JP5478082B2 - Interleukin-2 production inhibitor - Google Patents

Description

  The present invention relates to an interleukin-2 production inhibitor. The present invention also relates to a preventive or therapeutic agent for various diseases (interleukin-2 related diseases) that occur in association with increased production of interleukin-2, such as autoimmune diseases, rheumatism, and type IV allergy.

  In order to exclude foreign substances invading from the outside world, the living body has an immune system, but the immune system may react excessively to cause allergic diseases and autoimmune diseases. Allergic reactions are classified from type I to type IV depending on the reaction mechanism, and among these type IV allergies, allergic reactions start when T lymphocytes that have reacted with the antigen are activated and proliferated. Examples of diseases classified as type IV allergy include atopic dermatitis, contact dermatitis, thyroiditis, and allergic encephalitis. In addition, it is known that autoimmune diseases develop due to activation and proliferation of T lymphocytes as a cause by misrecognizing and reacting to their own biological components as foreign substances. Such autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis and the like.

  Incidentally, T cells play a central role in the immune response of living bodies, and the activities of a plurality of cytokines produced from activated T cells control the direction of the immune response (Non-patent Document 1). Furthermore, cytokine production from T cells is controlled by gene transcription activation after antigen stimulation (Non-patent Document 2).

  FK506 and cyclosporin A (Non-Patent Documents 3 to 4), which are known as immunosuppressants, bind to each intracellular receptor and then suppress the dephosphorylation activity of calcineurin, thereby interleukin- 2 (IL-2) It has been clarified that the activation of transcription factor NF-AT involved in gene transcription activation is suppressed, and as a result, IL-2 production is suppressed (Non-patent Document 5).

  In addition, it has been reported that cytokines such as IL-2 are deeply involved in bronchial asthma and the like (Non-Patent Documents 6 to 7). Furthermore, it is also known that FK506 and cyclosporin A are useful for atopic dermatitis.

  Furthermore, cytokine balance is involved in the development of rheumatoid arthritis, and in particular, Th1-type cytokines such as IL-2 are known to play a promoting role in the development of arthritis (Non-patent Document 8). (Nokahiro Okamoto et al., “Development of Rheumatoid Arthritis and Cytokine Balance”, Pharmaceutical Journal, 121 (2) 131-138 (2001)).

  Therefore, drugs that suppress the production of cytokines such as IL-2 in T cells are allergic diseases in general, specifically asthma, allergic rhinitis, atopic dermatitis, urticaria, food allergies, etc. Treatment for autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, type I diabetes mellitus, Hashimoto thyroiditis, myasthenia gravis, multiple sclerosis, and rejection associated with organ transplantation It is thought that it can be a drug or a preventive drug.

  Conventionally, various drug treatments have been tried for these diseases, but no effective and safe drug has been found yet. For example, non-steroidal anti-inflammatory analgesics are not effective against rheumatoid arthritis. Although steroids are effective, strong side effects may occur after long-term administration, which is problematic in terms of safety. In addition, the immunosuppressants (FK506 and cyclosporin A) described above have been reported to be highly effective against type IV allergies such as autoimmune diseases and atopic dermatitis, but these are problematic in terms of side effects. There is a need for the management of a doctor. Therefore, a safe drug having an IL-2 production inhibitory action is desired.

Powrie, F. et al., Cytokine regulation of T-cell function: potential for therapeutic intervention. Immunol. Today 1993, 14, 270. Fraser, J. D. et al., Signal transduction events leading to T-cell lymphokine gene expression. Immunol. Today 1993, 14, 357. Mattila, P. S. et al., The actions of cyclosporin A and FK506 suggest a novel step in the activation of T lymphocytes. EMBO J. 1990, 9, 4425. Liu, J. et al., Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 1991, 66, 807. Flanagan, W. M. et al., Nuclear association of a T-cell transcription factor blocked by FK506 and cyclosporin A. Nature 1991, 352, 803. Proc. Natl. Acad. Sci. USA 85: 2288-92, 1988 Akutsu, I. et al., Antibody against interleukin-5 prevents antigen-induced eosinophil infiltration and bronchial hyperreactivity in the guinea pig airways. Immunol. Lett. 1995, 45, 109. Nokahiro Okamoto et al., “Onset of Rheumatoid Arthritis and Cytokine Balance”, Pharmaceutical Journal, 121 (2) 131-138 (2001).

  An object of the present invention is to provide a novel IL-2 production inhibitor having an IL-2 production inhibitory action. The present invention also relates to the prevention of IL-2 related diseases, which is a composition having an IL-2 production inhibitory effect and effective for the prevention or treatment of diseases that develop or exacerbate in relation to increased production of IL-2. Another object is to provide a therapeutic agent.

  The inventors of the present invention have intensively studied to solve the above problems, and have found that the compound represented by the following formula (1):

Wherein R ¹ is a hydrogen atom or a hydroxy group; R ² and R ³ are the same or different and each represents a hydrogen atom, an amino group, a hydroxy group or a lower alkanoyloxy group. N is an integer of 2 to 7 means.)
The carboxylic acid represented by the above or its ester has an action of suppressing the production of interleukin-2, and by using such a compound as an active ingredient, the IL-2 production inhibitor that is the subject of the present invention, In addition, we were convinced that it would be possible to provide preventive or therapeutic agents for IL-2 related diseases. The present invention has been completed based on such findings.

The present invention includes the following embodiments.
Item 1. The following general formula (1):

Wherein R ¹ is a hydrogen atom or a hydroxy group; R ² and R ³ are the same or different and each represents a hydrogen atom, an amino group, a hydroxy group or a lower alkanoyloxy group. N is an integer of 2 to 7 means.)
An interleukin-2 production inhibitor comprising as an active ingredient a carboxylic acid represented by the formula (1) or a pharmaceutically acceptable salt or ester thereof.

  Item 2. The following general formula (1):

Wherein R ¹ is a hydrogen atom or a hydroxy group; R ² and R ³ are the same or different and each represents a hydrogen atom, an amino group, a hydroxy group or a lower alkanoyloxy group. N is an integer of 2 to 7 means.)
A prophylactic or therapeutic agent for interleukin-2 related diseases, which comprises a carboxylic acid represented by the formula (I) or a pharmaceutically acceptable salt or ester thereof as an active ingredient.

  Item 3. Item 3. The preventive or therapeutic agent according to Item 2, wherein the interleukin-2 related disease is an autoimmune disease or a type IV allergy.

  The compound provided by the present invention has an action of suppressing the production of IL-2, an IL-2 production inhibitor, and prevention of a disease that develops or worsens related to IL-2 (IL-2 related disease) Or it can be used effectively as an active ingredient of a therapeutic agent. Therefore, according to the present invention, it is possible to provide an IL-2 production inhibitor and an agent for preventing or treating IL-2 related diseases containing these compounds as active ingredients. The prophylactic or therapeutic agent for IL-2 related diseases can be effectively used for improvement and prevention of autoimmune diseases such as rheumatoid arthritis and type IV allergy such as atopic dermatitis.

  In particular, (E) -9,10-dihydroxy-2-decenoic acid, which is an active ingredient of the present invention, has an excellent IL-2 production inhibitory action and high safety, so there are few side effects and it can be used for a long time. is there.

In the step of isolating and purifying (2E) -9,10-dihydroxy-2-decenoic acid from royal jelly dry powder, the “Preparation of A3 fraction by ultrafiltration membrane” step is shown. In the process of isolating and purifying (2E) -9,10-dihydroxy-2-decenoic acid from dried royal jelly powder, the “fractionation by gel filtration from A3 fraction” step is shown. In the step of isolating and purifying (2E) -9,10-dihydroxy-2-decenoic acid from royal jelly dry powder, the “fractionation by reverse phase (ODS) medium pressure chromatography from D4 fraction” step is shown. In the process of isolating and purifying (2E) -9,10-dihydroxy-2-decenoic acid from dried royal jelly powder, the “fractionation by reverse phase (ODS) high performance liquid chromatography from D4-II fraction” step is shown. In the process of isolating and purifying (2E) -9,10-dihydroxy-2-decenoic acid from dried royal jelly powder, the "fractionation by reverse phase (ODS) high-performance liquid chromatography from D4-II-3 fraction" process Show. In the step of isolating and purifying (2E) -9,10-dihydroxy-2-decenoic acid from dried royal jelly powder, the step of “separation from D4-II-3-E fraction by normal phase chromatography” is shown. The flowchart of the isolation and purification method of (2E) -9,10-dihydroxy-2-decenoic acid from royal jelly dry powder employed in Preparation Example 2 is shown. The results of comparing the amount of IL-2 produced after adding DDA1 to DDA16 to cultured human lymphocytes and culturing for 48 hours with the amount of IL-2 produced when DDA is not added are shown (Test Example 1). A forced swimming test was performed on ovariectomized pathological model mice (test group) orally administered with DDA4, DDA6, DDA7, DDA14 + 15, or DDA16 and ovariectomized pathological model mice (control group: Cntl.) Orally administered with distilled water. This is a result showing the IL-2 concentration (pg / ml) contained in the collected blood serum. Each test result is an average of 6 animals.

(I) Active ingredient The active ingredient of the IL-2 production inhibitor and IL-2 related disease which are the subject of the present invention is carboxylic acid (1) represented by the following general formula (1):

は、シングル結合またはダブル結合を意味する。ｎは２〜７の整数を意味する。）。 Wherein R ¹ is a hydrogen atom or a hydroxy group; R ² and R ³ are the same or different and each represents a hydrogen atom, an amino group, a hydroxy group or a lower alkanoyloxy group.

Means a single bond or a double bond. n means an integer of 2 to 7. ).

がダブル結合である場合、好ましくは水素原子である。 Here, R ¹ means a hydrogen atom or a hydroxy group. Where

When is a double bond, it is preferably a hydrogen atom.

R ² and R ³ are the same or different and each represents a hydrogen atom, an amino group, a hydroxy group or a lower alkanoyloxy group. R ² is preferably a hydrogen atom or a hydroxy group, and R ³ is preferably a hydrogen atom, a hydroxy group or a lower alkanoyloxy group. As a combination of R ² and R ³ , when R ² is a hydroxy group, R ³ is a hydrogen atom or a hydroxy group: when R ² is a hydrogen atom, R ³ is a hydroxy group or a lower alkanoyloxy group: R ² When R is an amino group, R ³ is an amino group: when R ² is a lower alkanoyloxy group, R ³ is a lower alkanoyloxy group.

Here, the lower alkanoyloxy group refers to a group represented by R ⁴ —CO—O—, wherein R ⁴ is a lower alkyl group having 1 to ⁴ carbon atoms. The lower alkanoyloxy group is preferably an acetyloxy group in which R ⁴ is a methyl group, and a propanoyloxy group in which R ⁴ is an ethyl group, more preferably an acetyloxy group in which R ⁴ is a methyl group.

  n means any integer selected from 2 to 7. Preferably 2, 3, 5 and 7, more preferably 5.

  The compounds of the present invention specifically include those shown in Table 1.

The compound (1) targeted by the present invention may be either cis type or trans type, and the compound (1) of the present invention includes both these cis isomers and trans isomers. Of the compounds (1) of the present invention, R ³ is a hydrogen atom, a hydroxy group, an amino group or a lower alkanoyloxy group, and R ² is a hydroxy group, an amino group or a lower alkanoyloxy group. Enantiomers (R- and S-isomers) having a chiral center at the center carbon. That is, among the compounds (1) targeted by the present invention, a compound in which R ³ is a hydrogen atom, a hydroxy group, an amino group or a lower alkanoyloxy group and R ² is a hydroxy group, an amino group or a lower alkanoyloxy group May be any of an enantiomer having a chiral carbon at the 2-position, R-form or S-form, and may be a mixture containing these in an arbitrary ratio, for example, a racemate.

がダブル結合で、Ｒ^１が水素原子である化合物（(1a)〜(1f)）が含まれる： The compound (1) is not limited, but specifically shown below.

Are compounds in which R ¹ is a hydrogen atom ((1a) to (1f)):

(In the formula, R ² represents a hydroxy group, an amino group or a lower alkanoyloxy group, R ³ represents a hydrogen atom, a hydroxy group, an amino group or a lower alkanoyloxy group. (Asymmetric carbon), and the above formula includes both R and S compounds.)
Among them, preferably, among the compounds represented by the above formula (1e) or (1f), the following formula (2E) -9,10-dihydroxy-2-decenoic acid wherein R ¹ group and R ² group are hydroxy groups It is.

  The (2E) -9,10-dihydroxy-2-decenoic acid includes an R form ((2E, 9R) -9,10-dihydroxy-2-decenoic acid) represented by the following formula (i), There is an enantiomer of the S form ((2E, 9S) -9,10-dihydroxy-2-decenoic acid) represented by (ii).

  The (2E) -9,10-dihydroxy-2-decenoic acid targeted by the present invention is such an enantiomer, that is, R-form ((2E, 9R) -9,10-dihydroxy-2-decenoic acid) or S-form ( (2E, 9S) -9,10-dihydroxy-2-decenoic acid) or a mixture containing these in any proportion, for example, a racemic compound.

Further, as a preferable compound, among the compounds represented by the above formula (1e) or (1f), the following formula (2E) -9-hydroxy-2- wherein R ¹ group is a hydrogen atom and R ² group is a hydroxy group: Decenoic acid.

  The (2E) -9-hydroxy-2-decenoic acid includes an R form ((2E, 9R) -9-hydroxy-2-decenoic acid) represented by the following formula (iii) and a formula (iv) There are enantiomers (enantiomers) of the indicated S form ((2E, 9S) -9-hydroxy-2-decenoic acid).

  The (2E) -9-dihydroxy-2-decenoic acid targeted by the present invention is such an enantiomer, that is, R-form ((2E, 9R) -9,10-dihydroxy-2-decenoic acid) or S-form ((2E , 9S) -9,10-dihydroxy-2-decenoic acid), or a mixture containing these in an arbitrary ratio, for example, a racemic compound.

Further, as a preferable compound, among the compounds represented by the above formula (1a) or (1b), the following formula (2E) -7-acetoxy-2 wherein R ³ group is an acetyloxy group and R ² group is a hydrogen atom -Heptenoic acid.

  The compound (1) of the present invention may be in a carboxylic acid-free form or may have a pharmaceutically acceptable salt or ester form.

  Examples of the pharmaceutically acceptable salt include salts with inorganic bases or organic bases, and salts with basic amino acids. As an inorganic base. Examples thereof include alkali metal salts such as sodium and potassium; alkaline earth metal salts such as calcium and magnesium; ammonium salts and aluminum salts. Examples of the organic base include primary amines such as ethanolamine; secondary amines such as diethylamine, diethanolamine, dicyclohexylamine, and N, N′-dibenzylethylenediamine; trimethylamine, triethylamine, triethanolamine, pyridine, picoline, and the like. And tertiary amines. Examples of basic amino acids include lysine, arginine and ornithine.

  Further, as the ester, C1-6 lower alkyl ester of the corresponding carboxylic acid, preferably C1-3 lower alkyl ester (for example, methyl ester, ethyl ester, n-propyl ester) and the corresponding C1-6 lower alkylthioester, In addition to C1-3 lower alkylthioesters (for example, methylthioester, ethylthioester, n-propylthioester), di- or oligoesters of the corresponding carboxylic acid can be mentioned.

がダブル結合である化合物は、例えば、商業的に入手可能な4-(2-hydroxyethyl)-2,2-dimethyl-1,3-dioxolane（２）を出発原料として、下記反応式−１に示す方法により製造することができる。 Of the compounds represented by the general formula (1) of the present invention, R ¹ is a hydrogen atom, R ² and R ³ are both hydroxy groups,

Is a double bond, for example, commercially available 4- (2-hydroxyethyl) -2,2-dimethyl-1,3-dioxolane (2) as a starting material, shown in the following reaction formula-1. It can be manufactured by a method.

[In the above formula, Ts represents a tosyl group, X represents a halogen atom, R ³ and R ⁴ are the same or different and represent an alkyl group having 1 to 6 carbon atoms. n is the same as above. ].

  Compound (3) is produced by reacting compound (2) with p-tosyl halide. Examples of p-tosyl halide include p-tosyl chloride, p-tosyl bromide and the like. As a use ratio of the compound (2) and p-tosyl halide, the latter is usually about 0.5 to 2 mol relative to 1 mol of the former. This reaction is generally carried out in a suitable inert solvent. As the inert solvent, known solvents that do not adversely influence the reaction can be widely used, and examples thereof include pyridine and tetrahydrofuran. The reaction suitably proceeds at around −10 to 10 ° C., and the reaction is generally completed in about 5 to 10 hours.

  The compound (5) is produced by reacting the compound (3) with a known compound (4) that is easily available. X in the compound (4) includes, for example, chlorine, bromine and the like. As a use ratio of the compound (3) and the compound (4), it is preferable that the latter is usually about 0.5 to 2 mol with respect to 1 mol of the former. This reaction is generally carried out in a suitable inert solvent in the presence of a catalyst. As the inert solvent, known solvents that do not adversely influence the reaction can be widely used, and examples thereof include tetrahydrofuran and ether. Examples of the catalyst include copper iodide. The reaction suitably proceeds at room temperature to about 50 ° C., and the reaction is generally completed in about 2 to 5 hours.

  Compound (6) is produced by reacting compound (5) with ozone and then reacting the resulting compound with methyl sulfide. The reaction between compound (5) and ozone is generally carried out in a suitable inert solvent. As the inert solvent, known solvents that do not adversely influence the reaction can be widely used, and examples thereof include methylene chloride and methanol. The reaction suitably proceeds at around -80 to -70 ° C, and the reaction is generally completed in about 10 minutes to 1 hour. The subsequent reaction with methyl sulfide is carried out in the same solvent as the reaction between compound (5) and ozone. The reaction suitably proceeds from −80 ° C. to around room temperature, and the reaction is generally completed in about 2 to 8 hours.

  The compound (8) is produced by reacting the compound (6) with a known compound (7) that is easily available. As a use ratio of the compound (6) and the compound (7), it is usually preferable that the latter is about 0.5 to 2 mol with respect to 1 mol of the former. This reaction is generally carried out in a suitable inert solvent in the presence of a catalyst. As the inert solvent, known solvents that do not adversely influence the reaction can be widely used, and examples thereof include tetrahydrofuran, dimethyl sulfoxide and the like. Examples of the catalyst include sodium hydride and the like. The reaction suitably proceeds at around -10 ° C to 10 ° C, and the reaction is generally completed in about 2 to 5 hours.

がダブル結合である化合物）は、化合物（８）にトリフルオロ酢酸を反応させ、次いで得られる化合物に塩基を作用させることにより製造される。化合物（８）とトリフルオロ酢酸との反応は、一般に適当な不活性溶媒中で行われる。不活性溶媒としては、反応に悪影響を及ぼさない公知の溶媒を広く使用でき、例えば、塩化メチレン、ジクロロメタン、メタノール等が挙げられる。該反応は、−１０〜１０℃付近で好適に進行し、一般に３〜１０時間程度で反応は完了する。塩基としては、例えば、水酸化カリウム、水酸化ナトリウム等の公知のアルカリが使用される。塩基による処理は、例えば、メタノール、エタノール等のアルコール中で行われる。塩基による処理は、通常室温付近で１〜５時間程度で行われる。 Such a compound (1) (in the general formula (1), R ¹ is a hydrogen atom, R ² and R ³ are both hydroxy groups,

Is a double bond) is produced by reacting compound (8) with trifluoroacetic acid and then reacting the resulting compound with a base. The reaction of compound (8) with trifluoroacetic acid is generally performed in a suitable inert solvent. As the inert solvent, known solvents that do not adversely influence the reaction can be widely used, and examples thereof include methylene chloride, dichloromethane, methanol and the like. The reaction suitably proceeds at around −10 to 10 ° C., and the reaction is generally completed in about 3 to 10 hours. As the base, for example, a known alkali such as potassium hydroxide or sodium hydroxide is used. The treatment with a base is performed, for example, in an alcohol such as methanol or ethanol. The treatment with a base is usually performed at around room temperature for about 1 to 5 hours.

がダブル結合である化合物）のＲ^２及びＲ^３で示されるヒドロキシ基を、水素原子、低級アルカノイルオキシ基またはアミノ基に変換することにより製造される。より具体的には、後述する調製例に記載する方法に従って製造することができる。 Among the compounds represented by the general formula (1) of the present invention, a compound in which any of R ² and R ³ is a hydrogen atom, a lower alkanoyloxy group or an amino group is a compound (1) produced by the above method. (In the general formula (1), R ¹ is a hydrogen atom, R ² and R ³ are both hydroxy groups,

Is a double bond, and the hydroxy group represented by R ² and R ³ is converted to a hydrogen atom, a lower alkanoyloxy group or an amino group. More specifically, it can be produced according to the method described in the preparation examples described later.

がシングル結合である化合物は、例えば、商業的に入手可能な4-(2-hydroxyethyl)-2,2-dimethyl-1,3-dioxolane（２）を出発原料として、下記反応式−２に示す方法により製造することができる。 Of the compounds represented by the general formula (1) of the present invention, R ¹ is a hydrogen atom, R ² and R ³ are both hydroxy groups,

Is a single bond, for example, commercially available 4- (2-hydroxyethyl) -2,2-dimethyl-1,3-dioxolane (2) as a starting material is shown in the following reaction formula-2. It can be manufactured by a method.

がシングル結合である化合物（10）は、前記＜反応式−１＞に記載する方法と同様の方法で化合物（２）から化合物（８）を合成し、次いで、これを水素添加した後にアルカリ処理することで製造できる。より具体的には、後述する調製例１０に記載する方法に従って製造することができる。 Specifically, among the compounds represented by the general formula (1), R ¹ is a hydrogen atom, R ² and R ³ are both hydroxy groups,

Compound (10) in which is a single bond, compound (8) was synthesized from compound (2) by the same method as described in <Reaction Scheme-1>, and then hydrogenated, and then treated with alkali. Can be manufactured. More specifically, it can be produced according to the method described in Preparation Example 10 described later.

  The salt and ester form targeted by the present invention can be prepared from the carboxylic acid obtained by the above method according to a conventional method. For example, an ester body can be prepared by reacting the carboxylic acid with an alcohol or thiol.

  Moreover, the compound shown by General formula (1) of this invention can also be isolated and purified from the natural product containing these. Specifically, (E) -9,10-dihydroxy-2-decenoic acid represented by the above formula (i) or (ii) is isolated and purified from royal jelly as shown in Preparation Examples 1 and 2 described later. be able to. However, the method is not limited to these methods as long as the compound can be isolated.

(II) IL-2 production inhibitor, IL-2 related disease preventive / therapeutic agent IL-2 production inhibitor targeted by the present invention, and an agent for preventing or treating IL-2 related disease (hereinafter collectively referred to as The “formulation of the present invention” comprises a compound represented by the above general formula (1), or a pharmaceutically acceptable salt or ester thereof as an active ingredient. In addition, the said formulation of this invention may contain a compound (1) in the form of a solvate, for example, a hydrate.

  Compound (1) is preferably (2E) -9,10-dihydroxy-2-decenoic acid, (2Z, 9R) -9,10-dihydroxy-2-decenoic acid, (2E) -9-hydroxy-2- Decenoic acid, (2E) -7-acetoxy-2-heptenoic acid, (2E, 7R) -7,8-dihydroxy-2-octenoic acid, and (2E, 9R) -9,10-diamino-2-decenoic acid -Trifluoroacetate.

  The composition of the present invention may consist of 100% of these compounds, or pharmaceutically acceptable salts or esters thereof (hereinafter collectively referred to as “DDA”), and so on. Even if not, it may contain an effective amount of DDA that exerts an IL-2 production inhibitory effect. Although not limited, DDA is contained in the formulation of the present invention in a proportion of usually 0.001 to 99% by weight, preferably 0.01 to 80% by weight.

  The preparation of the present invention has an IL-2 production inhibitory effect, and can be effectively used to prevent or treat a disease that develops or worsens directly or indirectly due to increased IL-2 production.

  IL-2 production suppressive action is achieved when IL-2 producing cells such as lymphocytes isolated from blood, T cell-derived cell lines such as Jurkat, HUT78, and HL-60 are cultured in the presence of the test substance. Furthermore, it can be evaluated by measuring whether the IL-2 producing ability of the IL-2 producing cell is suppressed. In addition, methods for measuring IL-2 concentration in serum, plasma or culture supernatant by ELISA, Western blotting, multiplex assay, PCR, Northern blotting, CTLL-2 proliferation test, etc. are well known, and reagent kits are also commercially available Widely available.

  Diseases that develop or exacerbate directly or indirectly due to increased IL-2 production (IL-2 related diseases) include type IV such as atopic dermatitis, contact dermatitis, thyroiditis, and allergic encephalitis Allergic diseases; autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and lupus nephritis.

  The preparation of the present invention usually contains a pharmaceutically acceptable carrier or additive in addition to an effective amount of DDA for suppressing IL-2 production or preventing, improving or treating the above-mentioned IL-2 related diseases. Prepared. The amount of DDA compounded in the composition is appropriately selected according to the type and severity of the target disease, and the dosage form. Usually, in the case of a systemic preparation, the total weight of the preparation of the present invention. (100% by weight) of 0.001 to 50% by weight, particularly 0.01 to 10% by weight.

  Examples of the administration method of the preparation of the present invention include oral administration and parenteral administration such as intravenous administration, intramuscular administration, subcutaneous administration, transmucosal administration, transdermal administration, and rectal administration. Oral administration and intravenous administration are preferable, and oral administration is more preferable. The preparation of the present invention can be prepared into various forms of preparations (dosage forms) depending on the administration method. The preparations (dosage forms) will be described below, but the dosage forms used in the present invention are not limited to these, and various dosage forms that are usually used in the pharmaceutical preparation field can be used.

  Examples of dosage forms for oral administration include powders, granules, capsules, pills, tablets, elixirs, suspensions, emulsions, and syrups, and can be appropriately selected from these. . Moreover, modifications such as sustained release, stabilization, easy disintegration, poor disintegration, enteric solubility, easy absorption and the like can be applied to these preparations.

  In addition, dosage forms for intravenous administration, intramuscular administration, or subcutaneous administration include injections and infusions (including dried products prepared at the time of use), and can be selected as appropriate.

  In addition, dosage forms for transmucosal administration, transdermal administration, or rectal administration include mastication agents, sublingual agents, buccal agents, troches, ointments, patches, liquids, etc. Can be selected as appropriate. Moreover, modifications such as sustained release, stabilization, easy disintegration, difficulty disintegration, and easy absorption can be applied to these preparations.

  In the preparation of the present invention, pharmaceutically acceptable carriers and additives can be blended depending on the dosage form (oral dosage form or various parenteral dosage forms). Pharmaceutically acceptable carriers and additives include solvents, excipients, coating agents, bases, binders, lubricants, disintegrants, solubilizers, suspending agents, thickeners, emulsifiers, Stabilizers, buffers, tonicity agents, soothing agents, preservatives, flavoring agents, fragrances, and coloring agents can be mentioned. Specific examples of pharmaceutically acceptable carriers and additives are listed below, but the present invention is not limited thereto.

  Examples of the solvent include purified water, sterilized purified water, water for injection, physiological saline, peanut oil, ethanol, glycerin and the like. Excipients include starches (eg, potato starch, wheat starch, corn starch), lactose, glucose, sucrose, crystalline cellulose, calcium sulfate, calcium carbonate, sodium bicarbonate, sodium chloride, talc, titanium oxide, trehalose, xylitol Etc.

  Examples of binders include starch and derivatives thereof, cellulose and derivatives thereof (for example, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose), gelatin, sodium alginate, tragacanth, gum arabic and other natural polymer compounds, polyvinyl pyrrolidone, polyvinyl alcohol, etc. Synthetic polymer compounds, dextrin, hydroxypropyl starch and the like.

  Lubricants include light anhydrous silicic acid, stearic acid and its salts (eg magnesium stearate), talc, waxes, wheat denbun, macrogol, hydrogenated vegetable oil, sucrose fatty acid ester, polyethylene glycol, silicone oil, etc. be able to.

  Examples of disintegrants include starch and derivatives thereof, agar, gelatin powder, sodium bicarbonate, calcium carbonate, cellulose and derivatives thereof, hydroxypropyl starch, carboxymethylcellulose and salts thereof, and cross-linked products thereof, and low-substituted hydroxypropylcellulose. be able to.

  Examples of the solubilizer include cyclodextrin, ethanol, propylene glycol, polyethylene glycol and the like. Examples of the suspending agent include sodium carboxymethylcellulose, polypinyl pyrrolidone, gum arabic, tragacanth, sodium alginate, aluminum monostearate, citric acid, various surfactants and the like.

  Examples of the thickener include sodium carboxymethyl cellulose, polypinyl pyrrolidone, methyl cellulose, hydroxypropyl methyl cellulose, polyvinyl alcohol, tragacanth, gum arabic, sodium alginate and the like.

  Examples of the emulsifier include gum arabic, cholesterol, tragacanth, methylcellulose, lecithin, various surfactants (for example, polyoxyl 40 stearate, sorbitan sesquioleate, polysorbate 80, sodium lauryl sulfate) and the like.

  Stabilizers include tocopherols, chelating agents (eg, EDTA, thioglycolic acid), inert gases (eg, nitrogen, carbon dioxide), reducing substances (eg, sodium bisulfite, sodium thiosulfate, ascorbic acid, Rongalite) and the like. be able to.

  Examples of the buffer include sodium hydrogen phosphate, sodium acetate, sodium citrate, boric acid and the like.

  Examples of isotonic agents include sodium chloride and glucose. Examples of soothing agents include local anesthetics (procaine hydrochloride, lidocaine), pendyl alcohol, glucose, sorbitol, amino acids and the like.

  Examples of the corrigent include sucrose, saccharin, licorice extract, sorbitol, xylitol, glycerin and the like. Examples of fragrances include spruce tincture and rose oil. Examples of the colorant include water-soluble food dyes and lake dyes.

  Examples of the preservative include benzoic acid and its salts, paraoxybenzoic acid esters, chlorobutanol, reverse soap, benzyl alcohol, phenol, thimerosal, dehydroacetic acid, boric acid, and the like.

  Coating agents include sucrose, hydroxypropylcellulose (HPC), shellac, gelatin, glycerin, sorbitol, hydroxypropylmethylcellulose (HPMC), ethylcellulose, polyvinylpyrrolidone (PVP), hydroxypropylmethylcellulose phthalate (HPMCP), cellulose acetate phthalate (CAP) ), Methyl methacrylate-methacrylic acid copolymer and the above-described polymers.

  Bases include petrolatum, liquid paraffin, carnauba wax, beef tallow, hardened oil, paraffin, beeswax, vegetable oil, macrogol, macrogol fatty acid ester, stearic acid, sodium carboxymethylcellulose, bentonite, cacao butter, witepsol, gelatin, stearyl Alcohol, hydrolanolin, cetanol, light liquid paraffin, hydrophilic petrolatum, simple ointment, white ointment, hydrophilic ointment, macrogol ointment, hard fat, oil-in-water emulsion base, water-in-oil emulsion glaze etc. Can do.

  In addition, about each said dosage form, the technique of a well-known drug delivery system (DDS) is employable. The DDS preparations referred to in this specification include sustained release preparations, topical preparations (troches, buccal tablets, sublingual tablets, etc.), drug release control preparations, enteric preparations and gastric preparations, etc., administration routes, bioavailability Considering side effects and the like, it is a preparation in an optimal preparation form.

  The oral dosage of the preparation of the present invention is preferably in the range of 0.01 to 100 mg / kg, more preferably 0.02 to 10 mg / kg in terms of the amount of the carboxylic acid-free compound (1). When administered intravenously, the dosage should be such that the effective blood concentration of the carboxylic acid-free compound (1) is in the range of 0.01 to 1000 μg / mL, more preferably 0.02 to 100 μg / mL. Can do. Note that these doses may vary depending on age, sex, body type and the like.

Hereinafter, preparation examples, test examples, and examples are given to describe the present invention in more detail, but the present invention is not limited to these descriptions.
Preparation Example 1 Production of (2E, 9R) -9,10-dihydroxy-2-decenoic acid (hereinafter also referred to as “DDA1”)

First, 4-pentyn-1-ol (25) (manufactured by Sigma-Aldrich) was benzylated to give compound 26, and in the presence of BF ₃ OEt ₂ , compound 27 was made with n-BuLi and benzyl (R) -glycidyl ether. Subsequently, the alkyne was reduced by hydrogenolysis to give compound 28, which was then reacted with 2,2-dimethoxypropane to give acetal 29. Furthermore, this was oxidized with Swern, and ethyl ester 31 was synthesized through a Wittig reaction using triethyl phosphonoacetate. The acetonide of ethyl ester 31 was then removed, and the ethyl ester was removed by alkaline hydrolysis to synthesize the title (2E, 9R) -9,10-dihydroxy-2-decenoic acid 33 (DDA1).

The results of subjecting the obtained DDA1 to ¹ H-NMR measurement (JEOL: ECP500) are shown below.
¹ H-NMR (CD ₃ OD, 500 MHz): 1.36 (m, 4H), 1.50 (m, 4H), 2.23 (ddt, J = 7.1, 1.5, 7.0 Hz, 2H), 3.41 (dd, J = 11.1 , 6.5 Hz, 1H), 3.46 (dd, J = 11.1, 4.6 Hz, 1H), 3.56 (m, 1H), 5.79 (1H, dt, J = 15.6, 1.5 Hz), 6.95 (1H, dt, J = 15.6, 7.1 Hz).

Preparation Example 2 Production of (2E, 9S) -9,10-dihydroxy-2-decenoic acid (hereinafter also referred to as “DDA2”)

  First, Compound 26 produced by benzylation of 4-pentyn-1-ol (25) (manufactured by Sigma-Aldrich) was converted to Compound 34 using benzyl (S) -glycidyl ether. Aldehyde 37 was synthesized from compound 34 in three steps, and this was converted to t-butyl ester 38 by Wittig reaction using t-butyl P, P-dimethylphosphonoacetate. Then, the acetonide of t-butyl ester 38 was removed, and the t-butyl group was removed by TFA to synthesize the title (2E, 9S) -9,10-dihydroxy-2-decenoic acid 40 (DDA2).

The results of subjecting the obtained DDA2 to ¹ H-NMR measurement (JEOL: ECP500) are shown below.
¹ H-NMR (CD ₃ OD, 500 MHz): 1.36 (m, 4H), 1.50 (m, 4H), 2.23 (ddt, J = 7.1, 1.5, 7.0 Hz, 2H), 3.41 (dd, J = 11.1 , 6.5 Hz, 1H), 3.46 (dd, J = 11.1, 4.6 Hz, 1H), 3.56 (m, 1H), 5.79 (dt, J = 15.6, 1.5 Hz, 1H), 6.95 (dt, J = 15.6, 7.1 Hz, 1H).

Preparation Example 3 Preparation of (2E) -9,10-dihydroxy-2-decenoic acid (hereinafter also referred to as “DDA3”) (Part 1)
(2E) -9,10-dihydroxy-2-decenoic acid was isolated and purified from royal jelly according to the methods shown in the following (1) to (6) (see FIGS. 1 to 6).

The equipment used in the isolation and purification process is as follows.
High-speed cooling centrifuge: Hitachi CR-21 rotor: R10A
Ultrafiltration membrane: Prep / Scale-TFF Cartridge PLGC10k 1ft ² made by Millipore
Pump: Millipore filtration membrane dedicated roller pump freeze dryer: Tokyo Rika FDU-540 type.

(1) Preparation of A3 fraction by ultrafiltration membrane Add ion-exchanged water (1.5 L) to dry powder royal jelly (about 150 g), stir at room temperature for 2 hours, and then centrifuge using a high-speed cooling centrifuge (10,000 rpm, 18780 × g, 10 minutes) and fractionated into a water-soluble fraction and an insoluble fraction. Ion exchange water (0.5 L) was added to the insoluble fraction, and the same operation was performed twice (however, stirring was performed for 30 minutes).

  Finally, the remaining insoluble fraction was designated as the B1 fraction, and the three water-soluble fractions obtained were combined into one A1 fraction. The A1 fraction was fractionated with a 10 kDa ultrafiltration membrane, and the molecular weight of 10,000 or less was defined as the A3 fraction.

  The above operation was repeated 5 times to obtain 454 g of A3 fraction (lyophilized product) from 774 g of dried royal jelly powder.

(2) Fractionation by gel filtration from A3 fraction (Fig. 2)
Add ion-exchanged water (100 ml) to the A3 fraction prepared in (1) (lyophilized product: 30 to 35 g), stir at room temperature for 30 minutes, and then centrifuge (10,000 rpm, 4 ° C, 10 minutes) Fractionated into clear and precipitate (D5).

  The supernatant was injected into a gel filtration column (Tosoh Corporation: Toyopeal HW40F 2 liters, column size φ50 x 1000 mm), and the eluent (ion-exchanged water) was allowed to flow at a flow rate of 8 ml / min. Every 100 ml while detecting with UV at a wavelength of 210 nm Fractionation was performed. From 0.3 to 1.2 L, D1 fraction, from 1.3 to 2.6 L as D2 fraction, from 2.7 to 4.4 L as D3 fraction, and from 4.5 to 5.0 L as D4 fraction.

  This operation was repeated 13 times to obtain 4.66 g of the D4 fraction (lyophilized product) from 417.4 g of the A3 fraction (lyophilized product).

(3) Fractionation from D4 fraction by reverse phase (ODS) medium pressure chromatography (Figure 3)
The D4 fraction prepared in (2) is dissolved in an equal volume (1: 1) mixture of acetonitrile and 0.1% trifluoroacetic acid aqueous solution, and the preparative reversed phase column (Hi-Flash-Column ODS- Using an 8-50W-L (36 g, 2.6 × 100 mm)), fractionation was performed with a medium pressure chromatography fractionator (YFLC-Wprep2XY-W10V manufactured by Yamazen Co., Ltd.).

<Chromatography conditions>
Flow rate: 20 ml / min, fractional elution solvent: acetonitrile: 0.1% trifluoroacetic acid aqueous solution [98: 2 (8 minutes) → 0.01 minutes → 100: 0 (10 minutes)]
Detection: UV 254nm.

  The fraction that was not adsorbed by the (2:98) mixture of acetonitrile and 0.1% aqueous trifluoroacetic acid was designated as D4-I fraction, and the fraction eluted at 100% acetonitrile was designated as D4-II fraction. The same operation was repeated three times. From the D4 fraction (lyophilized product) (2.76 g), the D4-I fraction (lyophilized product) (0.81 g) and the D4-II fraction (lyophilized product) (2.26 g) Got.

(4) Fractionation of D4-II fraction by reversed phase (ODS) high performance liquid chromatography (Fig. 4)
The D4-II fraction prepared in (3) was dissolved in a (2: 5) mixture of acetonitrile and 0.1% aqueous trifluoroacetic acid and purified by high performance liquid chromatography (HPLC) under the following conditions.

<HPLC conditions>
HPLC system: Waters Delta 600
Detector: Waters 2996 (Photodiode array detector)
Preparative column: Nacalai Tesque Cosmosil 5C18-AR-II (φ10 × 250mm) + guard column (10 × 10mm)
Flow rate: 5ml / min
Detection: PDA Max plot Elution condition: Acetonitrile: 0.1% trifluoroacetic acid aqueous solution [0: 100 (10 minutes) → 50 minutes → 100: 0 (20 minutes)].

  Separation was performed by HPLC under the above elution conditions, and fractionation was performed every 10 minutes from the start of injection to obtain a total of 8 fractions (D4-II-1 to D4-II-8). This fractionation operation was repeated, and the D4-II-3 fraction (lyophilized product) (6.8 mg) was obtained from the D4-II fraction (lyophilized product) (49.3 mg).

(5) Fractionation from D4-II-3 fraction by reversed phase (ODS) high performance liquid chromatography (Figure 5)
The D4-II-3 fraction prepared in (4) was dissolved in a (1: 1) mixture of acetonitrile and 0.1% aqueous trifluoroacetic acid, and purified by high performance liquid chromatography (HPLC) under the following conditions.

<HPLC conditions>
HPLC system: Waters Delta 600
Detector: Waters 2996 (Photodiode array detector)
Preparative column: Nacalai Tesque Cosmosil 5C18-AR-II (φ10 × 250mm) + guard column (10 × 10mm)
Flow rate: 5ml / min
Detection: PDA Max plot Elution condition: Acetonitrile: 0.1% trifluoroacetic acid aqueous solution [13:87 (30 minutes) → 10 minutes → 0: 100 (10 minutes)].

  Separation by HPLC under the above elution conditions and fractionation using the chromatographic peak as an index gave a total of 8 fractions (D4-II-3A to D4-II-3H). This fractionation operation was repeated to obtain the D4-II-3E fraction (lyophilized product) (0.9 mg) from the D4-II-3 fraction (lyophilized product) (15.6 mg).

(6) Separation from D4-II-3E fraction by normal phase chromatography (Fig. 6)
D4-II-3E fraction (lyophilized product) (16.6 mg) prepared in (5) was dissolved in 0.5 ml of an equal volume (1: 1) mixture of chloroform and methanol, and normal phase column chromatography under the following conditions: Purification was performed by graphy.

<Normal phase column chromatography conditions>
Silica for separation: Fuji Silysia Chemical Silica Gel BW-300 (5g)
Size: φ15 × 44mm
Elution conditions: chloroform: methanol [95: 5 (35 ml) → 9: 1 (15 ml)].

  Under the above elution conditions, every 2 ml of the eluate was subjected to fractionation while confirming by thin-phase chromatography using anisaldehyde sulfuric acid coloration. As a result, a total of four fractions (D4-II-3E-1 to D4-II) were obtained. -3E-4) was obtained. This purification operation was repeated to obtain a D4-II-3E-4 fraction (lyophilized product) (3.3 mg).

(7) Identification of D4-II-3E-4 fraction The D4-II-3E-4 fraction obtained in (6) above was analyzed by FAB-MS (JEOL JMS-T100LC type (matrix: polyethylene glycol )), ¹ H-NMR measurement (manufactured by JEOL: ECP-500) and ¹³ C-NMR measurement (manufactured by Varian Technologies Japan Limited: Varian-Unity500 type), the following results were obtained.

[α] ²⁶ _D + 4.44 ° (c 0.495, MeOH)
FAB-MS: m / z 203 [M + H] ^-
1 H NMR (500 MHz, CD ₃ OD): 1.30 to 1.55 (6H, m, H-5 to H-8), 2.22 (2H, dq, J = 1.6, 7.3 Hz, H-4), 3.41 (1H , dd, J = 6.4, 11.0, H-10), 3.46 (1H, dd, J = 4.6, 110, H-10), 3.56 (1H, m, H-9), 5.79 (1H, dt, J = 15.6, 1.6 Hz, H-2), 6.95 (1H, dt, J = 15.6, 7.1 Hz, H-3).
¹³ C-NMR (125 MHz, CD ₃ OD): 23.4, 29.2, 30.3, 33.0, 34.3 (C-4 to C-8), 67.4 (C-10), 73.2 (C-9), 122.6 (C- 2), 151.1 (C-3), 177.7 (C-1).

  From this result, the compound contained in the D4-II-3E-4 fraction was identified as (2E) -9,10-dihydroxy-2-decenoic acid represented by the following formula. Since (2E) -9,10-dihydroxy-2-decenoic acid is a component contained in royal jelly that has been eaten, it is a highly safe compound without side effects even if taken orally. Conceivable.

The obtained (2E) -9,10-dihydroxy-2-decenoic acid was methylated by the method shown in the following formula and then treated with (S) -MTPACl to prepare (R) -MTPA ester. ¹ H-NMR of the obtained (R) -MTPA ester was measured, and the area ratio of the proton signal attributed to the 10-position was calculated. As a result, (2E) -9,10-dihydroxy-2-decenoic acid (DDA3) obtained above is a racemate, the abundance ratio of each steric is R: S = 3: 1, and calculation error is considered. It was found that at least R: S = 2.8 to 3.8: 1.

Preparation Example 4 Preparation of (2E) -9,10-dihydroxy-2-decenoic acid (hereinafter also referred to as “DDA3”) (Part 2) (see FIG. 7)
1500 g of methanol was added to 200 g of dry powder royal jelly (produced from Zhejiang Pinghu, Zhejiang Province) and extracted for 12 hours while stirring at room temperature. The eluate was filtered under reduced pressure, and the solvent of the filtrate was distilled off under reduced pressure. The obtained residue (108.9 g) was subjected to ODS column chromatography (column: Cosmosil 75C18-PREP, column size: φ80 × 205 mm, eluent: 10%, 50% aqueous methanol solution, 2500 mL each (1250 mL × 2 fractions) ) To fractions 1 (first half of 10% methanol elution zone), 2 (second half of 10% methanol elution zone), 3 (first half of 50% methanol elution zone), 4 (second half of 50% methanol elution zone) It was divided. Fraction 4 was purified by silica gel column chromatography (column: Daiso gel IR-60, column size: φ26 × 150 mm, eluent: chloroform containing 5% methanol, 500 mL (50 mL × 10 fraction)), 7- The 10th eluted fractions were combined and concentrated. This was purified again by silica gel column chromatography (column: Daiso gel IR-60, column size: φ26 × 150 mm, eluent: chloroform containing 5% methanol, 600 mL (60 mL × 10 fraction)), 5th The fraction eluted in HPLC was further HPLC (column: Cosmosil 5C18-AR column (Nacalai Tesque, 10 x 250 mm), eluent: 0.1% TFA containing 13% acetonitrile, flow rate: 5.0 mL / min, monitor: 220 nm) To obtain 13.3 mg of (2E) -9,10-dihydroxy-2-decenoic acid (DDA3).

Preparation Example 5 Production of (2E, 7R) -7,8-dihydroxy-2-octenoic acid (hereinafter also referred to as “DDA4”)

Propargyl alcohol (41) (manufactured by Fluka) is reacted with benzyl-protected compound 42 in the presence of BF ₃ OEt ₂ with benzyl (R) -glycidyl ether (43) (manufactured by Sigma-Aldrich) and n-BuLi. Carbon extended compound 44 was synthesized. This was converted to compound 45 by hydrogenolysis, and then reacted with 2,2-dimethoxypropane in the presence of PTS to obtain acetal 46. Next, acetal 46 was converted to aldehyde 47 by Swern oxidation, and compound 48 was synthesized by Wittig reaction using t-butyl P, P-dimethylphosphonoacetate. The acetonide and t-butyl groups were removed by TFA to synthesize the title (2E, 7R) -7,8-dihydroxy-2-octenoic acid 49 (DDA4).

The results of subjecting the obtained DDA4 to ¹ H-NMR measurement (manufactured by JEOL: Alpha-400) are shown below.
¹ H-NMR (400 MHz, DMSO): 1.12-1.59 (m, 4H), 2.16 (m, 2H), 3.12-3.55 (m, 3H), 5.76 (d, J = 15.6 Hz, 1H), 6.82 ( dt, J = 6.8, 7.1 Hz, 1H).

Preparation Example 6 Production of (2Z, 11R) -11,12-dihydroxy-2-dodecenoic acid (hereinafter also referred to as “DDA5”)

First, 6-Heptyn-1-ol (50) (manufactured by Sigma-Aldrich) was benzylated to obtain Compound 51. Next, in the presence of BF ₃ OEt _2, the compound 52 was synthesized by reacting with benzyl (R) -glycidyl ether (43) (manufactured by Sigma-Aldrich) and n-BuLi. The alkyne was further reduced by hydrogenation (compound 53), and then reacted with 2,2-dimethoxypropane in the presence of PTS to obtain acetal 54. Acetal 54 was converted to aldehyde 55 by Swern oxidation, and then compound 56 was obtained by Wittig reaction using triethyl phosphonoacetate. After removing acetonide of Compound 56, the title (2Z, 11R) -11,12-dihydroxy-2-dodecenoic acid 58 (DDA5) was synthesized by alkaline hydrolysis reaction.

The results of subjecting the obtained compound DDA5 to ¹ H-NMR measurement (manufactured by JEOL: Alpha-400) are shown below.
¹ H-NMR (400 MHz, DMSO): 1.14-1.43 (m, 12H), 2.15 (m, 2H), 3.18-3.45 (m, 3H), 5.75 (d, J = 15.6 Hz, 1H), 6.83 ( dt, J = 6.8, 7.1 Hz. 1H).

Preparation Example 7 Production of (2E, 9S) -9,10-diamino-2-decenoic acid trifluoroacid salt (hereinafter also referred to as “DDA6”)

The hydroxyl group of triol 29 obtained by the method of Preparation Example 1 was protected with a TBDPS group (Compound 61), then acetonide was removed with an acid (Compound 62), and then the hydroxyl group was protected with an Ms group to synthesize Compound 63. Compound 63 was reacted with NaN ₃ to give azide 64, which was protected with a Boc group to give compound 65. The TBDPS group of Compound 65 was removed by TBAF, and Compound 68 was obtained by Swit oxidation and Wittig reaction using t-butyl P, P-dimethylphosphonoacetate. The Boc group of Compound 68 was removed with TFA to synthesize (2E, 9R) -9,10-diamino-2-decenoic acid trifluoroacid salt (69) (DDA6).

The results of subjecting the obtained compound DDA6 to ¹ H-NMR measurement (manufactured by JEOL: Alpha-400) are shown below.
¹ H-NMR (400 MHz, DMSO): 1.21-1.69 (m, 8H), 2.19 (m, 2H), 3.02 (m, 2H), 3.28 (m, 1H), 5.77 (d, J = 15.6 Hz, 1H), 6.82 (dt, J = 6.8, 1.2 Hz. 1H).

Preparation Example 8 Production of (2Z, 9R) -9,10-dihydroxy-2-decenoic acid (hereinafter also referred to as “DDA7”)

  The acetonide 3 synthesized by the method described in Preparation Example 12 was swern oxidized to aldehyde 18 and then subjected to Wittig reaction using Ethyl ditrifluoroacetyl-phosphoacetate. Next, the acetonide is removed by alkaline hydrolysis with sodium hydride (compound 19), and finally the alkaline hydrolysis with sodium hydroxide is performed to give the title (2Z, 9R) -9,10-dihydroxy-2-decenoic acid. (20) (DDA7) was synthesized.

The results of subjecting the obtained DDA7 to ¹ H-NMR measurement (manufactured by JEOL Ltd .: Alpha-400) are shown below.
¹ H-NMR (400 MHz, DMSO): 1.39-1.18 (m, 8H), 2.58-2.50 (m, 2H), 3.19-4.34 (m, 3H), 5.71 (d, J = 11.6 Hz, 1H), 6.21 (dt, J = 11.6, 7.6 Hz, 1H).

Preparation Example 9 Production of 3,10-dihydroxydecenoic acid (hereinafter also referred to as “DDA9”) To 200 g of dry powder royal jelly (Zhejiang Pinghu, Zhejiang), add 1500 mL of methanol and stir at room temperature for 12 hours. Extracted. The eluate was filtered under reduced pressure, and the solvent of the filtrate was distilled off under reduced pressure. The obtained residue (108.9 g) was subjected to ODS column chromatography (column: Cosmosil 75C18-PREP, column size: φ80 × 205 mm, eluent: 10%, 50% aqueous methanol solution, 2500 mL each (1250 mL × 2 fractions) And purified into fractions 1 (first half of the 10% methanol elution zone), 2 (second half of the 10% methanol elution zone), 3 (first half of the 50% methanol elution zone), and 4 (second half of the 50% methanol elution zone) Fraction 4 was purified by silica gel column chromatography (column: Daiso gel IR-60, column size: φ26 × 150 mm, eluent: chloroform containing 5%, 10% methanol, 500 mL (50 mL × 10 fraction)) 10% of the 4th eluted fraction was concentrated, and this was again subjected to silica gel column chromatography (column: Daiso gel IR-60, column size: φ26 × 100 mm, eluent: 10, containing 15% methanol) Chloroform, 300 mL Purification was carried out (20 mL × 15 fractions), and the 13th to 19th eluting fractions were collected to obtain 23.7 mg of the title 3,10-dihydroxydecenoic acid (DDA9).

The results of subjecting the obtained compound DDA9 to ¹ H-NMR measurement (manufactured by JEOL Ltd .: ECA600) are shown below.
¹ H-NMR (600 MHz, CD ₃ OD): 1.34 (m, 4H), 1.35 (m, 2H), 1.47 (m, 4H), 1.52 (m, 2H), 2.36 (dd, J = 15.1, 8.0 Hz, 1H), 2.43 (dd, J = 15.1, 4.5 Hz, 1H), 3.53 (t, J = 6.6 Hz, 2H), 3.96 (m, 1H).

Preparation Example 10 Production of (9R) -9,10-dihydroxydecanoic acid (hereinafter also referred to as “DDA11”)

  Acetonide 3 synthesized by the method described in Preparation Example 12 was subjected to an ozonolysis reaction, followed by a Wittig reaction using triethyl phosphonoacetate to synthesize compound 70. Compound 70 was converted to Compound 72 through hydrogenation and removal of acetonide. Subsequently, the ethyl ester of Compound 72 was removed by an alkaline hydrolysis reaction to synthesize the title (9R) -9,10-dihydroxydecanoic acid 73 (Compound DDA11).

The results of subjecting the obtained compound DDA11 to ¹ H-NMR measurement (JEOL: Alpha-400) are shown below.
¹ H-NMR (400 MHz, CD ₃ OD): 1.30-1.20 (m, 6H), 1.41-1.40 (m, 2H), 1.53-1.50 (m, 2H), 2.07 (t, 7.6Hz, 2H), 3.32 (dd, 11.2, 6.2Hz, 1H), 3.38 (dd, 11.2, 4.3Hz, 1H), 3.48 (m, 1H).

Preparation Example 11 Production of (2E, 9R) -methyl-9,10-dihydroxy-2-decenoate (hereinafter also referred to as “DDA12”)

From the aldehyde 30 synthesized by the method described in Preparation Example 1, Wittig reaction with methyl (triphenylphosphoranylidene) acetate was performed to synthesize Compound 74. The acetonide was then removed by acid hydrolysis reaction to give the title (2E, 9R) -methyl-9,10-dihydroxy-2-decenoate (75) (DDA12).
The results of subjecting the obtained compound DDA12 to ¹ H-NMR measurement (JEOL: Alpha-400) are shown below.
¹ H-NMR (400 MHz, CDCl ₃ ): 1.38-1.29 (m, 2), 1.50-1.39 (m, 4H), 2.20 (dd, 14.0, 6.8Hz, 2H), 2.27 (m, 1H), 2.36 (m, 1H), 3.42 (dd, 10.8, 7.8Hz, 1H), 3.64 (dd, 10.8, 2.8Hz, 1H), 3.68 (m, 1H), 3.72 (s, 3H), 5.81 (dt, 15.6, 1.6Hz, 1H), 6.95 (dt, 15.6, 6.8Hz, 1H).

Preparation Example 12 Production of (2E, 9R) -9,10-diacetoxy-2-decenoic acid (hereinafter also referred to as “DDA13”)

  Grignard reaction using commercially available (4R) -4- (2-hydroxyethyl) -2,2-dimethyl-1,3-dioxolane (1) (Sigma-Aldrich) and 5-bromo-1-pentene To give (R) -2,2-dimethyl-4- (oct-7-enyl) -1,3-dioxolane 3. Diol 4 obtained by treating this with acetic acid was acetylated to give compound 76. Subsequently, an aldehyde was obtained by an ozonolysis reaction, followed by a Wittig reaction with t-buthyl diethylphosphonoacetate to obtain Compound 77. The t-butyl group was removed from compound 77 by acid hydrolysis reaction to synthesize the title (2E, 9R) -9,10-diacetoxy-2-decenoic acid (78) (DDA13).

The results of subjecting the obtained compound DDA13 to ¹ H-NMR measurement (JEOL: Alpha-400) are shown below.
¹ H-NMR (400 MHz, CDCl ₃ ): 1.40-1.29 (m, 4H), 1.62-1.54 (m, 2H), 2.06 (s, 3H), 2.07 (s, 3H), 2.27 -2.20 (m, 2H), 4.03 (dd, 11.6, 6.4Hz, 1H), 4.22 (dd, 11.6, 3.2Hz, 1H), 5.06 (m, 1H), 5.82 (dt, 15.6, 2.6Hz.1H), 7.05 (dt, 15.6, 7.0Hz, 1H).

Preparation Example 13 Production of (2E, 9S) -9-hydroxy-2-decenoic acid (hereinafter also referred to as “DDA14”)

  The diol 4 obtained by the method described in Preparation Example 12 was subjected to tosylation of the compound primary hydroxyl group. Next, ozonolysis and Wittig reaction using trimethyl phosphonoacetate were performed. After removing the THP group by acid hydrolysis with PPTS, alkaline hydrolysis with potassium hydroxide was performed to give the title (2E, 9S) -9-hydroxy -2-decenoic acid 11 (DDA14) was synthesized.

The results of subjecting the obtained DDA14 to ¹ H-NMR measurement (manufactured by JEOL: Alpha-400) are shown below.
¹ H-NMR (400 MHz, CD ₃ OD): 1.13 (d, J = 6.0 Hz, 3H), 1.50-1.34 (m, 8H), 2.23-2.19 (m, 2H), 3.69 (m, 1H), 5.79 (dt, J = 15.6, 1.4 Hz, 1H), 6.94 (dt, J = 15.6, 6.8 Hz, 1H).

Preparation Example 14 Production of (2E, 9R) -9-hydroxy-2-decenoic acid (hereinafter also referred to as “DDA15”)

  Compound 7 obtained by the method described in Preparation Example 13 was used as benzoate (12) whose steric structure was inverted by Mitsunobu reaction. Thereafter, the title (9R, 2E) -9-hydroxy-2-decenoic acid 17 (DDA15) was synthesized in the same manner as described in Preparation Example 13.

The results of subjecting the obtained DDA15 to ¹ H-NMR measurement (manufactured by JEOL Ltd .: Alpha-400) are shown below.
¹ H-NMR (400 MHz, CDCl ₃ ): 1.03 (d, J = 8.0 Hz, 3H), 1.44-1.20 (m, 8H), 2.17-2.11 (m, 2H), 3.62 (m, 1H), 5.71 (br.d, J = 15.6 Hz, 1H), 6.86 (dt, J = 15.6, 7.2 Hz, 1H).

Preparation Example 15 Synthesis of (2E) -7-acetoxy-2-heptenoic acid (hereinafter also referred to as “DDA16”)

  First, commercially available δ-valerolactone (22) (manufactured by Sigma-Aldrich) was converted to aldehyde 23 by DIBAL reduction. Furthermore, Wittig reaction using tert-butyl diethylphosphonoacetate, acetylation with acetic anhydride and pyridine, and finally removing the t-BuO group with trifluoroacetic acid, the title (2E) -7-acetoxy -2-Heptenoic acid 24 (DDA16) was synthesized.

The results of subjecting the obtained DDA16 to ¹ H-NMR measurement (manufactured by JEOL Ltd .: Alpha-400) are shown below.
¹ H-NMR (400 MHz, CDCl ₃ ): 1.71-1.52 (m, 4H), 2.05 (s, 3H), 2.30-2.21 (m, 2H), 4.07 (t, J = 6.2 Hz, 2H), 5.85 (dt, 15.6, 1.6 Hz, 1H), 7.06 (dt, 15.6, 7.0 Hz, 1H).

Preparation Example 16 Preparation of a mixture of (2E, 9R) -9,10-dihydroxy-2-decenoic acid (DDA1) and (2E, 9S) -9,10-dihydroxy-2-decenoic acid (DDA2) In Preparation Example 1 The synthesized (2E, 9R) -9,10-dihydroxy-2-decenoic acid (DDA1) and (2E, 9S) -9,10-dihydroxy-2-decenoic acid (DDA2) synthesized in Preparation Example 2 The mixture was mixed to a weight ratio of 3.3: 1 to prepare a mixed composition (hereinafter also referred to as “DDA10”).

Test example 1
Compounds (DDA1 to DDA7, DDA9, DDA11 to DDA16) and mixtures (DDA10) prepared in Preparation Examples 1 to 16 above, and (2E) -10-hydroxy-2-decenoic acid (Made) from Matsuura Pharmaceutical Co., Ltd. ( (Hereinafter also referred to as “DDA8”) was prepared using DMSO to a concentration of 2 mM (sample solutions 1 to 16).

Human peripheral lymphocytes were isolated from blood collected from one healthy male (28 years old) according to a standard method. The obtained human peripheral lymphocytes are suspended in RPMI-1640 medium (containing 10% FBS, 100 units / ml penicillin, 100 μg / ml streptomycin) to 1 × 10 ⁶ cells / ml and prepared as above. Each of the DDA1 to DDA16 sample solutions 1 to 16 was added to a concentration of 0.5% and cultured at 37 ° C. for 48 hours. After culture, the culture supernatant was obtained by centrifugation.

  The IL-2 content in the obtained culture supernatant was measured according to a manual using an IL-2 measurement kit (BD OptEIA ELISA Set, Human IL-2: Nippon Becton Dickinson Co., Ltd.).

  As a control test (-), DMSO not containing DDA as a test solution was added to human peripheral lymphocytes at a rate of 0.5%, and cultured at 37 ° C for 48 hours. IL-2 content was measured. With this IL-2 content as 100%, the relative ratio (%) of the IL-2 content in the culture supernatant cultured by adding each test solution 1-16 was calculated.

  The results are shown in FIG.

  As can be seen from FIG. 8, it was confirmed that all of DDA1 to DDA16 suppress the production of IL-2 in human lymphocytes. Among them, DDA8, DDA11, DDA13 and DDA14, especially DDA8 and DDA14, particularly DDA14, were found to have a high inhibitory effect on IL-2 production in human lymphocytes.

Test example 2
The compound (DDA4, DDA6, DDA7, DDA14 + 15, DDA16) was administered to an ovariectomized pathological model animal, and a forced swimming test (Nature, 266: 730-732, 1977) was performed. The IL-2 concentration in the medium was measured. As for DDA-14 and DDA-15, their racemates have been reported as natural products.Here, they are mixed at a weight ratio of 3: 1 so that the ratio is the same as this (2E, Used as 9S + 9R) -9-hydroxy-2-decenoic acid (racemic) (DDA14 + 15).

(1) Preparation of ovariectomized disease model animal An ICR female mouse (9 weeks old, body weight 29-33 g) was used as a test animal. All mice were housed in groups of 10 cages, and food (floor white flakes) was changed once a week. Ovariectomy was performed by the following method.

<Ovariectomy>
Under the pentobarbital (65mg / kg, ip) anesthesia, about 5mm incision in the dorsal abdomen of the mouse, the exposed adipose tissue around the ovary and ovary is temporarily exposed outside the body, after ligating between the ovary and the uterine end, Remove the ovaries. Thereafter, the tissue around the uterus and ovary is immediately returned to the abdominal cavity, and then the abdominal wall and skin are sutured.

(2) Administration compound of test substance (DDA4, DDA6, DDA7, DDA14 + 15, DDA16) 0.2 mg was dissolved in 50 ml of ion-exchanged water to prepare a suspension. This is the above test animal (ovariectomized pathological model mouse), using an oral sonde at a rate of 0.1 ml per 10 g of body weight for 14 days from the day after the ovary was removed (the day after the ovariectomy was taken as the first day of administration), Oral administration was carried out once a day (test group) (40 μg / kg / day). As a control experiment, distilled water for injection instead of DDA was orally administered to a test animal (an ovariectomized disease model mouse) at a rate of 0.1 ml per 10 g of body weight (control group).

(3) Forced swimming test Oral administration of test animals (ovariectomized pathological model mice) administered with each test substance (DDA4, DDA6, DDA7, DDA14 + 15, DDA16) for 14 days from the day after the ovary was removed Two hours after administration, the cells were placed in a water bath and subjected to forced swimming for 10 minutes (test group). In addition, as a control experiment, a similar test was performed on a test animal (an ovariectomized pathological model mouse) that was orally administered with distilled water for injection instead of DDA (control group).

(4) Measurement of serum IL-2 concentration Blood was collected from the test animals (test group, control group) subjected to forced swimming as described above, and the IL-2 concentration (pg / ml) in the blood was determined as IL-2. Measurement was performed according to the manual using a measurement kit (RSD M2000 IL-2 ELISA Kit mouse: R & S Systems).

(5) The results are shown in FIG. The results are average values of 6 test animals in both the test group and the control group.

  As can be seen from FIG. 9, the IL-2 content in the blood of the control group not administered with the test substance DDA was as high as 88.11 pg / ml, whereas each test substance (DDA4, DDA6, DDA7, DDA14 + 15, In the test group administered DDA16), the IL-2 content in the blood was 45 pg / ml or less, and it was confirmed that the IL-2 concentration in the blood decreased by oral administration of DDA.

  As can be seen from the above results, it was revealed that DDA1 to DDA16 have an IL-2 production inhibitory action.

Example 1 : Capsule
DDA1 (Preparation Example 1) 5 mg
Dry refined yeast (Asahi Beer Yakuhin Co., Ltd.) 344 mg
Sucrose fatty acid ester (Taiyo Kagaku Co., Ltd.) 1 mg
The above components (powder) were uniformly mixed, and 350 mg of the obtained powder was put into a No. 1 hard capsule to give a capsule (Example 1).

  Moreover, it replaced with DDA1 (preparation example 1), and DDA2-DDA16 was used and the capsule was prepared similarly.

Example 2 : Drink agent
DDA1 (Preparation Example 1) 5 mg
Lemon juice (Pokka Corporation) 20 ml
Propolis (Api Co., Ltd.) 0.2 g
Vitamin C (Takeda Pharmaceutical Co., Ltd.) 0.2 g
Honey (Api Co., Ltd.) 13.0 g
The above 6 components were dissolved in water and filled into a brown bottle to obtain a drink (100 ml for one bottle).

  Moreover, it replaced with DDA1 (preparation example 1) and prepared the drink agent similarly using DDA2-DDA16.

Example 3 : Hard capsule
DDA1 (Preparation Example 1) 5 mg
Lactose (Api Co., Ltd.) 150 mg
Theanine (Taiyo Kagaku Co., Ltd.) 50 mg
The above components (powder) were uniformly mixed, and 350 mg of the obtained powder was put into a No. 1 gelatin hard capsule to form a capsule.

  Moreover, it replaced with DDA1 (preparation example 1) and prepared the capsule in the same way using DDA2-DDA16.

Example 4 : Drink agent
DDA1 (Preparation Example 1) 5 mg
Honey (Api Co., Ltd.) 7,500 mg
Ezoukogi Extract (Yakuhan Pharmaceutical Co., Ltd.) 2,500 mg
Propolis extract (Api Co., Ltd.) 1,000 mg
Vitamin C (Takeda Pharmaceutical Co., Ltd.) 300 mg
Citric acid (API Corporation) 100 mg
The above six components were dissolved in water and filled into a brown bottle to obtain a drink (30 ml for one bottle).

  Moreover, it replaced with DDA1 (preparation example 1) and prepared the drink agent similarly using DDA2-DDA16.

  Since the compound (1) targeted by the present invention has an interleukin-2 production inhibitory action and can inhibit the activator and growth factor of T lymphocytes, an interleukin-2 production inhibitor As well as pharmaceutical compositions such as a therapeutic agent for type IV allergy and a therapeutic agent for autoimmune diseases.

Claims (1)

The following general formula (1):

Wherein R ¹ is a hydrogen atom or a hydroxy group; R ² and R ³ are the same or different,
It means a hydrogen atom, an amino group, a hydroxy group or a lower alkanoyloxy group. n means an integer of 2 to 7. )
An interleukin-2 production inhibitor comprising as an active ingredient a carboxylic acid represented by the formula (1) or a pharmaceutically acceptable salt or ester thereof.

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