JP2007191451A - Prolyl hydroxylase inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a prolyl hydroxylase inhibitor (PHD inhibitor) that ameliorates a hypoxic condition, is useful for protecting cells and tissues from damages by hypoxia, a pharmaceutical composition for ameliorating the hypoxic conditions of cells and tissues and a new compound useful as a PHD inhibitor and to provide a method for screening a compound effective as a PHD inhibitor. <P>SOLUTION: The compound containing at least one of a group having a backbone represented by formula (A) or (B) (R<SP>8</SP>and R<SP>9</SP>are mutually hydrogen atoms or may form a ring with an alkylene chain, an oxygen atom, a nitrogen atom or a sulfur atom bonded to them) in the compound or its salt is used as the active ingredient of the prolyl hydroxylase inhibitor or the pharmaceutical composition for ameliorating the hypoxic conditions of cells and tissues. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a prolyl hydroxylase inhibitor (hereinafter also referred to as “PHD inhibitor”). More specifically, the present invention has an action of inhibiting the action of PHD by inhibiting the binding between hypoxia-inducing factor (hereinafter also referred to as “HIF”) and prolyl hydroxylase (PHD). The present invention relates to a PHD inhibitor capable of stabilizing HIF by suppressing the degradation of HIF. The present invention also relates to a pharmaceutical composition which has a PHD inhibitory action and is effective for preventing or treating various diseases and pathologies (disorders) caused by hypoxia by stabilizing HIF. Furthermore, the present invention relates to a novel compound useful as a PHD inhibitor and a screening method effective for obtaining a compound useful as a PHD inhibitor.

  In diseases such as ischemic heart disease, chronic renal failure, and cerebral infarction, oxygen supply to cells and tissues decreases. If hypoxia continues due to such a decrease in oxygen supply, functional disorders occur in cells and tissues, so that cellular defense mechanisms work widely in vivo, including angiogenesis, erythropoiesis, glycolysis and oxidase inhibition. It has become.

  Hypoxia-inducible factor (HIF) is an important protein that mediates these biological defense mechanisms, and its expression increases when cells and tissues fall into hypoxia, and cells and tissues obtain more oxygen. As described above, it is known to promote angiogenesis (for example, see Non-Patent Documents 1 to 3). Such a hypoxia-inducing factor (HIF) is a protein consisting of a heterodimer of an α chain and a β chain, and is stable in a hypoxic state, but its stability is significantly reduced in the presence of oxygen. Specifically, in HIF, α-chain proline is hydroxylated by oxygen-dependent prolyl hydroxylase (PHD) in the presence of oxygen (see, for example, Non-Patent Documents 4 to 9), and as a result, von hipple lindau It binds to a protein and is ubiquitinated (see, for example, Non-Patent Documents 10 to 11), and as a result, it is targeted for proteasome and decomposed (for example, see Non-Patent Documents 12 to 13).

  However, under hypoxic conditions, HIF is not degraded because the α chain is not hydroxylated by PHD, and is bound to HRE (hypoxia response element) in the nucleus in the state of a dimer of α and β chains. However, it promotes transcription of many genes (for example, erythropoietin (erythropoiesis hormone), vascular endothelial growth factor (VEGF), glycolytic enzyme, glucose transporter, heme oxygenase) involved in adaptation to hypoxic stress located downstream. Thus, the living body functions to improve hypoxia (for example, see Non-Patent Document 14). For this reason, it is considered that stabilizing the HIF can activate the switch of the “master gene” that widely regulates downstream reactions, and can protect tissues and cells from the influence of hypoxia.

Currently, many treatments for diseases that result in hypoxia such as ischemia are used to improve symptoms and pathogenic factors, such as the use of thrombolytic drugs to improve blood flow during acute ischemia. It is the actual situation that is concentrated, and it is required to consider treatment methods from other angles.
Marx, J. Cell biology. How cells endure low oxygen. Science 303. 1454-1456 (2004): Semenza, GL & Wang, GL A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol. Cell Biol. 12. 5447-5454 (1992): Wang, GL & Semenza, GL General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia. Proc. Natl. Acad. Sci. 90. 4304-4308 (1993) Epstein, ACR et al. C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation.Cell 107. 43-54 (2001) Schofield, CJ & Ratcliffe, PJ Oxygen sensing by HIF hydroxylases. Nat. Rev. Mol. Cell Biol. 5. 343-354 (2004) Semenza, GL Hydroxylation of HIF-1: oxygen sensing at the molecular level.Physiology (Bethesda). 19. 176-182 (2004) Masson, N. & Ratcliffe, PJ HIF prolyl and asparaginyl hydroxylases in the biological response to intracellular O (2) levels. J. Cell Sci. 116. 3041-3049 (2003) Bruick, RK & McKnight, SL A conserved family of prolyl-4-hydroxylases that modify HIF. Science 294. 1337-1340 (2001) Semenza, GL HIF-1, O (2), and the 3 PHDs: how animal cells signal hypoxia to the nucleus.Cell 107. 1-3 (2001) Maxwell, PH et al. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399. 271-275 (1999) Ohh, M. et al. Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel-Lindau protein. Nat. Cell Biol. 2. 423-427 (2000) Maxwell, PH et al. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399. 271-275 (1999) Ohh, M. et al. Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel-Lindau protein. Nat. Cell Biol. 2. 423-427 (2000) Pugh, CW & Ratcliffe, PJ Regulation of angiogenesis by hypoxia: role of the HIF system. Nat. Med. 9. 677-684 (2003)

  The present invention provides a prolyl hydroxylase inhibitor (PHD inhibitor) that is effectively used to improve hypoxia caused by ischemic diseases and protect cells and tissues from damage caused by hypoxia. With the goal. The PHD inhibitor provided by the present invention is a PHD that hydroxylates proline of the HIFα chain by competitively inhibiting the binding of the hypoxia-inducing factor (HIF) α-chain proline and prolyl hydroxylase (PHD). It has the effect | action which inhibits the function of. Another object of the present invention is to provide a pharmaceutical composition using such a PHD inhibitor for the purpose of improving hypoxia of cells and tissues. It is another object of the present invention to provide a novel compound effective as a PHD inhibitor and a screening method for searching for an effective compound as a PHD inhibitor.

  As described above, hypoxia-inducible factor (HIF) plays a very important role in cell and tissue damage caused by hypoxia. Its activity is controlled by hydroxylation of the proline residue of the α chain by prolyl hydroxylase (PHD) in an oxygen-dependent manner. For this reason, HIF is stabilized by inhibiting PHD, thereby activating a biological defense function against a hypoxic state and protecting cells and tissues from damage caused by hypoxia.

  Based on this idea, the present inventors first used a drug design software based on the three-dimensional structure and structure of prolyl hydroxylase to develop a novel prolyl hydroxylase inhibitor. A docking simulation study was conducted using a prolyl hydroxylase inhibitor. As a result, prolyl hydroxylase has both a site that binds to HIFα proline and a site that binds to 2-oxyglutase, and the binding of HIF to proline hydroxylase through the site that binds to HIFα proline. It has been found that a compound having a specific group having an action of competitively inhibiting the activity is effective as a prolyl hydroxylase inhibitor that stably activates or stabilizes HIF. Furthermore, it was confirmed by in vivo tests that the compound having this specific group can stabilize HIF activity, promote angiogenesis, and prevent nerve death due to ischemia. The present invention has been completed based on such findings.

That is, the present invention includes the following embodiments:
(I) Prolyl hydroxylase inhibitor (PHD inhibitor)
Item 1. A prolyl hydroxylase inhibitor comprising, as an active ingredient, a compound containing at least one group having a skeleton represented by the following formula (A) or (B) or a salt thereof in one molecule:

（式中、Ｒ_８およびＲ_９は、互いに水素原子であるか、またはこれらが結合するアルキレン鎖、酸素原子、窒素原子または硫黄原子とともに環を形成してもよい。）。
項２．骨格Ａを有する基が、下式：

(In the formula, R ₈ and R ₉ may be hydrogen atoms with each other or may form a ring together with the alkylene chain, oxygen atom, nitrogen atom or sulfur atom to which they are bonded).
Item 2. A group having a skeleton A is represented by the following formula:

（Ｒ_１は水酸基、低級アルキル基、カルボキシル基で置換されたアルキル基、保護基を有していてもよいカルボキシアルキル基、またはアミノ基：Ｒ_２は水素原子、水酸基、低級アルキル基、カルボキシル基で置換されたアルキル基、またはアミノ基：Ｒ_３は水素原子、アミノ基、または低級アルキルアミノ基を意味する）
で示される基である、項１記載のプロリル水酸化酵素阻害剤。
項３．骨格Ｂを有する基が、下式：

(R ₁ is a hydroxyl group, a lower alkyl group, an alkyl group substituted with a carboxyl group, a carboxyalkyl group optionally having a protecting group, or an amino group: R ₂ is a hydrogen atom, a hydroxyl group, a lower alkyl group, a carboxyl group. An alkyl group substituted with or an amino group: R ₃ represents a hydrogen atom, an amino group, or a lower alkylamino group)
Item 2. The prolyl hydroxylase inhibitor according to Item 1, which is a group represented by:
Item 3. The group having the skeleton B has the following formula:

（Ｒ_４、Ｒ_５、Ｒ_６およびＲ_７は、同一または異なって、水素原子または低級アルキル基を意味する。Ｒ_４とＲ_５またはＲ_６とＲ_７は互いに結合して、それらが結合する炭素原子と共に、５または６員の飽和もしくは不飽和の置換基を有していてもよいシクロ環またはヘテロ環を形成してもよい。Ｒ_８およびＲ_９は、互いに水素原子であるか、またはこれらが結合するアルキレン鎖、酸素原子、窒素原子または硫黄原子とともに環を形成してもよい。）
で示される基である、項１または２に記載するプロリル水酸化酵素阻害剤。
項４．下式で示される化合物（Ｉ）：

(R ₄ , R ₅ , R ₆ and R ₇ are the same or different and each represents a hydrogen atom or a lower alkyl group. R ₄ and R ₅ or R ₆ and R ₇ are bonded to each other, and they are bonded. Together with the carbon atom it may form a cyclo or hetero ring optionally having 5 or 6 membered saturated or unsaturated substituents, R ₈ and R ₉ are each a hydrogen atom, or A ring may be formed together with an alkylene chain, an oxygen atom, a nitrogen atom or a sulfur atom to which they are bonded.)
Item 3. The prolyl hydroxylase inhibitor according to Item 1 or 2, which is a group represented by:
Item 4. Compound (I) represented by the following formula:

〔Ｒ_１は炭素数１〜６の低級アルキル基または保護基を有していてもよい炭素数１〜４のカルボキルアルキル基：Ｒ_２は水素原子、炭素数１〜６の低級アルキル基：Ｒ_１０は、Ｒ_１１−Ｓ−ＣＨ_２−（式中、Ｒ_１１は置換基を有していてもよいヘテロアリール基、または下式：

[R ₁ represents carboxyalkyl kill alkyl group having 1 to 4 carbon atoms which may have a lower alkyl group or a protective group of 1 to 6 carbon atoms: R ₂ is a hydrogen atom, a lower alkyl group having 1 to 6 carbon atoms: R ₁₀ represents R ₁₁ —S—CH ₂ — (wherein R ₁₁ is a heteroaryl group which may have a substituent, or the following formula:

（Ｒ_４、Ｒ_５、Ｒ_６およびＲ_７は、同一または異なって、水素原子または炭素数１〜６の低級アルキル基を意味する。Ｒ_４とＲ_５またはＲ_６とＲ_７は互いに結合して、それらが結合する炭素原子と共に、５または６員の飽和もしくは不飽和の置換基を有していてもよいシクロ環またはヘテロ環を形成してもよい。）で示される基を意味する。〕
および下式で示される化合物（II）：

(R ₄ , R ₅ , R ₆ and R ₇ are the same or different and each represents a hydrogen atom or a lower alkyl group having 1 to 6 carbon atoms. R ₄ and R ₅ or R ₆ and R ₇ are bonded to each other. And may form a cyclo or hetero ring optionally having a 5- or 6-membered saturated or unsaturated substituent together with the carbon atom to which they are bonded. ]
And compound (II) represented by the following formula:

（Ｒ_５およびＲ_７は、同一または異なって、水素原子または炭素数１〜６の低級アルキル基を意味する。）
からなる群から選択される少なくとも１つの化合物を有効成分とする項１乃至３のいずれかに記載するプロリル水酸化酵素阻害剤。
項５．下式で示される化合物１〜４：
（化合物１）

(R ₅ and R ₇ are the same or different and each represents a hydrogen atom or a lower alkyl group having 1 to 6 carbon atoms.)
Item 4. The prolyl hydroxylase inhibitor according to any one of Items 1 to 3, wherein the active ingredient is at least one compound selected from the group consisting of:
Item 5. Compounds 1 to 4 represented by the following formula:
(Compound 1)

（化合物２）

(Compound 2)

（化合物３）

(Compound 3)

（化合物４）

(Compound 4)

からなる群から選択される少なくとも１つを有効成分とする項１乃至４のいずれかに記載するプロリル水酸化酵素阻害剤。
項６．プロリル水酸化酵素と低酸素誘導因子との結合阻害剤である項１乃至５のいずかに記載するプロリル水酸化酵素阻害剤。
項７．プロリル水酸化酵素に結合して、プロリル水酸化酵素と低酸素誘導因子との結合を阻害する剤である項６に記載するプロリル水酸化酵素阻害剤。
項８．低酸素誘導因子分解阻害剤である項１乃至５のいずかに記載するプロリル水酸化酵素阻害剤。

Item 5. The prolyl hydroxylase inhibitor according to any one of Items 1 to 4, which comprises at least one selected from the group consisting of:
Item 6. Item 6. The prolyl hydroxylase inhibitor according to any one of Items 1 to 5, which is a binding inhibitor between prolyl hydroxylase and a hypoxia-inducing factor.
Item 7. Item 7. The prolyl hydroxylase inhibitor according to Item 6, which is an agent that binds to prolyl hydroxylase and inhibits binding between prolyl hydroxylase and a hypoxia-inducing factor.
Item 8. Item 6. The prolyl hydroxylase inhibitor according to any one of Items 1 to 5, which is a hypoxia-inducible factor degradation inhibitor.

(II) Pharmaceutical Composition Item 9. Item 9. A pharmaceutical composition comprising the prolyl hydroxylase inhibitor according to any one of Items 1 to 8 and a pharmaceutically acceptable carrier or additive.
Item 10. Item 10. The pharmaceutical composition according to Item 9, for improving hypoxia of cells or tissues.
Item 11. Item 11. The pharmaceutical composition according to Item 9 or 10, which is a prophylactic or therapeutic agent for a disease or condition caused by chronic or acute hypoxic conditions of cells or tissues.
Item 12. Item 11. The pharmaceutical composition according to Item 10, wherein the disease or condition caused by the hypoxic state of cells or tissues is a chronic or acute ischemic disease or a condition caused thereby.
Item 13. Item 13. The pharmaceutical composition according to any one of Items 9 to 12, which has an oral dosage form.

(III) Screening method for prolyl hydroxylase inhibitor 14. A screening method for a prolyl hydroxylase inhibitor comprising a step of selecting a compound having a competitive binding inhibitory action between prolyl hydroxylase and an α-chain proline of a hypoxia-inducing factor from test compounds.
Item 15. A compound having a competitive binding inhibitory action between prolyl hydroxylase and the alpha chain proline of the hypoxia-inducing factor has at least one group having a skeleton represented by the following formula (A) or (B) in one molecule. Item 15. The screening method according to Item 14, which is a compound comprising:

（式中、Ｒ_８およびＲ_９は、互いに水素原子であるか、またはこれらが結合するアルキレン鎖、酸素原子、窒素原子または硫黄原子とともに環を形成してもよい。）。
項１６．細胞または組織の慢性または急性の低酸素状態に関連して生じる疾患または病態の予防または治療薬の有効成分のスクリーニング方法である、項１４または１５に記載するスクリーニング方法。

(In the formula, R ₈ and R ₉ may be hydrogen atoms with each other or may form a ring together with the alkylene chain, oxygen atom, nitrogen atom or sulfur atom to which they are bonded).
Item 16. Item 16. The screening method according to Item 14 or 15, which is a method for screening an active ingredient of a prophylactic or therapeutic agent for a disease or condition caused in connection with a chronic or acute hypoxic condition of a cell or tissue.

(III) Novel Compound Item 17. A compound represented by the following formula or a salt thereof:

（式中、Ｒ_１２は水素原子または低級アルキル基を意味する。）

(In the formula, R ₁₂ represents a hydrogen atom or a lower alkyl group.)

  According to the present invention, a PHD inhibitor effective for stabilizing HIF can be provided. The PHD inhibitor can protect the living body from damage caused by hypoxia caused by ischemia or the like by stabilizing the HIF by inhibiting the inactivation of the HIF by PHD by hydroxylation. Therefore, the PHD inhibitor of the present invention can be effectively used as a pharmaceutical composition for preventing or ameliorating a disease or pathological condition (disorder) caused in connection with a hypoxic state. In particular, the PHD inhibitor of the present invention can be effectively used for the prevention or improvement of ischemic diseases of various organs of the kidney, heart, lower limbs or brain and the pathological conditions (disorders) that develop therefrom. The present invention also provides novel compounds effective as such PHD inhibitors.

  Furthermore, according to the screening method of the present invention, it is possible to select and obtain a novel PHD inhibitor having the above-mentioned action and useful for the prevention or improvement of ischemic diseases and pathological conditions (disorders) that develop from them.

(I) Prolyl hydroxylase inhibitor (PHD inhibitor)
The PHD inhibitor provided by the present invention is represented by the following formula (A) or (B):

（式中、Ｒ_８およびＲ_９は、互いに水素原子であるか、またはこれらが結合するアルキレン鎖、酸素原子、窒素原子または硫黄原子とともに環を形成してもよい。）
で示される骨格を有する基の少なくとも１つを、一分子中に含む化合物またはその塩を有効成分とするものである。

(Wherein R ₈ and R ₉ may be hydrogen atoms with each other, or may form a ring together with an alkylene chain, oxygen atom, nitrogen atom or sulfur atom to which they are bonded).
A compound containing at least one of the groups having a skeleton represented by the formula (1) in a molecule or a salt thereof is an active ingredient.

  The compound targeted by the present invention (hereinafter referred to as “the compound of the present invention”) contains at least one of the group having the skeleton A or the group having the skeleton B in one molecule, and the proline of the α chain of HIF is water. It has an action that hinders the function of oxidizing PHD. The compound of the present invention may be any compound as long as it has an effect of interfering with the function of PHD. The compound of the present invention chelates PFe Fe (II) to interfere with the function of PHD. There are no particular restrictions on the mechanism of action, such as those that competitively inhibit the binding of PHD to residues (hereinafter simply referred to as “HIFα proline”) and those that inhibit the hydroxylation activity of PHD. In particular, the compounds targeted by the present invention are preferably those having a HIF / PHD binding inhibitory action.

  The chelating action of the compound of the present invention on Fe (II) is not limited. For example, the method of Price et al. (Price DL, et al., “Chelating activity of advanced glycation end-product (AGE) inhibitors.” J Biol Chem 272: 5430-5437, 1997) or can be measured and evaluated accordingly.

  For example, as described in Experimental Example 2 described later, a method for evaluating the chelating action with copper as a transition metal can be used. Specifically, this method is a method in which copper chloride and ascorbic acid are incubated in the presence of a test compound, and the residual amount of ascorbic acid after the reaction is measured. If this can be prevented, it can be evaluated that the test compound has a metal chelating ability (chelating action on Fe (II)).

  Further, the inhibitory action of the compound of the present invention on HIF / PHD binding is not limited. For example, as described in detail in Experimental Example 4, in the presence of the test compound, the α chain domain of PHD and HIF labeled with a radioisotope or the like ( Preferably, the amino acid 557-576 region of HIF-1α is incubated to measure the amount of PHD bound to the α chain of HIF. If the presence of the test compound can prevent the binding of PHD to the α chain of HIF, It can be evaluated that the compound has a HIF / PHD binding inhibitory action.

Further PHD inhibitory activity of the compounds of the present invention is not limited, for example Kaule et al. Method (Cunhild Kaule and Volkmar Gunzler, " Assay for 2-Oxoglutarate Decarboxylating Enzymes Based on the Determination of [1- 14 C] Succinate: Application to Prolyl 4-Hydroxylase ”, Analytical Biochemistry 184, 291-297 (1990)) or can be measured and evaluated.

As an example, as described in Experimental Example 3 described later, a method for evaluating the catalytic ability to produce cis-3-hydroxyproline from 2-oxyglutarate (2-OG) and HIF proline can be used. Specifically, the method involves the PHD, [5- ¹⁴ C] 2-oxyglutarate (2-OG) and HIF α chain domain (preferably the amino acid 557-576 region of HIF-1α in the presence of the test compound. ) And measuring [1- ¹⁴ C] succinic acid after the reaction. If the production of [1- ¹⁴ C] succinic acid can be suppressed by the presence of the test compound, the test compound has PHD inhibitory activity. It can be evaluated that there is.

  The group having the skeleton A is more preferably a group represented by the following formula (A ′):

（式中、Ｒ_１は水酸基、低級アルキル基、保護基を有していてもよいカルボキシアルキル基、アミノ基：Ｒ_２は水素原子、水酸基、低級アルキル基、保護基を有していてもよいカルボキシアルキル基、アミノ基：Ｒ_３は水素原子、アミノ基または低級アルキルアミノ基を意味する）
式（Ａ’）において、Ｒ_１およびＲ_２の定義でみられる低級アルキル基としては、炭素数１〜６の直鎖または分枝鎖状のアルキル基を挙げることができる。具体的には、メチル基、エチル基、ｎ−プロピル基、イソプロピル基、ｎ−ブチル基、イソブチル基、ｓｅｃ−ブチル基、ｔｅｒ−ブチル基、ペンチル基、１，２−ジメチルプロピル基、１，１−ジメチルプロピル基、２，２−ジメチルプロピル基、２−エチルプロピル基、ｎ−ヘキシル基、１，２−ジメチルブチル基、２，３−ジメチルブチル基、１，３−ジメチルブチル基、１−エチル−２−メチルプロピル基、１−メチル−２−エチルプロピル基などを挙げることができる。好ましくは炭素数１〜４の直鎖または分枝鎖状のアルキル基であり、より好ましくはメチル基である。

(Wherein R ₁ is a hydroxyl group, a lower alkyl group, a carboxyalkyl group which may have a protecting group, and an amino group: R ₂ may have a hydrogen atom, a hydroxyl group, a lower alkyl group or a protecting group. Carboxyalkyl group, amino group: R ₃ represents a hydrogen atom, an amino group or a lower alkylamino group)
In the formula (A ′), examples of the lower alkyl group found in the definitions of R ₁ and R ₂ include linear or branched alkyl groups having 1 to 6 carbon atoms. Specifically, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, ter-butyl group, pentyl group, 1,2-dimethylpropyl group, 1, 1-dimethylpropyl group, 2,2-dimethylpropyl group, 2-ethylpropyl group, n-hexyl group, 1,2-dimethylbutyl group, 2,3-dimethylbutyl group, 1,3-dimethylbutyl group, 1 -Ethyl-2-methylpropyl group, 1-methyl-2-ethylpropyl group and the like can be mentioned. Preferably it is a C1-C4 linear or branched alkyl group, More preferably, it is a methyl group.

In the formula (A ′), the carboxyalkyl group found in the definition of R ₁ means one in which a carboxyl group is bonded to a carbon atom in any of the above lower alkyl groups. In this case, the carboxyl may have a protecting group. Here, examples of the protecting group include lower alkyl groups such as a methyl group, an ethyl group, and a tert-butyl group; a p-methoxybenzyl group, a p-nitrobenzyl group, a 3,4-dimethoxybenzyl group, a diphenylmethyl group, and a trityl group. A lower alkyl group substituted with an optionally substituted phenyl group such as a phenethyl group; a halogenated lower alkyl group such as a 2,2,2-trichloroethyl group and a 2-iodoethyl group; pivaloyloxy Methyl group, acetoxymethyl group, pupionyloxymethyl group, butyryloxymethyl group, valeryloxymethyl group, 1-acetoxyethyl group, 2-acetoxyethyl group, 1-pivaloyloxyethyl group, 2-pi Lower alkanoyloxy lower alkyl group such as valoyloxyethyl group; palmitoyloxyethyl group, heptadecanoyl group Lower alkoxycarbonyloxy lower alkyl such as higher alkanoyloxy lower alkyl group such as xymethyl group and 1-palmitoyloxyethyl group, methoxycarbonyloxymethyl group, 1-butoxycarbonyloxyethyl group and 1- (isopropoxycarbonyloxy) ethyl group Group; carboxy lower alkyl group such as carboxymethyl group and 2-carboxyethyl group; heteroaryl group such as 3-phthalidyl group; and benzoyloxy optionally having substituent such as 4-glycyloxybenzoyloxymethyl group Lower alkyl groups, (substituted dioxolene) lower alkyl groups such as (5-methyl-2-oxo-1,3-dioxolen-4-yl) methyl group; cycloalkyl substituted lower alkanoyloxy groups such as 1-cyclohexylacetyloxyethyl group Grade alkyl group, such as cycloalkyl alkyloxycarbonyl-lower alkyl groups such as 1-cyclohexyloxycarbonyloxy ethyl group can be cited. Further, various acid amides are also included in the carboxyl group having a protecting group in the present invention. In short, as long as it can be decomposed by some means in a living body to be a carboxylic acid, any can be a protecting group for a carboxyl group.

Of the lower alkylamino groups found in the definition of R _3, the lower alkyl groups described above can be similarly exemplified.

  Examples of the compound having a group having the skeleton A ′ include a compound (I) represented by the following formula.

ここで、Ｒ_１として前述する基を挙げることができるが、好ましくは炭素数１〜６の低級アルキル基、炭素数１〜４のカルボキシアルキル基、または炭素数１〜６の低級アルキル基で保護されている炭素数１〜４のカルボキシアルキル基を挙げることができる。炭素数１〜６の低級アルキル基としてより好ましくはメチル基、カルボキシアルキル基としてより好ましくはカルボキシエチル基、アルキル基で保護されているカルボキシアルキル基としてより好ましくは、メチル基で保護されたカルボキシエチル基を挙げることができる。

Here, examples of R ₁ include the above-mentioned groups, preferably protected with a lower alkyl group having 1 to 6 carbon atoms, a carboxyalkyl group having 1 to 4 carbon atoms, or a lower alkyl group having 1 to 6 carbon atoms. The C1-C4 carboxyalkyl group currently used can be mentioned. The lower alkyl group having 1 to 6 carbon atoms is more preferably a methyl group, more preferably a carboxyalkyl group, more preferably a carboxyethyl group, and more preferably a carboxyalkyl group protected with an alkyl group, and a carboxyethyl protected with a methyl group. The group can be mentioned.

Examples of R ₂ include the above-mentioned groups, preferably a lower alkyl group having 1 to 4 carbon atoms, more preferably a methyl group.

Examples of R ₁₀ include R ₁₁ —S—CH ₂ — (wherein R ₁₁ represents an optionally substituted heteroaryl group) or a group represented by the following formula:

（式中、Ｒ_４、Ｒ_５、Ｒ_６およびＲ_７は、同一または異なって、水素原子または炭素数１〜６の低級アルキル基を意味する。Ｒ_４とＲ_５またはＲ_６とＲ_７は互いに結合して、それらが結合する炭素原子と共に、５または６員の飽和もしくは不飽和の、置換基を有していてもよいシクロ環またはヘテロ環を形成してもよい。）で示される基を意味する。〕
Ｒ_１１の定義でみられるヘテロアリール基とは、酸素原子、窒素原子または硫黄原子の少なくとも１つを１〜４個含む単環または縮合環から誘導される基を意味する。例えば、チェニル基、フリル基、ベンゾチェニル基、ベンゾフラニル基、イソベンゾフラニル基、ピロリル基、イミダゾリル基、ピラゾリル基、イソチアゾリル基、イソキサゾリル基、テトラゾリル基、ピリジル基、ピラジニル基、ピリミジニル基、ピリダジニル基、インドリル基、イソインドリル基、キノリル基、イソキノリル基、フタラジル基、キノキサリル基、ナフチリジル基、キナゾリル基、アクリジニル基、イミダゾピリジル基などを挙げることができる。かかる基の置換基としては、水酸基：メチル基、エチル基、ｎ−プロピル基、イソプロピル基などの低級アルキル基；ハロゲン化アルキル基；メトキシ基、エトキシ基、ｎ−プロポキシ基、イソプロポキシ基などの低級アルコキシ基；フッ素原子、塩素原子、臭素原子、ヨウ素原子などのハロゲン原子；シアノ基、アセチル基、プロピオニル基、ベンゾイル基等のアシル基；アミノ基；ニトロ基；保護基を有していてもよいカルボキシル基；アシルアミノ基；スルホニルアミノ基；カルバモイル基；スルファモイル基；アミノスルホニル基；シアノアルキル基；ヘテロアリール基；カルボキシアルキル基；カルボキシアルコキシ基；ヘテロアリールアルキル基；ヘテロアリールアルコキシ基などを挙げることができる。Ｒ_１１として好ましくはピリジル基を挙げることができる。

(Wherein R ₄ , R ₅ , R ₆ and R ₇ are the same or different and represent a hydrogen atom or a lower alkyl group having 1 to 6 carbon atoms. R ₄ and R ₅ or R ₆ and R ₇ are And may form a 5- or 6-membered saturated or unsaturated, optionally substituted cyclo or heterocyclic ring together with the carbon atoms to which they are attached. Means. ]
The heteroaryl group found in the definition of R ₁₁ means a group derived from a monocyclic or condensed ring containing 1 to 4 of at least one of an oxygen atom, a nitrogen atom or a sulfur atom. For example, chenyl group, furyl group, benzocenyl group, benzofuranyl group, isobenzofuranyl group, pyrrolyl group, imidazolyl group, pyrazolyl group, isothiazolyl group, isoxazolyl group, tetrazolyl group, pyridyl group, pyrazinyl group, pyrimidinyl group, pyridazinyl group, Examples include indolyl group, isoindolyl group, quinolyl group, isoquinolyl group, phthalazyl group, quinoxalyl group, naphthyridyl group, quinazolyl group, acridinyl group, and imidazopyridyl group. Examples of such substituents include hydroxyl groups: lower alkyl groups such as methyl, ethyl, n-propyl, and isopropyl groups; halogenated alkyl groups; methoxy groups, ethoxy groups, n-propoxy groups, isopropoxy groups, and the like. Lower alkoxy group; halogen atom such as fluorine atom, chlorine atom, bromine atom and iodine atom; acyl group such as cyano group, acetyl group, propionyl group and benzoyl group; amino group; nitro group; Good carboxyl group; acylamino group; sulfonylamino group; carbamoyl group; sulfamoyl group; aminosulfonyl group; cyanoalkyl group; heteroaryl group; carboxyalkyl group; carboxyalkoxy group; heteroarylalkyl group; Can do. R ₁₁ is preferably a pyridyl group.

Examples of the lower alkyl group found in the definitions of R ₄ , R ₅ , R ₆ and R ₇ include the same C 1-6 linear or branched alkyl groups described for R ₁ and R _2. be able to.

The cyclo ring optionally having a substituent as defined in the definitions of R ₄ , R ₅ , R ₆ and R ₇ includes a 5- or 6-membered saturated monocycle such as cyclopentane or cyclohexane: cyclopentene, cyclohexene, 1 , 3-cyclohexadiene, 2,5-cyclohexadiene, benzene and the like 5- or 6-membered unsaturated monocycle. The hetero ring optionally having a substituent as defined in the definitions of R ₄ , R ₅ , R ₆ and R ₇ includes 1 or 2 of at least one of an oxygen atom, a nitrogen atom or a sulfur atom, 5 or Means a 6-membered saturated or unsaturated monocycle. Specific examples of such monocycles include furan, thiophene, pyrrole, thiazole, isothiazole, oxazole, isoxazole, imidazole, pyrazole, furazane, pyran, pyridine, pyridazine, pyrimidine, pyrazine, pyrrolidine, imidazolidine, pyrazolidine, piperidine, Mention may be made of piperazine and morpholine. In these cyclo rings and hetero rings, examples of the substituent include hydroxyl group: lower alkyl group such as methyl group, ethyl group, n-propyl group, isopropyl group; halogenated alkyl group; methoxy group, ethoxy group, n-propoxy. Group, lower alkoxy group such as isopropoxy group; halogen atom such as fluorine atom, chlorine atom, bromine atom and iodine atom; acyl group such as cyano group, acetyl group, propionyl group and benzoyl group; amino group; nitro group; A carboxyl group which may have a group; an acylamino group; a sulfonylamino group; a carbamoyl group; a sulfamoyl group; an aminosulfonyl group; a cyanoalkyl group; a heteroaryl group; a carboxyalkyl group; List arylalkoxy groups Door can be.

  Specific examples of compound (I) include compounds 1 to 3 represented by the following formula. In this specification, for convenience, Compound 1 (6-amuno-1,3-dimethyl-5- (2-pyridin-2-yl-quinoline-4-carbonyl) -1H-pyrimidine-2, 4-dione) is TM. -6008, Compound 2 (6-amino-1,3-di-methyl-5- [2- (pyridin-2-ylsulfanyl) -acetyl] -1H-pyrimidine-2,4-dione) TM-6089, and Compound 3 (3- {4-Amino-3-methyl-2,6-dioxo-5- [2- (pyridin-2-ylsulfanyl) -acetyl] -3,6-dihydro-2H-pyrimidin-1-yl} -propionic acid methyl ester) may be referred to as TM-6101.

(1) Compound 1:
6-amuno-1,3-dimethyl-5- (2-pyridin-2-yl-quinoline-4-carbonyl) -1H-pyrimidine-2,4-dione (TM-6008)

(2)化合物２：6-amino-1,3-di-methyl-5-[2-(pyridin-2-ylsulfanyl)-acetyl]-1H-pyrimidine-2,4-dione（TM-6089）;

(2) Compound 2: 6-amino-1,3-di-methyl-5- [2- (pyridin-2-ylsulfanyl) -acetyl] -1H-pyrimidine-2,4-dione (TM-6089);

(3)化合物３：
3-{4-Amino-3-methyl-2,6-dioxo-5-[2-(pyridin-2-ylsulfanyl)-acetyl]-3,6-dihydro-2H-pyrimidin-1-yl}-propionic acid methyl ester（TM-6101）

(3) Compound 3:
3- {4-Amino-3-methyl-2,6-dioxo-5- [2- (pyridin-2-ylsulfanyl) -acetyl] -3,6-dihydro-2H-pyrimidin-1-yl} -propionic acid methyl ester (TM-6101)

また上記骨格Ｂを有する基として、より好ましくは下式（Ｂ’）で示される基である：

The group having the skeleton B is more preferably a group represented by the following formula (B ′):

（Ｒ_４、Ｒ_５、Ｒ_６およびＲ_７は、同一または異なって、水素原子または低級アルキル基を意味する。Ｒ_４とＲ_５またはＲ_６とＲ_７は互いに結合して、それらが結合する炭素原子と共に、５または６員の飽和もしくは不飽和の、置換基を有していてもよいシクロ環またはヘテロ環を形成してもよい。Ｒ_８およびＲ_９は、互いに水素原子であるか、またはこれらが結合するアルキレン鎖、酸素原子、窒素原子または硫黄原子とともに環を形成してもよい。）
式（Ｂ’）中、Ｒ_４、Ｒ_５、Ｒ_６、およびＲ_７の定義にみられる低級アルキル基としては、Ｒ_１およびＲ_２に関して説明した炭素数１〜６の直鎖または分枝鎖状のアルキル基を同様に挙げることができる。好ましくは炭素数１〜４の直鎖または分枝鎖状の低級アルキル基であり、より好ましくはメチル基である。

(R ₄ , R ₅ , R ₆ and R ₇ are the same or different and each represents a hydrogen atom or a lower alkyl group. R ₄ and R ₅ or R ₆ and R ₇ are bonded to each other, and they are bonded. A 5- or 6-membered saturated or unsaturated, optionally substituted cyclo or hetero ring may be formed together with the carbon atom, and R ₈ and R ₉ are each a hydrogen atom, Or you may form a ring with the alkylene chain, oxygen atom, nitrogen atom, or sulfur atom which these couple | bond.
In formula (B ′), the lower alkyl group found in the definitions of R ₄ , R ₅ , R ₆ , and R ₇ is a straight or branched chain having 1 to 6 carbon atoms as described for R ₁ and R ₂ A similar alkyl group can be mentioned. Preferably it is a C1-C4 linear or branched lower alkyl group, More preferably, it is a methyl group.

The cyclo ring or hetero ring which may have a substituent found in the definitions of R ₄ , R ₅ , R ₆ and R ₇ , and the substituent are as described above. A benzene ring is preferred.

The alkylene chain found in the definitions of R ₈ and R ₉ means a linear or branched alkylene chain having 1 to 6 carbon atoms. Examples thereof include a methylene chain, an ethylene chain, a propylene chain, a butylene chain, and an ethylmethylene chain. A methylene chain is preferred. R ₈ and R ₉ are preferably hydrogen atoms or form a ring together with the oxygen atom to which they are bonded.

  Examples of the compound having a group having the skeleton B ′ include a compound (II) represented by the following formula.

（Ｒ_５およびＲ_７は、同一または異なって、水素原子または炭素数１〜６の低級アルキル基を意味する。）
より好ましくは、下式で示される化合物４（3,7-Dimethy-furo[3,2-b;4,5-b]dipyridine）である。本明細書において、便宜上、化合物４をTM-6010と称する場合がある。

(R ₅ and R ₇ are the same or different and each represents a hydrogen atom or a lower alkyl group having 1 to 6 carbon atoms.)
More preferably, it is the compound 4 (3,7-Dimethy-furo [3,2-b; 4,5-b] dipyridine) represented by the following formula. In this specification, for convenience, compound 4 may be referred to as TM-6010.

  (4) Compound 4: 3,7-Dimethy-furo [3,2-b; 4,5-b] dipyridine (TM-6010)

また、前述する化合物１（TM-6008）も骨格Ｂ’を有する基を有する化合物Ｉに該当する。

In addition, Compound 1 (TM-6008) described above also corresponds to Compound I having a group having a skeleton B ′.

  These compounds may be either free or salt forms. Here, examples of the salt include pharmaceutically acceptable salts such as salts with inorganic bases or organic bases, or acid addition salts such as inorganic acids, organic acids, or basic or acidic amino acids. Examples of the inorganic base include alkali metals such as sodium and potassium; alkaline earth metals such as calcium and magnesium; aluminum and ammonium. Examples of the organic base include primary amines such as ethanolamine; secondary amines such as diethylamine, diethanolamine, dicyclohexylamine, N, N′-dibenzylethylenediamine; trimethylamine, triethylamine, pyridine, picoline, triethanolamine and the like. And tertiary amines.

  Examples of inorganic acids that form acid addition salts include hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like. Examples of organic acids include formic acid, acetic acid, lactic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, benzoic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, ethanesulfonic acid, and benzenesulfone. An acid, p-toluenesulfonic acid, etc. can be mentioned. Examples of basic amino acids include arginine, lysine, ornithine and the like. Examples of acidic amino acids include aspartic acid and glutamic acid.

  The above compounds, especially compounds 1 (TM-6008), 2 (TM-6089) and 4 (TM-6010) are commercially available [eg Maybrige (UK), ASINEX (Russia), LaboTest (Germany) Specs (Netherlands), ENAMINE (Ukraine), Ambinter (France), ASDI (USA), Sigma (USA), Pharmeks (Russia), Life Chemical (Ukraine), Key Organics (UK), etc. Can be manufactured.

  Compound 3 (TM-6101) can be produced by the method described in Production Example 1 described later. Among the production steps, in step 1, compound 2 (TM-6089) can be synthesized by using Iodomethane instead of Ethyl 3-Bromopropinate. In step 1, Iodomethane is used in place of Ethyl 3-Bromopropinate, and in step 4, 2-Pyridine-2-yl-quinoline-4-carbonylchloride is used in place of 2-mercaptopyridine. ) Can be synthesized.

  As described above, the compound of the present invention has an action of hindering the function of PHD that hydroxylates proline of the α chain of HIF. Such an action may be any action that hinders the function of PHD as a result, such as a metal chelate action, an HIF / PHD binding inhibitory action, and a PHD activity inhibitory action. Any of these can be evaluated by the above-described in vitro assay system (for details, see Experimental Examples).

  Further, the compound of the present invention has an action of inhibiting the binding between PHD and HIF, and consequently has an action of stabilizing the HIF by inhibiting the binding and degradation of VIF due to hydroxylation of HIF. The HIF promoting action is not limited, but can be measured and evaluated according to the method of Experimental Example 1 described later or according to this method. In the in vitro assay, PHD-expressing cells into which a vector having a luciferase gene was introduced downstream of a promoter containing HRE (hypoxia responsive element) 7 times in tandem were incubated with a test compound, and luciferase of a cell lysate was obtained. This is a method for measuring activity. In the assay system, if the luciferase activity increases due to the presence of the test compound, it can be evaluated that the presence of the test compound stabilizes the HIF and activates the HRE system, that is, the test compound has an HIF stabilizing effect. it can.

  The compound of the present invention has an action of promoting angiogenesis as a result of inhibiting the function of PHD and stabilizing HIF. Such angiogenesis promoting action is not limited, but can be measured and evaluated according to the method of Experimental Example 5 (sponge angiogenesis analysis) described later or according to this method. The in vivo assay is a method in which a test compound is subcutaneously administered via a sponge disk embedded in the skin of a test animal, and the amount of hemoglobin and the number of blood vessels are measured over time. In the in vivo assay system, if the amount of hemoglobin and the number of blood vessels are increased by administration of the test compound, it can be evaluated that the test compound has an angiogenic effect.

  The compound of the present invention has an action of inhibiting the function of PHD. Among them, compounds 1 to 4, preferably compound 1, have an excellent PHD inhibitory action as shown in the experimental examples described later. Due to the PHD inhibitory action, the compound of the present invention prevents and stabilizes the degradation of HIF, and improves the hypoxia by stabilizing the HIF-HRE system, resulting in damage caused by hypoxia such as ischemia. Can protect cells and tissues. Therefore, according to the compound of the present invention, the following actions are expected.

(i) Renal chronic ischemia-improving action In the kidney, stagnation of blood flow in the peritubular capillaries induces chronic hypoxia, which is accompanied by promotion of tubulointerstitial damage and loss of capillaries around the tubules. It has been reported that the expression of HIF is enhanced by cobalt, which is a known PHD inhibitor, and as a result, renal interstitial disorder, which is a renal ischemic disorder, is improved (Matsumoto M, Tanaka T, Yamamoto T , Noiri E, Miyata T, Inagi R, Fujita T, Nangaku M. Hypoperfusion of peritubular capillaries induces chronic hypoxia before progression of tubulointerstitial injury in a progressive model of rat glomerulonephritis. J Am Soc Nephrol 15: 1574-1581 (2004)). From this, there is a possibility that stabilization of HIF promotes transcription of VEGF and erythropoietin, induces angiogenesis and improvement of blood flow, improves chronic ischemia, and suppresses progression to renal failure. is there.

(ii) Cerebral ischemia-improving action The cerebral infarction region that has fallen into cerebral infarction is ischemic, and ischemic damage to tissues is a serious problem. Thus, active angiogenesis that occurs in the cerebral infarction region has proven to be beneficial to tissues under ischemia (Krupinski J, Kaluza J, Kumar P, Kumar S & Wang JM. Role of angiogenesis in patients with cerebral ischemic stroke. Stroke 25: 1794? 1798 (1994)). It has also been demonstrated that VEGF reduces cerebral infarct size, edema formation, and cerebral blood barrier disruption by reducing neuronal damage caused by ischemia after temporary middle cerebral artery occlusion (Hayashi , T., Abe, K., Itoyama, Y. Reduction of ischemic damage by application of vascular endothelial growth factor in rat brain after transient ischemia. J. Cereb. Blood Flow Metab. 18: 887-895 (1998)). From these facts, it is considered that the enhancement of VEGF expression by stabilizing HIF is an effective treatment for cerebral infarction and ischemic stroke.

(iii) Ischemic heart disease improving action Ischemic heart disease includes angina pectoris and myocardial infarction. In either case, if appropriate treatment is delayed, heart failure and severe arrhythmia are caused and directly involved in life and death. For this reason, the development of a method for improving myocardial ischemia is regarded as an urgent issue. Recent studies have shown that administration of VEGF to a porcine progressive coronary occlusion model improves the ischemic area, and large doses improve myocardial perfusion blood volume (Pearlman JD, Hibberd MG, Chuang ML, Harada K, Lopez JJ, Glad-stone SR, Friedman M, Sellke FW, Simons M. Magnetic resonance mapping demonstrates benefits of VEGF-induced myocardial angiogenesis. Nat Med. 1: 1085-1089. (1995)) . In addition, it has been proven that angiogenesis is promoted when VEGF is administered to a porcine chronic myocardial ischemia model (Lopez JJ, Laham RJ, Stamler A, Pearlman JD, Bunting S, Kaplan A, Carrozza JP, Sellke FW). , Simons. M. VEGF administration in chronic myocardial ischemia in pigs. Cardiovasc Res .; 40: 272-281. (1998)). From these facts, it is considered that increased expression of VEGF by stabilizing HIF is an effective treatment for ischemic heart diseases such as myocardial infarction and angina.

(iv) Lower limb ischemia-improving effect In patients with lower limb ischemia, severe symptom leads to amputation of the lower limbs, which severely lowers the patient's ADL, causing serious problems not only medically but also socially. Yes. In recent studies, VEGF is considered effective for treatment in patients with lower limb ischemia. When VEGF and FGF-2 genes are introduced into ischemic muscle tissue, endogenous VEGF and HGF expression is increased by FGF-2 gene introduction, and histological examination and blood flow measurement increase neovascularization and collateral circulation. It has been reported that there is a clear improvement in lower limb ischemia (Onimaru M, Yonemitsu Y, Tanii M, Nakagawa K, Masaki I, Okano S, Ishibashi H, Shirasuna K, Hasegawa M, Sueishi K. Fibroblast growth factor-2 gene transfer can stimulate hepatocyte growth factor expression according to hypoxia-mediated downregulation in ischemic limbs. Circ. Res, 91: 723-730 (2002)). From these facts, it is considered that increased expression of VEGF by stabilizing HIF is an effective treatment for lower limb ischemia.

  The PHD inhibitor of the present invention comprises the aforementioned compound of the present invention or a salt thereof as an active ingredient. The PHD inhibitor of the present invention may be composed of 100% by weight of the above-described compound or a salt thereof, or may contain an effective amount of a compound that exhibits a PHD inhibitory action even if it is not. Although not limited, the PHD inhibitor usually contains the compound or a salt thereof in a proportion of 10 to 100% by weight.

(II) Pharmaceutical Composition The present invention provides a pharmaceutical composition containing the aforementioned PHD inhibitor as an active ingredient. In other words, the pharmaceutical composition of the present invention contains the compound of the present invention as an active ingredient. The pharmaceutical composition of the present invention has an effect of stabilizing HIF by containing an effective amount of PHD inhibitor (the compound of the present invention) that inhibits PHD. As a result, the pharmaceutical composition of the present invention improves hypoxia by activating the living HIF-HRE system, and as a result, protects cells and tissues from damage caused by hypoxia such as ischemia. be able to.

  For this reason, the pharmaceutical composition of the present invention is useful as a preventive or therapeutic agent for diseases and pathologies (disorders) caused in connection with hypoxic conditions of cells and tissues. Examples of such diseases or conditions include various ischemic diseases and disorders resulting from ischemia. Ischemic diseases include renal ischemic damage (tubulointerstitial damage), ischemic cerebrovascular disorders (cerebral embolism, cerebral infarction, transient ischemic attack, etc.), ischemic heart disease (anginal, Myocardial infarction), lower limb ischemic injury, and the like. Examples of the pathological condition caused by the development of these ischemic diseases include chronic renal failure, chronic heart failure, and limb gangrene.

  The pharmaceutical composition of the present invention is usually prepared by blending a pharmaceutically acceptable carrier or additive in addition to the compound of the present invention in an amount effective for PHD inhibition and thus effective for improving hypoxia. The The compounding amount of the compound of the present invention in the pharmaceutical composition is appropriately selected according to the type of disease or pathology to be targeted and the dosage form. Usually, in the case of a systemic preparation, the total weight of the pharmaceutical composition ( 100 wt%) to 0.001 to 50 wt%, particularly 0.01 to 10 wt%.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

  When the pharmaceutical composition of the present invention is used as a prophylactic or therapeutic agent for diseases and conditions associated with hypoxia, the oral dose is 0.03 to 300 mg / kg in terms of the amount of the compound of the present invention. A range is preferable, More preferably, it is 0.1-50 mg / kg. In the case of intravenous administration, there can be mentioned such a dose that the effective blood concentration of the compound of the present invention is in the range of 0.2 to 50 μg / mL, more preferably 0.5 to 20 μg / mL.

  Note that these doses may vary depending on age, sex, body type and the like.

(III) Screening Method The present invention provides a method for selecting a prolyl hydroxylase inhibitor (PHD inhibitor) that can be effectively used to improve hypoxia of cells and tissues from test substances. The PHD inhibitor searched and obtained by the method of the present invention has an action of inhibiting the binding of PHD to HIF and preventing αIF proline of HIF from being hydroxylated and decomposed, and as a result. The HIF-HRE system can be activated to improve the hypoxic state of cells and tissues. For this reason, according to the said substance, it is anticipated that the disease and pathological condition (disorder) which arise in relation to the hypoxic state of a cell or a tissue can be prevented or treated. Therefore, it can be said that the method of the present invention is a method for screening an active ingredient of a pharmaceutical composition for preventing or treating a disease or pathological condition (disorder) caused in connection with a hypoxic state of a cell or tissue.

The screening method of the present invention basically comprises searching for a substance having an action of inhibiting the competitive binding between prolyl hydroxylase (PHD) and alpha chain proline of hypoxia-inducing factor (HIF). Specifically, a method having the following steps (a) to (c) can be exemplified:
(a) contacting PHD with HIF α-chain proline in the presence of a test substance;
(b) measuring the binding between PHD and HIFα chain proline, and
(c) A step of selecting a test substance that inhibits the binding between PHD and HIFα chain proline as a PHD inhibitor.

  By this step, a substance that inhibits the competitive binding between PHD and α chain proline of HIF and suppresses the action of PHD that hydroxylates α chain proline of HIF can be obtained.

  Test substances include, but are not limited to, nucleic acids, peptides, proteins, organic compounds, inorganic compounds, and the like. Specifically, screening is performed using these test substances or compositions containing them (for example, cell extracts) A gene library expression product, etc.) can be brought into contact with the subject c-Src and ADAP.

  The contact conditions between the test substance, PHD, and HIFα chain proline employed for screening are not particularly limited, but it is usually preferable to perform in an in vitro experimental system in a physiological environment or in a cell.

  The origin of PHD used in the present invention is not particularly limited and is derived from vertebrates including mammals other than humans (for example, mice, rats, chickens, etc.) or various microorganisms (streptomysis) in addition to those derived from humans. PHD can be used. PHD may be a full-length protein or a partial fragment containing a domain involved in binding to HIFα proline.

  The origin of the HIFα chain used in the present invention is not particularly limited. In addition to those derived from humans, HIF derived from vertebrates including mammals other than humans (eg, mice, rats, chickens, etc.) can be used, but those derived from the same animal as the PHD used together It is preferable to use it. The HIF α chain may be a full-length protein or a partial fragment containing a proline residue located at position 564 of the α chain involved in binding to PHD. Specifically, a partial fragment having a region of 557 to 576 or 531 to 652 of the HIFα chain can also be used.

  The measurement of the binding between PHD and HIFα chain is not particularly limited as long as it can evaluate the presence or absence of the binding between PHD and HIFα chain. For example, after contacting PHD (or a partial fragment thereof) and HIFα chain (or a partial fragment containing a proline residue) in the presence of a test substance, the reaction solution (mixed solution) is subjected to electrophoresis to determine the binding from the molecular weight. Examples include a method for evaluating the presence or absence, a method for evaluating the presence or absence of coprecipitation of other proteins using an antibody of PHD (or a partial fragment of PHD) or HIFα chain (or a partial fragment containing a proline residue), and the like. it can.

Selection of a PHD inhibitor (candidate substance) by such a screening method can be performed by evaluating the presence or absence of binding between cPHD (or a partial fragment thereof) and a HIFα chain (or a partial fragment containing a proline residue). In this case, contrast with the binding (control) of PHD and HIFα chain produced by contacting PHD (or a partial fragment thereof) with HIFα chain (or a partial fragment containing a proline residue) in the absence of the test substance. Can also be evaluated. When PHD and HIFα chain do not bind at all, and when binding is reduced compared to control, the test substance used can be selected as a candidate substance for PHD inhibitor.

  The substance selected by the above screening has an action of stabilizing HIF by inhibiting the action of PHD that hydroxylates α-chain proline of HIF, and is reduced through activation of the HIF-HRE system. It can be used as an active ingredient of a pharmaceutical composition for improving oxygen state and preventing or treating diseases caused by hypoxia and pathological conditions (disorders) developed therefrom.

  Examples of diseases caused by hypoxia include various ischemic diseases and disorders caused by ischemia, specifically, renal ischemic disorder (tubulointerstitial disorder), ischemic cerebrovascular disorder ( Cerebral embolism, cerebral infarction, transient cerebral ischemic attack, etc.), ischemic heart disease (angina pectoris, myocardial infarction), lower limb ischemic disorder and the like. Examples of the pathological condition caused by the development of these ischemic diseases include chronic renal failure, chronic heart failure, and limb gangrene.

  Candidate substances selected by the above screening method use sponge angiogenesis analysis or model non-human animals in hypoxia to evaluate angiogenesis as one action to further improve the hypoxia. Can also be screened. The candidate substance thus selected is a drug effect test using a non-human animal having an ischemic disease, a safety test, and a patient (human) in a hypoxic state such as an ischemic disease or a patient in a previous state thereof It may be used for clinical trials on (human), and by conducting these tests, it is possible to select and acquire the active ingredients of a pharmaceutical composition for the prevention or treatment of diseases associated with more practical hypoxia it can.

  The material selected in this way is subjected to structural analysis as necessary, and then, depending on the type of the material, chemical synthesis, biological synthesis (including fermentation), or genetic engineering operations can be used for industrialization. And can be used for the preparation of a pharmaceutical composition for the prevention and treatment of diseases associated with hypoxia.

  As described above, the above screening method basically comprises searching for a substance having an action of inhibiting the binding between PHD and HIFα proline. Therefore, the above screening method can be defined from another angle as a method for screening a substance (PHD / HIF binding inhibitor) having an action of inhibiting the binding between PHD and HIFα proline.

  The thus obtained compound having a competitive binding inhibitory action between prolyl hydroxylase and α chain proline of a hypoxia-inducing factor includes at least one group having a skeleton represented by the following formula (A) or (B): In one molecule.

（式中、Ｒ_８およびＲ_９は、互いに水素原子であるか、またはこれらが結合するアルキレン鎖、酸素原子、窒素原子または硫黄原子とともに環を形成してもよい。）
好ましくは、本発明化合物、特に化合物１〜３を含む化合物（Ｉ）ならびに化合物４を含む化合物（II）が含まれる。

(Wherein R ₈ and R ₉ may be hydrogen atoms with each other, or may form a ring together with an alkylene chain, oxygen atom, nitrogen atom or sulfur atom to which they are bonded).
Preferably, the compound of the present invention, particularly the compound (I) containing the compounds 1 to 3 and the compound (II) containing the compound 4 are included.

Experimental example

  Hereinafter, the present invention will be described more specifically with reference to production examples and experimental examples, but the present invention is not limited to these examples.

Production Example 1 Synthesis of TM-6101 As shown in the following formula, using 6-amino-1-methyluracil (compound (a)) as a starting material, the desired TM-6101 (compound 4) (molecular weight: 378.41) was obtained. Manufactured.

（１）工程１
アルゴン雰囲気下、6-アミノ-1-メチルウラシル（化合物(a)）3.53ｇ（25.0mmol）とN,N’-ジメチルホルムアミドジメチルアセチル11.9g（0.10mmol）の脱水ＤＭＦ懸濁溶液（125mL）を、40℃にて3時間攪拌した。炭酸カリウム5.18ｇ（37.5mmol）とエチル-3-ブロモプロピネート6.79g（37.5mmol）を加え、80℃にて3時間攪拌した。減圧下濃縮し無機化合物を濾過した。酢酸エチル洗浄濾液を、減圧下濃縮し、スラリー状でヘキサンを加えて析出結晶を濾過し、化合物(b)を3.00g取得した（収率40％）。

(1) Step 1
In an argon atmosphere, a dehydrated DMF suspension solution (125 mL) of 3.53 g (25.0 mmol) of 6-amino-1-methyluracil (compound (a)) and 11.9 g (0.10 mmol) of N, N′-dimethylformamidedimethylacetyl was added. The mixture was stirred at 40 ° C. for 3 hours. 5.18 g (37.5 mmol) of potassium carbonate and 6.79 g (37.5 mmol) of ethyl-3-bromopropinate were added and stirred at 80 ° C. for 3 hours. The mixture was concentrated under reduced pressure, and the inorganic compound was filtered. The ethyl acetate washing filtrate was concentrated under reduced pressure, hexane was added as a slurry, and the precipitated crystals were filtered to obtain 3.00 g of compound (b) (yield 40%).

(2) Step 2
To a dehydrated methanol solution (127 mL) of compound (b) (2.50 g, 8.44 mmol) was added 4.89 g (35.9 mmol) of zinc chloride, and the mixture was refluxed for 5 days under an argon atmosphere. After cooling, water was added and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate and then concentrated under reduced pressure. The concentrated residue was washed with methylene chloride to obtain 0.93 g of compound (c) (yield 48%).

(3) Process 3
Under an argon atmosphere, 0.36 g (4.55 mmol) of pyridine and 0.51 g (4.52 mmol) of chloroacetyl chloride were added to a dehydrated dioxane solution (15 mL) of compound (c) (0.69 g, 3.04 mmol), and the mixture was stirred at room temperature for 30 minutes. After confirming the disappearance of the raw material, it was poured into water and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The concentrated residue was purified by silica gel column chromatography (eluent: methanol / methylene chloride = 1/9) to obtain 0.88 g of colorless crystal compound (d) (yield 95%).

(4) Step 4
To a toluene solution (80 mL) of compound (d) (0.80 g, 2.63 mmol), 0.44 g (3.95 mmol) of 2-mercaptopyridine and 0.53 g (5.27 mmol) of triethylamine were added and stirred at room temperature for 24 hours. Then, after stirring at 40 ° C. for 7 hours and at room temperature for 19 hours, 0.53 g (5.27 mmol) of triethylamine was added, and the mixture was further stirred at room temperature for 16 hours. Water was added and extracted with ethyl acetate. The organic layer was concentrated under reduced pressure, and the concentrated residue was purified by silica gel column chromatography (eluent: ethyl acetate) and washed again with strategy ethyl to give 0.60 g of compound 4 (TM-6101) as colorless to pale yellow crystals. Obtained (yield 60%).

The NMR data of TM-6101 (Compound 4) is as follows:
1H NMR (CDCl3) δ = 2.68 (2H, t, J = 9 Hz), 3.46 (3H, s), 3.70 (3H, s), 4.29 (2H, t, J = 9 Hz), 4.81 (2H, s ), 6.93-6.97 (1H, m), 7.26-7.29 (1H, m), 7.44-7.50 (1H, m), 8.37-8.40 (1H, m).

  Compound 4 is a carboxylic acid by adding 1 mole of NaOH in a 1 to 1 solution of THF: water, adding equimolar to 3 times mole of Compound 4 as NaOH, and stirring at room temperature. (e) can be prepared.

Experimental example 1
(1) Analysis of three-dimensional structure of Prolyl-3-hydroxylase (PHD3)
Prolyl-3-hydroxylase (PHD3) is an enzyme that produces cis-3-hydroxyproline from 2-oxyglutarate (2-OG) and HIF proline in the presence of Fe (II). The X-ray crystal structure has already been reported for PHD3 derived from Streptomyces sp. (Clifton, IJ et al. Structure of proline 3-hydroxylase. Evolution of the family of 2-oxoglutarate dependent oxygenases. Eur. J Biochem. 268. 6625-6636 (2001)). However, the binding sites for 2-OG and HIF proline in the PHD3 molecule are still unknown. Therefore, the following experiment was performed to determine these binding sites.

  X-ray crystal structure of PHD3 derived from Streptomyces was obtained from Protein Data Bank (PDB code: 1E5RS) (Bernstein, FC et al. The Protein Data Bank: a computer-based archival file for macromolecular structures. J. Mol. Biol. 112. 535-542 (1977)). After removing water molecules and sulfate ions, hydrogen atoms were added to optimize the position of the protons in accordance with the standard protonation state of acidic and basic residues in the protein. The research included software system MOE (Molecular Operating Environment, version 2004.04) and MMFF94s force field (Halgren, TA Merck molecular force field. I. Basis, form, scope, parameterization, and performance of MMFF94. J. Comp. Chem. 17. 490-519 (1996)) was adopted.

The binding site is the MOE alpha site finder function (Edelsbrunner, H., Facello, M., Fu, R., & Liang, J. Measuring Proteins and Voids in Proteins.Proceedings of the 28th Hawaii International Conference on Systems Science, 256 -264 (1995)). Docking of small molecules and target sites is the program Ph4Dock (Goto, J., Kataoka, R. & Hirayama, N. Ph4Dock-Pharmacophore-based protein-ligand docking. J. Med. Chem. 47. 6804-6811 (2004)) It went by. At the final stage of docking, the positions of hydrogen atoms in the PHD3 molecule and all atoms in the small molecule were optimized. The obtained docking result was judged by the following interaction energy.
U _total = U _ele + U _vdw + U _ligand
Here, U _ele and U _{vdw mean} an electrostatic interaction and a Wanderworth interaction between PHD3 and a small molecule, respectively. U _ligand means the constituent energy of a small molecule.

  From the above docking simulation, it was found that there was a relatively large pocket in the PHD3 molecule, and Fe (II) was present therein (see FIG. 1a). The sphere (blue) in FIG. 1a indicates Fe (II). There appears to be a binding site for 2-OG and HIF proline residues around this Fe (II). In FIG. 1a, the proline residues of 2-OG and HIF are shown by a branch model (green), and 2-OG and HIF proline bind to sites opposite to each other with respect to Fe (II) [in FIG. , HIF proline binds to the upper part (B site) of Fe (II), and 2-OG binds to the lower part (A site). 2-OG is bonded to Fe (II) via a carboxyl group.

(2) Molecular docking of known PHD inhibitors
Targeting the binding site in the pocket around Fe (II) in the PHD3 molecule (A site: 2-OG binding site, B site: HIF proline residue binding site), using a known PHD inhibitor, A docking simulation was performed.

  As a known PHD inhibitor, 3,4-dehydroxybenzoate (hereinafter referred to as “3,4-DHB”) shown in the following formula: Warnecke, C. et al. Activation of the hypoxia-inducible factor-pathway and stimulation of angiogenesis by application of prolyl hydroxylase inhibitors. FASEB. J. 17. 1186-1188 (2003)), 6-chloro-3-hydroxychinoline-2-carbonic acid-N- (carboxymethyl) -amide, a derivative of 3-hydroxychinolin 2-carbonic acid (Hereinafter referred to as “S956711”. Warnecke, C. et al. Activation of the hypoxia-inducible factor-pathway and stimulation of angiogenesis by application of prolyl hydroxylase inhibitors. FASEB. J. 17. 1186-1188 (2003)), and 4 -Oxo-1,4-dihydro- [1,10] phenanthroline-3-carboxylic acid (hereinafter referred to as “FG-0041”. Ivan, M. et al. Biochemical purification and pharmacological inhibition of a mammalian prolyl hydroxylase acting on hypoxia -inducible factor. Proc. Natl. Acad. Sci. 99. 13459-13464 (2002)).

その結果、3,4-DHBは、そのカルボキシル基を介して、PHD3分子内のFe(II)周囲のＡ部位（2-OG結合部位）と結合することがわかった(図１ｂ)。S956711は、比較的分子構造が大きくまた環構造を有することから、Ａ部位とＢ部位の両方（2-OG結合部位とHIFのプロリン残基結合部位）に結合すると考えられた。さらにFG-0041は、Ｂ部位（HIFのプロリン残基結合部位）に結合することが明らかになった。

As a result, 3,4-DHB was found to bind to the A site (2-OG binding site) around Fe (II) in the PHD3 molecule via its carboxyl group (FIG. 1b). Since S956711 has a relatively large molecular structure and a ring structure, it was considered to bind to both the A site and the B site (2-OG binding site and HIF proline residue binding site). Furthermore, FG-0041 was found to bind to the B site (HIF proline residue binding site).

  These three PHD inhibitors all have a motif that forms a chelate with an iron ion in addition to the ability to bind to the A site or / and the B site. However, since iron is a cofactor necessary for exerting many important cellular functions in relation to oxidative phosphorylation and arachidonic acid signaling, its chelating agent suppresses PHD for long-term therapy. It is not considered suitable as an agent. For this reason, we searched for PHD inhibitors that act independently of iron chelation. Since the A site (2-OG binding site) in the PHD3 molecule is surrounded by bulky residues such as Arg168 and His135, the PHD inhibitor may be difficult to access. Therefore, it was decided to search for a molecule that binds to the B site in competition with the proline residue of HIF as a PHD inhibitor.

(3) Identification of novel HIF stabilizing compounds
Under the above concept, 19 derivatives of FG-0041 that bind to the B site in the PHD3 molecule were purchased and the stabilization of HIF was investigated. 12 of the 19 derivatives have a [2,2] bipyridinyl skeleton, and the other 7 have other sites (4-oxo-1,4-dihydro-quinoline-3-carboxylic acid) . The luciferase production promoting activity of these 19 derivatives by HIF stabilization was measured by an in vitro screening assay described later. Cobalt (cobalt chloride), a known compound that produces hypoxia by stabilizing HIF-1α (HIF α chain), was used as a positive control (Wang, GL & Semenza, GL Purification and characterization of hypoxia-inducible factor 1. J. Biol. Chem. 20. 1230-1237 (1995)).

<In vitro screening assay>
A reporter vector having a luciferase gene downstream of a promoter having HRE repeated 7 times in tandem was introduced into immortalized renal proximal tubule (IRPTC) cells, and this was transferred to the above 19 derivatives (test compounds). It was used to measure luciferase production promoting activity by stabilizing HIF. Specifically, IRPTC cells into which the reporter vector has been introduced are lysed after incubation for 24 hours at 37 ° C. with a test compound (final concentration: 6.7 to 100 μM), and the luciferase activity in the cell lysate is determined using Lumat 9507. Measurements were made using a luminometer (EG and Berthold, Germany). In addition, luciferase activity in a cell lysate obtained by similarly incubating IRPTC cells introduced with a reporter vector in the absence of a test compound was used as a control (negative control).

  The negative control luciferase activity was set to 1, and the relative ratio of the luciferase activity of each test compound to this was evaluated as the luciferase production promoting activity by HIF stabilization of each test compound. The results are shown in FIG. As shown in FIG. 2, among the derivatives having a [2,2] bipyridinyl skeleton, two compounds represented by the following formula: 6-amino-1,3-dimethyl-5- (2-pyridin-2-yl- quinoline-4-carbonyl) -1H-pyridine-2,4-dione (TM-6008) (molecular weight: 387.40)

および3,7-Dimethy-furo[3,2-b;4,5-b]dipyridine(TM-6010) （分子量：198.23）

And 3,7-Dimethy-furo [3,2-b; 4,5-b] dipyridine (TM-6010) (Molecular weight: 198.23)

が、HIF安定化によるルシフェラーゼ産生促進活性を示した。

However, the luciferase production promotion activity by HIF stabilization was shown.

  Both TM-6008 and TM-6010 have [2,2] bipyridinyl which is an iron chelate motif. Therefore, this motif was removed from TM6008, and 18 compounds having a 5-acetyl-6-amino-1,3-di-methyl-1H-pyrimidine-2,4-dione structure were obtained from the library.

  One of those compounds, 6-amino-1,3-di-methyl-5- [2- (pyridin-2-ylsulfanyl) -acetyl] -1H-pyrimidine-2,4-dione (TM-6089) (Molecular weight: 306.35)

は、鉄のキレート化モチーフ部（[2、2]bipyridinyl）を欠如しているにもかかわらず、強いHIF安定化によるルシフェラーゼ産生促進活性を示した(図２)。

Despite lack of iron chelation motif ([2,2] bipyridinyl), it showed luciferase production promoting activity by strong HIF stabilization (FIG. 2).

  The docking structure of these TM-6008, TM-6010 and TM-6089 is shown in FIG. 1c (in FIG. 1c, yellow indicates TM-6008, green indicates TM-6010 and red indicates TM-6089). This shows that these compounds all bind to the B site in the PHD3 molecule.

(4) Luciferase production promoting activity by TM6101 HIF stabilization
The luciferase activity of TM-6089 carboxylic acid derivative TM6101 (compound 5) synthesized in Production Example 1 was measured by in vitro screening assay in the same manner as (3) above, and the luciferase production promoting activity by HIF stabilization was evaluated. did. For positive control and comparison, luciferase activity was measured in the same manner using cobalt chloride and TM6008 as test compounds, respectively, and luciferase production promoting activity by HIF stabilization was evaluated. The results are shown in FIG. FIG. 3 shows the relative ratio of the luciferase activity of each test compound (cobalt chloride, TM6008, TM6101) with the negative control luciferase activity as 1, as in FIG. As a result, TM6101 showed a higher luciferase production promoting activity due to HIF stabilization than TM6008, although it was lower than the known PHD inhibitor cobalt chloride.

Experimental Example 2 Metal Chelate Activity As described above, the chemical structure suggested that a motif chelating iron ions exists in 3,4-DHB, S965711, FG-0041, TM-6008 and TM-6010. . Therefore, this was confirmed by an in vitro assay using copper-catalyzed oxidation of ascorbic acid (an evaluation system assay for transition metal chelation).

Specifically, with respect to the compounds (TM-6008, TM-6010 and TM-6089) in which the HIF promoting action was observed in Experimental Example 1 and known PHD inhibitors 3,4-DHB and S965711, transition metals The chelating activity for ions was modified by the method of Price et al. (Price DL, et al., “Chelating activity of advanced glycation end-product (AGE) inhibitors.” J Biol Chem 272: 5430-5437, 1997). Measured. Briefly, 15 μl of 50 μM CuCl ₂ and 30 μl of various concentrations of test compound were incubated in 1.38 ml of phosphate buffer at 30 ° C. for 5 minutes. The reaction was started by adding 75 μl of 10 mM ascorbic acid (CuCl ₂ and ascorbic acid final concentrations: 500 nM and 500 μM, respectively). Incubation was performed at 30 ° C. for 0-60 minutes. The amount of ascorbic acid in the reaction solution was measured by detecting the absorbance at 244 nm using reverse phase high performance liquid chromatography (HPLC).

As a result, 3,4-DHB, S965711, TM-6008 and TM-6010 chelated to transition metal (copper) and inhibited ascorbic acid autoxidation in a dose-dependent manner (IC ₅₀ were 330 μM, 31.4 μM, respectively) 0.57 μM and 4.84 μM). On the other hand, TM-6089 did not chelate transition metal (copper) even at a concentration of 100 μM. This revealed that TM-6008 and TM-6010 certainly chelate with metals. On the other hand, TM-6089 does not have a metal chelating ability, and similarly TM6101 structurally similar to TM-6089 seems not to have a metal chelating ability.

Experimental Example 3 PHD Inhibitory Activity PHD inhibitory activity was examined for the compounds (TM-6008, TM-6010 and TM-6089, and cobalt chloride as a positive control) in which HIF promoting action was observed in Experimental Example 1 above.

PHD inhibitory activity was determined by the method of Kaule et al. (Cunhild Kaule and Volkmar Gunzler, “Assay for 2-Oxoglutarate Decarboxylating Enzymes Based on IHDTC cells (renal proximal tubule cells) homogenate of mitochondrial fraction rich in PHD). on the Determination of [1- ¹⁴ C] Succinate: Application to Prolyl 4- Hydroxylase ”, Analytical Biochemistry 184, 291-297, (1990))
Measured according to Specifically, IRPTC cells (renal proximal tubule cells) suspended in 10 mM Tris-HCl (pH 6.7) containing 0.15 mM MgCl ₂ and 10 mM KCl were first dextrose (final concentration: 0.25 M) on ice. ) And centrifuged at 1500 × g for 15 minutes at 4 ° C. to remove the nuclear fraction. Subsequently, the supernatant was centrifuged at 20000 × g for 10 minutes at 4 ° C. to obtain a mitochondrial fraction (30 μg). The mitochondrial fraction was reacted with various concentrations of test compounds (TM-6008, TM-6010, TM-6089 and cobalt chloride) at 37 ° C for 30 minutes, and then 2.7 mM Tris-HCl (pH 7.5), 3.3 μM FeSO ₄ 13.3 μM oxygen-dependent degradation domain (ODD) dissolved in a buffer containing 6.7 μM [5- ¹⁴ C] -2-OG (Amersham), 66 μM ascovirate, 27 μg / ml catalase, and 33 μM dithiothreitol The peptide (HIF-1α amino acid 557-576 region) was reacted at 37 ° C. for 1 hour. After 1 hour incubation, 3.3 mM succinate, 3.3 mM 2-OG and 27 mM 2,4-dinitrophenylhydrazine were added. The mixture was left at room temperature for 30 minutes, and 0.3 M 2-OG was further added. Centrifuged for 5 min the supernatant was separated by 7000 × g, and the radioactivity was measured in [1- ¹⁴ C] succinate by liquid scintillation counter. As a control (negative control), the above reaction was carried out without a test compound, and the radioactivity of [1- ¹⁴ C] succinate was measured in the same manner, and this was defined as 100% hydroxylase activity.

The results are shown in FIG. The hydroxylase activity of each test compound shown in FIG. 4 is a relative ratio to the radioactivity (hydroxylase activity 100%) of [1- ¹⁴ C] succinate obtained from the negative control.

  As shown in FIG. 4, the test compounds (TM-6008, TM-6010 and TM-6089, and cobalt chloride as a positive control) all inhibited PHD activity in a dose-dependent manner. TM-6008 and TM-6089 had higher PHD inhibitory activity than cobalt chloride, a known PHD inhibitor, and TM-6008 had the highest PHD inhibitory activity.

Experimental Example 4 In vitro binding assay of HIF-1α (α chain) peptide and PHD
The docking simulation in Experimental Example 1 revealed that three compounds (TM-6008, TM-6010, and TM-6089) compete with the proline residue of HIF and bind to the B site in the PHD3 molecule. In order to confirm this, using the above compounds (TM-6008, TM-6010 and TM-6089) and known PHD inhibitors 3,4-DHB and S965711, HIF-1α peptide and An in vitro binding assay for PHD was performed.

<In vitro binding assay>
PHD3 is transcribed in vitro from the expression plasmid pBOS-FLAG-mEGLN3 and transformed in the presence of [ ³⁵ S] methionine (Amersham) using the reticulocyte lysate TnT quick kit (Promega) did. GST-HIF-1 (531-652) cloned in the pGEX4T-1 vector was expressed in E. coli (DH5α), and affinity purification was performed using GST-coupled beads (Glutathion Sepharose 4B: Amersham). PHD3 labeled with ³⁵ S was converted to 20 mM Tris- containing 100 mM KCl, 10% glycerin, 1.4 mM β-mercaptoethanol, 100 mM FeCl ₂ , 0.2% NP-40 and 0.5 mg / ml bovine serum albumin. Pre-incubation with test compounds (final concentrations: 50 and 500 μM) for 15 minutes in 50 μl of HCl (pH 8.0). Next, GST-HIF-1α (531-652) -bound beads (12.5 μg) were added to the mixture and incubated at 4 ° C. for 2 hours. After washing, ³⁵ S-labeled PHD3 bound to GST-HIF-1α (531-652) was washed with 120 mM Tris-HCl (pH 6.8) containing 120 mM SDS, 12% glycerin, 240 mM β-mercaptoethanol and 230 μM bromophenol blue. ). The eluate was subjected to SDS-PAGE and subjected to autoradiography.

The inhibitory effect of the test compound on ³⁵ S-labeled PHD3 binding to GST-HIF-1α (531-652) was evaluated by a pull-down assay. The result of the above binding assay in the absence of the test compound (binding activity) was defined as 100% (control), and the binding activity of each test compound relative thereto was expressed as%.

  The results are shown in FIG. Similar to the known PHD inhibitor S965711, all three novel PHD inhibitors (TM-6008, TM-6010 and TM-6089) compete with the HIF peptide, and GST-HIF-1α (531- 652) was inhibited (FIG. 5). On the other hand, 3,4-DHB, a known PHD inhibitor, did not compete with the HIF peptide and did not inhibit the binding with GST-HIF-1α (531-652) (FIG. 5). Moreover, even when an excessive amount of 2-OG was added, this result did not change.

Experimental Example 5 In Vivo Test of Promoting Angiogenesis (1) Sponge Angiogenesis Analysis In vivo test of whether angiogenesis is promoted by local administration of the above test compounds (TM-6008, TM-6010 and TM-6089) did. For this purpose, a small sponge disc is inserted into the mouse subcutaneously, and the test compound is administered subcutaneously via this sponge. After 10 days, the amount of hemoglobin and the number of blood vessels are measured to promote the HIF-HRE system. Evaluation (sponge angiogenesis analysis).

  Sponge angiogenesis analysis was performed as described in Muramatsu et al. (Muramatsu, M., Katada, J., Hayashi, I. & Majima, M. Chymase as a proangiogenic factor. A possible involvement of chymase-angiotensin-dependent pathway in the hamster sponge angiogenesis model. J. Biol. Chem. 275. 5545-5552 (2000)). Specifically, a circular sponge disc (5 mm thickness and 12 mm diameter) was implanted into the subcutaneous air sac on the back of 6 week old male ICR mice (n = 2) anesthetized with ether. 5 days after transplantation, a test compound dissolved in DMSO and diluted with physiological saline (50 μg TM-6008, TM-6010 or TM-6089 or 30 μg cobalt per mouse) was given a day for one week. Every 4 times, it was subcutaneously administered to the sponge. bFGF (10 μg) (Genzyme) was used as a positive control. Moreover, what administered only the liquid (it describes as "vehicle" in a figure) which diluted DMSO with the physiological saline was used as negative control.

  Mice were killed after the experiment and immediately granulated tumor tissue was excised with the sponge graft. Measure the amount of hemoglobin in tissues collected using the kit (hemoglobin B test: Wako Pure Chemical Industries), and use anti-mouse CD31 antibody (Pharmingen) to determine the number of blood vessels in the tissues (number of CD31 positive blood vessels) Was quantitatively measured by immunohistological techniques. The number of blood vessels was measured in 400 × field of 5 non-overlapping sites arbitrarily selected per mouse.

  The amount of hemoglobin is shown in FIG. 6a, and the relative fluorescence intensity corresponding to the number of blood vessels is shown in FIG. 6b. FIG. 6c shows an immunostained image of endothelial cells. As can be seen from the results in FIG. 6a, it was recognized that the amount of hemoglobin was increased by administration of TM-6008, TM-6010 and TM-6089 in the same manner as bFGF. Moreover, from FIG. 6c, the number of blood vessels increased by administration of TM-6008, TM-6010 and TM-6089, and angiogenesis was recognized. In particular, significant angiogenesis was observed by the administration of TM-6008 (FIGS. 5b and 5c).

(2) Activation of the HIF-HRE system Whether or not the test compound (TM-6008, TM-6089) promotes the HIF-HRE system in various organs, hypoxia gene recombination expressing HRE luciferase Mice (Tanaka, T. et al. Hypoxia in renal disease with proteinuria and / or glomerular hypertension. Am. J. Pathol. 165. 1979-1992 (2004)) were examined.

  Mice were orally administered TM-6008 or TM-6089 suspended in an aqueous solution of 0.5% carboxymethylcellulose sodium salt at a rate of 100 mg / kg 2 or 4 hours before sacrifice (each n = 3). Only 0.5% carboxymethylcellulose solution was orally administered to control mice (n = 3). After the mice were killed, tissues were collected for examination of reporter gene expression. Hypoxia-responsive luciferase gene expression was assessed by semi-quantitative RT-PCR according to a previously reported method (Tanaka, T. et al. Hypoxia in renal disease with proteinuria and / or glomerular hypertension. Am. J. Pathol 165. 1979-1992 (2004)). The protocol was performed according to the animal experiment guidelines of the University of Tokyo.

  As a result, expression of the reporter gene in the kidney was not observed under basic conditions (40 amplifications), but was observed after single oral administration of TM-6008 or TM-6089 at a rate of 100 mg / kg. (Detected at 31.0 ± 0.85 times and 31.0 ± 2.05 times, respectively). Reporter gene expression in the liver was not observed under basic conditions, but was detected by administration of TM-6008 (detected at 32.3 ± 0.35). However, administration of TM-6089 had no effect. Reporter gene expression in the heart was detected under basic conditions and increased with the administration of TM-6008 or TM-6089 (1.37 ± 1.00-fold and 6.69 ± 5.45-fold, respectively).

  These results suggest that TM-6008 plays an important role in vascular network formation by activating the HIF-HRE system in all organs.

Experimental Example 6 Prevention of neuronal cell death due to hypoxia The PHD inhibitor (TM-6008) was evaluated for its protective effect against cell damage due to hypoxia.

  The experiment was conducted using a delayed neuronal death model (male, 70-80 g) of a gerbil mouse. Mice were randomly divided into three experimental groups, and group 1 and 2 mice were artificially caused transient ischemia.

  Specifically, for mice in groups 1 and 2, under anesthesia, the two common carotid arteries were occluded with a small aneurysm clip (Sugita Clip # 07-940-12: Mizuho Medical) for 5 minutes. It caused transient ischemia over time (Kirino, T. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 239. 57-69 (1982)). The clip was then released, and microreflux of the surface layer was confirmed by laser Doppler flowmetry (Omega Flow FLO-C1: Neurosci). Group 1 mice (n = 20) were forcibly orally administered TM-6008 (100 mg / kg / day) dissolved in 0.5% sodium carboxymethylcellulose for 7 days after carotid occlusion. Group 2 mice (n = 18) were orally administered only 0.5% carboxymethylcellulose solution for 7 days after carotid occlusion. Group 3 (n = 8) was fake and used as a control.

All mice were killed by injecting pentobarbital 7 days after occlusion, the brains were removed, coronal brain sections were fixed with 4% paraformaldehyde, and stained with hematoxylone and eosin. TUNEL staining was performed using an indirect immunohistochemical staining method (TdT-Bule label of in situ apoptosis detection kit: manufactured by Trevigen). CA1 hippocampal neurons were observed using an optical microscope to evaluate ischemic damage in the hippocampus CA1. The evaluation was made by counting the number of normal pyramidal nerves present in the hippocampus along a predetermined area (0.62 mm ² ) for a high performance field (400 ×).

  A representative photomicrograph (hematoxylone eosin staining) in the CA1 hippocampus is shown in FIG. Compared to non-ischemic mouse neurons (FIG. 7a), mice that were exposed to ischemia and then administered only carboxymethylcellulose (denoted as “vehicle” in the figure) had pyramid-shaped nerves. In many cases, cytoplasm and glial cells that were wilt and darkly stained were observed to be concentrated and accumulated, and it was found that the cells were damaged by ischemia (FIG. 7b). On the other hand, in mice treated with TM-6008 after ischemia, only a small number of nerves showed ischemia (FIG. 7c).

  Cells showing nuclei and nucleoli were detected as viable neurons. The number of neurons surviving in the CA1 hippocampus was significantly higher in mice treated with TM6008 (166 ± 73) than in mice treated with post-ischemic carboxymethylcellulose alone (61 ± 55), non-ischemic control group There was no statistically significant difference from the number of surviving nerves observed in (227 ± 50) (FIG. 7d).

  The final plasma concentration of TM-6008 in these experiments was 7.8 ± 2.9 μg / ml. From the above results, it became clear that TM-6008 has a significant protective effect against nerve death caused by ischemia.

Experimental Example 7 Toxicity (1) Cytotoxicity
HeLa cells were cultured in Dulbecco's MEM medium for 24 hours in the presence of test compounds (TM6008, TM6010 and TM6089, 50-250 μM). Cytotoxicity is determined by measuring the amount of lactate dehydrogenase (LDH) released in the medium using a kit (Promega) and measuring the percentage of total LDH activity released after lysis of all cells. Indicated. TM-6008, TM-6010 and TM-6089 did not show cytotoxicity at concentrations lower than 50 μM, 250 μM and 100 μM, respectively.

(2) In vivo acute or subacute toxicity
Acute toxicity was evaluated using 30-35 g ICR mice (obtained from Clare Japan). TM6008, TM6010 and TM6089 were administered by gavage to ICR mice at single doses of 500, 1000, 1500 and 2000 mg / kg. The toxicity results of this acute administration were monitored for 2 weeks. No acute toxicity was observed after administration of 2000 mg / kg for TM-6008 and 1500 mg / kg for TM-6010 for 2 weeks. On the other hand, the 50% lethal dose of TM-6089 was 500 mg / kg. In addition, the subacute toxicity test was carried out by forcibly administering 100 mg / kg of the test compound (TM6008, TM6010 or TM6089) to mice for 30 days. Even when each test compound was administered at a rate of 100 mg / kg / day for 30 days, the mice showed almost no subacute toxicity.

(3) Biochemical analysis
Blood and urine samples were collected for biochemical analysis (glucose in plasma, total cholesterol, triglycerides, GOT, GPT, creatinine, urea nitrogen, total protein, and albumin; hemoglobin in blood, red blood cell count and hematocrit; urine Protein and creatinine). The amount of plasma erythropoietin was measured using a kit (F. Hoffmann-La, manufactured by Roche). As a result of histological analysis and serum test of brain, lung, heart, kidney and liver, no abnormality was observed after one month. There was also no significant change in plasma erythropoietin levels.

(4) Pharmacokinetics
Test compounds (TM6008, TM6010 and TM6089, 50 mg / kg) were administered by gavage to 180-200 g male Wister rats. Heparinized blood samples were collected before dosing (0 h) and 1, 2, 6 and 24 h after dosing. Plasma concentrations were analyzed by reverse phase HPLC to determine maximum drug concentration time (Tmax), maximum drug concentration (Cmax), and half-life (T1 / 2).

  The results of pharmacokinetics (maximum drug concentration time (Tmax), maximum drug concentration (Cmax), and half-life (T1 / 2)) in mice that were orally administered each compound at a rate of 50 mg / kg were as follows: there were:

TM-6010は平面構造を有している。ドッキングシミュレーションはDNAとのインターカレーションの可能性を示した。 このため、in vivoでの実験はTM-6008とTM-6089でのみ行なった。

TM-6010 has a planar structure. Docking simulation showed the possibility of intercalation with DNA. For this reason, in vivo experiments were performed only with TM-6008 and TM-6089.

FIG. A is a diagram showing an X-ray crystal structure of PHD3 derived from streptomysis. In the figure, the sphere (blue) in the middle pocket indicates Fe (II). In the figure, the stick model (branch) located around this Fe (II) shows the proline residue of HIF on the top and 2-OG on the bottom. FIG. B shows the binding mode of 3,4-DHB, a known PDH inhibitor, in PHD3. It can be seen that it binds to the A site which is the binding site of 2-OG. FIG. C shows the binding mode of TM6008 (shown in yellow in the figure), TM6010 (shown in green in the figure) and TM6089 (shown in red in the figure) in PHD3. The figure was drawn with software PyMOL version 0.97 (DeLano Scientific LLC). When IRPTC cells transformed with 7xHRE / Luc plasmid with HIF-dependent luciferase reporter gene were incubated with test compounds (positive control: cobalt chloride, TM6008, TM6010 and TM6089, final concentration: 20 μM or 60 μM) for 24 hours Shows luciferase activity. The result is evaluated as an average of three independent experiments, which is an increase in the number of times that of the control result (taken as 1). Data are expressed as mean ± SD (* P <0.05, ** P <0.01 vs control). The luciferase activity when incubated for 24 hours with cobalt chloride, TM6008 and TM6101 as test compounds is shown. The result is evaluated as an average of three independent experiments, which is an increase in the number of times that of the control result (taken as 1). Data are expressed as mean ± SD (* P <0.05, ** P <0.01 vs control). The mitochondrial fraction of IRPTC cell homogenate (30 μg) was reacted with the test compound and HIF-1α oxygen-dependent degradation domain (ODD) peptide (amino acids 556-575). ODD-dependent hydroxylase activity was assessed by counting the radioactivity of [1- ¹⁴ C] succinate converted from [5- ¹⁴ C] -2-OG. Results are shown as% inhibition of ODD-dependent hydroxylase activity as an average of three independent experiments. Data were expressed as mean ± SD. * P <0.01 vs control. The inhibitory effect of the test compound on ³⁵ S-labeled PHD3 binding to GST-HIF-1α (531-652) was evaluated by a pull-down assay. Average values were taken from the results of three independent experiments and expressed as% of binding activity. Data are shown as mean ± SD. * P <0.01 vs control Polyurethane sponges were implanted subcutaneously in mice, and test compounds (48 μg TM6008 or 30 μg cobalt per mouse) were injected into the sponge every other day for 1 week. In order to evaluate the degree of angiogenesis, the amount of hemoglobin in the sponge (a) was measured, and the endothelial cell marker CD31 (b, c) was stained. b is a representative photograph of CD31 immunostaining (x200). c indicates the average number of results caused by each reagent. CA1 hippocampus of non-ischemic animals (a), animals treated with vehicle (carboxymethylcellulose) only after ischemic condition (b), and animals treated with TM-6008 after rejection condition (c), Representative photomicrographs of hematoxylone-eosin staining are shown. In the figure, the bar indicates 0.1 mm. d is the normal nerve in the CA1 hippocampus of the non-ischemic group (No ischemia), the post-ischemic vehicle (carboxymethylcellulose) treated group (Ischemia treated with vehicle), and the post-ischemic TM6008 treated group (Ischemia treated with TM6008). Indicates the number of

Claims (16)

A prolyl hydroxylase inhibitor comprising, as an active ingredient, a compound containing at least one group having a skeleton represented by the following formula (A) or (B) or a salt thereof in one molecule:

(In the formula, R ₈ and R ₉ may be hydrogen atoms with each other or may form a ring together with the alkylene chain, oxygen atom, nitrogen atom or sulfur atom to which they are bonded). A group having a skeleton A is represented by the following formula:

(R ₁ is a hydroxyl group, a lower alkyl group, an alkyl group substituted with a carboxyl group, a carboxyalkyl group optionally having a protecting group, or an amino group: R ₂ is a hydrogen atom, a hydroxyl group, a lower alkyl group, a carboxyl group. An alkyl group substituted with or an amino group: R ₃ represents a hydrogen atom, an amino group, or a lower alkylamino group)
The prolyl hydroxylase inhibitor of Claim 1 which is group shown by these. The group having the skeleton B has the following formula:

(R ₄ , R ₅ , R ₆ and R ₇ are the same or different and each represents a hydrogen atom or a lower alkyl group. R ₄ and R ₅ or R ₆ and R ₇ are bonded to each other, and they are bonded. Together with the carbon atom it may form a cyclo or hetero ring optionally having 5 or 6 membered saturated or unsaturated substituents, R ₈ and R ₉ are each a hydrogen atom, or A ring may be formed together with an alkylene chain, an oxygen atom, a nitrogen atom or a sulfur atom to which they are bonded.)
The prolyl hydroxylase inhibitor according to claim 1 or 2, which is a group represented by the formula: Compound (I) represented by the following formula:

[R ₁ represents carboxyalkyl kill alkyl group having 1 to 4 carbon atoms which may have a lower alkyl group or a protective group of 1 to 6 carbon atoms: R ₂ is a hydrogen atom, a lower alkyl group having 1 to 6 carbon atoms: R ₁₀ represents R ₁₁ —S—CH ₂ — (wherein R ₁₁ is a heteroaryl group which may have a substituent, or the following formula:

(R ₄ , R ₅ , R ₆ and R ₇ are the same or different and each represents a hydrogen atom or a lower alkyl group having 1 to 6 carbon atoms. R ₄ and R ₅ or R ₆ and R ₇ are bonded to each other. And may form a cyclo or hetero ring optionally having a 5- or 6-membered saturated or unsaturated substituent together with the carbon atom to which they are bonded. ]
And compound (II) represented by the following formula:

(R ₅ and R ₇ are the same or different and each represents a hydrogen atom or a lower alkyl group having 1 to 6 carbon atoms.)
The prolyl hydroxylase inhibitor according to any one of claims 1 to 3, wherein the active ingredient is at least one compound selected from the group consisting of: Compounds 1 to 4 represented by the following formula:
(Compound 1)

(Compound 2)

(Compound 3)

(Compound 4)

The prolyl hydroxylase inhibitor according to any one of claims 1 to 4, wherein the active ingredient is at least one selected from the group consisting of:   The prolyl hydroxylase inhibitor according to any one of claims 1 to 5, which is a binding inhibitor between prolyl hydroxylase and a hypoxia-inducing factor.   The prolyl hydroxylase inhibitor according to any one of claims 1 to 5, which is a hypoxia-inducible factor degradation inhibitor.   A pharmaceutical composition comprising the prolyl hydroxylase inhibitor according to claim 1 and a pharmaceutically acceptable carrier or additive.   The pharmaceutical composition according to claim 8 for improving hypoxia of a cell or tissue.   10. The pharmaceutical composition according to claim 8 or 9, which is a prophylactic or therapeutic agent for a disease or condition caused in connection with chronic or acute hypoxic conditions of cells or tissues.   10. The pharmaceutical composition according to claim 9, wherein the disease or condition caused in connection with the hypoxic state of cells or tissues is a chronic or acute ischemic disease or a condition caused thereby.   The pharmaceutical composition according to any one of claims 8 to 11, which has an oral dosage form.   A screening method for a prolyl hydroxylase inhibitor comprising a step of selecting a compound having a competitive binding inhibitory action between prolyl hydroxylase and an α-chain proline of a hypoxia-inducing factor from test compounds. A compound having a competitive binding inhibitory action between prolyl hydroxylase and the alpha chain proline of the hypoxia-inducing factor has at least one group having a skeleton represented by the following formula (A) or (B) in one molecule. A screening method according to claim 13, which is a compound comprising:

(In the formula, R ₈ and R ₉ may be hydrogen atoms with each other or may form a ring together with the alkylene chain, oxygen atom, nitrogen atom or sulfur atom to which they are bonded).   The screening method according to claim 13 or 14, which is a screening method for an active ingredient of a prophylactic or therapeutic drug for a disease or pathology that occurs in association with a chronic or acute hypoxic state of cells or tissues. A compound represented by the following formula or a salt thereof:

(In the formula, R ₁₂ represents a hydrogen atom or a lower alkyl group.)

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