JP2003137894A - Phosphorylated amino alcohol, method of producing the same and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a medicine that antagonizes Edg (endothelial differentiation gene) receptor. SOLUTION: This medicine is a threo type (2S, 3S) phosphorylated amino alcohol represented by general formula I [wherein R is a straight-chain or branched CH3 Cn H(2n-2m) - (n is an integer of 2-19; m is an integer of 0-3) which may be substituted or an aryl which may be substituted] or pharmacologically acceptable salts thereof.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel amino alcohol phosphate compound, a method for producing the same, and a method for utilizing the same. This compound is an endothelial differentiation gene end
By antagonizing othelial differentiation gene (Edg) receptor, anti-circulatory system disease (eg, anti-arteriosclerosis, anti-cardiac disease (eg, anti-arrhythmic, anti-myocardial infarction)), anti-rheumatic, anti- It is useful as a drug exhibiting cancerous, antidiabetic retinopathy and antirespiratory system diseases.

[0002]

2. Description of the Related Art Recent researches in the fields of medicine and biochemistry have revealed the importance of sphingolipids in living organisms. Glycosphingolipids are known to be mutual recognition between cells, regulation of cell proliferation, regulation of development and differentiation. It has been suggested that it plays an important role in infection, malignant transformation of cells and the like.

On the other hand, sphingosine-1-phosphate (SPN-1P), which is a degradation metabolite of sphingolipids

[Chemical 10] , Its role was unknown, but in recent years, the orphan receptor endothelial dile with SPN-1P as an endogenous ligand has been investigated.
The fferentiation gene (Edg) was found, and the physiological effects have gradually become clear.

Edg was cloned in 1990 as an orphan receptor [Edg-1 (JBC, `90, 265,
p. 9308)], but then as Edg-1 homologue, Ed
g -3 (BB RC, `96, 227, p. 608), Edg-5 (AGR 16
/ H 218) (JCB, `96, 135, p. 1071) was obtained, but its physiological role was unknown. However, in '98, S
It is suggested that PN-1P may be an endogenous ligand of Edg-1 (Science, `98, 279, p. 1552), and then E
dg-3 and Edg-5 were also shown to be SPN-1 P-specific receptors (BBRC `99, 260, p. 263, JBC` 99, 27
4, p. 18997).

Edg-1 on vascular endothelial cells upregulates adhesion proteins such as cadherin by stimulation of SPN-1P (Science, `98, 279, p. 1552), and T lymphocyte-derived cell line SPN-1P stimulation in vit
Enhance vascular layer invasion in a pseudo-vascular model (EMBO J.,
`98, vol. 17, No. 14, p. 4066). Also, Okajima et al. Edg
--1 or Edg-3 forcibly expressed CHO cells, a pseudovascular migration test was carried out. In both cases, SPN-1P concentration-dependent migration was promoted (`99th Annual Meeting of the Japanese Biochemical Society Abstracts p. 883). On the other hand, Igarashi et al. Reported that the cancer cell line F10 was SPN-1 P 10 <sup>-8</sup> ~ 10 in a pseudo blood vessel model.
<sup>It</sup> was shown that migration was suppressed by up to 80% in a concentration-dependent manner at <sup>-6</sup> M, but Edg-5 was expressed in F10 cells with almost no expression of Edg-1 or Edg-3. (`99 Japanese Biochemical Society). SP due to different expression variants
It has been pointed out that N-1P may have suppressed migration (`9
9th Annual Meeting of the Chemical Society of Japan, p. 675, 883).

Vascular smooth muscle cells (Eur. J. Biochem. `98,
257, p. 403) or airway smooth muscle cells (Biochem. J. `9
9, 338, p. 643), and both have MAP for SPN-1 P responsiveness.
Kinase activation has been observed and it has been pointed out that SPN-1P may act in the direction of vascular smooth muscle proliferation.

Sugiyama et al. Observed that SPN-1P was administered to rats via the tail vein route and observed hemodynamics. As a result, a significant decrease in systolic blood pressure and two indices of left ventricular pressure differential was observed.
-1P has been shown to have a possibility of acting in the direction of decreased cardiac function in vivo (`00 Annual Meeting of the Pharmacological Society of Japan P. 12
7). Muscarinic receptor-directed K ⁺ rectification in guinea pig, mouse, and human coronary artery smooth muscle cell cultures
It has been pointed out that SPN-1P is activated via Edg-3 and that SPN-1P may stimulate the vagus nerve that causes dyspnea (Mollecular Pharmacology, '00, 58, 44.
9). In addition, in the culture of rabbit sinoatrial cells, SPN-1P extends the cycle of spontaneous pacemakers,
-It has been suggested that 1 P may have a negative effect on the beating cycle of the heart (Pfuger Arch-Eur J Physiol
'99, 438, 642).

The action of SPN-1P on vascular endothelial cells
As a result of examination using an angiogenesis animal model, VEGF and FGF
-SPN-1P induces angiogenesis by growth factors such as 2
-1, synergistically promoted by combining with Edg-3,
It has been pointed out that Edg may be involved in the progression of rheumatism, solid tumors and diabetic retinopathy (Cell `99, p. 30.
1).

As a result of excessive inflammation and airway remodeling caused by the binding of SPN-1P and Edg receptor, pneumonia and chronic obstructive airway disease (COPD)
(airway disease), respiratory hypertension may progress (Pulmonary Pharmacology & Therapeut
ics, 2000, 13, p. 99).

[0010] Suramin, a eradication drug for the protozoan trypanosomes,
It has been reported to exhibit Edg-3 specific antagonism and block the signal of SPN-1P-Edg binding (JBC `9
9, 274, 27, p. 18997). Suramin has been shown to be curatively effective in a model of arteriosclerosis (Circulation,
`99, 100, p. 861, Cardiovascular Res.,` 94, 28,
p. 1166), but it is possible that Edg antagonisticity is involved in the mechanism of this drug effect.

From the above findings, SPN-1P is Edg
When combined with, it acts to promote arteriosclerosis such as inflammatory cell activation, vascular smooth muscle cell proliferation, and hemodynamic deterioration. In addition, it has been shown that it may promote angiogenesis and act in the direction of progression of rheumatism, solid cancer, and diabetic retinopathy. That is, the substance that antagonizes Edg is anti-atherogenic,
Anti-cardiovascular disease, anti-cardiovascular disease, anti-rheumatic,
It may have anti-cancer properties, anti-diabetic retinopathy properties, and anti-respiratory disease properties. Therefore, it is extremely important to synthesize various sphingolipids and find a substance having an Edg antagonistic activity in order to create a therapeutic drug for these diseases. However, since many steps are required for the synthesis of sphingolipids, almost no structure-activity relationship studies have been conducted so far.

Up to now, the chemical synthesis method of D-erythro-type (2S, 3R-form) sphingosine corresponding to the stereoisomer of the 3-position hydroxyl group, which is the most abundant natural type basic skeleton, has been studied and many reports have been made. Has been done. However, it is not easy to selectively synthesize an L-threo type (2S, 3S form) having a configuration different from that of the natural type, and its phosphorylated form has not been synthesized, as described below. .

As a synthesis example up to now, a method utilizing an asymmetric aldol type reaction (Ito et al., Tetrahedron Lett., 1
988, Vol. 29, p. 239; EJ Corey et al., Tetrahedron L.
et al., 2000, Vol. 41, p. 2765), but there were problems that the reagents used in the intermediate steps were special or required complicated operations. A method of inverting the natural 3-hydroxy group at the erythro type is also known, but there is a problem that a positional isomer is likely to occur due to a double bond at the adjacent 4-position.

A method for producing threo-type sphingosine using the amino acid L-serine as a starting material is also known (P.
Herold, Helv. Chim. Acta, 1988, Vol. 71, p. 354;
I. Van Overmeire et al., J. Med. Chem., 1999, Vol. 42,
(p. 2697), this method is not convenient because threo-type propargyl alcohols are obtained and then the triple bond is reduced to form an alkene (double bond).

A method for directly acting an alkenylating agent (alkenylating metal reagent) on an aldehyde compound to produce two stereoisomers of the protected sphingosine at the 3-hydroxyl group, erythro and threo, is known. Has been. However, since the production ratio of these isomers easily changes depending on the metal species, solvent, catalyst to be added, reaction temperature, etc., it is difficult to set the conditions for selectively producing the isomers, and the isomers obtained by chromatography, etc. Is difficult to separate. Further, a method for selectively obtaining erythro is known (Japanese Patent Laid-Open No. 7-291904, J. Chem. S.
oc., Perkin Trans.1, 1994, p. 1257), and a method of obtaining threo selectively is not known. Therefore, a simple method for obtaining a threo body by a reaction with high stereoselectivity is desired.

[0016]

DISCLOSURE OF THE INVENTION The object of the present invention is to identify Edg.
It is intended to provide a novel amino alcohol phosphate compound having a receptor antagonistic activity, a simple method for producing the same, and a method for using the same.

[0017]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that threo-type aminoalcohol compounds have no antagonism of Edg receptors, but their phosphoric acid compounds The following general formula I compound (hereinafter referred to as “the compound of the present invention”) has an Edg receptor antagonistic action, and it was found that the compound can be easily produced by a synthetic route excellent in stereoselectivity. The invention was completed.

The present invention includes the following general formula I

[Chemical 11] (In the formula, R is an optionally substituted linear or branched CH ₃ C _n H _(2n-2m) -(n is any integer from 2 to 19, and m is , An unsaturated number, which is an integer between 0 and 3) or an aryl group which may be substituted), a threo type (2S, 3S) aminoalcohol-1-phosphate. It relates to a compound or a pharmaceutically acceptable salt thereof.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The substituents in the general formula I will be described. “CH ₃ C _n H _(2n-2m) which may be substituted
- substituted CH ₃ C _n H a _'(2n-2m) - and is, for example,
CH ₃ C _n H _(2n-2m) substituted with one or more selected from the group consisting of a hydroxyl group, a halogen atom, a linear or branched alkyl group having 1 to 10 carbon atoms or a branched chain, and an aryl group. - Specific examples of the "aryl group" include a phenyl group, a 1-naphthyl group, a 2-naphthyl group and the like. The substituted aryl group of the “aryl group which may be substituted” is, for example, selected from the group consisting of a linear or branched alkyl group or alkoxy group having 1 to 10 carbon atoms, a nitro group and a halogen atom. Aryl group substituted with one or more of

The following are preferred examples of R. n is preferably 5-16, more preferably 9-12. m is preferably 0-1 and more preferably 0. The position of the unsaturated bond is preferably the 8-position when n is 12.

Particularly preferred compounds of formula I compounds of the present invention are listed below.

[Chemical 12]

[Chemical 13]

[Chemical 14]

The salt of the compound of the present invention is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include lower alkyls such as methanesulfonate, trifluoromethanesulfonate and ethanesulfonate. Aryl sulfonates such as sulfonates, benzene sulfonates, p-toluene sulfonates, acetates, fumarates, succinates, citrates, tartrates, oxalates, and maleates. Examples thereof include acid salts, glycine salts, alanine salts, glutamate salts, amino acid salts such as aspartate salts, and alkali metal salts such as sodium salts and potassium salts.

All of the compounds of the present invention exhibit endothelial differentiation gene (Edg) receptor antagonism, and SPN-1P for Edg.
It can antagonize the binding of Edg receptor agonists such as and block the intracellular signal transduction system by these. It was also found that when the compounds of the present invention are used as essential components of medicines, the above properties are effectively exhibited even when they are mixed with other usual ingredients of medicines. Therefore, the present invention provides a pharmaceutical which contains the compound of the present invention as an active ingredient and antagonizes the endothelial differentiation gene (Edg) receptor.
In addition, the present invention, inflammatory cell activation and vascular smooth muscle proliferation,
Diseases caused by hemodynamic deterioration and further angiogenesis, for example, cardiovascular disease (for example, arteriosclerosis, heart disease (for example, myocardial infarction, arrhythmia)), rheumatism (for example, rheumatoid arthritis), cancer, diabetic retina The present invention provides the above-mentioned medicine for preventing or treating illness and respiratory disease (eg, pneumonia, chronic obstructive airway disease, respiratory hypertension). Here, the "circulatory system disease" refers to a disease in which circulatory states such as blood and lymph are impaired and causes damage to tissues and cells, and examples thereof include arteriosclerotic diseases (for example,
Atherosclerosis), heart disease (eg myocardial infarction, arrhythmia). Here, "respiratory system disease" refers to a disease in which respiratory organs such as the trachea, bronchus, and lungs are impaired and symptoms related thereto, and examples thereof include asthma (immediate type, delayed type, allergic disease). Asthma, etc.), bronchial asthma, allergic rhinitis, eosinophil infiltration, bronchitis (chronic bronchitis, etc.), airway inflammation, emphysema, pneumonia, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome, respiratory hypertension , Dyspnea, pain, cough, sputum, vomiting, shortness of breath.

In order to use the compound of the present invention as a medicine, it may be in any form of solid composition, liquid composition and other compositions, and the optimum one is selected according to need. The pharmaceutical composition is prepared by adding a pharmaceutically acceptable carrier to the compound of the present invention, that is, a conventional excipient, a bulking agent, a binder, a disintegrating agent, a pH adjusting agent, a solubilizing agent, etc. It can be prepared into tablets, pills, capsules, granules, powders, solutions, emulsions, suspensions, injections and the like depending on the technique. Examples of excipients and extenders include lactose, magnesium stearate, starch, talc, gelatin, agar, pectin, gum arabic, olive oil,
Examples include sesame oil, cocoa butter, ethylene glycol, and other commonly used substances. In order to prevent the oxidation of the preparation, it is possible to add an antioxidant (tocopherol or the like), to include with an inclusion agent such as cyclodextrin, or to encapsulate with a film such as gelatin.
Further, the compound can be prepared as an O / W type preparation by using a phospholipid or a nonionic surfactant as an emulsifier as described in JP-A-6-298642. The emulsifiers may be used alone or in combination of two or more, and the addition amount may be appropriate, but 0.001 to 10%
(W / V), preferably 0.01 to 5% (W / V). As phospholipids, soybean-derived phospholipids, egg yolk-derived phospholipids, lysolecithin, phosphatidylcholine (lecithin), phosphatidylserine and the like can be used alone or in combination. As the non-surfactant, a polyoxyethylene-polyoxypropylene block copolymer having a molecular weight of 500 to 15,000 (for example, Pluronic F-68), a polyalkylene glycol having a molecular weight of 1,000 to 10,000, and a polyoxyalkylene copolymer having a molecular weight of 1,000 to 20,000 are used. Polymer, hydrogenated castor oil polyoxyalkylene derivative, castor oil polyoxyalkylene derivative, glycerin fatty acid ester, polyglycerin fatty acid ester, sorbitan fatty acid ester, polyoxyethylene castor oil, hydrogenated castor oil, polyoxyethylene alkyl ether, sucrose fatty acid The esters and the like are preferably used alone or in combination, but are not limited thereto. The compounding amount of each compound in the present invention may be about 0.0001 to about 100 mg / kg body weight / day based on the drug, and may be administered orally or parenterally once or several times a day. This dose can be appropriately increased or decreased depending on the type of disease, the age, weight and symptoms of the patient.

The compound of the present invention can be produced by the following production method. The method for producing the compound of the formula I will be described below, including an example of preparation of reaction raw materials. (1) Preparation Example of Reaction Raw Material (A) Synthesis of Alkenylating Agent General Formula V

[Chemical 15] (In the formula, R is an optionally substituted linear or branched CH ₃ C _n H _(2n-2m) -(n is any integer from 2 to 19, and m is Represents an unsaturated number and is an integer between 0 and 3) or an optionally substituted aryl group, and Cp represents a cyclopentadienyl group)
A dialkylzinc is allowed to act on the organozirconium compound represented by the formula (1) as follows to prepare an alkenylating agent.

[Chemical 16] The above reaction can be carried out with respect to the organozirconium compound represented by the general formula V, preferably using an equimolar amount of dialkylzinc. As the dialkyl zinc, lower dialkyl zinc (eg, dimethyl zinc or diethyl zinc) is preferable. The organozirconium compound represented by the general formula V is prepared by a known method (J. Schwartz et al., Org. Synth.,
1992, Vol. 71, p. 83), by reacting hydrogenated zirconocene chloride with 1-alkyne or aryl acetylenes in an organic solvent. As the hydrogenated zirconocene chloride used for forming the organozirconium compound represented by the general formula V, a commercially available product can be used, and a known method (SL Buchwald et al., Org. Synth.,
It can also be prepared from inexpensive zirconocene dichloride according to 1992, Vol. 71, p. 77). The 1-alkyne used for producing the organozirconium compound represented by the general formula V may be a linear or branched one having 5 to 22 carbon atoms, and has a double bond in the molecule. Good.
Specifically, it is 1-octyne, 1-dodecine, 1-pentadecine, etc. In particular, in the case of 1-pentadecine having 15 carbon atoms, C18 sphingosine which is the most abundant in natural products can be obtained. It is also possible to use aryl acetylenes,
It may have a substituent on the aryl. Specifically, phenylacetylene, 4-chlorophenylacetylene, 1-
Naphthyl acetylene and the like.

(B) N-protected (S) -formyloxy
Synthesis of Sazolidine Derivatives N-protected (S) -formyloxazolidine derivatives of formula III below can be synthesized by conventional methods, eg,
(S) -Serine to Garner et al. (J. Org. Chem., 19
87, Vol. 52, 2361, Org. Synth., 1991, Vol. 70, p.
18), or a commercially available product can be used.

[Chemical 17] (In the formula, A is a protecting group for N, B and C are alkyl groups (eg, methyl group, ethyl group, etc.), or B and C
May form a cyclic alkyl group (for example, a cyclopentyl group, a cyclohexyl group, etc.) together with the carbon atom to which they are attached. Here, the protecting group A for N is, for example, benzyloxycarbonyl (Z ), T-butoxycarbonyl (Boc), t-amyloxycarbonyl (Aoc), isobornyloxycarbonyl, p-methoxybenzyloxycarbonyl, 2-chloro-benzyloxycarbonyl, adamantyloxycarbonyl,
Groups such as. Preferably Boc is used.

(2) Synthesis of Compound of Formula I A synthetic scheme of the compound of Formula I is shown below.

[Chemical 18]

First step: The alkenylating agent obtained in (1) (A) above and N-of formula III obtained in (1) (B) above.
Reaction with a protected (S) -formyloxazolidine derivative selectively gives the threo of formula II.

[Chemical 19] As the reaction solvent, a halogenated hydrocarbon such as methylene chloride is suitable for selectively obtaining a threo compound. The reaction temperature is preferably -30 to 30 ° C, particularly preferably -20 to 0 ° C. A reaction time of about several hours is sufficient. As a result, the threo body II having a different configuration of the hydroxyl group from the natural type is selectively obtained (threo / erythro = 9/1 or more).

Next, with respect to the compound of the formula II, the removal of the acetal type protecting group corresponding to the following steps 2-4,
The present invention has established a simple production method for obtaining a threo body by performing a three-step sequence of selective phosphorylation of primary hydroxyl groups and removal of all protecting groups. The details will be described below. In addition, Japanese Patent Publication No. 8-500816, Bioorg. Me
Although threo type I can be produced from compound II by using a method similar to the method for producing the erythro body described in d. Chem. Lett., 1992, Vol. 2, p. There are 5 steps because protection of the primary hydroxyl group is required, and purification by chromatography is required between each step.
Since the operation is extremely complicated, the following method is simpler.

Step 2: The oxazoline of compound II is opened to remove the acetal-type protecting group, and then the compound of general formula VI

[Chemical 20] An amino group-protected alcohol compound represented by the formula (R and A in the formula are the same as above) is obtained. The reaction is hydrochloric acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid in a solvent,
Using an acid such as a strongly acidic ion exchange resin, -20 to 100
It can be carried out at ℃ for several tens of minutes to several tens of hours.
However, it is important to choose reaction conditions that do not remove the amino protecting group. About 50 in about 90% acetic acid aqueous solution
It is particularly preferable to carry out heating at 0 ° C., and the reaction is completed in a few hours.

Third step: The amino group-protected alcohol compound (Compound VI) obtained in the second step is converted to the following 1-phosphate analog (Compound VII).

[Chemical 21] (In the formula, R and A are the same as above, and R ′ is a lower alkyl group (for example, a methyl group or an ethyl group).) The above reaction selectively reacts the primary hydroxyl group of the two hydroxyl groups of compound VI. It can be carried out using a known phosphorylating agent effective for that purpose. The most convenient and preferred method is the method described by A. Bielawska et al. (Tetrahedron Lett., 2000, Vol. 41, p. 7), which is described in detail below.
821). The phosphoramidite method has also been proposed as a selective phosphorylation method (A. Boumendjel et al., J.
Lipid. Res., 1994, Vol. 35, p. 2305), the following method is preferable because it requires preparation of a phosphorus reagent and an oxidation step. According to the method of A. Bielawska et al., Compound VI is treated with a trialkyl phosphite and carbon tetrabromide in an organic base (eg, pyridine). Trimethyl phosphite or triethyl phosphite is suitable as the phosphorylating agent, and 1 to 3 equivalents relative to compound VI are sufficient. Also, the amount of carbon tetrabromide used is preferably 1 to 3 equivalents to VI. The reaction is -20 ° C
It can be carried out at -50 ° C, preferably 0 ° C-room temperature for several tens of minutes to tens of hours.

Fourth Step: The protecting groups for the amino group and the phosphate group of compound VII are removed. The reaction of the fourth step can be carried out by reacting an acidic substance in a solvent. As the acidic substance, trimethyl silane bromide, trimethyl silane iodide, or a combination of trimethyl silane chloride and sodium iodide is suitable, and the amount used is 3 to VII.
10 equivalents, especially 5-6 equivalents are preferred. A halogenated hydrocarbon such as methylene chloride is suitable as the solvent. The reaction temperature is preferably room temperature to 40 ° C. In addition, the protecting group on the phosphate group may be different from other groups (for example,
When converted to a trimethylsilyl (TMS) group, the converted group can be removed by distilling off the reaction solvent, acidifying the reaction solution, and hydrolyzing it. Finally, after concentration, the volatile acidic substance is distilled off azeotropically and recrystallized (for example, from a THF-water mixed solvent) to obtain a pure compound I. If crystals are difficult to precipitate, purification by silica gel chromatography is performed, and if necessary, recrystallization is further performed.

[0033]

EXAMPLES The present invention will be described in more detail by way of examples, which should not be construed as limiting the technical scope of the present invention. Synthesis of (Example 1) threo -C15- sphingosine-1-phosphate (in a compound _{I, R = nC 10 H 21} ) 1. Threo -C15-N-Boc-N, 1O- isopropylidene - sphingosine (Compound II in _{R = nC 10 H 21, A} =
Synthesis of Boc, B = CH ₃ , C = CH ₃ ) Under an argon atmosphere, hydrogenated zirconocene chloride 490 mg (1.8
mmol) in 2 ml of methylene chloride, and while cooling with ice,
1- dodecyne _{(R = nC 10 H 21)} 310 mg of methylene solution 2 ml chloride (1.8 mmol) was added, to prepare a compound V was stirred at room temperature for 1 hour. After cooling the reaction solution to -20 ° C,
1.8 ml (1.8 mmol) of a 1.0 M hexane solution of diethyl zinc was added, and the mixture was stirred for about 10 minutes. Aldehyde compound II
I (A = Boc, B = CH ₃ , C = CH ₃ ) 118 mg (1.0
(2 mmol) of methylene chloride solution was added, and the mixture was stirred at -20 to 0 ° C for 3 hours. Add saturated ammonium chloride solution,
Ethyl acetate was added and stirred for a while. The generated precipitate was filtered and washed with ethyl acetate. The layers were separated, the organic layer was washed with saturated brine, and the aqueous layers were combined and extracted twice with ethyl acetate. The organic layers are combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to give a pale yellow oil.
600 mg remained. This was purified by silica gel chromatography (hexane / ethyl acetate = 9: 1 → 6: 1 → 4: 1) to give the title compound as a colorless oily substance.
0 mg (88% yield) was obtained. [α] _D −41.4 ° (c 1.26, CHCl ₃ ) ¹ H-NMR (C ₆ D ₆ , 80 ° C.): δ (ppm) 0.89 (3H, t, J = 6.
6 Hz), 1.28 (16H, s), 1.40 (9H, s), 1.45 (3H, s),
1.64 (3H, s), 1.99 (2H, q, J = 6.7 Hz), 3.69 (1H,
d, J = 6.3, 9.3 Hz), 3.90 (1H, dd, J = 1.8, 9.3 H
z), 3.95 (1H, dt, J = 1.5, 6.6 Hz), 4.41 (1H, t, J
= 7.0 Hz), 5.53 (1H, dd, J = 7.1, 15.4Hz), 5.71 (1
H, dt, J = 6.7, 15.4 Hz), m / z (CI): C ₂₃ H ₄₄ NO ₄ (M + H)
Calculated value as ⁺ , 398.3200, measured value 398.3212

2. Threo -C15-Boc-sphingosine (R = nC ₁₀ H ₂₁ in compound VI, A = Boc) of the compound obtained in 1 of 95 mg (0.24 mmol) of acetic acid 0.9ml of water
It was dissolved in 0.1 ml and stirred at 50 ° C. for 5 hours. After evaporation of the solvent,
When 1 ml of heptane was added and concentrated to dryness, a pale yellow oil remained. This was purified by silica gel chromatography (hexane / ethyl acetate = 2: 1 → 1: 1) to obtain 70 mg (yield 82%) of the title compound as a colorless oily substance. [α] _D −1.6 ° (c 1.78, CHCl ₃ ) ¹ H-NMR (270 MHz, CDCl ₃ ): δ (ppm) 0.87 (3H, t, J =
6.7 Hz), 1.25 (14H, s), 1.34 (2H, m), 1.43 (9H,
s), 2.02 (2H, q, J = 6.7 Hz), 2.82 (1H, broad),
3.61 (1H, m), 3.77 (2H, d-like, 3.7 Hz), 4.33 (1H,
m), 5.18 (1H, d, J = 7.6 Hz), 5.50 (1H, dd, J = 6.
3, 15.6 Hz), 5.74 (1H, dt, J = 6.7, 15.4 Hz) ¹³ C-NMR (CDCl ₃ ) 14.1, 22.6, 28.3, 28.4, 29.0, 29.
2, 29.3, 29.5, 29.6, 31.9, 32.3, 55.5, 64.2, 73.4,
79.7, 128.9, 134.0, 156.6 Elemental analysis (as C ₂₀ H ₃₉ NO ₄₎ Found (%): C 67.08; H11.15 ; N 3.87 Calculated (%): C 67.19; H10.99 ; N 3.92

3. Threo-C15-sphingosine-1-
Phosphate ester (in compound VII, R = nC ₁₀ H ₂₁ ,
A = H, R ′ = CH ₃ ) under a nitrogen atmosphere 65 mg (0.18 mmol) of the compound obtained in 2
And 135 mg (0.4 mmol) of carbon tetrabromide in 1 ml of pyridine, and while cooling, trimethyl phosphite 50 mg (0.4 mmo
l) was added and the mixture was stirred at room temperature for 5 hours. A saturated aqueous ammonium chloride solution and ethyl acetate were added thereto, and the mixture was stirred for a while, and then the layers were separated. The organic layer was washed with saturated saline, and then the aqueous layers were combined and extracted twice with ethyl acetate. The organic layers are combined, dried over anhydrous sodium sulfate and filtered.
When it was concentrated to dryness, a pale yellow oil remained. This was subjected to silica gel chromatography (hexane / ethyl acetate = 1: 1).
By purification with 1 → 1: 2), 59 mg (yield 70%) of the title compound was obtained as a colorless oil, and 9 mg (14%) of the starting material (compound obtained in 2) was recovered. [α] _D −5.4 ° (c 1.4, CHCl ₃ ) ¹ H-NMR (270 MHz, CDCl ₃ ): δ (ppm) 0.87 (3H, t, J =
6.6 Hz), 1.25 (14H, s), 1.33 (2H, m), 1.42 (9H,
s), 2.02 (2H, q, J = 6.7 Hz), 3.77 (3H, d, J = 11.2)
Hz), 3.78 (3H, d, J = 11.2 Hz), 3.79 (1H, m), 4.0
9 (2H, m), 4.33 (1H, m), 5.03 (d, J = 9.0 Hz), 5.46
(1H, dd, J = 6.0, 15.4 Hz), 5.76 (1H, dt, J = 6.
7, 15.4 Hz) ¹³ C-NMR (CDCl ₃ ) 14.1, 22.7, 28.3, 29.1, 29.2, 29.
3, 29.5, 29.57, 29.61, 29.7, 31.9, 32.3, 54.0, 54.
4, 54.7, 66.1, 70.0, 79.7, 128.4, 134.0, 156.0 m / z
(CI): Calculated as C ₂₂ H ₄₅ NO ₇ P (M + H) ⁺ , 466.2863,
Found, 466.2855

4. Threo-C15-sphingosine-1-
Phosphoric acid (in compound I, R = n-C_TenH _{twenty one}) Synthesis 23 mg (0.05 mmol) of the compound obtained in Step 3 was added to methylene chloride.
Dissolve in 1 ml and cool, trimethylsilane bromide 50 micro
L (0.3 mmol) was added, and the mixture was stirred at room temperature for 3 hours. This melt
After concentrating the solution, add 0.5 ml of dioxane and 0.5 ml of water to about 2
Stir for hours. Concentrate this and add 1 ml of methanol again.
And concentrated. The remaining 20 mg was reconstituted from THF-water (2: 1).
12 mg of the title compound as a colorless solid by crystallization.
(Yield 71%) was obtained.¹ H-NMR (270 MHz, CD₃OD-CD₃CO₂D, 3: 1): δ (ppm) 0.8
7 (3H, t, J = 6.7 Hz), 1.27 (14H, s), 1.39 (2H, m),
 2.07 (2H, q, J = 6.6 Hz), 3.41 (1H, m), 3.92 (1H,
dd, J = 7.0, 12.1 Hz), 4.06 (1H, m), 4.17 (1H, t,
J = 7.8 Hz), 5.45 (1H, dd, J = 7.7, 15.3 Hz), 5.86
 (1H, dt, J = 6.6, 15.1 Hz) m / z (CI): C₁₅H₃₄NO_FiveP (M +
H)⁺As calculated, 338.2026, measured value, 338.2051.

[0037] Synthesis of (Example 2) threo -C18- sphingosine-1-phosphate (in a compound _{I, R = nC 13 H 27} ) 1. Threo -C18-N-Boc-N, 1O- isopropylidene - in sphingosine (compound _{II, R = nC 13 H 27} , A
= Boc, B = CH ₃ , C = CH ₃ ) was synthesized in the same manner as in Example 1-1. Colorless oil. Yield 86% [α] _D −37.8 ° (c 0.84, CHCl ₃ ) ¹ H-NMR (C ₆ D ₆ , 80 ° C.): δ (ppm) 0.89 (3H, t, J = 6.
7 Hz), 1.31 (22H, s), 1.40 (9H, s), 1.45 (3H, s),
1.64 (3H, s), 1.99 (2H, q, J = 6.8 Hz), 3.69 (1H, d
d, J = 6.3, 9.0 Hz), 3.90 (1H, dd, J = 1.5, 9.3 H
z), 3.95 (1H, dt, J = 1.5, 6.6 Hz), 4.41 (1H, t, J
= 7.0 Hz), 5.53 (1H, dd, J = 6.8, 15.3 Hz), 5.71
(1H, dt, J = 6.7, 15.4 Hz), ¹³ C-NMR (C ₆ D ₆ ) 14.1, 23.0, 24.3, 27.1, 28.4, 28.5,
29.5, 29.6, 29.7, 29.9, 30.1, 32.3, 32.6, 62.5, 6
4.8, 74.8, 80.3, 94.6, 130.6, 134.0, 156.6 m / z (C
I): Calcd as C ₂₆ H ₅₀ NO ₄ (M + H) ⁺ : 440.3670, found: 440.3652

2. (In the compounds _{VI, R = nC 13 H 27} , A = Boc) threo -C18-Boc-sphingosine was synthesized in the same manner as 2 of Example 1. Colorless solid. Yield 80% Melting point: 58-60 ° C [α] _D −0.7 ° (c 1.6, CHCl ₃ ) ¹ H-NMR (270 MHz, CDCl ₃ ): δ (ppm) 0.87 (3H, t, J =
6.7 Hz), 1.25 (20H, s), 1.34 (2H, m), 1.44 (9H,
s), 2.03 (2H, q, J = 6.8 Hz), 2.60 (1H, broad),
3.61 (1H, m), 3.78 (2H, d-like, 3.7 Hz), 4.34 (1H,
dd, J = 2.9, 6.1Hz), 5.18 (1H, d, J = 7.6 Hz), 5.5
0 (1H, dd, J = 6.5, 15.5 Hz), 5.74 (1H, dt, J = 6.
8, 15.4 Hz) ¹³ C-NMR (CDCl ₃ ) 14.1, 22.7, 28.3, 28.4, 29.0, 29.
2, 29.3, 29.5, 29.58, 29.63, 29.7, 31.9, 32.3, 55.
5, 64.3, 73.4, 79.7, 128.9, 134.0, 156.6 Elemental analysis (as C ₂₃ H ₄₅ NO ₄₎ Found (%): C 69.22; H11.45 ; N 3.47 Calculated (%): C 69.13; H11 .35; N 3.51

3. Threo-C18-sphingosine-1-
Phosphate ester (in compound VII, R = nC
₁₃ H ₂₇ , A = H, R ′ = CH ₃ ) was synthesized in the same manner as in Example 1-3. Colorless oil. Yield 65% [α] _D −3.5 ° (c 1.0, CHCl ₃ ) ¹ H-NMR (270 MHz, CDCl ₃ ): δ (ppm) 0.87 (3H, t, J =
6.6 Hz), 1.25 (22H, s), 1.42 (9H, s), 2.02 (2H,
q, J = 6.7 Hz), 3.77 (3H, d, J = 11.2 Hz), 3.78 (3
H, d, J = 11.2 Hz), 3.79 (1H, m), 4.09 (2H, m), 4.
33 (1H, m), 5.03 (d, J = 9.0 Hz), 5.46 (1H, dd, J =
6.0, 15.4 Hz), 5.76 (1H, dt, J = 6.7, 15.4 Hz) ¹³ C-NMR (CDCl ₃ ) 14.1, 22.7, 28.3, 29.1, 29.2, 29.
3, 29.4, 29.5, 29.58, 29.63, 29.7, 31.9, 32.3, 54.
3, 54.5, 54.6, 66.2, 70.1, 79.7, 128.4, 134.0, 15
6.0 m / z (CI): Calcd for C ₂₅ H ₅₁ NO ₇ P (M + H) ⁺ , 50
8.3333, Found, 508.3335

4. Threo-C18-sphingosine-1-
(In the compounds _{I, R = nC 1 3 H} 27) phosphoric acid was synthesized in the same manner as 4 of Example 1. Colorless solid. Yield 62% ¹ H-NMR (270 MHz, CD ₃ OD-CD ₃ CO ₂ D, 3: 1): δ (ppm) 0.8
7 (3H, t, J = 6.6 Hz), 1.26 (20H, s), 1.38 (2H, m),
2.06 (2H, q, J = 6.6 Hz), 3.37 (1H, m), 3.95 (1H,
dd, J = 7.0, 12.1 Hz), 4.07 (1H, m), 4.19 (1H, t,
J = 7.8 Hz), 5.44 (1H, dd, J = 7.7, 15.3 Hz), 5.86
(1H, dt, J = 6.6, 15.2 Hz) Elemental analysis value (as C ₁₈ H ₃₈ NO ₅ P) Actual value (%): C 56.82; H11.35; N 3.57 Calculated value (%): C 56.97; H10 .09; N 3.69

Example 3 Synthesis of threo-2-amino-3-cinnamyl-1,3-diol-1-phosphate (in compound I, R = C ₆ H ₅ ) 1. Threo-cinnamyl alcohol derivative (Compound I
In I, R = C ₆ H ₅ , A = Boc, B = CH ₃ , C =
CH ₃₎ Synthesis phenylacetylene 103 mg (1.0 mmol) of hydrogenated zirconocene dichloride 265 mg (1.0 mmol), diethylzinc 1.0 M hexane solution 0.9 ml (0.9 mmol), aldehyde compound III
(A = Boc, B = CH ₃ , C = CH ₃ ) 135 mg (0.6 mm
was synthesized in the same manner as in Example 1, 1 to obtain 180 mg (yield 92%) of the title compound as a colorless oily substance. [α] _D −89.1 ° (c 1.48, CHCl ₃ ) ¹ H-NMR (C ₆ D ₆ , 80 ° C.): δ (ppm) 1.39 (9H, s), 1.42
(3H, s), 1.59 (3H, s), 3.67 (1H, dd, J = 6.3, 9.3 H
z), 3.94 (1H, dd, J = 2.0, 9.3 Hz), 4.02 (1H, dt,
J = 2.0, 6.4 Hz), 4.60 (1H, t, J = 6.7 Hz), 6.22
(1H, dd, J = 7.1, 15.9 Hz), 6.62 (1H, d, J = 15.9
Hz), 7.00-7.15 (3H, m), 7.26 (2H, m) ¹³ C-NMR (C ₆ D ₆ , 80 ° C) 24.3, 27.1, 28.5, 62.5, 64.
5, 74.4, 80.4, 94.6, 127.0, 127.1, 128.8, 128.9, 1
29.9, 132.7, 156.6m / z (CI): Calcd as C ₁₉ H ₂₈ NO ₄ (M + H) ⁺ : 334.1948, Found: 334.1962

2. In cinnamyl type threo -Boc amino alcohol derivative (compound _{VI, R = C 6 H 5} , A =
Boc) was synthesized in the same manner as in Example 1-2. Compound II (R = C
66 mg (yield 89%) of the title compound as a colorless solid was obtained from 84 mg (0.25 mmol) of ₆ H ₅ ). Melting point: 78-80 ° C [α] _D + 11.3 ° (c 1.32, CHCl ₃ ) ¹ H-NMR (270 MHz, CDCl ₃ ): δ (ppm) 1.43 (9H, s), 2.
03 (2H, q, J = 6.8 Hz), 3.75 (1H, broad), 3.79
(1H, m), 3.85 (2H,), 4.64 (1H, m), 5.37 (1H, d, J
= 8.3 Hz), 6.27 (1H, dd, J = 5.9, 15.9 Hz), 6.69
(1H, d, J = 15.9Hz), 7.22-7.42 (5H, m) ¹³ C-NMR (CDCl ₃ ) 28.2, 55.6, 63.5, 72.6, 79.9, 126.
5 (2C), 127.7, 128.5 (2C), 128.8, 131.4, 136.5, 15
6.6 Elemental analysis value (as C ₁₆ H ₂₃ NO ₄ ) Actual value (%): C 65.24; H 8.03; N 4.67 Calculated value (%): C 65.51; H 7.90; N 4.77

3. Cinnamyl type threo-Boc amino alcohol phosphate (in compound VII, R =
C ₆ H ₅ , A = H, R ′ = CH ₃ ) was synthesized in the same manner as in Example 1-3. Colorless oil. Yield 65% [α] _D + 0.9 ° (c 0.9, CHCl ₃ ) ¹ H-NMR (270 MHz, CDCl ₃ ): δ (ppm) 1.37 (9H, s), 3.
76 (3H, d, J = 11.2 Hz), 3.78 (3H, d, J = 11.1 H
z), 3.93 (1H, m), 4.15 (2H, m), 4.58 (1H, m), 5.12
(d, J = 8.3 Hz), 6.20 (1H, dd, J = 5.4, 16.0 Hz),
6.70 (1H, d, J = 16.0 Hz), 7.18-7.40 (5H, m) ¹³ C-NMR (CDCl ₃ ) 28.2, 54.3, 54.5, 54.6, 66.0, 66.
9, 79.8, 126.5 (2C), 127.6, 128.3, 128.5 (2C), 13
1.6, 136.6, 156.0m / z (CI): Calculated as C ₁₈ H ₂₉ NO ₇ P (M + H) ⁺ : 402.1611, Found: 402.1632

4. Threo-2-amino-3-cinnamyl-1,3-diol-1-phosphate (in compound I,
R = C ₆ H ₅ ) was synthesized in the same manner as in Example 1-4. Since it could not be crystallized from the concentrate after the reaction, silica gel chromatography (developing solvent: n-butanol / acetic acid / water = 6: 1: 1)
Purified by. Colorless solid. Yield 41% ¹ H-NMR (270 MHz, CD ₃ OD-CD ₃ CO ₂ D, 4: 1): δ (ppm) 3.6
5 (1H, m), 4.03 (1H, m), 4.14 (2H, m), 6.22 (1H, d
d, J = 6.0, 16.0 Hz), 6.80 (1H, d, J = 16.0Hz), 7.
22-7.40 (5H, m)

Example 4 Edg Receptor Responsiveness Test HL 60 cells were obtained from a cell bank, and BBRC `98, 263, p. 2
RPM containing 10% fetal bovine serum according to the method described in 53.
The promyeloblastoma cell line HL60 expressing the Edg receptor on the cell surface was prepared by subculturing about 50 times using I-1640 medium (Gibco). HL expressing this Edg receptor on the cell surface
Using 60, the cell responsiveness of the test substance was examined. As an index of cell response, increase in intracellular Ca <sup>2+</sup> concentration was measured. Edg receptors on the surface of HL60 cells have been reported to increase intracellular Ca <sup>2+</sup> concentration after binding to SPN-1 P, phosphorylating G protein and activating IP <sub>3</sub> kinase. (FEBS Letter `96, 379, p. 260, BBRC` 98, 253, p.
253), the intracellular Ca ²⁺ concentration is an index of Edg receptor responsiveness. The Ca ²⁺ chelating reagent Fura-2 AM was incorporated into HL 60 cells. A quartz cell was filled with 1.2 ml of cell suspension, mounted on a Fluorometer LS-50B (Perkin Elmer, for cell measurement), and the excitation wavelength was 340 nm (Ca ²⁺ chelate) every 0.5 seconds. Fura-2 excited) and 380 nm (unreacted Fura-
Alternately, 2 was excited) and the fluorescence intensity at 510 nm was measured. The test substance (compound of Example 1, 2 or 3) was added to 30
At the final concentration of microM, the fluorescence intensity was traced after the addition using a microsyringe to examine whether Ca ²⁺ increases.
In addition, it was confirmed whether Ca ²⁺ increased when 1 microM SPN-1P was added after addition of the test substance, and SPN-
We examined 1P antagonism. After the addition of the compound of Example 1 or 2, the increase in intracellular Ca ²⁺ concentration due to the addition of SPN-1P was blocked, suggesting that these substances may be Edg antagonistic. Next, the dose dependence of the intracellular Ca ²⁺ increase inhibitory action by the compound of Example 1, 2 or 3 in which the possibility of antagonism was suggested was examined. As a control, suramin for which an antagonistic action has already been confirmed was used. The experimental method is
The procedure was the same as that described above, except that SPN-1 P (1 microM) was added after addition of the test substance at each concentration. Further, the increase in Ca ²⁺ concentration was evaluated by the Ca ²⁺ increase concentration% as a relative value with respect to the Ca ²⁺ increase concentration in the negative control group (without drug addition). As a result, the compound of Example 1 was added in an amount of 0.01-0.03 to 0.03 microM at a concentration of 0.003-0.03 microM.
SPN-1P dose-dependently at 0.1 microM concentration
The increase in Ca ²⁺ concentration was suppressed. The result is shown in FIG. Moreover, the 50% inhibitory concentration (ED ₅₀ value) of intracellular Ca ²⁺ increase was 0.015 ± 0.007 microM for the compound of Example 1, 0.030 ± 0.002 microM for the compound of Example 2, and 0.037 for the compound of Example 3.
Although it was ± 0.014 microM, the potency of the compound of Example 1 was about 100 times that of the ED ₅₀ value of suramin of 1.8 ± 0.1 microM for which Edg antagonism was already reported.
The potency of the compound was about 60 times stronger. The results are shown in Table 1.

[Table 1]

(Example 5) Action on vascular smooth muscle The action of a test substance on vascular smooth muscle proliferation was examined.
It is considered that vascular smooth muscle cells are transformed from contractile type to synthetic type with the progress of arteriosclerosis, and vascular smooth muscle cells proliferate while secreting inflammatory cytokines and the arteriosclerotic lesion develops. hypothesis). On the surface of vascular smooth muscle cells
It has been reported that the Edg receptor is expressed (The A
merican Society for Pharmacology and Experimental T
herapeutics' 00, Vol. 58, p. 449), similar to SPN-1 P, it has been reported that vascular smooth muscle cells proliferate in response to sphingosylphosphorylcholine acting on Edg receptors (The American). Physiological Society 9
8, C1255). Therefore, this time, the effect of the compound of Example 1 on vascular smooth muscle proliferation was examined. As a positive control
Suramin whose Edg receptor antagonism was confirmed was used.
The carotid intima of the rat was scraped by ballooning, and after 2 weeks, vascular smooth muscle cells prepared by the Explant culture were cultured in DMEM medium (Gibco) containing 10% fetal bovine serum and subcultured several times. Then stabilize and then 5 x
The cells were seeded at a cell density of 10 ³ cells / cm ² and used in the experiment. Growth Factor Sphingosylphosphorylcholine (10 microM
) Together with the compound of Example 1 or suramin, and 24 hours later, BrdU assay (Science '82, 2
18, p.474) to determine the cell density. As a result, the compound of Example 1 suppressed vascular smooth muscle cell proliferation in a dose-dependent manner at a concentration of 0.3 to 3 microM. Suramin used as a positive control inhibited vascular smooth muscle cell proliferation at concentrations of 30 and 100 microM. The result is shown in FIG.

(Example 6) Competition experiment using ³ H-SPN-1 P The promyeloblastoma cell line HL60 expressing the same Edg receptor as in Example 4 on the cell surface was used. The HL60 cells were collected by centrifugation, suspended in F-12 medium (stored at 4 ° C, 10 ml), and transferred to the RI laboratory. Cell suspension 200 micro
l (1 x 10 ⁶ cells / ml F-12) at a final concentration of 1 nM ³ H-SPN
-1 P (15 micro Ci / 1 mM) and a final concentration of 100 nM unlabeled compound (Example 1 compound, Example 2 compound, Example 3 compound) were added, and the mixture was added at 4 ° C for 30 minutes (with occasional stirring). ) A binding test was performed. After centrifugation at 12,000 rpm for 7 minutes, the supernatant is quickly discarded (without damaging the cell pellet) with a micropipettor and the cell pellet is ready-solved.
(Beckman) After suspending with 1.5 ml, it was transferred to a vial and the radioactivity was measured with a liquid scintillation counter L 2100 (Beckman). The results are shown in Table 2 below. From these results, the compounds of Example 1 and Example were the same as SPN-1P.
Since 2 compounds and the compound of Example 3 competed with ³ H-SPN-1 P, it was considered that these substances were specifically bound to Edg.

[Table 2]

Example 7 Anti-Inflammatory Test Using Pseudovascular Model At the damaged site in the body, exposed collagen (extracellular matrix) is targeted as a damage signal, and platelets are aggregated. It is believed that inflammatory cytokines such as PDGF released from activated platelets promote inflammation, and severe inflammation causes cardiovascular homeostasis to break and arteriosclerosis to proceed. SPN-1P
Is also considered to have similar effects to PDGF. Therefore, using SPN-1P as an inflammation inducing agent,
Whether an in vitro model is established and whether or not the compound of the present invention exhibits an anti-inflammatory effect by using the model, and whether it may act to maintain homeostasis of the circulatory organ and improve pathology I examined.

(1) Establishment of Pseudovascular In Vitro Inflammation Model Using a transwell divided into an upper chamber and a lower chamber by a porous membrane having a pore size of 3 μm, one layer of bovine endothelial cells was formed on the porous membrane at the bottom of the transwell upper chamber. Were cultured, a fluorescently labeled neutrophil suspension was added to the upper chamber of the transwell, and SPN-1P was suspended in the lower chamber to a final concentration of 0.1 to 10 microM. That is, the upper and lower chambers of the transwell are separated by the endothelial layer,
The model is a pseudo-vascular in vitro inflammation model in which the upper chamber corresponds to the inside of the blood vessel and the lower chamber corresponds to the outside of the blood vessel. The number of neutrophils penetrating from the upper chamber to the lower chamber through the endothelial layer and the number of neutrophils adhering to the endothelial layer were measured from the fluorescence intensity of 530 nm, and the relative number of neutrophils was calculated as follows. It was calculated. Relative neutrophil count% = [number of neutrophils (permeation and adhesion) in experimental group] / [neutrophils in control (permeation and adhesion)]
Number] × 100 (control is the case where SPN-1P is not added). As a result, SPN-1P 10 microM significantly promoted neutrophil permeation into the endothelial layer and adhesion (see FIG. 3). That is, SPN-1P was considered to act as an inflammation-inducing substance.

(2) Effect of the Compound of the Present Invention on Inflammatory Cell-Vascular Endothelial Cell Interaction Next, using the above pseudovascular in vitro inflammation model, the interaction between neutrophils and vascular endothelial cells was examined. It was examined how the two compounds affect. That is, the compound of Example 2 was added to the Transwell lower chamber at 0.01 to 1 microM, and SPN-1P 10 microM was placed in the lower chamber to induce inflammation. As a control, the one in which no drug was added and inflammation was induced as described above was used. The number of neutrophils adhered to the endothelium layer or the number of neutrophils that passed through the endothelium layer and penetrated to the lower chamber was 53
The relative neutrophil count was calculated from the above formula by measuring the fluorescence intensity at 0 nm. The result is shown in FIG. As shown in the figure, the compound of Example 2 suppressed neutrophil permeation and adhesion. Therefore, in the pseudo blood vessel in vitro model, SP
When N-1P is used as an inflammation-inducing agent, the compound of Example 2 acts anti-inflammatoryly, and therefore, it is considered that the homeostasis of the circulatory organ is maintained and the action may be improved.

[0051]

INDUSTRIAL APPLICABILITY The compound of the present invention exhibits an excellent Edg receptor antagonistic action, and pharmaceuticals containing the compound of the present invention as an active ingredient are circulatory diseases (for example, arteriosclerosis, heart disease), cancer, It has an excellent preventive or therapeutic effect on rheumatism, diabetic retinopathy and respiratory diseases.

[Brief description of drawings]

FIG. 1 is a graph showing that the compound of the present invention shows Edg antagonism in a dose-dependent manner (suramin: control).

FIG. 2 is a graph showing that the compound of the present invention has a dose-dependent inhibitory effect on vascular smooth muscle cell proliferation (suramin: control). In the figure, the open squares are the data when the test substance was not added, and *: risk rate p ≦ 5% with respect to the negative control.
Significantly suppressed by **, risk ratio p ≦ 1 compared to negative control
% Indicates significant inhibition.

FIG. 3: SPN-1 on endothelial cell-neutrophil interaction
The action of P is shown. *: Significantly promoted at a risk rate of 5% compared to the control without drug addition

FIG. 4 is a graph showing that the compound of the present invention suppressively acts on the migration of neutrophils to vascular endothelial cells.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 11/00 A61P 11/00 27/02 27/02 29/00 101 29/00 101 35/00 35 / 00 (72) Inventor Masazumi Nishikawa 16-2 Wadai, Tsukuba City, Ibaraki Maruha Co., Ltd. Central Research Institute (72) Inventor Teiichi Murakami 1-1-1 East, Tsukuba City, Ibaraki Prefecture F-term in Tsukuba Center (reference) 4C086 AA01 AA02 AA03 AA04 DA34 MA01 MA04 NA14 ZA33 ZA36 ZA37 ZA40 ZA45 ZA59 ZA96 ZB15 ZB26 4H050 AA01 AA02 AA03 AB20 AB23 AB25 AB27 AB28 AC40 AC80 BD70

Claims (13)

[Claims] 1. The general formula I: (In the formula, R is an optionally substituted linear or branched CH ₃ C _n H _(2n-2m) -(n is any integer from 2 to 19, and m is , An unsaturated number, which is an integer between 0 and 3) or an aryl group which may be substituted), a threo type (2S, 3S) aminoalcohol-1-phosphate. A compound or a pharmaceutically acceptable salt thereof. 2. The general formula I is represented by the following formula: The compound according to claim 1 represented by or a pharmaceutically acceptable salt thereof. 3. The general formula I is represented by the following formula: The compound according to claim 1 represented by or a pharmaceutically acceptable salt thereof. 4. The general formula I is represented by the following formula: The compound according to claim 1 represented by or a pharmaceutically acceptable salt thereof. (1) General formula V: (In the formula, R is an optionally substituted linear or branched CH ₃ C _n H _(2n-2m) -(n is any integer from 2 to 19, and m is Represents an unsaturated number and is an integer between 0 and 3) or an optionally substituted aryl group, and Cp represents a cyclopentadienyl group)
An alkylating agent prepared by allowing a dialkylzinc to act on an organozirconium compound represented by:
N-protected (S) -formyloxazolidine compound of: (Wherein A is a protecting group for N and B and C are alkyl groups) to react with the threo compound of the general formula II (2S, 3
Selectively obtaining S), (Wherein R, A, B and C are the same as defined above) (2) The general formula VI in which the amino group is protected by ring-opening the oxazoline of the compound II to remove the acetal-type protecting group.
Obtaining the alcohol compound of: (In the formula, R and A are the same as defined above.) (3) From the compound of the general formula VI, 1-of the following general formula VII
Converting to a phosphoric acid analog compound: (In the formulae, R and A are the same as defined above, and R ′ is a lower alkyl group.) (4) Including removal of the respective protecting groups of the amino group and the phosphate group of the compound of the general formula VII, A method for producing the compound of claim 1. 6. A drug which antagonizes an endothelial differentiation gene (Edg) receptor, which comprises the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof as an active ingredient. . 7. The medicine according to claim 6 for treating or preventing cardiovascular diseases. 8. The cardiovascular disease is arteriosclerosis.
Medicine. 9. A method for treating or preventing cancer according to claim 6.
Medicine. 10. The medicine according to claim 6 for treating or preventing rheumatism. 11. The pharmaceutical according to claim 6, which is used for treating or preventing diabetic retinopathy. 12. The medicine according to claim 6 for treating or preventing respiratory diseases. 13. The cardiovascular disease is heart disease.
Medicine.