JP2011042639A - Biphenylpyrazine compound, and erythropoietin production promoter containing the same as active ingredient - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a low-molecular compound having EPO(erythropoietin) production-promoting activity. <P>SOLUTION: The low-molecular compound is a biphenylpyrazine compound represented by formula (1) (wherein, R<SP>1</SP>is H or amino; and R<SP>2</SP>, R<SP>3</SP>, R<SP>4</SP>, R<SP>5</SP>, R<SP>6</SP>ad R<SP>7</SP>may be the same as or different from one another, each representing H, 1-6C alkoxy or carboxy). <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a novel erythropoietin production promoter. More specifically, the present invention relates to a preventive and / or therapeutic agent for diseases caused by decreased production of erythropoietin, such as anemia. The present invention also relates to a novel compound having an erythropoietin production promoting action.

  Erythropoietin (EPO) is a glycoprotein hormone involved in the maturation and differentiation of erythroid progenitor cells into mature erythrocytes, and is a naturally occurring monomeric polypeptide consisting of 165 amino acids (Non-patent Document 1). ).

  Human EPO is essential for the growth and differentiation of erythrocytes and is useful for the treatment of blood diseases characterized by a decrease in erythrocyte production. Clinically, EPO is found in patients with chronic renal failure (CRF) who are treated for anemia, self-contained blood, and premature infant anemia (Non-Patent Documents 2-4), in cancer patients undergoing AIDS and chemotherapy. (Non-patent Document 5). EPO has also been shown to be effective in chronic anemia.

  EPO is produced mainly in the kidney in adults, but it is also produced in astrocytes and neurons in the central nervous system, and EPO and EPO receptors are expressed in capillaries at the brain-periphery boundary. In addition, systemic administration of EPO has been reported to cross the blood brain barrier and reduce neuronal cell loss in response to brain and spinal cord ischemia, mechanical trauma, epilepsy, excitotoxins and neuroinflammation. (Non-Patent Documents 6 to 10).

  In the therapy using a protein such as EPO, in order to maintain a therapeutically effective concentration of the compound in the circulation and a short plasma half-life due to being susceptible to protease degradation (Non-patent Documents 11 and 12) There are problems such as having to make internal injections frequently. In addition, there is subcutaneous injection as an administration route instead of intravenous injection, but since the absorption from the administration site is slow, there is a sustained release effect, but the plasma concentration is significantly lower than that of intravenous injection. Therefore, in order to obtain an equivalent therapeutic effect, the same number of injections as in the case of intravenous injection must be performed, which is a burden on the patient. In addition, human serum EPO is a glycoprotein, and the structure of the sugar chain bound to the surface of EPO is complex, and its glycosylation is extensive and diverse. However, human serum EPO cannot be produced with good reproducibility.

  Therefore, there is a need in the art for methods and compounds that increase endogenous EPO, rather than low bioavailability EPO, in the treatment of diseases caused by reduced EPO production including anemia as described above.

  On the other hand, it is known that the expression level of EPO is controlled by the oxygen concentration via a hypoxia-inducible factor (HIF) that is a transcription factor (Non-patent Document 13). That is, under normal atmospheric conditions, the HIF subunit (HIF-1α) in which the proline residue is hydroxylated by 2-oxoglutarate dioxygenase enzyme is degraded by the ubiquitin-proteasome system, and the production of EPO is not promoted. Below, hydroxylation of the proline residue of HIF-1α by the 2-oxoglutarate dioxygenase enzyme is suppressed, and as a result, the stabilized HIF-1α moves from the cytoplasm into the nucleus and dimerizes with HIF-1β. It forms a body and binds to the hypoxia responsive element (HRE) sequence of the EPO gene to promote transcription and promote production of EPO.

  Enzyme inhibitors for HIF prolyl hydroxylase such as 2-oxoglutarate dioxygenase enzyme utilizing such an EPO production mechanism have been reported as EPO production promoters (Patent Documents 1 to 4).

  However, genes whose production is controlled by HIF include not only genes encoding EPO but also genes encoding vascular endothelial growth factor (VEGF). It has also been reported that VEGF has an angiogenesis-promoting action and can cause malignant tumors to deteriorate through this function (Non-Patent Documents 14 and 15). In addition, since anemia is also caused by cancer chemotherapy, and an anemia treatment drug may be administered to a cancer patient receiving such chemotherapy (Non-patent Document 6), VEGF worsens cancer. Such a risk is included in a compound having an inhibitory action on HIF prolyl hydroxylase enzyme activity that may also promote the production of and the like.

  Production of EPO is controlled by a promoter located on the 5 'side of EPO and an enhancer located on the 3' side, and HIF is thought to bind to the HRE sequence in the enhancer and promote expression of EPO. In addition, GATA-2, NFκB, and the like are also considered to control EPO production (Non-patent Documents 16 and 17), and it is considered that EPO production promotion can be achieved by an action mechanism other than inhibition of HIF prolyl hydroxylase enzyme activity. Therefore, a compound having an EPO production promoting action independent of inhibition of HIF prolyl hydroxylase enzyme activity is considered useful in the treatment of anemia.

  In addition, as described above, EPO promotes proliferation and maturation of erythroid progenitor cells, but a compound having an action of promoting maturation / differentiation of erythroid progenitor cells without involving production of EPO is also a therapeutic agent for anemia. Useful as. Compounds that have an activity to enhance blood cell proliferation promoting action of EPO and compounds that have an inhibitory action on hematopoietic cell phosphatase that catalyzes dephosphorylation, which is one of the important control mechanisms of EPO signaling, have been reported. (Patent Documents 5 to 7), the activity is not always sufficient. Furthermore, although synthetic peptides and hematides that act on the EPO receptor have been reported (Non-patent Document 18), a high dose is required for the expression of activity equivalent to EPO, and it is not suitable for oral administration. There is.

  Therefore, an orally administrable low-molecular-weight anemia treatment agent having an EPO production promoting action is considered useful in future anemia treatment.

  On the other hand, various pharmacological actions have been reported for compounds having a biphenylpyrazine skeleton related to the present invention. For example, it can be used as an apo AI expression enhancer useful for arteriosclerosis (patent document 8), platelet aggregation inhibitor (patent documents 9, 10, non-patent document 19), antioxidant (patent document 11), etc. It is described in the literature. However, these documents have no description or suggestion regarding the action of enhancing the production of erythropoietin such as the compound of the present invention or the treatment of anemia, and no therapeutic agent for anemia having a biphenylpyrazine skeleton has been known.

JP 2006-137763 A WO2003 / 53997 pamphlet WO2005 / 11696 pamphlet WO2007 / 38571 pamphlet Special Table 2000-536365 JP-A-11-171774 JP 2002-275159 A JP 2001-335476 A JP-A-63-208520 Japanese Patent Laid-Open No. 3-074370 WO2001 / 87853 pamphlet

Lin F-K et al., Proc. Natl. Acad. Sci. USA, 82: 7580-7584 (1985) Eschbach JW, et al., N. Engl. J. Med, 316: 73-78 (1987) Eschbach JW, et al., Ann. Intern. Med., 111: 992 (1989) Lim VS, et al., Ann. Intern. Med., 110: 108-114 (1989) Danna RP, et al., Erythropoietin in Clinical Applications-An International Perspective, New York: Marcel Dekker; p301-324 (1990) Sakanaka M, et al., Proc. Natl. Acad. Sci. USA., 95, 4635-4640 (1998) Celik M, et al., Proc. Natl. Acad. Sci. USA., 99, 2258-2263 (2002) Brines ML, et al., Proc. Natl. Acad. Sci. USA., 97, 10526-10531 (2000) Calapai G, et al., Eur. J. Pharmacol., 401, 349-356 (2000) Siren A-L, et al., Proc. Natl. Acad. Sci. USA., 98, 4044-4049 (2001) Spivack JL and Hogans BB, Blood, 73: 90 (1989) McMahon FG, et al., Blood, 76: 1718 (1990) Jelkman W, Internal Medicine 43, 649-659 (20 04) Maxwell PH, et al., Proc. Natl. Acad. Sci. USA. 94, 15, 8104-8109 (1997) Fang J, et al., Cancer Res., 61, 15, 5731-5735 (2001) Imagawa S, et al., Blood 89, 1430-1439 (1997) La Ferla K, et al., FASEB J, 16, 1811-1813 (2002) Stead RB, et al., Blood 108, 1830-1834 (2006) Ohta A, et al., Biol. Pharm. Bull. 20, 1076-1081 (1997)

  An object of the present invention is to provide a compound having a low molecular weight EPO production promoting action. More specifically, the object is to provide a medicament useful for the prevention and / or treatment of anemia.

  In view of the above circumstances, the present inventors have intensively searched for a compound having an EPO production promoting action. As a result, the biphenylpyrazine compound represented by the following general formula (1) was tested using HepG2 cells derived from human liver cancer. And found that the production of EPO was promoted, and the present invention was completed.

  That is, the present invention provides the following general formula (1):

[Where:
R ¹ represents a hydrogen atom or an amino group,
R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ may be the same or different and each represents a hydrogen atom, a C _1-6 alkoxy group or a carboxyl group]
The EPO production promoter which uses the biphenylpyrazine compound represented by these, its salt, or those solvates as an active ingredient.

More particularly, the present invention provides the following group of compounds:
2- (4-Carboxyphenyl) -6- (3,4,5-trimethoxyphenyl) pyrazine (Compound 1), and 2-amino-3,5-biphenylpyrazine (Compound 2)
The EPO production promoter selected from the group consisting of:

  Moreover, this invention relates to the EPO production promoter which uses the biphenyl pyrazine compound represented by the said General formula (1), its salt, or those solvates as an active ingredient. More specifically, the present invention provides a prophylactic and / or therapeutic agent for anemia comprising a biphenylpyrazine compound selected from the group of compounds described above, or a salt thereof, or a solvate thereof as an active ingredient.

  The present invention also relates to the use of the biphenylpyrazine compound compound represented by the general formula (1) or a salt thereof or a solvate thereof for producing a preparation for promoting EPO production. More specifically, the present invention provides use of a biphenylpyrazine compound selected from the group of compounds described above, or a salt thereof, or a solvate thereof for producing a preparation for promoting EPO production. Is.

  Furthermore, the present invention provides administration of an effective amount of the biphenylpyrazine compound represented by the above general formula (1), or a salt thereof, or a solvate thereof to a patient who is required to promote production of EPO. The present invention relates to a method for promoting EPO production. More specifically, the present invention is to administer an effective amount of a biphenylpyrazine compound selected from the above-mentioned compound group, or a salt thereof, or a solvate thereof to a patient in need of promotion of EPO production. The present invention provides a method for promoting EPO production.

  In addition, the present invention provides a method for promoting EPO production in a cell by contacting the cell with an effective amount of the biphenylpyrazine compound represented by the general formula (1), or a salt thereof, or a solvate thereof. About. More specifically, the present invention relates to a method for promoting the production of EPO in a cell by contacting the cell with an effective amount of a biphenylpyrazine compound selected from the group of compounds described above, or a salt thereof, or a solvate thereof. Is to provide. Here, the term “contact” as used herein means that the biphenylpyrazine compound or the like of the present invention is in vitro or in vivo due to the uptake action of the compound or the like by a cell or the interaction on the cell surface. It means that the aforementioned compound or the like is added to cells so as to regulate cell functions such as proliferation, differentiation and secretion of physiologically active substances.

From another point of view, the present invention provides the following group of compounds:
2- (4-Carboxyphenyl) -6- (3,4,5-trimethoxyphenyl) pyrazine (Compound 1), and 2-amino-3,5-biphenylpyrazine (Compound 2)
A biphenylpyrazine compound compound, or a salt thereof, or a solvate thereof is selected from the group consisting of:

  Furthermore, the present invention promotes the production of EPO comprising the biphenylpyrazine compound represented by the general formula (1), or a salt thereof, or a solvate thereof, and a pharmaceutically acceptable carrier. The present invention relates to a pharmaceutical composition. More specifically, the present invention contains a biphenylpyrazine compound selected from the aforementioned compound group, or a salt thereof, or one or more compounds of solvates thereof, and a pharmaceutically acceptable carrier. Thus, a pharmaceutical composition for promoting the production of EPO is provided.

  The present invention has found a biphenylpyrazine compound, a salt thereof, or a solvate thereof having an excellent EPO production promoting action, and a disease whose symptoms are improved by promoting EPO production. (E.g., anemia in patients with chronic renal failure, anemia in premature infants, anemia in cancer patients undergoing AIDS and chemotherapy, chronic anemia, iron deficiency anemia, aplastic anemia, hemolytic anemia, giant red bud It is useful as a pharmaceutical composition for the prevention and / or treatment of anemia such as angiocytic anemia). The present invention also provides a preventive and / or therapeutic agent for anemia comprising an orally administrable low molecular weight compound having an EPO production promoting action as an active ingredient.

  Hereinafter, the present invention will be described in detail.

  The definitions of terms in the present invention are as follows.

  As used herein, “halogen atom” means a halogeno group, specifically a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

As used herein, an “alkyl group” may be linear or branched. Accordingly, the “C _1-6 alkyl group” specifically includes methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n -Pentyl group, isopentyl group, neopentyl group, 4-methylbutyl group, 1-ethylpropyl group, n-hexyl group, isohexyl group, 3-methylpentyl group, 2-methylpentyl group, 1-methylpentyl group, 3, 3 -Dimethylbutyl group, 2,2-dimethylbutyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2,3-dimethylbutyl group, 1-ethylbutyl group, Examples thereof include a linear or branched alkyl group having 1 to 6 carbon atoms such as a 2-ethylbutyl group.

As used herein, an “alkoxy group” may be linear or branched. Therefore, as the “C _1-6 alkoxy group”, specifically, methyloxy group, ethyloxy group, n-propyloxy group, isopropyloxy group, n-butyloxy group, isobutyloxy group, sec-butyloxy group, tert- Butyloxy group, n-pentyloxy group, isopentyloxy group, neopentyloxy group, 4-methylbutyloxy group, 1-ethylpropyloxy group, n-hexyloxy group, isohexyloxy group, 3-methylpentyloxy group 2-methylpentyloxy group, 1-methylpentyloxy group, 3,3-dimethylbutyloxy group, 2,2-dimethylbutyloxy group, 1,1-dimethylbutyloxy group, 1,2-dimethylbutyloxy group 1,3-dimethylbutyloxy group, 2,3-dimethylbutyloxy group, 1-ethylbutyl Examples thereof include straight-chain or branched alkoxy groups having 1 to 6 carbon atoms such as tiloxy group and 2-ethylbutyloxy group.

  Other groups not defined here follow the usual definitions.

In the general formula (1), as the “C _1-6 alkoxy group” in R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , a “C _1-4 alkoxy group” is preferable, and a methoxy group is More preferred.

Specific examples of the biphenylpyrazine compound represented by the general formula (1) of the present invention include the following compounds.
2- (4-Carboxyphenyl) -6- (3,4,5-trimethoxyphenyl) pyrazine (Compound 1), and 2-amino-3,5-biphenylpyrazine (Compound 2)

  The biphenylpyrazine compound represented by the general formula (1) of the present invention, or a salt thereof, or a solvate thereof includes not only the biphenylpyrazine compound of the present invention but also a pharmaceutically acceptable salt thereof, It includes hydrates, solvates, substances having crystalline polymorphs, and substances that are prodrugs of these substances.

  As the pharmaceutically acceptable salt of the biphenylpyrazine compound represented by the general formula (1) of the present invention, specifically, when the compound is treated as a basic compound, an inorganic acid (for example, hydrochloric acid, hydrogen bromide) is used. Acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, etc.) and organic acids (eg formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, Examples include acid addition salts with citric acid, methane sulfonic acid, ethane sulfonic acid, p-toluene sulfonic acid, aspartic acid, glutamic acid and the like. When the compound is treated as an acidic compound, an inorganic salt (for example, sodium salt) , Potassium salt, lithium salt, barium salt, calcium salt, magnesium salt, etc.) and organic salt (eg pyridinium salt, picolinium salt, triethylammonium salt) ), And the like.

  Examples of the solvate of the biphenylpyrazine compound represented by the general formula (1) of the present invention or a pharmaceutically acceptable salt thereof include hydrates and various solvates (for example, solvates with alcohols such as ethanol). Product).

  The biphenylpyrazine compound represented by the general formula (1) of the present invention can be produced by a known method. For example, although it can manufacture by the method shown below or the method according to this, it is not limited to this. Moreover, you may perform each reaction, protecting a functional group as needed. The protection and deprotection conditions can be carried out with reference to commonly used methods (Protective Groups in Organic Synthesis Third Edition, John Wiley & Sons, Inc.).

[Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ are the same as those described above, R ⁸ and R ⁹ are a hydroxyl group, a C _1-6 alkyl group, C _1-6 represents an alkoxy group, and X ¹ and X ^{2 each} represent a halogen atom. ]

[Steps 1 and 2] The biphenylpyrazine compound (1) is obtained by reacting the dihalogenopyridine compound (I) with the boronic acid derivatives (a) and (b) in the presence of a catalyst and a base in a solvent in a Suzuki-Miyaura coupling reaction. It can manufacture by attaching | subjecting to. Examples of the solvent used here include benzene, toluene, xylene, diethyl ether, tetrahydrofuran, dimethoxyethane, dioxane, acetonitrile, dimethylformamide, N-methylpiperidone, methanol, ethanol, water, and the like. Moreover, you may use combining these solvents. Examples of the catalyst include tetrakis (triphenylphosphine) palladium (0), tris (bisbenzylideneacetone) dipalladium (0), palladium (II) acetate, palladium (II) chloride, dichloro [1,2-bis (diphenylphosphino). ) Ethane] palladium (II), dichloro [1,2-bis (diphenylphosphino) butane] palladium (II), dichloro [1,1′-bis (diphenylphosphino) ferrocene] palladium (II) and the like. . In addition, tri (t-butyl) phosphine, triphenylphosphine, tri (o-tolyl) phosphine, tri (2-furyl) phosphine, 2,2′-bis (diphenylphosphino), if necessary for these catalysts. A ligand such as -1,1′-binaphthyl, 1,2-bis (diphenylphosphino) ethane may be combined. Examples of the base include sodium acetate, potassium carbonate, sodium carbonate, sodium hydrogen carbonate, potassium phosphate, sodium hydroxide, potassium hydroxide, barium hydroxide, sodium methoxide, sodium ethoxide, cesium fluoride, triethylamine and the like. . The reaction conditions are room temperature to 200 ° C. for 30 minutes to 4 days, preferably room temperature to 100 ° C. for 4 to 24 hours. Further, this reaction may be a multi-stage reaction for isolating intermediate (II) or a one-pot reaction in which Step 1 and Step 2 are carried out successively.

  Intermediates and target products obtained in the above reactions are necessary for purification methods commonly used in organic synthetic chemistry, such as filtration, extraction, washing, drying, concentration, recrystallization, various chromatography, etc. Can be isolated and purified. In addition, the intermediate can be subjected to the next reaction without any particular purification.

  Furthermore, various isomers can be isolated by applying a conventional method using the difference in physicochemical properties between the isomers. For example, a racemic mixture is obtained by a general racemic resolution method such as a method of optically resolving a diastereomeric salt with a general optically active acid such as tartaric acid or a method using optically active column chromatography. Can lead to optically pure isomers. Further, the diastereomeric mixture can be divided by, for example, fractional crystallization or various chromatography. An optically active compound can also be produced by using an appropriate optically active raw material.

  The EPO production promoter or anemia treatment agent of the present invention comprises a biphenylpyrazine compound represented by the general formula (1), a salt thereof, or a solvate thereof as an active ingredient, and a pharmaceutical composition Can be used as In that case, the compound of the present invention may be used alone, but it is usually used in combination with a pharmaceutically acceptable carrier and / or diluent.

  The administration route is not particularly limited, but can be appropriately selected depending on the purpose of treatment. For example, any of oral preparations, injections, suppositories, inhalants and the like may be used. Pharmaceutical compositions suitable for these dosage forms can be produced by utilizing known preparation methods.

  When preparing an oral solid preparation, the biphenylpyrazine compound represented by the general formula (1) is a pharmaceutically acceptable excipient, and if necessary, a binder, a disintegrant, a lubricant, a colorant, After adding a flavoring agent, a flavoring agent, etc., a tablet, a coated tablet, a granule, a powder, a capsule, etc. can be manufactured using a conventional method. Additives may be those commonly used in the art. Examples of excipients include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid and the like. Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, polyvinylpyrrolidone and the like. Examples of the disintegrant include dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, and lactose. Examples of the lubricant include purified talc, stearate, borax, and polyethylene glycol. Examples of the corrigent include sucrose, orange peel, citric acid, and tartaric acid.

  When preparing oral liquid preparations, oral liquids and syrups are added to the biphenylpyrazine compound represented by the general formula (1) by adding a corrigent, a buffer, a stabilizer, a corrigent and the like using conventional methods. An elixir or the like can be produced. As the corrigent, those mentioned above may be used. Examples of the buffer include sodium citrate, and examples of the stabilizer include tragacanth, gum arabic, and gelatin.

  When preparing injections, add pH adjusters, buffers, stabilizers, isotonic agents, local anesthetics, etc. to the biphenylpyrazine compound represented by the general formula (1), and use conventional methods. Subcutaneous, intramuscular and intravenous injections can be manufactured. Examples of the pH adjusting agent and buffer include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid and the like. Examples of local anesthetics include procaine hydrochloride and lidocaine hydrochloride. Examples of isotonic agents include sodium chloride and glucose.

  When preparing a suppository, a known suppository carrier for the biphenylpyrazine compound represented by the general formula (1), for example, polyethylene glycol, lanolin, cocoa butter, fatty acid triglyceride, and the like, and a surfactant (for example, , Tween (registered trademark)) and the like can be added and then manufactured using a conventional method.

  In addition to the above, it is possible to appropriately prepare a preferable preparation using a conventional method.

  The dose of the biphenylpyrazine compound represented by the general formula (1) of the present invention varies depending on age, body weight, symptom, dosage form, number of administrations, etc., but is usually a compound represented by the general formula (1) for adults 1 mg to 1000 mg per day is preferably administered orally or parenterally in 1 or several divided doses.

  Next, although an example and a test example are given and the present invention is further explained, the present invention is not limited to these examples.

Example 1: Preparation of 2- (4-carboxyphenyl) -6- (3,4,5-trimethoxyphenyl) pyrazine (Compound 1)

[Step 1] Under an argon atmosphere, 58 mg (0.38 mmol) of 2,6-dichloropyrazine, 21 mg (0.097 mmol) of 3,4,5-trimethoxyphenylboronic acid, 43 mg of tetrakis (triphenylphosphine) palladium (0. 038 mmol) 0.3 mL of 2M sodium carbonate aqueous solution was stirred at 70 ° C. for 1.5 hours in a mixed solvent of toluene-methanol (2: 1) 1.5 mL. After completion of the reaction, water and 1N aqueous hydrochloric acid solution were added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: hexane = 1: 10 to 1: 5), and 13 mg (58%) of 6-chloro-2- (3,4,5-trimethoxyphenyl) pyrazine was added to a white needle. Obtained as a crystal.

[Step 2] Under argon atmosphere, 13 mg (0.055 mmol) of 6-chloro-2- (3,4,5-trimethoxyphenyl) pyrazine, 12 mg (0.072 mmol) of 4-carboxyphenylboronic acid, tetrakis (triphenyl) Phosphine) palladium (7 mg, 0.0055 mmol), 2M aqueous sodium carbonate solution (0.2 mL) was stirred in a mixed solvent of toluene-tetrahydrofuran (1: 1) (2 mL) at 70 ° C. for 16 hours. After completion of the reaction, water and 1N aqueous hydrochloric acid solution were added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform: methanol = 50: 1) to obtain 5.0 mg (25%) of the title compound as a pale yellow solid.

MS (FAB); M ⁺ + 1 = 367

Example 2: Preparation of 2-amino-3,5-biphenylpyrazine (compound 2)

  2-amino-3,5-dibromopyrazine 154 mg (0.61 mmol), phenylboronic acid 220 mg (1.8 mmol), tetrakis (triphenylphosphine) palladium 35 mg (0.030 mmol), sodium carbonate 515 mg (4.9 mmol). , Water-toluene-ethanol (5: 15: 3) was stirred at 80 ° C. for 12 hours in 11.5 mL of a mixed solvent. After completion of the reaction, water and 1N aqueous hydrochloric acid solution were added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: hexane = 1: 3 to 1: 1) and crystallized from ethyl acetate-hexane to give 122 mg (81%) of the title compound as a pale yellow crystalline powder.

¹ H-NMR (400 MHz, CDCl ₃ ) δ: 4.82 (2 H, br. S), 7.36 (1 H, tt, J = 7.2, 1.2 Hz), 7.42-7.49 (3 H, m), 7.53 (2 H, dd, J = 7.2, 7.2 Hz), 7.83 (2 H, dd, J = 7.2, 1.2 Hz), 7.97 (2 H, dd, J = 7.2, 1.2 Hz), 8.45 (1 H, s)

[Test example]
The compounds used in the test examples were those prepared by the method described in the examples.

Test Example The EPO production promoting activity of the biphenylpyrazine compound of the present invention was measured based on the EPO promoter activity.

<Materials and methods>
A vector in which the 5 ′ promoter region and 3 ′ enhancer region of human EPO were linked to the luciferase gene of pGL3 basic (Promega) was introduced into a cell line HepG2 derived from human liver cancer by lipofection. The obtained stable expression strain (HepG2 EPO-luc) was used for the evaluation of the test compound. Next, 5 × 10 ³ cells of HepG2 EPO-luc were seeded in each well of a 96-well plate using a minimum essential medium (MEM) (Sigma) containing 10% fetal bovine serum. Thereafter, the test compound dissolved in DMSO was added to each well so as to have a final concentration of 30 μM, and the amount of the medium was adjusted to 100 μl per well. After incubation for 24 hours in a CO ₂ incubator with an oxygen concentration of 4%, Bright-Glo ™ Luciferase Assay System (Promega) was added at 100 μl / well as EPO promoter activity, and immediately using ARVO (Perkin Elmer) The luciferase activity was measured. The above method was in accordance with the instruction manual attached to the Bright-Glo (trademark) Luciferase Assay System.

  The EPO promoter activity (% of control) induced by each compound was determined with the EPO promoter activity in an unstimulated state without addition of the compound as 100%. The results are shown in Table 1.

<Result>
EPO production was promoted by adding a test compound at a concentration of 30 μM (see Table 1). From this, it became clear that these compounds have an EPO production promoting action, and were found to be useful as an anemia treatment agent.

  The present invention for the first time finds that the biphenylpyrazine compound represented by the general formula (1) or a salt thereof, or a solvate thereof has an excellent EPO production promoting action, and an excellent EPO production promoting action. An orally administrable low-molecular-weight anemia-preventing and / or treating agent is provided. The present invention provides a novel low-molecular-weight anemia prevention and / or treatment agent, which is useful in the pharmaceutical industry and has industrial applicability.

Claims (7)

The following general formula (1):

[Where:
R ¹ represents a hydrogen atom or an amino group,
R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ may be the same or different and each represents a hydrogen atom, a C _1-6 alkoxy group or a carboxyl group]
The EPO production promoter which uses the biphenylpyrazine compound represented by these, its salt, or those solvates as an active ingredient.   A prophylactic and / or therapeutic agent for anemia comprising a compound represented by the general formula (1), a salt thereof, or a solvate thereof as an active ingredient. The following group of compounds:
A biphenylpyrazine compound selected from the group consisting of 2- (4-carboxyphenyl) -6- (3,4,5-trimethoxyphenyl) pyrazine and 2-amino-3,5-biphenylpyrazine, or a salt thereof, or The agent according to claim 1 or 2, which is a solvate thereof. The following group of compounds:
A biphenylpyrazine compound selected from the group consisting of 2- (4-carboxyphenyl) -6- (3,4,5-trimethoxyphenyl) pyrazine and 2-amino-3,5-biphenylpyrazine, or a salt thereof, or Their solvates.   A pharmaceutical composition comprising a biphenylpyrazine compound selected from the group of compounds according to claim 4, or a salt thereof, or one or more compounds of solvates thereof, and a pharmaceutically acceptable carrier. .   The pharmaceutical composition according to claim 5, which is for promoting production of EPO.   The pharmaceutical composition according to claim 5, which is used for prevention and / or treatment of anemia.