butyl ester (213 mg, 0.55 mmol), and tetrabromo methane atmosphere the parent (470 mg, 1.25 mmol) argon - (1 R) -1-hydroxymethyl-1-hexahydro to Dorsett acid tert And cooled to 0 ° C. Trimethyl phosphite (0.19 ml, 1.56 mmol) was slowly added dropwise, followed by stirring at room temperature for 2.5 hours. Water was added to the reaction solution, and the organics obtained by extraction with ethyl acetate were washed sequentially with 2N hydrochloric acid, saturated sodium bicarbonate solution, and brine, and then dried over magnesium sulfate. The residue obtained by concentration under reduced pressure was separated by column chromatography (n-hexane: ethyl acetate = 3: 1) to give 221 mg (yield: 81%) of the title compound as a colorless oil.

[a] _D ²⁵ = + 8.26 (c 0.64, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 4.57 (1H, br), 3.69-3.51 (3H, m), 2.37 (1H, br), 1.57-1.25 (39H, m), 0.88 (3H, t).

(4) (1 R ) -1-hydroxymethyl-1-hexadecylcarbamic acid-1-phosphate

(1 R) -1-hydroxymethyl-1-hexahydro to Dorsett acid tert - butyl ester in dry dichloromethane under-1- (O, O'- dimethyl phosphate) argon atmosphere a (116 mg, 0.25 mmol) ( 3.5 ml ) And cooled to 0 ° C. Bromotrimethylsilane (3.4 ml, 25 mmol) was slowly added dropwise and then stirred at room temperature for 4 hours. The reaction solution was concentrated under reduced pressure, methanol (2 ml) and water (1 ml) were added thereto, and then concentrated under reduced pressure. The obtained residue was recrystallized from methanol / water mixed solvent to give 49 mg (yield: 60%) of the title compound as a white solid.

[a] _D ²⁵ = -7.80 (c 0.26, AcOH).

¹ H-NMR (300 MHz, CD ₃ CO ₂ D); δ 3.69-3.51 (3H, m), 1.57-1.25 (30H, m), 0.88 (3H, t).

Example 11: (1 S ) -1-hydroxymethyl-1-hexadecylcarbamic acid-1-phosphate (method (V))

(1) (4 R ) -4- (hexadecanyl) -2,2-dimethyl-oxazolidine-3-carboxylic acid tert -Butyl ester

(4 R) -4- formyl-2,2-dimethyl-oxazolidine-3-carboxylic acid tert-butyl ester The title compound was prepared in the same manner as in Example 10 (1) as a starting material was obtained.

[a] _D ²⁵ = + 17.58 (c 0.56, CHC1 ₃ ).

¹ H-NMR (300 MHz, CD ₃ CO ₂ D); δ 3.91 (1H, dd), 3.73 (2H, d), 1.59-1.26 (48H, m), 0.88 (3H, t).

(2) (1 S ) -1-hydroxymethyl-1-hexadecylcarbamic acid tert -Butyl ester

(4 R) -4- (hexahydro decanyl) -2,2-dimethyl-oxazolidine-3-carboxylic acid tert-butyl ester to give the title compound in the same manner as in (2) of Example 10 as a starting material by It was.

[a] _D ²⁵ = -9.38 (c 0.54, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 4.57 (1H, br), 3.70-3.51 (3H, m), 2.37 (1H, br), 1.57-1.25 (39H, m), 0.88 (3H, t).

(3) (1 S ) -1-hydroxymethyl-1-hexadecylcarbamic acid tert -Butyl ester-1- (O, O'-dimethylphosphate)

(1 S ) -1-hydroxymethyl-1-hexadecylcarbamic acid tert -butyl ester was used as a starting material to obtain the title compound in the same manner as in Example 10 (3).

[a] _D ²⁵ = -8.94 (c 1.07, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 4.57 (1H, br), 3.69-3.51 (3H, m), 2.37 (1H, br), 1.57-1.25 (39H, m), 0.88 (3H, t).

(4) (1 S ) -1-hydroxymethyl-1-hexadecylcarbamic acid-1-phosphate

The title 1-butyl ester as the same method as in (4) of Example 10 to the (O, O'- dimethyl phosphate) as a starting material - (1 S) -1-hydroxymethyl-1-hexahydro to Dorsett acid tert The compound was obtained.

[a] _D ²⁵ = + 9.76 (c 0.33, AcOH).

¹ H-NMR (300 MHz, CD ₃ CO ₂ D); δ 3.69-3.51 (3H, m), 1.57-1.25 (30H, m), 0.88 (3H, t).

Example 12: (2 S ) -2-Amino-3-hydroxy-N-tetradecyl-propionamide-3-phosphate (method (VI))

(1) (1 S )-[2-hydroxy-1- (tetradecylcarbamoyl) -ethyl] -carbamic acid tert -Butyl ester

L-serine (2.5 g, 23.8 mmol) was dissolved in dioxane (90 ml) and water (45 ml) mixed solvent, and 1N sodium hydroxide solution (45 ml) was added. After cooling to 0 ° C., Boc ₂ O (5.7 g, 26.1 mmol) was slowly added dropwise, followed by stirring at room temperature. After 10 hours, the reaction solution was concentrated under reduced pressure, ethyl acetate (20 ml) was added thereto, and the mixture was cooled to 0 ° C. The reaction solution was acidified to pH 3 with aqueous potassium hydrogen sulfate solution and extracted with ethyl acetate. The extracted residue was washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a colorless oil. This colorless oil was dissolved in dichloromethane (140 ml) and DIC (3.73 ml, 23.8 mmol) and HOBT (3.22 g, 23.8 mmol) were added dropwise. The reaction solution was cooled to 0 ° C. and tetradecylamine (4.06 g, 19.04 mmol) was slowly added dropwise and stirred at room temperature. After 10 hours, water was added to the reaction solution, and the organic material obtained by extraction with ethyl acetate was washed with water and brine, and dried over magnesium sulfate. The residue obtained by concentration under reduced pressure was separated by column chromatography (n-hexane: ethyl acetate = 3: 1) to give 5.8 g (yield: 76%) of the title compound as a colorless oil.

[a] _D ²⁵ = -26.9 (c 0.74, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 6.65 (1H, br), 5.56 (1H, br), 4.11 (2H, m), 3.63 (1H, m), 3.30-3.14 (3H, m), 1.49-1.23 (33H, m), 0.88 (3H , t).

(2) (1 S )-[2-hydroxy-1- (tetradecylcarbamoyl) -ethyl] -carbamic acid tert -Butyl ester-2- (O, O'-dimethylphosphate)

( 1S )-[2-hydroxy-1- (tetradecylcarbamoyl) -ethyl] -carbamic acid tert -butyl ester (620 mg, 1.55 mmol) and tetrabromocarbon (1.03 g, 3.1 mmol) were nitrogen Dissolve in anhydrous pyridine (7 ml) under air. After cooling to 0 ° C. and trimethyl phosphite (0.4 ml, 3.41 mmol) was slowly added dropwise, the mixture was stirred at room temperature for 2 hours. Water was added to the reaction solution, and the organics obtained by extraction with ethyl acetate were washed sequentially with 2N hydrochloric acid, saturated sodium bicarbonate solution, and brine, and then dried over magnesium sulfate. The residue obtained by concentration under reduced pressure was separated by column chromatography (n-hexane: ethyl acetate = 3: 2) to give 418 mg (yield: 53%) of the title compound as a colorless oil.

[a] _D ²⁵ = + 4.15 (c 0.66, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 6.46 (1H, br), 5.65 (1H, br), 4.46-4.37 (2H, m), 4.15 (1H, m), 3.80 (3H, d), 3.77 (3H, d), 3.26 (2H, m ), 1.50-1.25 (33H, m), 0.88 (3H, t).

(3) (2 S ) -2-amino-3-hydroxy-N-tetradecyl-propionamide-3-phosphate

(1 S )-[2-hydroxy-1- (tetradecylcarbamoyl) -ethyl] -carbamic acid tert -butyl ester-2- (O, O'-dimethylphosphate) (135 mg, 0.27 mmol) It was dissolved in anhydrous acetonitrile (4 ml) under air and cooled to 0 ° C. Bromotrimethylsilane (3.4 ml, 25 mmol) was slowly added dropwise and stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure, methanol (2 ml) and water (1 ml) were added thereto, and then concentrated under reduced pressure. The obtained residue was recrystallized from acetic acid / methanol / water mixed solvent to give 63 mg (yield: 62%) of the title compound as a white solid.

[a] _D ²⁵ = -2.27 (c 0.34, AcOH).

¹ H-NMR (300 MHz, CD ₃ CO ₂ D); δ 4.57 (2H, m), 4.26 (1H, m), 3.31 (2H, m), 1.55-1.29 (24H, m), 0.89 (3H, t).

Example 13: (2 R ) -2-Amino-3-hydroxy-N-tetradecyl-propionamide-3-phosphate (method (VI))

(1) (1 R )-[2-hydroxy-1- (tetradecylcarbamoyl) -ethyl] -carbamic acid tert -Butyl ester

The title compound was obtained in the same manner as in Example 12 (1), using D-serine as a starting material.

[a] _D ²⁵ = + 25.1 (c 0.91, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 6.65 (1H, br), 5.56 (1H, br), 4.11 (2H, m), 3.63 (1H, m), 3.30-3.14 (3H, m), 1.49-1.23 (33H, m), 0.88 (3H , t).

(2) (1 R )-[2-hydroxy-1- (tetradecylcarbamoyl) -ethyl] -carbamic acid tert -Butyl ester-2- (O, O'-dimethylphosphate)

(1 R )-[2-hydroxy-1- (tetradecylcarbamoyl) -ethyl] -carbamic acid tert -butyl ester was used as starting material to obtain the title compound in the same manner as in Example (2). .

[a] _D ²⁵ = -1.90 (c 0.76, CHC1 ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ); δ 6.46 (1H, br), 5.65 (1H, br), 4.46-4.37 (2H, m), 4.15 (1H, m), 3.80 (3H, d), 3.77 (3H, d), 3.26 (2H, m ), 1.50-1.25 (33H, m), 0.88 (3H, t).

(3) (2 R ) -2-amino-3-hydroxy-N-tetradecyl-propionamide-3-phosphate

(1 R) - [2- hydroxy-1- (tetradecyl-carbamoyl) -ethyl] - butyl ester -2- (O, O'- dimethyl phosphate) Example 12 as a starting material to -carbamic acid tert The title compound was obtained in the same manner as in (3).

[a] _D ²⁵ = + 5.34 (c 0.36, AcOH).

¹ H-NMR (300 MHz, CD ₃ CO ₂ D); δ 4.57 (2H, m), 4.26 (1H, m), 3.31 (2H, m), 1.55-1.29 (24H, m), 0.89 (3H, t).

Test Example: Affinity Test for EDG Receptor

Tests prepared in Examples 1 to 13 above with known affinity for EDG-1, 3 and 5 receptors of known compound D-erythro-sphingosine-1-phosphate (S1P) present in vivo at 100 The relative affinity of each compound for EDG-1, 3, and 5 receptors was measured in the following manner.

Chinese hamster ovary (CHO) cells were transfected with the EDG-1 expression vector gene. CHO cells expressing EDG-1 were cultured in 6-well plates of HAM's F-12 culture containing 10% FBS (fetal bovine serum). Twenty four hours prior to drug administration, the cultures were changed to fresh HAM's F-12 without FBS. Each well of the 6-well plate incubated for one day was washed twice with a binding buffer, and then a buffer solution containing 1 nM [ ³ H] S1P and 100 nM of test compound was added. After incubation on ice for 30 minutes, it was washed twice with cold binding buffer again. Then, 500 μl of solubilizing solution (0.1% SDS, 0.4% NaOH, 2% Na ₂ CO ₃ ) was added to each well to dissolve and mixed with 5 ml of cocktail solution, followed by radioactivity by light scintillation counter. (radioactivity) was measured. Experiments were performed in the same manner for CHO cells transfected with EDG-3 and -5. The relative affinity for S1P was determined from the measured results and is shown in Table 1 below.

compound EDG-1 receptor EDG-3 Receptor EDG-5 Receptor In vivo S1P 100 100 100 Example 1 25.8 44.6 22.4 Example 2 <5 17.0 <5 Example 3 9.9 20.3 40.1 Example 4 <5 <5 <5 Example 5 11.4 50.1 29.2 Example 6 <5 <5 <5 Example 7 <5 <5 <5 Example 8 9.6 <5 <5 Example 9 <5 <5 <5 Example 10 8.3 25.3 <5 Example 11 9.3 <5 <5 Example 12 <5 <5 <5 Example 13 <5 7.5 <5

From Table 1, it can be seen that the sphingosine-1-phosphate derivatives prepared simply and selectively according to the present invention and their pharmaceutically acceptable salts exhibit selective and useful degrees of affinity for EDG receptors.

As mentioned above, the sphingosine-1-phosphate derivatives of formula 1 and pharmaceutically acceptable salts thereof of the present invention are structural features and activities as effective and selective agonists and antagonists for EDG (endothelial differentiation gene) receptors. It can be usefully used as a therapeutic agent for various refractory diseases such as wound healing, cancer proliferation and metastasis, angiogenesis related diseases such as rheumatoid arthritis, inflammation related diseases and circulatory diseases.

Claims (14)

A sphingosine-1-phosphate derivative of Formula 1 or a pharmaceutically acceptable salt thereof: Formula 1 Where Represents a single bond or a double bond; R ₁ and R ₂ are each independently hydrogen, C _1-6 alkyl, sugars or inositols; R ₃ and R ₄ are each independently hydrogen, C _1-6 alkyl, Or together with the nitrogen atom to which it is attached form a 3-8 membered heterocyclic ring, wherein m is 0, 1, 2 or 3, p is 0, 1 or 2 and R ₇ is hydroxy, Amino, C _1-6 alkyl, halogen, C _1-6 alkyloxy or C _1-6 alkylamino); R ₅ is Is hydrogen or OH when is a single bond, Is O when is a double bond; L is O, S, CH ₂ or NH; R ₆ is -NR ₁₀ R ₁₁ or -OR ₁₂ { Represents a single bond or a double bond, R ₈ is hydrogen, C _1-20 alkyl, 1 to 9 halogen atoms substituted C _1-20 alkyl, 3 to 8-membered cyclic alkyl, one to nine halogen atoms 3 to 8-membered cyclic alkyl, or R ₉ is hydrogen, amino, hydroxy, halogen, C _1-20 alkylamino, 3 to 8 membered cyclic alkylamino, C _1-20 alkyloxy, 3 to 8 membered cyclic alkyloxy, 1 to C _1-20 alkyl substituted with 9 halogen atoms or 3-8 membered cyclic alkyl substituted with 1 to 9 halogen atoms, R ₁₀ , R ₁₁ and R ₁₂ are each independently hydrogen, C _1-20 alkyl, 3 to 8-membered cyclic alkyl or Wherein R ₁₃ is as defined above for R ₉ , X is N or CH, n is 0, 1, 2, 3, 4 or 5 and q is 0, 1 or 2). The method of claim 1, R ₁ and R ₂ are each independently hydrogen or C _1-4 alkyl; R ₃ and R ₄ are each independently hydrogen, C _1-4 alkyl, Or together with the nitrogen atom to which it is attached form a 3-6 membered heterocyclic ring, wherein m is 0, 1, 2 or 3, p is 0, 1 or 2 and R ₇ is hydroxy, Amino, C _1-3 alkyl, halogen, C _1-3 alkyloxy or C _1-3 alkylamino); L is O, S or CH ₂ , characterized in that Derivatives or pharmaceutically acceptable salts thereof. 1) reacting a compound of formula 2 with lithium dimethylmethylphosphonate to produce a compound of formula 3, 2) reacting the resulting compound of Formula 3 with a compound of Formula 4 to produce a compound of Formula 5, 3) reducing the compound of Formula 5 to produce a compound of Formula 6, 4) deprotecting the compound of formula 6 to produce a compound of formula 7, 5) reacting the resulting compound of Formula 7 with a compound of Formula 8 and then reducing to form a compound of Formula 9, 6) reacting the resulting compound of formula 9 with a compound of formula 10 followed by hydrolysis, A process for preparing the derivative of claim 1 or a pharmaceutically acceptable salt thereof: R ₈ CHO R'CHO P (OR ₁ ) ₃ Wherein R ₁ , R ₃ , R ₄ , R ₈ and L are as defined in claim 1, P ₁ and P ₂ are each independently hydrogen or a hydroxy- or amine-protecting group, R 'is hydrogen, C _1-6 alkyl or Where m, p and R ₇ are as defined in claim 1. The method of claim 3, wherein Instead of steps 5) and 6), protecting the amine group of the compound of formula 7 produced in step 5) 5)) to produce a compound of formula 11, 6 ') reacting the resulting compound of formula 11 with a compound of formula 10 to produce a compound of formula 12 and then deprotecting it; A process for preparing the derivative of claim 1 or a pharmaceutically acceptable salt thereof: Formula 7 Formula 10 P (OR ₁ ) ₃ Wherein R ₁ , R ₃ , R ₈ , L and P ₂ are each as defined in claim 3. The method of claim 4, wherein Hydrogenating the compound of formula 12 prior to deprotection to produce a compound of formula 13, thereby deprotecting it A process for preparing the derivative of claim 1 or a pharmaceutically acceptable salt thereof: Formula 12 Wherein R ₁ , R ₃ , R ₈ , L and P ₂ are each as defined in claim 3. 1) deprotecting the compound of formula 5 to produce a compound of formula 14, 2) reducing the compound of Formula 14 to produce a compound of Formula 7, 3) reacting the resulting compound of formula 7 with a compound of formula 8 and reducing, followed by reacting with a compound of formula 10 and hydrolyzing, A process for preparing the derivative of claim 1 or a pharmaceutically acceptable salt thereof: Formula 5 Formula 7 Formula 8 R'CHO Formula 10 P (OR ₁ ) ₃ Wherein R ₁ , R ₃ , R ₄ , R ₈ and L are as defined in claim 1, P ₁ and P ₂ are each independently hydrogen or a hydroxy- or amine-protecting group, R 'is hydrogen, C _1-6 alkyl or Where m, p and R ₇ are as defined in claim 1. The method of claim 6, Instead of step 3), 3 ') protecting the amine group of the compound of formula 7 produced in step 2) to give a compound of formula 11, 4 ') reacting the resulting compound of Formula 11 with a compound of Formula 10 to produce a compound of Formula 12, 5 ') comprising deprotecting the resulting compound of formula 12, A process for preparing the derivative of claim 1 or a pharmaceutically acceptable salt thereof: Formula 7 Formula 10 P (OR ₁ ) ₃ Formula 11 Formula 12 Wherein R ₁ , R ₃ , R ₈ , L and P ₂ are each as defined in claim 6. The method of claim 7, wherein Hydrogenation of the compound of formula 12 produced in step 5 ′) followed by deprotection, A process for preparing the derivative of claim 1 or a pharmaceutically acceptable salt thereof: Formula 12 Wherein R ₁ , R ₃ , R ₈ , L and P ₂ are each as defined in claim 6. 1) deprotecting the compound of formula 5 to produce a compound of formula 14, 2) protecting the amine group of the resulting compound of Formula 14 to produce a compound of Formula 15, 3) reacting the resulting compound of Formula 15 with a compound of Formula 10 and then deprotecting, A process for preparing the derivative of claim 1 or a pharmaceutically acceptable salt thereof: Formula 5 Formula 10 P (OR ₁ ) ₃ Formula 14 Wherein R ₁ , R ₃ , R ₈ , L and P ₂ are each as defined in claim 6. 1) reacting a compound of Formula 27 with a compound of Formula 28 to produce a compound of Formula 29, 2) deprotecting the resultant compound of formula 29 to produce a compound of formula 30, 3) reacting the resulting compound of Formula 30 with a compound of Formula 25 and then reacting with a compound of Formula 8 to produce a compound of Formula 31, 4) reacting the resulting compound of formula 31 with a compound of formula 10 and then deprotecting, A process for preparing the derivative of claim 1 or a pharmaceutically acceptable salt thereof: Formula 8 R'CHO Formula 10 P (OR ₁ ) ₃ RCHO Wherein R ₁ , R ₃ , R ₄ , R ₉ , L and X are as defined in claim 1, P ₁ and P ₂ are each independently hydrogen or a hydroxy- or amine-protecting group, R and R 'are each independently hydrogen, C _1-6 alkyl or Where m, p and R ₇ are as defined in claim 1. 1) reacting a compound of Formula 27 with a compound of Formula 32 and then reducing to form a compound of Formula 33, 2) deprotecting the resulting compound of Formula 33 to produce a compound of Formula 34, 3) reacting the resulting compound of Formula 34 with a compound of Formula 25 and then reacting with a compound of Formula 8 to produce a compound of Formula 35, 4) reacting the resulting compound of formula 35 with a compound of formula 10 and then deprotecting, A process for preparing the derivative of claim 1 or a pharmaceutically acceptable salt thereof: Formula 8 R'CHO Formula 10 P (OR ₁ ) ₃ Formula 25 RCHO Formula 27 Ph ₃ P ⁺ R ₆ Br ^- Wherein R ₁ , R ₃ , R ₄ , R ₆ , R ₇ and L are as defined in claim 1, P ₁ and P ₂ are each independently hydrogen or a hydroxy- or amine-protecting group, R and R 'are each independently hydrogen, C _1-6 alkyl or Where m, p and R ₇ are as defined in claim 1. The method of claim 11, Instead of steps 3) and 4), protecting the amine group of the compound of formula 34 produced in 3 ') step 2) to give a compound of formula 36, 4 ') reacting the resulting compound of formula 36 with a compound of formula 10 and then deprotecting, A process for preparing the derivative of claim 1 or a pharmaceutically acceptable salt thereof: Formula 10 P (OR ₁ ) ₃ Formula 34 Wherein R ₁ , R ₆ , L and P ₂ are each as defined in claim 11. 1) reacting a compound of Formula 37 with a compound of Formula 38 to produce a compound of Formula 39, 2) deprotecting the compound of formula 39 to produce a compound of formula 40, 3) reacting the resulting compound of Formula 40 with a compound of Formula 25 and then reacting with a compound of Formula 8 to produce a compound of Formula 41, 4) reacting and deprotecting the resulting compound of formula 41 with the compound of formula 10, A process for preparing the derivative of claim 1 or a pharmaceutically acceptable salt thereof: Formula 8 R'CHO Formula 10 P (OR ₁ ) ₃ Formula 25 RCHO HNR ₁₀ R ₁₁ Wherein R ₁ , R ₃ , R ₄ , R ₁₀ , R ₁₁ and L are as defined in claim 1, P ₁ and P ₂ are each independently hydrogen or a hydroxy- or amine-protecting group, R and R 'are each independently hydrogen, C _1-6 alkyl or Where m, p and R ₇ are as defined in claim 1. Efficacy and antagonistic pharmaceutical composition for EDG (endothelial cell differentiation gene) receptor, comprising as an active ingredient the derivative of formula 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.