JP2004210768A - Novel compound and pharmaceutical composition containing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel compound and a pharmaceutical composition containing the same. <P>SOLUTION: A formyl-substituted phenyl or pyrrole compound is represented by any one of formulae I to IV (wherein R<SP>1</SP>is a group to be selected from the group consisting of H, OR<SP>3</SP>, OR<SP>4</SP>, NHR<SP>3</SP>, NHR<SP>4</SP>, SR<SP>3</SP>, SR<SP>4</SP>, SO<SB>3</SB>R<SP>3</SP>, SO<SB>3</SB>R<SP>4</SP>, (CH<SB>2</SB>)<SB>n</SB>R<SP>3</SP>, (CH<SB>2</SB>)<SB>n</SB>R<SP>4</SP>, (CH=CH)<SB>n</SB>R<SP>3</SP>, (CH=CH)<SB>n</SB>R<SP>4</SP>, C(=O)R<SP>3</SP>, C(=O)R<SP>4</SP>, PhR<SP>3</SP>, PhR<SP>4</SP>, and (CH<SB>2</SB>)<SB>n</SB>CHR<SP>3</SP>R<SP>4</SP>; R<SP>2</SP>is a group to be selected from the group consisting of the same groups as defined in the R<SP>1</SP>and C(NHR<SP>3</SP>)R<SP>3</SP>; R<SP>3</SP>is a substituted or non-substituted straight-chain 1-6C alkyl; R<SP>4</SP>is a straight-chain 1-6C alkyl substituted with a carboxyl group; and n is an integer of 0-6). <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to novel compounds. More specifically, the present invention relates to a compound having a novel formylphenyl structure or a compound having a formylpyrrole structure, and a pharmaceutical composition containing the compound as an active ingredient.

  In recent years, as the desire to maintain health has increased, many people have taken health foods and health supplements. As health foods and health supplements, natural materials containing components with biological activity such as immunostimulatory activity and anticancer activity, which have almost no side effects, and the components contained in them, have attracted attention, and have immunostimulatory and carcinogenic effects Numerous health foods are commercially available. These health foods are generally provided in the form of a hot water extract of a natural material. Examples of such natural materials containing physiologically active components include mushrooms and agaricus mushrooms.

In general, what is called the mushroom is a mushroom belonging to the Basidiomycetes agaricaceae of "Kawariharatake" and "Agaricus blazei Murill A garicus b lazei M urll" Japanese name the scientific name. Agaricus mushroom (hereinafter generally referred to as Kawariharatake or ABM) is traditionally used as a medicine in the Piedade region, located in the State of Sao Paulo, Brazil. Kawariharatake is said to have various immunostimulatory activities, carcinogenic preventive effects, and tumor growth inhibitory effects, and is currently widely taken as a health food.

  Many polysaccharides having antitumor activity have been isolated from mushrooms (Hamuro J et al. (1978), Cancer Res. 38: 3080-3085, Non-Patent Document 1; Mizuno T et al. (1992), Biosci. Biotechnol). Biochem. 56: 347-348, Non-Patent Document 2).

  The polysaccharide contained in Kawariharatake has antitumor activity against sarcoma 180 and contains a β-1,6-glucopyranosyl residue (Ebina T et al. (1986), Jpn. J. Cancer Res 77: 1034-). 1042, Non-Patent Document 3). Kawariharatake extract contains (1 → 4) -α-D-glucan with (1 → 6) -β branch and has selective antitumor activity mediated via natural killer cell activation and apoptosis. (Fujimiya Y et al. (1998), Cancer Immunol Immunother 46: 147-159, Non-Patent Document 4). In the double transplanted tumor system, peptidoglycan contained in Kawariharatake has a direct cytotoxic effect on Meth A tumor cells and an indirect immunopotentiating effect on tumor-bearing mice. (Ebina T et al. (1998), Biotherapy 11: 259-265, Non-Patent Document 5). The polysaccharide contained in Kawariharatake changed the ratio of spleen Thy1,2- and L3T4 positive cells in T cell subsets in mice (Mizuno M et al. (1998), Biosci. Biotechnol. Biochem. 62: 434-437, non-patented). Reference 6). These reports suggest that the polysaccharide contained in Kawariharatake has a cytotoxic effect on tumor cells through immunomodulatory activity.

Except for polysaccharides, there are almost no reports on the components of Kawariharatake that exhibit physiological activity. In general, the quality of health foods and health supplements derived from natural materials is due to differences in the production area, harvest time, extraction method, etc. of the materials, or due to the variety of components contained. It is difficult to provide certain products.
Hamuro J et al. (1978), Cancer Res. 38: 3080-3085 Mizuno T et al. (1992), Biosci. Biotechnol. Biochem. 56: 347-348 Ebina T et al. (1986), Jpn. J. Cancer Res 77: 1034-1042 Fujimiya Y et al. (1998), Cancer Immunol Immunother 46: 147-159. Ebina T et al. (1998), Biotherapy 11: 259-265. Mizuno M et al. (1998), Biosci. Biotechnol. Biochem. 62: 434-437

  The present invention provides a novel compound having a physiological activity including an immunostimulatory activity and a superoxide dismutase-like activity, whereby a natural product is used as a raw material and a constant quality can be obtained in place of a conventional product in which the quality tends to fluctuate. And a composition having the physiological activity of

  The present inventors have purified and separated low molecular components derived from Agaricus blazei Murill (Agaricus blazei Murill), determined the chemical structure thereof, and found that a compound having a formylphenyl structure exists. The inventors of the present invention have synthesized and studied various derivatives based on their chemical structures. As a result, compounds having a formylphenyl structure represented by formulas (I) to (X) or formylphenyl The present inventors have found that a compound having a pyrrole-type structure has various physiological activities including an immunostimulatory activity and a superoxide dismutase-like activity, in particular, an ability to induce dendritic cells (DCs) derived from human peripheral blood monocytes. The invention has been completed.

  The present invention provides the following to achieve the above object.

  (1) A formyl-substituted phenyl or pyrrole compound represented by any of the following formulas I to IV,

ここで、Ｒ^１は、Ｈ、ＯＲ^３、ＯＲ^４、ＮＨＲ^３、ＮＨＲ^４、ＳＲ^３、ＳＲ^４、ＳＯ_３Ｒ^３、ＳＯ_３Ｒ^４、（ＣＨ_２）_ｎＲ^３、（ＣＨ_２）_ｎＲ^４、（ＣＨ＝ＣＨ）_ｎＲ^３、（ＣＨ＝ＣＨ）_ｎＲ^４、Ｃ（＝Ｏ）Ｒ^３、Ｃ（＝Ｏ）Ｒ^４、ＰｈＲ^３、ＰｈＲ^４、および（ＣＨ_２）_ｎＣＨＲ^３Ｒ^４からなる群から選択される基であり、
Ｒ^２は、上記Ｒ^１で定義した基と同意義の基およびＣ（ＮＨＲ^３）Ｒ^３からなる群から選択される基であり、
Ｒ^３は、置換または非置換の直鎖Ｃ_１〜Ｃ_６アルキルであり、
Ｒ^４は、カルボキシル基で置換された直鎖Ｃ_１〜Ｃ_６アルキルであり、
ｎは、０〜６の整数である、化合物。

Here, R ¹ is H, OR ³ , OR ⁴ , NHR ³ , NHR ⁴ , SR ³ , SR ⁴ , SO ₃ R ³ , SO ₃ R ⁴ , (CH ₂ ) _n R ³ , (CH ₂ ) _{n ^{R 4, (CH = CH)}} n R 3, (CH = CH) n R 4, C (= O) R 3, C (= O) R 4, PhR 3, PhR 4, and _{(CH 2)} n CHR ^A group selected from the group consisting of ³ R ⁴ ,
R ² is a group having the same meaning as the group defined for R ¹ and a group selected from the group consisting of C (NHR ³ ) R ³ ;
R ³ is a substituted or unsubstituted linear C ₁ -C ₆ alkyl;
R ⁴ is a linear C ₁ -C ₆ alkyl substituted with a carboxyl group;
The compound wherein n is an integer from 0 to 6.

(2) The compound according to item (1), which has a 4-formylphenyl structure represented by the above formula II,
Here, the compound wherein R ¹ is a straight-chain C ₁ -C ₆ alkyl substituted with a terminal carboxyl group, or an amino acid substituted with a terminal hydrazino group or a terminal amino group.

(3) In the above formula II, ^{R 1} represents _{NH-NH-R a} or _{NH-R a,} where, _{R a} is, alpha-or β- aspartyl or alpha-, with β- or γ- glutamyl, A compound according to item (2).

  (4) The compound according to item (3) represented by the following formula V:

（５）項（１）に記載の化合物、および薬学的に受容可能なキャリアを含む、薬学的組成物。

(5) A pharmaceutical composition comprising the compound according to (1) and a pharmaceutically acceptable carrier.

  (6) The pharmaceutical composition according to item (5), which has an immunostimulatory activity.

  (7) The composition according to item (6), wherein the immunostimulatory activity is a dendritic cell-inducing activity-inducing ability.

  (8) The composition according to item (5), which has superoxide dismutase (SOD) -like activity.

(9) The compound according to item (1), which has a 2-formylpyrrole structure represented by the above formula IV,
Here, R ¹ and R ² are independently OR ⁴ , NHR ⁴ , SR ⁴ , SO ₃ R ⁴ , (CH ₂ ) _n R ³ , (CH ₂ ) _n R ⁴ , (CH = CH) a _{^{n R 4, C (= O}} ) R 4, PhR 4 and _{(CH 2)} n ^CHR 3 groups selected from the group consisting of ^{R 4,}
R ³ is a substituted or unsubstituted linear C ₁ -C ₆ alkyl;
R ⁴ is a linear C ₁ -C ₆ alkyl substituted with a carboxyl group;
The compound wherein n is an integer from 0 to 6.

(10) The compound according to (9), wherein R ² is linear C ₁ -C ₆ alkyl substituted with a hydroxyl group.

  (11) The compound according to item (10) represented by the following formula VI:

（１２）項（９）に記載の化合物、および薬学的に受容可能なキャリアを含む、薬学的組成物。

(12) A pharmaceutical composition comprising the compound according to (9) and a pharmaceutically acceptable carrier.

  (13) The pharmaceutical composition according to item (12), which has an immunostimulatory activity.

  (14) The composition according to item (13), wherein the immunostimulatory activity is a dendritic cell-inducing activity-inducing ability.

  (15) The compound according to item (1) represented by the following formula IX.

（１６）以下の式Ｘで表される、項（１）に記載の化合物。

(16) The compound according to item (1), represented by the following formula X:

  A novel compound having a physiological activity including an immunostimulatory activity and a superoxide dismutase-like activity is provided, whereby a natural product is used as a raw material, and instead of a conventional product in which quality tends to fluctuate, a physiological activity of a certain quality is provided. Is provided.

  It is to be understood that the terms used herein are used in definitions commonly used in the art unless otherwise specified.

“Alkyl” refers to a monovalent group resulting from the loss of one hydrogen atom from an aliphatic hydrocarbon such as methane, ethane, and propane, and is generally represented by C _n H _{2n + 1} − (wherein n is a positive integer). Alkyl can be straight or branched.

  "Substituted alkyl" means an alkyl in which one or more hydrogen atoms of the alkyl are replaced with a group selected from the group consisting of amino, carboxyl, hydroxyl, formyl, and hydrazino. "Unsubstituted alkyl" refers to alkyl in which any hydrogen atom is not replaced by another group.

  “Formyl” refers to a group represented by —CHO.

  “Carboxyl” refers to a group represented by —COOH.

“Hydrazino” refers to a group represented by —HN—NH ₂ .

“Amino” refers to a group represented by —NH ₂ .

  "Hydroxyl" refers to the group represented by -OH.

  “Formyl-substituted phenyl” refers to phenyl in which a hydrogen atom of phenyl is substituted with a CHO group.

  "Formyl-substituted pyrrole" refers to pyrrole in which a hydrogen atom of pyrrole is substituted with a CHO group.

  The "4-formylphenyl type structure" refers to a structure of phenyl in which one hydrogen atom of phenyl is substituted with a CHO group and further has another substituent at the p-position to the CHO group.

  The “2-formylpyrrole-type structure” refers to a pyrrole structure in which one hydrogen atom at the 2-position of pyrrole is substituted with a CHO group.

  "Terminal carboxyl group" means a carboxyl group for replacing a hydrogen atom in a terminal (or tip) functional group of a molecule.

  "Terminal hydrazino group" means a hydrazino group for replacing a hydrogen atom in a terminal (or tip) functional group of a molecule.

  “Terminal amino group” means an amino group for replacing a hydrogen atom in a terminal (or head) functional group of a molecule.

  "Amino acid" refers to a compound having a carboxyl group and an amino group in a molecule, and a compound in which an amino group and a carboxyl group are bonded to the same carbon atom is referred to as an α-amino acid. , Γ-, δ-, ... amino acids.

  "Aspartyl" means a group formed by removing the hydroxyl group from one of the two carboxyl groups of aspartic acid, which is a monoaminodicarboxylic acid.

  “Glutamyl” means a group formed by removing a hydroxyl group from one of two carboxyl groups of glutamic acid, which is a monoaminodicarboxylic acid.

It is not known that the compounds represented by the above formulas (I) to (X) have physiological activities including, for example, immunostimulatory activity and superoxide dismutase-like activity. Therefore, the present invention provides novel compounds represented by the general formulas (I) to (X). Without being bound by a particular theory, the compounds represented by the above formulas (I) to (X) may have a combination of a formyl group and an R ¹ group and the distance between them may have immunostimulating activity and superoxide. It is thought to be related to dismutase-like activity. The present invention further provides a pharmaceutical composition such as a novel immunopotentiator containing the compounds represented by the general formulas (I) to (X).

  The compounds represented by the above formulas (I) to (III), (V), (IX) or (X) can be typically synthesized using, for example, phenylhydrazine or aniline as a starting material. Phenylhydrazine is a monoclinic crystal or a pale yellow transparent liquid obtained by diazotizing aniline with sodium nitrite and hydrochloric acid and then reacting with sodium sulfite (GH Coleman, Org. Syn., Coll. Vol., 1, 442 (1951)). Phenylhydrazine is used as a dye intermediate, a raw material for antipyrine (antipyretic agent), and a reagent for detecting sugars, aldehydes, and ketones. The novel compounds of the present invention can be prepared starting from phenylhydrazine or aniline. Phenylhydrazine or aniline produces various derivatives by condensation reactions known to those skilled in the art. The novel compound of the present invention can be synthesized utilizing such a condensation reaction.

A novel compound characterized by a formylphenyl-type chemical structure is obtained by condensing phenylhydrazine and a monoaminodicarboxylic acid (eg, glutamic acid) using a known method, and adding HCl / AlCl ₃ under pressurized CO. By performing a formylation in the presence of

  The compound having a 2-formylpyrrole-type chemical structure represented by the above formula (IV) or (VI) is typically a commercially available pyrrole or a pyrrole-type compound, and is typically used in a chemical reaction. , An alkylation reaction, an arylation reaction and an acylation reaction.

In the formula (IV), the substituent represented by R ² can be a saturated or unsaturated alkyl with or without various substituents. These compounds are obtained by adding a pyrrole-type compound as a starting material, to which an alkylating agent usually used in a chemical reaction, for example, an alkyl halide having a corresponding alkyl, is added, and a suitable alkali such as potassium carbonate, n is added. It can be obtained by adding a hexane solution of -butyllithium or the like and, if necessary, stirring under a predetermined temperature condition in the presence of an absorbent for an acid generated during the reaction (for example, dimethylformamide).

Alternatively, in the formula (IV), the substituent represented by R ² may be an aliphatic or aromatic acyl with or without various substituents. These compounds are obtained by dissolving a pyrrole-type compound as a starting material in a suitable solvent, for example, pyridine, methylene chloride, tetrahydrofuran or the like, and adding an acylating agent, for example, an acid chloride or an acid anhydride. , In the presence of a suitable solvent, for example, a hexane solution of pyridine, triethylamine, n-butyllithium, or the like, under predetermined temperature conditions. In addition, by appropriately combining the above reactions, a compound represented by the formula (IV) or (VI) can be obtained.

  Alternatively, the compound represented by the above formula (IV) or (VI) may be isolated and purified from the fruiting body or cultured mycelium of basidiomycete, in addition to the above-mentioned organic synthesis method. For example, an extract is obtained by heating and extracting a fruiting body or a cultured mycelium of a basidiomycete, and the obtained extract is separated and purified using a gel filtration, a hydrophobic gel column, an ion-exchange resin or the like to obtain 1- ( 3-carboxypropyl) -2-formyl-5-hydroxymethyl-pyrrole may be obtained. Further, the fraction extract in the course of this separation / purification also contains the compound represented by the formula (IV) or (VI).

  Can the compounds represented by the above formulas (I) to (X) be used as they are as active ingredients in pharmaceutical compositions having various physiological activities including immunostimulatory activity and superoxide dismutase-like activity according to the present invention? Or with a suitable carrier in the composition of the invention.

  As used herein, the term "immunostimulatory activity" generally refers to normal or reduced activity through immunocompetent cells such as macrophages, T cells, B cells, A cells (accessory cells), and plasma cells. Activating or enhancing the immune response of an individual. T cells include helper T cells, suppressor T cells, killer T cells, T cells that cause delayed-type hypersensitivity (DTHT cells), and the like. B cells generally include progenitor cells of antibody-producing cells. A cells include Ia-positive macrophages, dendritic cells of lymphoid tissue, Langerhans cells of epidermis, and the like. Plasma cells include antibody-producing cells distributed in secondary lymphoid tissues such as spleen and lymph nodes, bone marrow, and connective tissues throughout the body.

As used herein, the term "superoxide dismutase-like activity (or SOD-like activity)" refers to the ability to carry out an enzymatic reaction similar to superoxide dismutase (SOD), in the presence of which SOD reacts with active oxygen, for example, superoxide anion _{(O 2} ^-) to catalyze the substrate _2O ² - ⁺ 2H + → _H can be assessed by inhibiting the second reaction of _O 2 + _{O 2} (dismutation of superoxide).

  In general, when living organs perform aerobic life activities, they cannot avoid exposure to reactive oxygen species, especially oxygen free radicals and hydrogen peroxide in living organs. This is because cells of a living organ generate reactive oxygen species by aerobic life activity. Therefore, not only harmful substances from the outside world, such as chemical substances and environmental pollutants, but also all cells of living organs are also considered to be one of the living attack species. When the pro-oxidant becomes predominant in the living body and the balance with the antioxidant is lost, the living body may be damaged.

Active oxygen includes types such as hydroxy radical, peroxy radical, and nitric oxide. SOD is typically an enzyme that catalyzes the reaction of 2O ₂ ⁻ + 2H ⁺ → H ₂ O ₂ + O ₂ using O ₂ ⁻ as a substrate, and protects a living body from oxygen damage by eliminating O ₂ ^−. are doing. SOD is classified into five types, Cu, Zn-SOD, Mn-SOD, Fe-SOD, Extracellular (EC) -SOD, Fe, and Zn-SOD, depending on the metal contained in the molecule. Human tissues and erythrocytes are known to have high Cu, Zn-SOD, Mn-SOD, and Fe-SOD activities.

When the distribution of SOD activity is compared among living organs, the highest activity is observed in the liver where metabolism is active, followed by the high activity in the adrenal gland and kidney. The adrenal, O ₂ under physiological conditions ^- and lipid peroxide is generated. This, P-450 involved in the production of various adrenal hormones O ₂ ^- are believed to generate and SOD activity in order to prevent its influence is rising.

  Looking at SOD activity by disease, the value increases in acute renal failure because kidney cells are destroyed, and the value decreases in diabetes. This is thought to be due to the fact that glucose increased by diabetes causes SOD to be glucosylated and its activity to be reduced. A similar phenomenon occurs with aging. Thus, SOD-like activity is one indicator of various diseases, and compositions with SOD-like activity trigger three major adult diseases by catalyzing the disproportionation of active oxygen. It can be used to treat diseases including various diseases, for example, liver disorders, adrenal disorders, kidney disorders, various cancers, hypertension, anti-aging, and the like.

  The present invention also provides a composition comprising the compounds represented by the above formulas (I) to (X) alone or in combination with a stabilizing compound, a carrier such as a diluent, or other components or drugs. The composition of the present invention is such that the compounds represented by the above formulas (I) to (X) produce or increase a physiological activity including an immunostimulatory activity and a superoxide dismutase-like activity in an administered organism. Used in various forms.

  The compounds of formulas (I)-(X) above are generally sterile and biocompatible carriers, including but not limited to saline, buffered saline, dextrose, and water. ) Can be administered to a subject. Alternatively, the compounds of the above formulas (I) to (X) can be used alone or in the form of a composition mixed with suitable excipients, adjuvants and / or pharmaceutically acceptable carriers. It can be administered to a subject in combination with other agents.

  Administration of the compounds or compositions of the present invention is accomplished orally or parenterally. Methods for parenteral delivery include topical, dermal application, intraarterial (eg, directly on tumor, aneurysm), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or Intranasal administration may be mentioned.

  The composition of the present invention may contain, in addition to the compounds represented by the above formulas (I) to (X), a suitable pharmaceutically acceptable carrier in order to prepare a pharmaceutically usable formulation. Further details of techniques for formulation and administration are described, for example, in the final version of "REMINGTON'S PHARMACEUTICAL SCIENCES" (Macack Publishing Co., Easton, PA).

  Hereinafter, the immunostimulating preparation containing the composition of the present invention will be described in detail by exemplifying a pharmaceutically acceptable carrier to be used for each administration form. The composition of the present invention shown below is an exemplification of the present invention and does not limit the present invention.

  Formulations for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art as dosage forms suitable for administration. Such carriers enable the compositions of the present invention to be formulated into tablets, pills, dragees, capsules, liquids, gels, ointments, syrups, slurries, suspensions, and the like, which are suitable for consumption by a patient. And

  Formulations for oral use are prepared by combining the compounds of formulas (I) to (X) with a solid excipient as a carrier, pulverizing the resulting mixture, if desired, and, if desired, tablets or In order to obtain a sugar-coating core, it can be obtained by adding a suitable further compound and then treating the mixture of granules. As representative excipients, the following carbohydrates, protein fillers, etc. may be used: sugars including lactose, sucrose, mannitol, or sorbitol; corn, wheat, rice, potato, or other plant-derived Starch; cellulose, such as methylcellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose; gums, including gum arabic and tragacanth; gelatin; and proteins, such as collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof (eg, sodium alginate).

  Dragee cores are provided with suitable coatings, such as concentrated sugar solutions. It may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solution, and a suitable organic solvent or solvent mixture. Dyestuffs or pigments may be added to the tablets or dragees for product identification or to characterize the amount (ie, dosage) of the active compound.

  Formulations that can be used orally can be, for example, gelatin capsules, soft sealed capsules consisting of gelatin and a coating, such as glycerol or sorbitol. Gelatin capsules may contain the active ingredient in admixture with fillers or binders such as lactose or starch, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In the soft capsule, the compounds represented by the above formulas (I) to (X) can be used by being dissolved or suspended in a suitable liquid such as fatty oil, liquid paraffin or liquid polyethylene glycol.

  Preparations for parenteral administration include aqueous solutions of the compounds of formulas (I)-(X). For injection, the compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Such suspensions may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles may include fatty acids such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. If desired, suspensions may contain stabilizers or suitable agents or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

  For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated can be included in the formulation. Such penetrants are generally known in the art.

  The formulations of the present invention can be prepared in a manner similar to that known in the art (eg, by means of conventional mixing, dissolving, granulating, dragee-making, elutriating, emulsifying, encapsulating, entrapping, or lyophilizing). Can be manufactured.

  The composition of the present invention contains the compounds represented by the above formulas (I) to (X) in an amount effective to achieve the intended purpose. “Therapeutically effective amount” or “pharmacologically effective amount” is a term well recognized by those skilled in the art and refers to an amount of an agent effective to produce the intended pharmacological result. Thus, a therapeutically effective amount is an amount sufficient to reduce the symptoms of the disease to be treated. One useful assay for determining an effective amount (eg, a therapeutically effective amount) for a given application is to measure the immune activity of the target disease. The actual amount administered will depend on the individual to whom the treatment is to be applied, and will preferably be an amount that is optimized so that the desired effect is achieved without significant side effects. Determination of an effective dose is well within the capability of those skilled in the art.

  For any compound of formulas (I)-(X) above, a therapeutically effective dose can be estimated initially either in cell culture assays or in any suitable animal model. Animal models are also used to achieve a desired concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

  A therapeutically effective amount refers to that amount of a compound of Formulas (I)-(X) that reduces the symptoms or condition of the disease. Therapeutic efficacy and toxicity of such compounds will depend on standard pharmaceutical procedures in cell cultures or experimental animals (eg, ED50, therapeutically effective dose in 50% of the population; and LD50, 50% of the population). Dose that is lethal). The dose ratio between therapeutic and toxic effects is the therapeutic index and it can be expressed as the ratio ED50 / LD50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from the cell culture assays and animal studies is used in formulating a range of quantities for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. This dose will vary within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration. As an example, the dose of the compounds represented by the above formulas (I) to (X) is appropriately selected depending on the age and other conditions of the patient, the type of the disease, and the like. Intra-articular administration, generally 1 μg to 100 mg per dose, can be administered once to several times daily.

  The exact dose will be chosen by the individual clinician, taking into account the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors that may be considered include the severity of the disease state (eg, size and location of the tumor; age, weight, and sex of the patient; dietary time and frequency of administration, drug combination, response sensitivity, and resistance to treatment / Response). Depending on the half-life and clearance rate of the particular formulation, a long acting pharmaceutical composition may be administered every 3 to 4 days, every week, or once every two weeks. Guidance as to particular dosages and methods of delivery is provided in the literature known in the art.

  The thus-obtained pharmaceutical containing the compounds represented by the above formulas (I) to (X) as a main component can be administered by various methods depending on the type of disease to be controlled.

  The compounds represented by the above formulas (I) to (X) or the compositions containing the same of the present invention can be used as liquid, gel or solid food materials without any limitation. For example, added to soft drinks, juices, teas, jellies, puddings, breads, cookies, caramels, oysters, etc., and, if necessary, excipients such as starch, dextrin, lactose, and other edible composition extracts It can be processed into powders, granules, and tablets together with agents, pigments, flavors, etc., or formed into capsules using a coating agent such as gelatin, and used as health foods or dietary supplements. It goes without saying that the composition of the present invention is not limited to these examples.

  Further, the composition of the present invention can be added in a range of approximately 0.1 to 100% by weight depending on the type and condition of the final product. The standard of the addition amount is about 0.005 to 1%, preferably 0.01 to 0.5%.

  Hereinafter, the present invention will be described in more detail with reference to Examples, but the following Examples are exemplifications of the present invention, and do not limit the present invention.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Synthesis of Compound
1.1 . Synthesis of β-N- (γ-glutamyl) -4-formylphenylhydrazine (Formula VII) A large excess of glutamic acid was added to 940 mg of phenylhydrazine, and the mixture was condensed using a known method to give β-N- (γ- Glutamyl) -phenylhydrazine was formed and this condensation reaction was carried out until the phenylhydrazine completely disappeared. Next, the product was subjected to formylation in the presence of HCl / AlCl ₃ under a pressurized CO atmosphere to obtain a final product 1 (150 mg).

1.2 . Synthesis of N- (3-carboxypropyl) -2-formyl-5-hydroxymethylpyrrole (Formula VIII) To 625 mg of 2-formyl-5-hydroxymethylpyrrole was added 674 mg of 4-chloro-butyric acid and 600 mg of potassium carbonate, and the mixture was added for 18 hours. Heated to reflux. After cooling, the reaction solution was filtered, the filtrate was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (silica gel: 30 g, eluent: chloroform-methanol 2: 1) to give the final product 2. (Colorless oil, 60 mg) was obtained.

  The above two final products 1 and 2 were subjected to mass spectrometry and NMR analysis under the following conditions, and the structure thereof was analyzed. As a result, these two components were respectively β-N- (γ- Glutamyl) -4-formylphenylhydrazine (having a structure represented by the following formula VII), and N- (3-carboxypropyl) -2-formyl-5-hydroxymethylpyrrole (represented by the following formula VIII) Has a structure that is

［質量分析］
得られた最終生成物１および２をそれぞれ、１０ｍｇ／ｍｌの濃度になるように、ｍｉｌｉ−Ｑ水を加えて水溶液とした後に測定に供した。測定装置として、ＪＥＯＬ ＨＸ１１０／１１０Ａタンデム型質量分析計を用い、以下のＦＡＢ、ＥＩ、ＣＩおよびＨＲＦＡＢの質量分析において、それぞれ、ポジティブ（ＦＡＢ、ＥＩ、ＣＩ）、ネガティブ（ＦＡＢ、ＣＩ）およびネガティブ（ＨＲＦＡＢ）の測定モードで測定した。なお、各質量分析における分解能は、ＦＡＢ、ＥＩおよびＣＩについて１０００、そしてＨＲＦＡＢについて５０００で行った。

[Mass spectrometry]
The resulting final products 1 and 2 were each subjected to measurement after adding mili-Q water to an aqueous solution so as to have a concentration of 10 mg / ml. Using a JEOL HX110 / 110A tandem mass spectrometer as a measuring device, in the following mass spectrometry of FAB, EI, CI and HRFAB, positive (FAB, EI, CI), negative (FAB, CI) and negative (FAB, CI), respectively HRFAB). The resolution in each mass spectrometry was 1000 for FAB, EI and CI, and 5000 for HRFAB.

1. FAB-MS
Glycerol was used as the matrix. The measurement was performed immediately after glycerol and the sample were mixed at 1: 1 (v / v) on the sample mounting table. For mass calibration, a mixture of alkali ions was used in the positive mode, and a glycerol solution of cesium iodide (CsI) was used in the negative mode.

2. EI-MS and CI-MS
The sample itself was introduced into the ion source. In CI, isobutane was used as ion gas.

3. HRFAB-MS
PEG-500 was used as a mass calibration sample. After mixing glycerol and the sample at 1: 1 (v / v) on the sample mounting table, an appropriate amount of PEG-500 was added, and the measurement was immediately performed in a negative mode. Mass calibration was performed by selecting two peaks of PEG-500 sandwiching the target peak ([MH] ⁻ = 210).

[NMR analysis]
Bruker DMX500 nuclear magnetic resonance apparatus ⁽¹ H500MHz) and JEOL JNM-A400 nuclear magnetic resonance apparatus ⁽¹ H400MHz) was performed using.

  The sample after mass spectrometry was freeze-dried and then dissolved in heavy water (0.3 ml) for measurement. In addition, measurement was performed using a sample in which the solvent was replaced with deuterated chloroform.

[Result Analysis]
Final product 1:
In FAB-MS, observe the false molecular ion MH + at m / z 266, in the EI-MS, for trifluoroacetic acid and acetic acid derivatives, respectively, with _{m / z 649 (C 20 H} 11 F 12 N 3 O 3) the M ⁺ ions were observed _[M-H 2 ^{O] +} ion at m / z 331. H ¹ NMR characterized the aldehyde proton, the p-substituted aromatic ring and the glutamic acid residue —CO—CH ₂ —CH ₂ —CH (NH ₂ ) —COOH. Unshielding of two aromatic protons (d 7.91 ppm), UV absorption in neutral solution (313 nm) and alkaline solution (385 nm), of important ions in m / z 136 (C ₇ H ₈ N ₂ O) The occurrence was consistent with the p-formylphenyl hydrazine unit. The following ¹ H-NMR was measured in heavy water using tetramethylsilane as an internal standard. The δ value is in ppm and the coupling constant (J) is in Hz. In the data, d represents a singlet, d represents a doublet, t represents a triplet, and m represents a multiplet.

UVλ ¹²⁰ _max nm (log ε); 231 (3.79), 313 (4.19); + NaOH 248 (3.76), 385 (4.30); + HCl 231 (3.79), 313 (4.19). ): FAB ⁺ MS m / z (relative intensity): 266 (MH ⁺ , 100), 201 (62), 166 (16), 137 (20), 136 (15), 132 (39), 121 (16) _{^{; 1 H NMR (400 MHz,}} D 2 O) 9.73 (1H, s, -CHO), 7.91 (2H, d, J = 8.5Hz), 7.03 (2H, d, J = 8 0.5 Hz), 3.91 (1H, t, J = 6 Hz, Hα), 2.67 (2H, m, Hγ), 231 (2H, m, Hβ).

Final product 2:
A peak corresponding to a molecular weight of 211 was observed in any of the spectra in the various mass spectrometry modes, and the molecular weight of this compound was determined to be 211. Next, when the HRFAB-MS measurement was performed in a negative mode having a better peak sensitivity, the observed accurate mass (m / z = 210.0757) was good to be C ₁₀ H ₁₂ O ₄ N (−1.0 mmu). Based on the agreement, the molecular formula of the final product 2 was determined to be C ₁₀ H ₁₈ O ₄ N.

  Next, various NMR measurements were performed for the purpose of obtaining structural information, and analysis was performed based on cross-peak information of the HMBC spectrum. As a result, it was determined to have the structure represented by the above formula VI.

1.3 . Synthesis of β-N- (γ-glutamyl) -4-formylaniline (or 4- (4-glutamyl) aminobenzaldehyde) (Formula V: hereinafter referred to as ATO-1) Synthesis of ATO-1 The scheme and the synthesis method are shown below.

Ｎ−Ｂｏｃ−１−ベンジルグルタミン酸（９６４ｍｇ、２．８６ｍｍｏｌ、２）をＴＨＦ３０ｍｌに溶解させて０℃に冷却し、ジシクロヘキシルカルボジイミド（ＤＣＣ）（６１９ｍｇ、３ｍｍｏｌ）を加え、０℃にて３０分攪拌した。次に、パラアミノベンズアルデヒド塩酸塩（６３０ｍｇ、４ｍｍｏｌ、１）、ピリジン０．４ｍｌおよびＮ，Ｎ’−ジメチルアミノピリジン（ＤＭＡＰ）（３４９ｍｇ、２．８６ｍｍｏｌ）を加え、水浴を外して室温で1時間攪拌した。反応液にトリエチルアミン０．３ｍｌを加えてさらに１６時間攪拌した。その後、反応液を減圧濾過し、酢酸エチル３０ｍｌを加えて１Ｍ ＨＣｌ、５％ＮａＨＣＯ_３および水で洗浄した後、乾燥し減圧濃縮した。残渣（１．３ｇ）をシリカゲルクロマトグラフィーで精製することによりアミド３（１１５．２ｍｇ）を得た。次に、アミド３（５６．２ｍｇ）をＭｅＯＨ（０．１４ｍｌ）に溶解し、４Ｍ ＮａＯＨ（０．０４ｍｌ）を加えて室温で３０分攪拌した。反応混合物に水４ｍｌを加え、酢酸エチルで洗浄後水層にＤｏｗｅｘ５０ＷＸ８（１ｍｌ）を加えて５分間攪拌した後、Ｄｏｗｅｘ５０ＷＸ８を濾別、水層を酢酸エチルで抽出した。酢酸エチルを留去した残渣を９８％ギ酸（１．５ｍｌ）で処理することにより（ＡＴＯ−１、１８．６ｍｇ、２からの収率６．７％）を得た。
４−（４−グルタミル）アミノベンズアルデヒド（ＡＴＯ−１）
ＭＳ ｍ／ｚ ２５１（Ｍ^＋＋１）；ＩＲ（ＫＢｒ）ν３４００，１６７０，１５９０，１５４０ｃｍ^−１；^１Ｈ ＮＭＲ（Ｄ_２Ｏ，５００ＭＨｚ）δ２．２０（２Ｈ，ｄｄｄ，Ｊ＝６．８），２．６０（２Ｈ，ｄｄ，Ｊ＝６．８，７．３），３．７９（１Ｈ，ｔ，Ｊ＝６．８），７．６５（２Ｈ，ｄ，Ｊ＝８．３），７．８８（２Ｈ，ｄ，Ｊ＝８．３），９．７９（１Ｈ，ｓ）。

N-Boc-1-benzylglutamic acid (964 mg, 2.86 mmol, 2) was dissolved in 30 ml of THF, cooled to 0 ° C., dicyclohexylcarbodiimide (DCC) (619 mg, 3 mmol) was added, and the mixture was stirred at 0 ° C. for 30 minutes. . Next, para-aminobenzaldehyde hydrochloride (630 mg, 4 mmol, 1), 0.4 ml of pyridine and N, N'-dimethylaminopyridine (DMAP) (349 mg, 2.86 mmol) were added, and the water bath was removed, followed by stirring at room temperature for 1 hour. did. 0.3 ml of triethylamine was added to the reaction solution, and the mixture was further stirred for 16 hours. Thereafter, the reaction solution was filtered under reduced pressure, added with 30 ml of ethyl acetate, washed with 1 M HCl, 5% NaHCO ₃ and water, dried, and concentrated under reduced pressure. The residue (1.3 g) was purified by silica gel chromatography to give amide 3 (115.2 mg). Next, amide 3 (56.2 mg) was dissolved in MeOH (0.14 ml), 4M NaOH (0.04 ml) was added, and the mixture was stirred at room temperature for 30 minutes. After adding 4 ml of water to the reaction mixture and washing with ethyl acetate, Dowex50WX8 (1 ml) was added to the aqueous layer, and the mixture was stirred for 5 minutes. The Dowex50WX8 was filtered off, and the aqueous layer was extracted with ethyl acetate. The residue obtained by distilling off ethyl acetate was treated with 98% formic acid (1.5 ml) to give (ATO-1, 18.6 mg, 6.7% yield from 2).
4- (4-glutamyl) aminobenzaldehyde (ATO-1)
^{MS m / z 251 (M +} +1); IR (KBr) ν3400,1670,1590,1540cm -1; 1 H NMR (D 2 O, 500MHz) δ2.20 (2H, ddd, J = 6.8), 2.60 (2H, dd, J = 6.8, 7.3), 3.79 (1H, t, J = 6.8), 7.65 (2H, d, J = 8.3), 7 .88 (2H, d, J = 8.3), 9.79 (1H, s).

1.4 . Synthesis of N- (4-carboxybutyl) -2-formyl-5-hydroxymethylpyrrole (Formula VI) Except for using 5-chloro-valeric acid instead of the starting 4-chloro-butyric acid. Alkylation and purification were carried out in the same manner as in the above to give the final product 4 (colorless oil, 50 mg).

  The two final products 3 and 4 were subjected to mass spectrometry and NMR analysis under the same conditions as described above, and the structures thereof were analyzed. As a result, these two substances had β-N- (Γ-glutamyl) -4-formylaniline (having a structure represented by the following formula V), and N- (4-carboxybutyl) -2-formyl-5-hydroxymethylpyrrole (the following formula VI Having the structure represented).

１．５．４−（４−フォルミルフェニルカルバモイル）ブタン酸（以下の式ＩＸで表される構造をもつ：以下、本明細書ではＡＴＯ−３と称する）の合成
ｐ−ニトロベンズアルデヒド１．５１ｇを無水エタノール２０ｍｌ中に懸濁し、塩化スズ（ＩＩ）１１．２８ｇを添加して還元反応を行った。得られた反応液を水５０ｍｌに添加し、水酸化ナトリウムを用いて中和した後、反応産物を酢酸エチルで抽出した。得られた有機層を飽和食塩水で洗浄し、無水硫酸ナトリウムを用いて乾燥した後濾別した。得られた濾液に１Ｎ ＨＣｌ／ＭｅＯＨ １１ｍｌを添加して造塩し、析出した結晶を濾別、洗浄および乾燥してｐ−アミノベンズアルデヒド塩酸塩０．８８ｇを得た。

1.5 . Synthesis of 4- (4-formylphenylcarbamoyl) butanoic acid (having a structure represented by the following formula IX: hereinafter referred to as ATO-3 in the present specification) 1.51 g of p-nitrobenzaldehyde and 20 ml of absolute ethanol The mixture was suspended in the solution, and 11.28 g of tin (II) chloride was added to perform a reduction reaction. The obtained reaction solution was added to 50 ml of water, neutralized with sodium hydroxide, and the reaction product was extracted with ethyl acetate. The obtained organic layer was washed with a saturated saline solution, dried using anhydrous sodium sulfate, and then filtered. To the obtained filtrate, 11 ml of 1N HCl / MeOH was added to form a salt, and the precipitated crystal was separated by filtration, washed and dried to obtain 0.88 g of p-aminobenzaldehyde hydrochloride.

  0.8 g of the obtained p-aminobenzaldehyde hydrochloride was suspended in 10 ml of methylene chloride, and 0.53 g of triethylamine was added to obtain a p-aminobenzaldehyde solution. This solution was added to 10 ml of methylene chloride in which 2.9 g of glutaric anhydride was suspended to synthesize a peptide. After completion of the synthesis, the precipitated crystals were separated by filtration, and water was added to the filtrate for crystallization. This was separated by filtration, washed and dried to obtain 0.81 g of slightly yellow crystals. As a result of analyzing the slightly yellow crystal using NMR, IR, LC, and TLC, it was confirmed that the crystal was 4- (4-formylphenylcarbamoyl) butanoic acid represented by the following structural formula.

The result to which the absorption peak was assigned in the IR analysis is shown below.
3350 cm ^-1 (-NH), 3102 to 2981 cm ^-1 (-CO OH ), around 2842 cm ^-1 (-CH _2- ), 1716 cm ^-1 (-CHO), around 1664 to 1525 cm ^-1 (-NH), 1581 cm ^{-1 (- CO OH), 1317cm} -1 vicinity ^{_{(-CH 2), 1164cm -1 (}} Ph · N).

ＭＳ ｍ／ｚ ２３４（Ｍ^＋−１）；ＩＲ（ＫＢｒ）ν３２００、１７１０、１６８０、１５７５、１５２５ｃｍ^−１；^１Ｈ ＮＭＲ（ＣＤ_３ＯＤ、５００ＭＨｚ）δ１．９８（２Ｈ、ｔｔ、Ｊ＝７．４）、２．３９（２Ｈ、ｔ、Ｊ＝７．４）、２．４８（２Ｈ、ｔ、Ｊ＝７．４）、７．７８（２Ｈ、ｄ、Ｊ＝８．８）、７．８５（２Ｈ、ｄ、Ｊ＝８．８）、９．８６（１Ｈ、ｓ）。

MS m / z 234 (M ⁺ -1); IR (KBr) ν 3200, 1710, 1680, 1575, 1525 cm ^-1 ; ¹ H NMR (CD ₃ OD, 500 MHz) δ 1.98 (2H, tt, J = 7. 4), 2.39 (2H, t, J = 7.4), 2.48 (2H, t, J = 7.4), 7.78 (2H, d, J = 8.8), 7. 85 (2H, d, J = 8.8), 9.86 (1H, s).

1.6 . 2. Synthesis of 4- (4-formylphenylcarbamoyl) propylamine (having a structure represented by the following formula X: hereinafter referred to as ATO-4 in the present specification) N-Boc gamma aminobutyric acid (GABA) 66 g was dissolved in 10 ml of THF, and after cooling, a solution of 4.1 g of DCC / 5 ml of THF was added thereto, and the mixture was reacted overnight. The precipitated crystals (DCU) were separated by filtration, and the obtained filtrate was solvent-substituted with methylene chloride to obtain an N-Boc GABA-free aqueous solution. To this N-Boc GABA anhydride solution, 0.63 g of p-aminobenzaldehyde hydrochloride and 0.42 g of triethyleneamine were added to form a peptide. The obtained reaction solution was concentrated, the residue was dissolved in ethyl acetate, washed with a 5% aqueous sodium bicarbonate solution, water, and saturated saline in this order, and then dried using sodium sulfate. This was filtered, concentrated, purified using silica gel chromatography, and concentrated to obtain a reddish brown oil. This was dissolved in methylene chloride, and a TFA / methylene chloride solution was added dropwise under ice-cooling to remove the Boc group. The obtained reaction solution was concentrated to obtain a reddish brown oil. This was analyzed using NMR, IR, LC, and TLC, and as a result, it was confirmed to be 4- (4-formylphenylcarbamoyl) propylamine represented by the following structural formula.

ＭＳ ｍ／ｚ２０７（Ｍ^＋＋１）；ＩＲ（ＫＢｒ）ν３４００、１６７２、１５８７、１５２６ｃｍ^−１；^１Ｈ ＮＭＲ（Ｄ_２Ｏ、５００ＭＨｚ）δ２．０２（２Ｈ、ｔｔ、Ｊ＝７．３）、２．５８（２Ｈ、ｔ、Ｊ＝７．３）、３．０５（２Ｈ、ｔ、Ｊ＝７．３）、７．６６（２Ｈ、ｄ、Ｊ＝８．３）、７．９１（２Ｈ、ｄ、Ｊ＝８．３）、９．８０（１Ｈ、ｓ）。

^{MS m / z207 (M + +1} ); IR (KBr) ν3400,1672,1587,1526cm -1; 1 H NMR (D 2 O, 500MHz) δ2.02 (2H, tt, J = 7.3), 2 .58 (2H, t, J = 7.3), 3.05 (2H, t, J = 7.3), 7.66 (2H, d, J = 8.3), 7.91 (2H, d, J = 8.3), 9.80 (1H, s).

(Example 2) Measurement of immunostimulatory activity N- (3-carboxypropyl) -2-formyl-5-hydroxymethylpyrrole (final product 2) synthesized in Example 1 above and β-N- (γ- Glutamyl) -4-formylphenylhydrazine (final product 1) was measured for immunostimulatory activity using dendritic cells.

  Dendritic cells are cells involved in establishing immunity. Also called dendritic cells, dendritic leukocytes, D cells. It is a collective term for a group of cells having a dendritic morphology derived from bone marrow cells distributed in various tissue organs in a living body except the brain. This cell group includes lymphoid dendritic cells, Langerhans cells, veil cells, interconnected cells, interstitial cells, and the like. Along with an inflammatory response initiated by invasion of foreign matter in a living body, the foreign matter is taken up (pinocytosis) and travels from a local area via an imported lymphatic vessel to a regional lymph node or blood stream to the spleen. After reaching the T cell-dependent region, it is known to initiate an immune response by presenting the antigen in bound form with a class II antigen and activating specific T cells. These cells are the cells responsible for the immunity. In the present application, the immunostimulatory activity of the compound of the present invention was confirmed by assaying the ability of monocytes to induce dendritic cells.

N- (3-carboxypropyl) -2-formyl-5-hydroxymethylpyrrole and β-N- (γ-glutamyl) -4-formylphenylhydrazine have a formyl group and a terminal carboxyl as the R ₁ group. And the combination of these groups, and the spacing between them, is thought to be related to immunostimulatory activity.

2 . Materials and methods
2.1 . Materials Peripheral blood of two healthy subjects who could obtain consent was used as a material.

2.2 . Separation of Peripheral Blood Mononuclear Cell (PBMC) PBMC was separated by Ficoll-Paque specific gravity centrifugation from a whole blood sample collected with heparin.

2.3 . Test samples N- (3-carboxypropyl) -2-formyl-5-hydroxymethylpyrrole (final product 2) synthesized in Example 1 above and β-N- (γ-glutamyl) -4-formyl Phenylhydrazine (final product 1) was used as a test sample.

  Each of these compounds was dissolved at a concentration of 200 mg / ml in a culture solution of cells used for each experiment (RPMI-1640 containing 10% FCS and RPMI-1640 containing 5% human AB serum), and a filter having a pore size of 0.22 μm was dissolved. What was sterilized by filtration was used as a test sample.

2.4 . Measurement of Immunostimulatory Activity The immunostimulatory effect of the above compounds was examined based on the ability to induce human peripheral blood monocyte-derived dendritic cells (DCs) in vitro.

  PBMC was dish-treated in a carbon dioxide incubator for 1 hour to obtain a monocyte-rich adherent cell fraction. The obtained cells were used in the presence of 500 U / ml of GM-CSF (manufactured by DIACLONE Research) and IL-4 (manufactured by BD Biosciences), respectively. Cultured in

  A 3.5 cm diameter dish (manufactured by NUNC) was used for the culture. On day 6 of culture, picibanil and the two compounds were added to give final concentrations of 0.1 KE / ml for picibanil and 200 μg / ml for the two compounds, respectively. Picibanil (manufactured by Chugai Pharmaceutical) was used as a positive control having DC inducibility. Negative controls are cells similarly cultured using 5% human AB serum-supplemented RPMI-1640.

  All DCs in the dish were collected on day 7 after the start of culture, and their surface antigens were analyzed by flow cytometry using a monoclonal antibody (anti-CD80; manufactured by BD Bioscience). In addition, the significant difference test was performed using Student's t-test.

3 . Results In the subjects examined, by adding the above compound on the 6th day from the start of DC culture, remarkably strong induction of expression of the CD80 molecule was observed. One example showing a typical pattern is shown in FIGS.

  FIG. 1 shows the test results for β-N- (γ-glutamyl) -4-formylphenylhydrazine (final product 1). The chart shown in the upper part of FIG. 1 shows the results of flow cytometry analysis of cells to which no test compound was added, and the chart shown in the lower part of FIG. 1 shows the addition of β-N- (γ-glutamyl) -4-formylphenylhydrazine. 4 shows the results of flow cytometry analysis of cultured cells after the above. In the figure, the horizontal axis of the chart indicates the fluorescence intensity (10 g), the vertical axis indicates the number of cells, and M1 indicates the region set from the measured value of the control antibody (IgG1FITC).

  As shown in FIG. 1, the addition of β-N- (γ-glutamyl) -4-formylphenylhydrazine increased the expression of CD80 on DC by about 92% (measured values not shown). It has been shown that the addition of a novel compound induces the activation of dendritic cells.

  Similarly, FIG. 2 shows the test results for N- (3-carboxypropyl) -2-formyl-5-hydroxymethylpyrrole (final product 2). The chart shown in the upper part of FIG. 2 shows the results of flow cytometry analysis of cells to which no test compound was added, and the chart shown in the lower part of FIG. 2 shows N- (3-carboxypropyl) -2-formyl-5-hydroxymethylpyrrole. 4 shows the results of flow cytometry analysis of cultured cells after addition of. The horizontal axis and the vertical axis of the chart shown are the same as those in FIG.

  As shown in FIG. 2, the novel compound N- (3-carboxypropyl) -2-formyl-5-hydroxymethylpyrrole increased the expression of CD80 on DC by about 20% (measured values not shown). It was shown that the addition of N- (3-carboxypropyl) -2-formyl-5-hydroxymethylpyrrole induced the activation of dendritic cells.

  On the other hand, FIG. 3 shows the results obtained by adding picibanil (manufactured by Chugai Pharmaceutical Co., Ltd.) on the 6th day of starting DC culture for the same PBMC. Picibanil is a substance known to have the ability to induce DC. In the upper part of FIG. 3, the expression of CD80 on DC was increased by about 26% (measured values are not shown) by addition of picibanil, and in the lower part of FIG. 3, the activity of dendritic cells was increased by the addition of picibanil. This indicates that the transformation was induced.

  According to the results of FIGS. 1, 2 and 3, N- (3-carboxypropyl) -2-formyl-5-hydroxymethylpyrrole and β-N- (γ-glutamyl) -4-formylphenylhydrazine were DC It has been shown that β-N- (γ-glutamyl) -4-formylphenylhydrazine (final product 1) has an ability to induce DC more than picicibanil.

(Example 3) Growth inhibitory action of leukemia cell line N- (3-carboxypropyl) -2-formyl-5-hydroxymethylpyrrole (final product 2) synthesized in Example 1 above and β-N- ( γ-glutamyl) -4-formylphenylhydrazine (final product 1) was measured for its growth inhibitory effect on leukemia cell lines. The growth inhibitory effect of the leukemia cell line was measured using a TUNEL method (terminal deoxynucleotidyl transferase (TdT) -mediated deoxyuridine triphosphate (dUTP) -biotin nick).

  The TUNEL method points to apoptosis on tissue sections, reported by Gavrieli et al. In 1992 (Yael Gavrieli et al., The Journal of Cell Biology, Vol. 119, No. 3, November 1992, p. 493-501). Is the way. It is known as a method that enables observation of apoptosis at the level of individual cells in various disease states and comparison with morphological changes. The TUNEL method was performed on the leukemia cell line HL-60 using the Apoptosis in situ Detection Kit wako (Wako Pure Chemical Industries, Ltd.) according to the method of Gavrieli et al.

N- (3-carboxypropyl) -2-formyl-5-hydroxymethylpyrrole is present in a concentration range from 2.07 × 10 ⁻⁶ M to 2.07 × 10 ⁻⁴ M and and β-N- (γ- Glutamyl) -4-formylphenylhydrazine was tested in the 2.7 × 10 ⁻⁶ M to 2.7 × 10 ⁻⁴ M concentration range. The results are shown in FIGS.

  FIG. 4 shows various concentrations of N- (3-carboxypropyl) -2-formyl-5-hydroxymethylpyrrole (final product 2: abbreviated as F-Py in the upper left of FIG. 4), and Growth curve of the HL-60 cell line in the presence of β-N- (γ-glutamyl) -4-formylphenylhydrazine (final product 1: abbreviated as F-φ in the upper left of FIG. 4) is there. Each symbol in FIG. 4 indicates the measurement result of the number of cells in the presence of the component having the concentration described corresponding to each symbol shown in the upper left of FIG. 4 (cells / ml). Open circles are actinomycin D (ACD), a positive control for apoptosis-inducing ability, and solid circles are control plots without drug.

As shown in FIG. 4, both N- (3-carboxypropyl) -2-formyl-5-hydroxymethylpyrrole and β-N- (γ-glutamyl) -4-formylphenylhydrazine were substantially Inhibits the growth of HL-60 cells in a concentration-dependent manner, especially in the presence of 2.07 × 10 ⁻⁴ M of N- (3-carboxypropyl) -2-formyl-5-hydroxymethylpyrrole (F-Py) , And in the presence of 2.7 × 10 ⁻⁴ M of β-N- (γ-glutamyl) -4-formylphenylhydrazine (F-φ), the proliferation of HL-60 cells was remarkably suppressed. Was done.

FIG. 5 shows N- (3-carboxypropyl) -2-formyl-5-hydroxymethylpyrrole (F-Py) in the presence of 2.07 × 10 ⁻⁴ M and β-N- (γ) after 2 days of culture. FIG. 4 is a graph showing the ratio of apoptosis-positive cells in the presence of 2.7 × 10 ⁻⁴ M of (glutamyl) -4-formylphenylhydrazine (F-φ). The horizontal axis in FIG. 5 shows each test plot, and the vertical axis shows the percentage of apoptotic cells. As shown in FIG. 5, in the presence of 2.07 × 10 ⁻⁴ M of N- (3-carboxypropyl) -2-formyl-5-hydroxymethylpyrrole (F-Py), in about 35% of the cells, In the presence of 2.7 × 10 ⁻⁴ M of β-N- (γ-glutamyl) -4-formylphenylhydrazine (F-φ), apoptosis was induced in about 70% of the cells, respectively. It was confirmed that (3-carboxypropyl) -2-formyl-5-hydroxymethylpyrrole and β-N- (γ-glutamyl) -4-formylphenylhydrazine have apoptosis-inducing ability.

(Example 4)
Β-N- (γ-glutamyl) -4-formylaniline synthesized in Example 1 (final product 3) and N- (4-carboxybutyl) -2-formyl-5-hydroxymethylpyrrole (final product) Regarding 4), the immunostimulatory activity is measured in the same manner as in Example 2. The surface antigen on dendritic cells cultured in the presence of β-N- (γ-glutamyl) -4-formylaniline and N- (4-carboxybutyl) -2-formyl-5-hydroxymethylpyrrole was determined by monoclonal antibody By analyzing by flow cytometry using (anti-CD80; manufactured by BD Bioscience), the ability to induce DC cells is assayed to confirm the immunostimulatory activity.

(Example 5)
6 mg of each of ATO-1, ATO-3, and ATO-4 synthesized in Examples 1.3, 1.6, and 1.7 were placed in an Eppendorf tube, and added to DMSO to a final concentration of 100 μl / ml. After dissolution, a solution of each test sample was prepared. Each of the resulting test sample solutions was further diluted with DMSO to create a series of dilutions of different concentrations for each test sample. A series of dilution series of each of the obtained test samples was diluted 10-fold with freshly collected purified water (miliQ), and SOD activity was measured as a test solution.

The SOD activity was measured using a commercially available kit (DOJINDO MOLECULAR TECHNOLOGIES, INC.) According to the manufacturer's instructions. Briefly, test solutions were placed in microplates and reaction reagents were added. Finally, an enzyme (SOD) solution was added and incubated at 37 ° C. for 20 minutes. The absorbance of the microplate after the reaction was measured, and the SOD-like activity (enzyme inhibitory activity) was determined by the following equation.
SOD activity value (% inhibition) = [(A _blank1 -A _blank3 )-(A _sample -A _blank2 )] / (A _blank1 -A _blank3 ) × 100
Here, A _blank1 indicates the total amount of color development without inhibition, A _blank2 indicates the blank of the test solution itself, and A _blank3 indicates the blank of the SOD enzyme solution.

  Table 1 shows the results.

表１に示されるように、ＡＴＯ−１、ＡＴＯ−３、およびＡＴＯ−４のいずれも、その濃度が増加するにつれて、ＳＯＤの活性を阻害する割合が増加し、８３．３μｇ／ｍｌの濃度で、ＳＯＤ活性を、それぞれ約３０％、４０％および２１％阻害することが示された。

As shown in Table 1, as the concentration of ATO-1, ATO-3, and ATO-4 increased, the rate of inhibition of SOD activity increased, and at a concentration of 83.3 μg / ml. , SOD activity were shown to inhibit about 30%, 40% and 21%, respectively.

  Novel compounds having immunostimulatory activity and superoxide dismutase-like activity are provided. Instead of conventional products prepared from conventional natural materials and having a tendency to fluctuate in quality, formulations having a certain quality of biological activity are provided. These new compounds have the effect of promoting the induction of monocytes into dendritic cells, and the effect of decomposing reactive oxygen species, so that they efficiently promote the inflammatory response initiated by invasion of foreign substances into the living body. And a pharmaceutical composition capable of preventing aging and carcinogenesis associated with active oxygen.

FIG. 2 shows the immunostimulatory activity of the compound of the present invention. FIG. 2 shows the immunostimulatory activity of the compound of the present invention. It is a figure which shows the immunostimulatory activity of the immunostimulant picibanil. The figure which shows the growth curve of the leukemia cell line HL-60 in presence of N- (3-carboxypropyl) -2-formyl-5-hydroxymethylpyrrole and (beta) -N-([gamma] -glutamyl) -4-formylphenylhydrazine. It is. FIG. 4 is a diagram showing apoptosis of leukemia cell line HL-60 in the presence of N- (3-carboxypropyl) -2-formyl-5-hydroxymethylpyrrole and β-N- (γ-glutamyl) -4-formylphenylhydrazine. is there.

Claims (16)

Formyl-substituted phenyl or pyrrole compounds of any of formulas I-IV below:

Here, R ¹ is H, OR ³ , OR ⁴ , NHR ³ , NHR ⁴ , SR ³ , SR ⁴ , SO ₃ R ³ , SO ₃ R ⁴ , (CH ₂ ) _n R ³ , (CH ₂ ) _{n ^{R 4, (CH = CH)}} n R 3, (CH = CH) n R 4, C (= O) R 3, C (= O) R 4, PhR 3, PhR 4, and _{(CH 2)} n CHR ^A group selected from the group consisting of ³ R ⁴ ,
R ² is a group having the same meaning as the group defined for R ¹ and a group selected from the group consisting of C (NHR ³ ) R ³ ;
R ³ is a substituted or unsubstituted linear C ₁ -C ₆ alkyl;
R ⁴ is a linear C ₁ -C ₆ alkyl substituted with a carboxyl group;
n is an integer of 0-6. The compound according to claim 1, which has a 4-formylphenyl type structure represented by the formula II:
Here, R ¹ is a linear C ₁ -C ₆ alkyl substituted with a terminal carboxyl group, or an amino acid substituted with a terminal hydrazino group or a terminal amino group. In Formula II, ^{R 1} represents _{NH-NH-R a} or _{NH-R a,} wherein, _{R a} is alpha-or beta-aspartyl or alpha-, beta-or γ- glutamyl, wherein Item 3. The compound according to Item 2. 4. A compound according to claim 3, having the formula V:

A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 5, which has immunostimulatory activity. The composition according to claim 6, wherein the immunostimulatory activity is a dendritic cell-inducing activity-inducing ability. The composition according to claim 5, which has superoxide dismutase (SOD) -like activity. The compound according to claim 1, which has a 2-formylpyrrole type structure represented by the formula IV:
Here, R ¹ and R ² are independently OR ⁴ , NHR ⁴ , SR ⁴ , SO ₃ R ⁴ , (CH ₂ ) _n R ³ , (CH ₂ ) _n R ⁴ , (CH = CH) a _{^{n R 4, C (= O}} ) R 4, PhR 4 and _{(CH 2)} n ^CHR 3 groups selected from the group consisting of ^{R 4,}
R ³ is a substituted or unsubstituted linear C ₁ -C ₆ alkyl;
R ⁴ is a linear C ₁ -C ₆ alkyl substituted with a carboxyl group;
n is an integer of 0-6. Wherein R ² is C ₁ -C ₆ straight-chain alkyl substituted by a hydroxyl group, a compound of claim 9. 11. The compound of claim 10, which is represented by the following formula VI:

A pharmaceutical composition comprising a compound according to claim 9 and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 12, which has immunostimulatory activity. 14. The composition according to claim 13, wherein the immunostimulatory activity is a dendritic cell-inducing activity-inducing ability. The compound according to claim 1, wherein the compound is represented by the following formula IX.

The compound according to claim 1, which is represented by the following formula X:

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