JP5107589B2 - Indole derivatives - Google Patents

Description

  The present invention relates to a novel indole derivative having an action for maintaining undifferentiation of embryonic stem (ES) cells, a method for producing the same, and use thereof.

  Organs and tissues damaged by trauma, illness, and aging need to promote regeneration and restore their function. In particular, since organs such as the heart, liver, kidney, and pancreas are essential for life support, their functional decline and abolition are directly linked to death, and transplantation medical treatment for saving lives by organ transplantation is actively performed. However, a new approach is needed to solve this problem due to the permanent shortage of donors.

  Recently, tissues and organs have been created using stem cells that are present in embryos or adults and are considered to have the ability to divide indefinitely and differentiate in one or more directions. Regenerative medicine is attracting attention as a treatment that surpasses the drawbacks of conventional organ transplantation.

  Specifically, stem cells are proliferated and then differentiated and used for cell transplantation, or artificial tissue construction is performed in conjunction with the use of artificial support tissue, which is then transplanted in vivo or used as an artificial organ Things are considered. If stem cells can be used for cell transplantation treatment and tissue engineering, it is expected that problems associated with conventional transplantation treatments including autologous transplantation, such as tissue defects after donor graft removal and donor shortage, are expected.

  Stem cells have been identified in various fields such as blood vessels, nerves, blood, cartilage, bone, liver, pancreas, among which totipotent stem cells that have the ability to differentiate into all cell types are the above-mentioned regenerative medicine. In addition to the field, it has attracted particular attention as a cell that can easily provide cells and tissues for use in drug discovery and gene therapy.

  As examples of totipotent stem cells, embryonic stem (Embryonic Stem, hereinafter referred to as ES) cells and embryonic germ (Embryonic Germ, hereinafter referred to as EG) cells are known. ES cells are cell lines isolated from the inner cell mass (ICM) of the mouse blastocyst stage (Non-patent Document 1). Individual cells are derived from the inner cell mass (ICM) at the blastocyst stage or the primary ectoderm derived from the epiblast (epiblast). ICM and epiblast Can be said to be a totipotent stem cell group. ES cells retain the ability to differentiate into various individual forming tissues, and have the ability to differentiate into any mature adult cell by forming normal and chimeric embryos. Further, it has the ability to generate various cells such as blood cells, cardiomyocytes, vascular endothelial cells, nerve cells, pigment cells, pancreatic endocrine cells depending on the differentiation induction conditions in the test tube (Non-patent Document 2).

  EG cells are cells established by culturing primordial germ cells in the presence of LIF (Leukemia Inhibitory Factor) and bFGF (basic Fibroblast Growth Factor) (Non-patent Documents 3 and 4), and various tissues similar to ES cells. Has the ability to differentiate into

  Recently, the establishment of ES cell lines other than mice has been reported, and it has been shown to have pluripotency similar to mouse ES cells (bovine ES cells: Non-patent document 5, porcine ES cells: Non-patent document 6, sheep ES cell: non-patent document 7, hamster ES cell: non-patent document 8, rhesus monkey ES cell: non-patent document 9, marmoset ES cell: non-patent document 10, human ES cell: non-patent document 11 14, cynomolgus monkey ES cells: Non-patent document 15).

  Recently, an example of infection of an endogenous virus between different animals has been reported (Non-patent Document 16). In the culture method aiming at the utilization of human ES cells for medical use, development of a culture method that avoids contact between different animal cells as much as possible is desired. Therefore, the above ES cell undifferentiated maintenance culture method using mouse-derived cells is not suitable for the culture of ES cells for medical purposes.

  As a method for culturing primate ES cells that do not use mouse-derived feeder cells, a method in which secreted components of mouse primary fibroblasts are added to the culture medium has been reported (for example, see Patent Document 1). However, even in this case, ES cells cultured in such an environment are not suitable for use in medical applications because ES cells in culture are exposed to unidentified factors secreted from mouse cells. In addition, there is a risk of infection with endogenous viruses. Accordingly, the disadvantages caused by co-culture with mouse primary line cells have not been solved at all.

  In addition, as another ES cell culture method not using mouse-derived feeder cells, a method using a compound (BIO) having a GSK-3β inhibitory action has been reported (Non-patent Document 17). However, the undifferentiated maintenance effect of BIO is effective only in short-term culture, and the action of maintaining undifferentiated ES cells in long-term culture by subculture has not been confirmed.

  We have already reported a method using a low molecular weight compound as a method for culturing ES cells without using mouse-derived feeder cells (Patent Document 2). A dihydroisoquinoline derivative and an indole derivative are compounds that exhibit an undifferentiated maintenance activity against ES cells without using feeder cells. Considering the use for medical applications, development of new low molecular weight compounds with higher undifferentiation maintenance activity is desired, but so far, new indole derivatives showing the undifferentiation maintenance effect of ES cells are known. It wasn't.

  To date, the mechanisms involved in maintaining undifferentiated ES cells have not been fully elucidated. If a target protein or binding protein of a certain low molecular weight compound is identified, it is considered to be very useful for elucidating the mechanism relating to maintenance of undifferentiation. As one of the techniques, for example, an affinity purification method for immobilizing a low-molecular compound in a carrier and purifying the bound protein can be mentioned (for example, Non-Patent Document 18). However, so far, no indole derivative that can be immobilized and exhibits an undifferentiated maintenance activity of ES cells has not been known.

Evans et al., Nature, 292, p154, 1981 Toru Nakano, latest medical supplement-regenerative medicine, p81-89, 2000 Matsui et al., Cell, 70, p841, 1992 Resnic et al., Nature, 359, p550, 1992 Schellander et al., Theriogenology, 31, p15-17, 1989 Strojek et al., Theriogenology, 33: p901, 1990 Handyside, Roux ’s Arch.De v.Biol., 196: p185, 1987 Doetschman et al., Dev. Biol., 127, p224, 1988 Thomson et al., Proc. Natl. Acad. Sci. USA, 92, p7844, 1995 Thomson et al., Biology of Production, 55, p254, 1996 Thomson et al., Science, 282, p1145, 1998 Reubinoff et al., Nature Biotech, 18, p399, 2000 Mitalipova et al., Stem Cells, 21, p521, 2003 Heins et al., Stem Cells, 22, p367, 2004 Suemori et al., Dev. Dyn. 222, P273, 2001 van der Laan et al., Nature, 407, p90, 2000 Sato et al., Nature Medicine, vol. 10, p55, 2004 Handahiro, Kawaguchi Haruma, Nanoaffinity Beads, 2003, Nakayama Shoten JP 2001-17163 A WO2005 / 007838

  An object of the present invention is to provide an indole derivative capable of culturing embryonic stem cells in an undifferentiated state without using feeder cells or feeder cell-derived components. Another object of the present invention is to provide an indole derivative that can be specifically immobilized on a carrier in order to analyze the mechanism for maintaining undifferentiation of embryonic hepatocytes.

As a result of intensive studies on compounds having an ES cell undifferentiation maintenance activity, the present inventors have found that indole derivatives include novel compounds represented by the following general formulas (1), (2) and (3) or salts thereof. The compound that was specifically produced for the first time, surprisingly, based on its unique chemical structure, exhibits excellent ES cell undifferentiation maintenance activity and is useful for the proliferation of ES cells in regenerative medicine applications Based on this, this research was completed.
That is, the present invention has the following configuration.

(1) General formula (1)
[Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ may be the same or different and each represents an electron-withdrawing group, an electron-donating group or a hydrogen atom. X represents an alkylene group having 0 to 10 atoms in the main chain. The alkylene group having 0 atoms represents a single bond. One or more ethylene constituting the alkylene group may be replaced by a -C = C- group and / or -N = N- group and / or -NHCO- group. Furthermore, the alkylene group may have one or more electron-withdrawing groups, electron-donating groups, or hydrogen atoms as a substituent. G represents an electron-withdrawing group, an electron-donating group, an aromatic group that may have a hydrogen atom, or a cyclic molecule that includes at least one heteroatom that may have an electron-withdrawing group, an electron-donating group, or a hydrogen atom. To express. Or a salt thereof.

(2) The compound or salt thereof according to (1), wherein X represents an alkylene group having 0 or 1 main chain atoms.

(3) General formula (2)
[Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ may be the same or different and each represents an electron-withdrawing group, an electron-donating group or a hydrogen atom. A is a hydrogen atom, a methyl group or
Represents. Here, R ⁷ , R ⁸ , R ⁹ , R ¹⁰ and R ¹¹ may be the same or different and each represents an electron withdrawing group, an electron donating group or a hydrogen atom. Or a salt thereof.

(4) General formula (3)
[Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ may be the same or different and each represents an electron-withdrawing group, an electron-donating group or a hydrogen atom. B represents an electron withdrawing group, an electron donating group or a cyclic molecule containing at least one hetero atom which may have a hydrogen atom. ]
Or a salt thereof.

(5) R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ and R ¹¹ are each independently an alkyl group, an unsaturated hydrocarbon group, Acyl group, aroyl group, aryl group, alkoxy group, alkoxyalkyl group, acyloxy group, alkoxycarbonyl group, hydroxyalkyl group, amino group, acetamido group, aminocarbonyl group, alkylaminocarbonyl group, alkylamino group, alkylaminoalkyl group , Alkylaminovinyl group, aminoalkyl group, hydroxyalkylamino group, hydroxyalkylaminoalkyl group, hydroxyalkylaminovinyl group, arylamino group, arylaminovinyl group, nitro group, hydroxyl group, alkylthio group, sulfonamido group, sulfone Group, carboxy group, mercapto group, oxalo group, hydroxyimino A group or an atom selected from the group consisting of an alkyl group, a thiocarbamoyl group, a pyrrolyl group, an oxazol group, a halogen atom and a hydrogen atom, and the alkyl contained therein includes an electron-withdrawing group, an electron-donating group or The compound or a salt thereof according to (3), which may have one or more hydrogen atoms.

(6) R ¹ , R ² , R ³ , R ⁴ , R ⁵ , and R ⁶ are each independently an alkyl group, unsaturated hydrocarbon group, acyl group, aroyl group, aryl group, alkoxy group, alkoxyalkyl. Group, acyloxy group, alkoxycarbonyl group, hydroxyalkyl group, amino group, acetamide group, aminocarbonyl group, alkylaminocarbonyl group, alkylamino group, alkylaminoalkyl group, alkylaminovinyl group, aminoalkyl group, hydroxyalkylamino group , Hydroxyalkylaminoalkyl group, hydroxyalkylaminovinyl group, arylamino group, arylaminovinyl group, nitro group, hydroxyl group, alkylthio group, sulfonamido group, sulfo group, carboxy group, mercapto group, oxalo group, hydroxyiminoalkyl Group, thiocarba Benzoimidazole, imidazole, which is a group or atom selected from the group consisting of yl group, pyrrolyl group, oxazol group, halogen atom and hydrogen atom, and B may have an electron-withdrawing group, electron-donating group or hydrogen atom , Pyrrolidine, piperidine, pyridine, pyrimidine, dihydrothiophen-2-one, tetrahydrothiophene-1,1-dioxide, isoxazole, benzoxazole, isothiazole, indole, quinoline, benzofuran, quinoxaline, chroman-4-one, 2 The compound or salt thereof according to (4), which is 1,3-dihydrobenzo [1,4] dioxacin or benzothiazole.

(7) R ¹ , R ² , R ¹⁰ and R ¹¹ are hydrogen atoms, R ³ is a hydroxyl group, a lower alkoxy group or an acetyloxy group, R ⁴ is a hydrogen atom, a lower alkylamino group, hydroxy A lower alkyl group, a hydroxyl group, a lower alkyl group, or an amino lower alkyl group, and R ⁵ is a hydrogen atom, a lower alkyl group, a lower alkylaminovinyl group, a hydroxy lower alkylaminovinyl group, or an arylaminovinyl group, R ⁶ is a nitro group, and R ⁷ , R ⁸ and R ⁹ may be different from each other, hydrogen atom, acetyl group, hydroxyl group, hydroxy lower alkyl group, lower alkoxy lower alkyl group, amino group, lower alkylamino Group, acetamide group, sulfamoyl group, nitrile group, formyl group, mercapto group, acetoxy group, lower alkoxycarbonyl Or a carboxyl group, the compound or salt thereof according to (3).

(8) R ¹ , R ² , R ⁹ , R ¹⁰ and R ¹¹ are hydrogen atoms, R ³ is a hydroxyl group, a lower alkoxy group or an acetyloxy group, R ⁴ is a hydrogen atom, a lower alkylamino group , A hydroxy lower alkyl group, a hydroxyl group, a lower alkyl group, or an amino lower alkyl group, and R ⁵ is a hydrogen atom, a lower alkyl group, a lower alkylaminovinyl group, a hydroxy lower alkylaminovinyl group, or an arylaminovinyl group. The compound or a salt thereof according to (3), wherein R ⁶ is a nitro group, and R ⁷ and R ⁸ may be different from each other, and are a hydrogen atom, a lower alkyl group, or a halogen atom.

(9) R ¹ , R ² , R ⁷ , R ⁹ , R ¹⁰ and R ¹¹ are hydrogen atoms, R ³ is a hydroxyl group, a lower alkoxy group or an acetyloxy group, R ⁴ is a hydrogen atom, lower An alkylamino group, a hydroxy lower alkyl group, a hydroxyl group, a lower alkyl group, or an amino lower alkyl group, wherein R ⁵ is a hydrogen atom, a lower alkyl group, a lower alkylaminovinyl group, a hydroxy lower alkylaminovinyl group, or an arylamino; The compound or a salt thereof according to (3), which is a vinyl group, R ⁶ is a nitro group, and R ⁸ is a lower alkoxy group.

(10) R ¹ is a hydrogen atom, a halogen atom, or a lower alkoxy group, and R ² is a carbamoyl group, an ethynyl group, a lower alkylamino group, a lower alkyl group, a lower alkoxy group, or an optionally substituted phenyl A group, an arylamino group, or a halogen atom, R ³ is a hydroxyl group, a lower alkoxy group, or an acetyloxy group, and R ⁴ is a hydrogen atom, a lower alkylamino group, a hydroxy lower alkyl group, a hydroxyl group, or a lower alkyl group Or an amino lower alkyl group, R ⁷ , R ⁸ , R ¹⁰ and R ¹¹ are hydrogen atoms, R ⁵ is a hydrogen atom, a lower alkyl group, a lower alkylaminovinyl group, a hydroxy lower alkylaminovinyl group, or aryl amino vinyl group, R ⁶ is a nitro group, R ⁸ is a lower alkoxy group, or a hydroxyl group, according to (3) The compound or a salt thereof.

(11) R ¹ , R ² , R ⁷ , R ⁸ , R ¹⁰ and R ¹¹ are hydrogen atoms, R ³ is a hydroxyl group, a lower alkoxy group or an acetyloxy group, R ⁴ is a hydrogen atom, lower An alkylamino group, a hydroxy lower alkyl group, a hydroxyl group, a lower alkyl group, or an amino lower alkyl group, wherein R ⁵ is a lower alkyl group, hydroxyl group, acetyl group, carboxyl group, lower alkylamino group, lower alkylthio group, lower An alkoxyalkyl group, a hydroxy lower alkyl group, or a lower alkoxycarbonyl group, R ⁶ is a hydrogen atom, a halogen atom, an acetamido group, a carboxyl group, an acetyl group, or a hydroxyl group, and R ⁸ is a lower alkoxy group or a hydroxyl group The compound or a salt thereof according to (3), which is a group.

(12) R ¹ , R ³ , R ⁷ , R ⁸ , R ¹⁰ and R ¹¹ are hydrogen atoms, R ² is a hydroxyl group, a lower alkoxy group or an acetyloxy group, R ⁴ is a hydrogen atom, lower An alkylamino group, a hydroxy lower alkyl group, a hydroxyl group, a lower alkyl group, or an amino lower alkyl group, wherein R ⁵ is a lower alkyl group, hydroxyl group, acetyl group, carboxyl group, lower alkylamino group, lower alkylthio group, lower An alkoxyalkyl group, a hydroxy lower alkyl group, or a lower alkoxycarbonyl group, and R ⁶ is a hydrogen atom, a halogen atom, a nitro group, a nitrile group, an alkoxycarbonyl group, an acetamide group, a carboxyl group, an acetyl group, or a hydroxyl group. , R ⁸ is a lower alkoxy group or a hydroxyl group, or a compound or a salt thereof according to (3) .

(13) R ¹ and R ² are hydrogen atoms, R ³ is a hydroxyl group, a lower alkoxy group, or an acetyloxy group, R ⁴ is a hydrogen atom, a lower alkylamino group, a hydroxy lower alkyl group, a hydroxyl group, A lower alkyl group or an amino lower alkyl group, R ⁵ is a hydrogen atom, a lower alkyl group, a lower alkylaminovinyl group, a hydroxy lower alkylaminovinyl group, or an arylaminovinyl group, and R ⁶ is a nitro group. R ⁷ , R ⁸ , R ¹⁰ and R ¹¹ are an amino group, lower alkoxy group, acetyl group, aminoalkyl group, lower alkoxycarbonyl group, lower alkyl group, carboxyl group, sulfo group, nitrile group, hydroxyl group, acetamide Group, sulfamoyl group, vinyl group, isopropylidene group, hydroxyalkyl group, nitro group, hydroxyimid Alkyl group, oxaloacetic group, a sulfonamido group, a thiocarbamoyl group, a hydrogen atom, or a halogen atom, R ⁹ is a lower alkoxy group, or a hydroxyl group, the compound or salt thereof according to (3).

(14) R ¹ , R ² , R ⁷ , R ¹⁰ and R ¹¹ are hydrogen atoms, R ³ is a hydroxyl group, a lower alkoxy group or an acetyloxy group, R ⁴ is a hydrogen atom, a lower alkylamino group , A hydroxy lower alkyl group, a hydroxyl group, a lower alkyl group, or an amino lower alkyl group, and R ⁵ is a hydrogen atom, a lower alkyl group, a lower alkylaminovinyl group, a hydroxy lower alkylaminovinyl group, or an arylaminovinyl group. The compound or a salt thereof according to (3), wherein R ⁶ is a nitro group, and R ⁸ and R ⁹ are a hydroxyl group or a lower alkoxy group which may be the same or different.

(15) R ¹ , R ² , R ⁷ , R ⁸ , R ¹⁰ and R ¹¹ are hydrogen atoms, R ³ is a hydroxyl group, a lower alkoxy group or an acetyloxy group, R ⁴ is a hydrogen atom, lower An alkylamino group, a hydroxy lower alkyl group, a hydroxyl group, a lower alkyl group, or an amino lower alkyl group, wherein R ⁵ is a hydrogen atom, a lower alkyl group, a lower alkylaminovinyl group, a hydroxy lower alkylaminovinyl group, or an arylamino; The compound or a salt thereof according to (3), which is a vinyl group, R ⁶ is a nitro group, and R ⁹ is a hydroxyl group.

(16) R ¹ , R ² and R ⁷ are hydrogen atoms, R ³ is a hydroxyl group or a substituent represented by formula (5), R ⁴ is a lower alkylaminoalkyl group, or formula (6) R ⁵ is a lower alkylaminovinyl group or a substituent represented by formula (7), R ⁶ is a nitro group, R ⁸ is a hydrogen atom, or a hydroxyl group, The compound or a salt thereof according to (3), wherein R ⁹ is a hydrogen atom, a lower alkoxy group, or a substituent represented by formula (8).
(In the formula, C represents an alkylene group having 1 to 10 atoms in the main chain. One or more ethylene constituting the alkylene group is replaced by a —C—O— group and / or a —NHCO— group. D may represent biotin, amino group, mercapto group, hydroxyl group, carboxyl group, or bromoacetamide group.)

(17) 1- (4-Methoxy-2-methyl-phenyl) -2-methyl-3-nitro-1H-indole-6-ol, 1- (4-methoxy-phenyl) -2,5-dimethyl-3 -Nitro-1H-indol-6-ol, 4- (6-hydroxy-2-methyl-3-nitro-indol-1-yl) -benzoic acid ethyl ester, 1- (3-chloro-4-methoxy-phenyl) ) -2-Methyl-3-nitro-1H-indole-6-ol, 5-chloro-1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol, 4- (6-hydroxy-2-methyl-3-nitro-indol-1-yl) -benzoic acid, 1- (4-methoxy-phenyl) -2-methyl-3-nitro-6-propoxy-1H-indole, [ 1- (4-Methoxy-phenyl) -2-methyl-3-nitro-1H-indol-6-yloxy] -acetic acid, (2-methyl-3-nitro-1-phenyl-1H-indol-6-yloxy) -Acetic acid, 7-[(2-amino-ethylamino) -methyl] -1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H Ndol-6-ol, N- (2- [2- [1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indol-6-yloxy] -acetylamino] -ethyl) -2 , 2-dimethyl-propionamide, N- (2-amino-ethyl) -2- [1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indol-6-yloxy] -acetamide, Or N- (2-[[6-hydroxy-1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indol-7-ylmethyl] -amino] -ethyl) -2,2-dimethyl -The compound or salt thereof according to (3), which is propionamide.

(18) 1- (4-Methoxy-phenyl) -2-methyl-3-nitro-7-propylaminomethyl-1H-indole-6-ol, 1- (4-hydroxy-phenyl) -2-methyl-3 -Nitro-1H-indole-6-ol, 1- (3-hydroxy-4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol, 1- (4-ethoxy-phenyl) 2-Methyl-3-nitro-1H-indole-6-ol, 1- (3-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol, 7-aminomethyl-1- (3-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol, 1- (3,4-dimethoxy-phenyl) -2-methyl-3-nitro-1H-indole-6- All, 2-methyl-3-nitro-1- (3,4,5-trimethoxy-phenyl) -1H-indole-6-ol, 5- (2-oxo-hexahydro-thieno [3,4-d] imidazole -4-yl) -pentenoic acid or (2- [2- [2- (2-[[6-hydroxy-1- (4-methoxy-phenyl) -2-methyl-3-ni The compound or a salt thereof according to (3), which is tro-1H-indol-7-ylmethyl] -amino] -ethoxy) -ethoxy] -ethoxy] -ethyl) -amide.

(19) A stem cell differentiation inhibitor comprising the compound or salt thereof according to any one of (1) to (18) as an active ingredient.
(20) A stem cell culture method comprising culturing stem cells in an undifferentiated state using the compound or salt thereof according to any one of (1) to (18).
(21) A method for culturing embryonic stem cells, comprising culturing embryonic stem cells in an undifferentiated state using the compound or salt thereof according to any one of (1) to (18).
(22) A medium for growing stem cells in an undifferentiated state containing the compound or salt thereof according to any one of (1) to (18) as an active ingredient.
(23) The medium according to (22), wherein the concentration of the stem cell differentiation inhibitor is 10 ng / ml to 100 μg / ml.

(24) A stem cell cultured in an undifferentiated state using the compound or salt thereof according to any one of (1) to (18).
(25) A cell or tissue obtained by differentiating a stem cell cultured in an undifferentiated state using the compound or salt thereof according to any one of (1) to (18).
(26) A treatment method for transplanting the cell or / and tissue according to (24) or (25) into a living body.
(27) A prodrug of the compound or salt thereof according to any one of (1) to (18).
(28) A pharmaceutical composition comprising the compound according to any one of (1) to (18) or a salt thereof or a prodrug thereof.

  The indole derivative of the present invention has an effect of proliferating stem cells in an undifferentiated state. Also provided are indole derivatives that can be immobilized specifically and regiospecifically on a carrier believed to be necessary for identification of the binding protein.

Hereinafter, the present invention will be described in detail.
The following terms are defined as provided below unless otherwise stated.
All other terms used herein are defined with respect to their terminology in the particular field relating to that term, unless stated otherwise.
Stem cell: A stem cell refers to another cell type with a specific function, ie, a cell that can differentiate into a terminally differentiated cell or other stem cell type that can differentiate into a narrower range of cell types. .
Totipotent stem cells: Totipotent stem cells are cells that can differentiate into any cell type, including pluripotent cells and fully differentiated cells (ie, cells that can no longer differentiate into various cells). Say that.

Pluripotent stem cell: A pluripotent stem cell refers to a cell that is not necessarily of all types, but can differentiate into one of a number of different cell types. One example of a pluripotent cell is a hematopoietic stem cell, which can differentiate into various blood cell types such as lymphocytes and erythrocytes.
Embryonic stem cells: Embryonic stem cells are totipotent cells obtained from the morula or blastocyst stage of a stem cell, especially an embryo at the pre-implantation stage, and are also called ES cells. In addition, embryonic stem cells may refer to pluripotent stem cells derived from primordial germ cells of embryos or fetuses (fetuses) that are determined to be sperm or eggs. However, this cell is sometimes called an embryonic germ (EG) cell and is sometimes distinguished from an embryonic stem cell. The embryonic stem cells used in the present specification may be of any animal species, and examples include embryonic stem cells of primates including humans, mammals other than primates, birds and the like.

Totipotency: Totipotency refers to a state that can differentiate into any cell type, including pluripotent cells and fully differentiated cells (ie, cells that can no longer differentiate into various cells).
Pluripotency: Pluripotency refers to a condition that can differentiate into one of a number of different cell types, although not necessarily all types.
Undifferentiated: Undifferentiated is a cell in which one cell or an arbitrary cell population composed of a plurality of cells is capable of differentiating into one or more further differentiated cells, Or it is the state which is a cell population containing this cell.
Feeder: A feeder used for the purpose of describing the present invention refers to one in which totipotent stem cells are plated thereon and provides an environment that aids in the growth of the plated totipotent stem cells.
Feeder cells: Feeder cells used for purposes of describing the present invention are non-totipotent stem cells on which totipotent stem cells are plated, and non-totipotent stem cells are a proliferation of plated totipotent stem cells. Providing an environment that helps.
Cell-derived components: All components derived from cells such as components secreted from cells, contents, and cell membrane components.

  The indole derivative of the present invention provides a differentiation inhibitor capable of proliferating and maintaining stem cells in an undifferentiated state, a culture method using the same, a culture medium using the same, and a cell line produced by culturing the same. To do.

The indole derivative of the present invention can be used as long as it is a chemically stable low molecular weight compound and has an activity of maintaining stem cells in an undifferentiated state. Preferably, the indole derivative is represented by the following general formula (1). The low molecular compound represented is mentioned.
In the formula, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ may be the same or different and each represents an electron-withdrawing group, an electron-donating group or a hydrogen atom. The electron donating group is a substituent capable of donating electrons to the benzene ring, and the electron withdrawing group is a substituent having a property of attracting π electrons on the benzene ring. Also, using Hammett's substituent constant σ, σ <0 can be defined as an electron donating group and σ> 0 as an electron withdrawing group (Basic Organic Reaction Theory, written by Shizunobu Hashimoto, Sankyo Publishing, 1997) ). X represents an alkylene group having 0 to 10 atoms in the main chain. The alkylene group having 0 atoms represents a single bond. One or more ethylene constituting the alkylene group may be replaced by a -C = C- group and / or -N = N- group and / or -NHCO- group. Furthermore, the alkylene group may have one or more electron-withdrawing groups, electron-donating groups, or hydrogen atoms as a substituent. G represents an electron-withdrawing group, an electron-donating group, an aromatic group that may have a hydrogen atom, or a cyclic molecule that includes at least one heteroatom that may have an electron-withdrawing group, an electron-donating group, or a hydrogen atom. To express. Further, the alkylene group represented by X preferably has 0 or 1 main chain atoms.

For example, examples of the general formula (1) include indole derivatives represented by the formula (2) or the formula (3).
However, A is a hydrogen atom, a methyl group, or
Represents. In the formula, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are as defined above. R ⁷ , R ⁸ , R ⁹ , R ¹⁰ and R ¹¹ may be the same or different and each represents an electron withdrawing group, an electron donating group or a hydrogen atom.
In the formula, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are as defined above. B represents an electron withdrawing group, an electron donating group or a cyclic molecule containing at least one hetero atom which may have a hydrogen atom.

Examples of the indole derivative represented by the general formula (2) include compounds represented by the following formula (4).
In the formula, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ and R ¹¹ are each independently an alkyl group, an unsaturated hydrocarbon group, Acyl group, aroyl group, aryl group, alkoxy group, alkoxyalkyl group, acyloxy group, alkoxycarbonyl group, hydroxyalkyl group, amino group, acetamido group, aminocarbonyl group, alkylaminocarbonyl group, alkylamino group, alkylaminoalkyl group , Alkylaminovinyl group, aminoalkyl group, hydroxyalkylamino group, hydroxyalkylaminoalkyl group, hydroxyalkylaminovinyl group, arylamino group, arylaminovinyl group, nitro group, hydroxyl group, alkylthio group, sulfonamido group, sulfone Group, carboxy group, mercapto group, oxalo group, hydroxyimino A group or an atom selected from the group consisting of an alkyl group, a thiocarbamoyl group, a pyrrolyl group, an oxazol group, a halogen atom and a hydrogen atom, and the alkyl contained therein includes an electron-withdrawing group, an electron-donating group as a substituent. Alternatively, one or more hydrogen atoms may be included.

In the formula, R ¹ , R ² and R ⁷ are hydrogen atoms, R ³ is a hydroxyl group or a substituent represented by the formula (5), and R ⁴ is a lower alkylaminoalkyl group or a formula ( 6), R ⁵ is a lower alkylaminovinyl group, or a substituent represented by formula (7), R ⁶ is a nitro group, and R ⁸ is a hydrogen atom or a hydroxyl group. R ⁹ is a hydrogen atom, a lower alkoxy group, a substituent represented by the formula (8), or the like.

  However, C represents an alkylene group having 1 to 10 atoms in the main chain. One or more ethylene constituting the alkylene group may be replaced by a —C—O— group and / or a —NHCO— group. D represents biotin, amino group, mercapto group, hydroxyl group, carboxyl group, or bromoacetamide group.

Moreover, the following are illustrated as a specific compound contained in the said General formula (4) which is an indole derivative.
1- (4-Methoxy-phenyl) -2-methyl-3-nitro-7-propylaminomethyl-1H-indole-6-ol, 1- (4-hydroxy-phenyl) -2-methyl-3-nitro- 1H-indole-6-ol, 1- (3-hydroxy-4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol, 1- (4-ethoxy-phenyl) -2- Methyl-3-nitro-1H-indole-6-ol, 1- (4-methoxy-phenyl) -2-methyl-7-methylaminomethyl-3-nitro-1H-indole-6-ol, 7-ethylamino Methyl-1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol, 5- (2-oxo-hexahydro-thieno [3,4-d] imidazol-4-yl ) -Pentanoic acid (2- [2- [2- (2-[[6-hydroxy-1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indol-7-ylmethyl] -amino ] -Ethoxy) -ethoxy] -ethoxy] -ethyl) -amide, 5- (2-oxo-hexahydro-thieno [3,4-d] i Dazol-4-yl) -pentanoic acid (2- [2- [2- (2- [2- [6-hydroxy-1- (4-methoxy-phenyl) -3-nitro-1H-indol-2-yl] ] -Vinylamino] -ethoxy) -ethoxy] -ethoxy] -ethyl) -amide, 5- (2-oxo-hexahydro-thieno [3,4-d] imidazol-4-yl) -pentanoic acid (2- [ 2- [2- (2-[[6-hydroxy-1- (3-hydroxy-4-methoxy-phenyl) -2-methyl-3-nitro-1H-indol-7-ylmethyl] -amino] -ethoxy) -Ethoxy] -ethoxy] -ethyl) -amide, 5- (2-oxo-hexahydro-thieno [3,4-d] imidazol-4-yl) -pentanoic acid (2- [2- [2- (2- [[6-hydroxy-1- (4-hydroxy-phenyl) -2-methyl-3-nitro-1H-indol-7-ylmethyl] -amino] -ethoxy) -ethoxy] -ethoxy] -ethyl) -amide, 5- (2-Oxo-hexahydro-thieno [3,4-d] imidazol-4-yl) -pentanoic acid (2- [2- [2- (2- [2- [6-hydroxy-1- (3 -Hydroxy-4- Toxi-phenyl) -3-nitro-1H-indol-2-yl] -vinylamino] -ethoxy) -ethoxy] -ethoxy] -ethyl) -amide, 5- (2-oxo-hexahydro-thieno [3,4 -d] imidazol-4-yl) -pentanoic acid (2- [2- [2- (2- [2- [6-hydroxy-1- (4-hydroxy-phenyl) -3-nitro-1H-indole- 2-yl] -vinylamino] -ethoxy) -ethoxy] -ethoxy] -ethyl) -amide, 5- (2-oxo-hexahydro-thieno [3,4-d] imidazol-4-yl) -pentanoic acid ( 2- [2- [2- (2-[[2- (2-Dimethylamino-vinyl) -6-hydroxy-1- (4-methoxy-phenyl) -3-nitro-1H-indol-7-ylmethyl] -Amino] -ethoxy) -ethoxy] -ethoxy] -ethyl) -amide, 5- (2-oxo-hexahydro-thieno [3,4-d] imidazol-4-yl) -pentanoic acid (2- [2- [2- (2-[[2- (2-Dimethylamino-vinyl) -6-hydroxy-3-nitro-1-p-tolyl-1H-indol-7-ylmethyl] -amino No] -ethoxy) -ethoxy] -ethoxy] -ethyl) -amide, 5- (2-oxo-hexahydro-thieno [3,4-d] imidazol-4-yl) -pentanoic acid (2- [2- [ 2- (2-[[2- (2-Dimethylamino-vinyl) -6-hydroxy-1- (3-hydroxy-4-methoxy-phenyl) -3-nitro-1H-indol-7-ylmethyl] -amino ] -Ethoxy) -ethoxy] -ethoxy] -ethyl) -amide, 5- (2-oxo-hexahydro-thieno [3,4-d] imidazol-4-yl) -pentanoic acid (2- [2- [2 -(2-[[2- (2-Dimethylamino-vinyl) -6-hydroxy-1- (4-hydroxy-phenyl) -3-nitro-1H-indol-7-ylmethyl] -amino] -ethoxy)- Ethoxy] -ethoxy] -ethyl) -amide, 5- (2-oxo-hexahydro-thieno [3,4-d] imidazol-4-yl) -pentanoic acid (2- [2- [2- (2- [ 2- [7-Dimethylaminomethyl-6-hydroxy-1- (4-methoxy-phenyl) -3-nitro-1H-indol-2-yl] -vinylamino] -eth C) -Ethoxy] -ethoxy] -ethyl) -amide, 5- (2-oxo-hexahydro-thieno [3,4-d] imidazol-4-yl) -pentanoic acid (2- [2- [2- ( 2- [2- [7-Dimethylaminomethyl-6-hydroxy-1- (3-hydroxy-4-methoxy-phenyl) -3-nitro-1H-indol-2-yl] -vinylamino] -ethoxy)- Ethoxy] -ethoxy] -ethyl) -amide, 5- (2-oxo-hexahydro-thieno [3,4-d] imidazol-4-yl) -pentanoic acid (2- [2- [2- (2- [ 2- [7-Dimethylaminomethyl-6-hydroxy-1- (4-hydroxy-phenyl) -3-nitro-1H-indol-2-yl] -vinylamino] -ethoxy) -ethoxy] -ethoxy] -ethyl ) -Amide, 5- (2-oxo-hexahydro-thieno [3,4-d] imidazol-4-yl) -pentanoic acid [2- [2- (2- [2- [2- (7-dimethylamino] Methyl-6-hydroxy-3-nitro-1-p-tolyl-1H-indol-2-yl) -vinylamino] -ethoxy] -ethoxy) -ethoxy] -d Til] -amide, 4- (6-hydroxy-2-methyl-3-nitro-indol-1-yl) -N- [2- [2- (2- [2- [5- (2-oxo-hexahydro -Thieno [3,4-d] imidazol-4-yl) -pentanoylamino] -ethoxy] -ethoxy) -ethoxy] -ethyl] -benzamide, 5- (2-oxo-hexahydro-thieno [3,4- d] imidazol-4-yl) -pentanoic acid (2- [2- [2- (2- [2- [1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6 -Yloxy] -acetylamino] -ethoxy) -ethoxy] -ethoxy] -ethyl) -amide, 5- (2-oxo-hexahydro-thieno [3,4-d] imidazol-4-yl) -pentanoic acid [2 -[2- (2- [2- [2- (2-Methyl-3-nitro-1-phenyl-1H-indol-6-yloxy) -acetylamino] -ethoxy] -ethoxy) -ethoxy] -ethyl] -Amide, 7-dimethylaminomethyl-1- (3-hydroxy-4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol, 1- ( 3-hydroxy-4-methoxy-phenyl) -7-hydroxymethyl-2-methyl-3-nitro-1H-indole-6-ol, 2- (2-dimethylamino-vinyl) -1- (3-hydroxy- 4-Methoxy-phenyl) -3-nitro-1H-indole-6-ol, 7-dimethylaminomethyl-2- (2-dimethylamino-vinyl) -1- (3-hydroxy-4-methoxy-phenyl)- 3-Nitro-1H-indole-6-ol, 2- (2-dimethylamino-vinyl) -1- (3-hydroxy-4-methoxy-phenyl) -7-hydroxymethyl-3-nitro-1H-indole- 6-ol, 7-aminomethyl-1- (4-hydroxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol, 7-hydroxymethyl-1- (4-hydroxy-phenyl)- 2-Methyl-3-nitro-1H-indole-6-ol, 2- (2-dimethylamino-vinyl) -1- (4-hydroxy-phenyl) -3-nitro-1H-indole-6-ol, 7 -Dimethylaminomethyl-2- (2-dimethylamino- Vinyl) -1- (4-hydroxy-phenyl) -3-nitro-1H-indole-6-ol, 2- (2-dimethylamino-vinyl) -7-hydroxymethyl-1- (4-hydroxy-phenyl) -3-Nitro-1H-indole-6-ol, 2- [2- (2-hydroxy-ethylamino) -vinyl] -1- (4-methoxy-phenyl) -3-nitro-1H-indole-6- All, 7-dimethylaminomethyl-2- [2- (2-hydroxy-ethylamino) -vinyl] -1- (4-methoxy-phenyl) -3-nitro-1H-indole-6-ol, 7- [ (2-Hydroxy-ethylamino) -methyl] -2- [2- (2-hydroxy-ethylamino) -vinyl] -1- (4-methoxy-phenyl) -3-nitro-1H-indole-6-ol 7-[(2-hydroxy-ethylamino) -methyl] -1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol, 2- (2-dimethylamino- Vinyl) -7-[(2-hydroxy-ethylamino) -methyl] -1- (4-methoxy-phenyl) -3-nitro-1H- Ndol-6-ol, 7-[(2-hydroxy-ethylamino) -methyl] -1- (3-hydroxy-4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol 2- (2-Dimethylamino-vinyl) -7-[(2-hydroxy-ethylamino) -methyl] -1- (3-hydroxy-4-methoxy-phenyl) -3-nitro-1H-indole-6 -Ol, 7-[(2-hydroxy-ethylamino) -methyl] -2- [2- (2-hydroxy-ethylamino) -vinyl] -1- (3-hydroxy-4-methoxy-phenyl) -3 -Nitro-1H-indole-6-ol, 7-dimethylaminomethyl-2- [2- (2-hydroxy-ethylamino) -vinyl] -1- (3-hydroxy-4-methoxy-phenyl) -3- Nitro-1H-indole-6-ol, 2- [2- (2-hydroxy-ethylamino) -vinyl] -1- (3-hydroxy-4-methoxy-phenyl) -3-nitro-1H-indole-6 -Ol, 7-[(2-hydroxy-ethylamino) -methyl] -1- (4-hydroxy-phenyl) -2- Methyl-3-nitro-1H-indole-6-ol, 2- (2-dimethylamino-vinyl) -7-[(2-hydroxy-ethylamino) -methyl] -1- (4-hydroxy-phenyl)- 3-Nitro-1H-indole-6-ol, 7-[(2-hydroxy-ethylamino) -methyl] -2- [2- (2-hydroxy-ethylamino) -vinyl] -1- (4-hydroxy -Phenyl) -3-nitro-1H-indole-6-ol, 7-dimethylaminomethyl-2- [2- (2-hydroxy-ethylamino) -vinyl] -1- (4-hydroxy-phenyl) -3 -Nitro-1H-indole-6-ol, 2- [2- (2-hydroxy-ethylamino) -vinyl] -1- (4-hydroxy-phenyl) -3-nitro-1H-indole-6-ol, 1- (3-Chloro-4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol, 1- (4-methoxy-phenyl) -2-methyl-3-nitro-6- Propoxy-1H-indole, [1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ylo Xyl] -acetic acid, (2-methyl-3-nitro-1-phenyl-1H-indol-6-yloxy) -acetic acid, acetic acid 1- (3-hydroxy-4-methoxy-phenyl) -2-methyl-3- Nitro-1H-indol-6-yl ester, acetic acid 7-dimethylaminomethyl-1- (3-hydroxy-4-methoxy-phenyl) -2-methyl-3-nitro-1H-indol-6-yl ester, acetic acid 1- (3-hydroxy-4-methoxy-phenyl) -7-hydroxymethyl-2-methyl-3-nitro-1H-indol-6-yl ester, 2-methoxy-5- (6-methoxy-2-methyl) -3-nitro-indol-1-yl) -phenol, 5- (7-dimethylaminomethyl-6-methoxy-2-methyl-3-nitro-indol-1-yl) -2-methoxy-phenol, 5- (7-hydroxymethyl-6-methoxy-2-methyl-3-nitro-indol-1-yl) -2-methoxy-phenol, acetic acid 2- (2-dimethylamino-vinyl) -1- (3-hydroxy- 4-methoxy-phenyl) -3-ni B-1H-Indol-6-yl ester, acetic acid 7-dimethylaminomethyl-2- (2-dimethylamino-vinyl) -1- (3-hydroxy-4-methoxy-phenyl) -3-nitro-1H-indole -6-yl ester, acetic acid 2- (2-dimethylamino-vinyl) -1- (3-hydroxy-4-methoxy-phenyl) -7-hydroxymethyl-3-nitro-1H-indol-6-yl ester, 5- [2- (2-Dimethylamino-vinyl) -6-methoxy-3-nitro-indol-1-yl] -2-methoxy-phenol, 5- [7-dimethylaminomethyl-2- (2-dimethyl) Amino-vinyl) -6-methoxy-3-nitro-indol-1-yl] -2-methoxy-phenol, 5- [2- (2-dimethylamino-vinyl) -7-hydroxymethyl-6-methoxy-3 -Nitro-indol-1-yl] -2-methoxy-phenol, acetic acid 1- (4-hydroxy-phenyl) -2-methyl-3-nitro-1H-indol-6-yl ester, acetic acid 7-dimethylaminomethyl -1 -(4-Hydroxy-phenyl) -2-methyl-3-nitro-1H-indol-6-yl ester, acetic acid 7-hydroxymethyl-1- (4-hydroxy-phenyl) -2-methyl-3-nitro- 1H-Indol-6-yl ester, 4- (6-methoxy-2-methyl-3-nitro-indol-1-yl) -phenol, 4- (7-dimethylaminomethyl-6-methoxy-2-methyl- 3-Nitro-indol-1-yl) -phenol, 4- (7-hydroxymethyl-6-methoxy-2-methyl-3-nitro-indol-1-yl) -phenol, acetic acid 2- (2-dimethylamino) -Vinyl) -1- (4-hydroxy-phenyl) -3-nitro-1H-indol-6-yl ester, acetic acid 7-dimethylaminomethyl-2- (2-dimethylamino-vinyl) -1- (4- Hydroxy-phenyl) -3-nitro-1H-indol-6-yl ester, acetic acid 2- (2-dimethylamino-vinyl) -7-hydroxymethyl-1- (4-hydroxy-phenyl) -3-nitro-1H -Indole-6- Ester, 4- [2- (2-dimethylamino-vinyl) -6-methoxy-3-nitro-indol-1-yl] -phenol, 4- [7-dimethylaminomethyl-2- (2-dimethylamino) -Vinyl) -6-methoxy-3-nitro-indol-1-yl] -phenol, 4- [2- (2-dimethylamino-vinyl) -7-hydroxymethyl-6-methoxy-3-nitro-indole- 1-yl] -phenol, 4- (6-hydroxy-2-methyl-3-nitro-indol-1-yl) -benzoic acid ethyl ester, 4- (6-hydroxy-2-methyl-3-nitro-indole -1-yl) -benzoic acid, 1- (4-methoxy-phenyl) -2,5-dimethyl-3-nitro-1H-indole-6-ol, 5-chloro-1- (4-methoxy-phenyl) 2-Methyl-3-nitro-1H-indole-6-ol, 1- (4-methoxy-phenyl) -2,5-dimethyl-3-nitro-1H-indole-6

-All.

The compound represented by the general formula (3) which is an indole derivative includes the following. R ¹ , R ² , R ³ , R ⁴ , R ⁵ , and R ⁶ are each independently an alkyl group, unsaturated hydrocarbon group, acyl group, aroyl group, aryl group, alkoxy group, alkoxyalkyl group, acyloxy Group, alkoxycarbonyl group, hydroxyalkyl group, amino group, acetamide group, aminocarbonyl group, alkylaminocarbonyl group, alkylamino group, alkylaminoalkyl group, alkylaminovinyl group, aminoalkyl group, hydroxyalkylamino group, hydroxyalkyl Aminoalkyl group, hydroxyalkylaminovinyl group, arylamino group, arylaminovinyl group, nitro group, hydroxyl group, alkylthio group, sulfonamido group, sulfo group, carboxy group, mercapto group, oxalo group, hydroxyiminoalkyl group, thio Carbamoyl , A pyrrolyl group, an oxazol group, a group or an atom selected from the group consisting of a halogen atom and a hydrogen atom, and B may have an electron-withdrawing group, an electron-donating group or a hydrogen atom, benzimidazole, imidazole, pyrrolidine , Piperidine, pyridine, pyrimidine, dihydrothiophen-2-one, tetrahydrothiophene-1,1-dioxide, isoxazole, benzoxazole, isothiazole, indole, quinoline, benzofuran, quinoxaline, chroman-4-one, 2,3 -A compound or salt thereof which is dihydrobenzo [1,4] dioxacin or benzothiazole.

  Examples of the lower alkyl group referred to in the present invention include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, 1-methylpropyl, n -Hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 3,3-dimethylpropyl, 2-ethylbutyl and the like. Preferably, it is methyl or butyl.

  Examples of the lower alkoxy group in the present invention include methoxy, ethoxy, propoxy, isopropoxy, butoxy, sec-butoxy, tert-butoxy, pentoxy, hexyloxy, heptyloxy, octyloxy and the like. Preferably it is methoxy.

  Examples of the halogen atom referred to in the present invention include fluorine, chlorine, bromine, iodine and the like, preferably chlorine or bromine.

  Examples of the lower acyl group referred to in the present invention include formyl, acetyl, propionyl, butyryl and the like, preferably acetyl.

  Examples of the lower alkyl group that may form a cyclic structure in the present invention include cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like, and cyclopentyl, cyclohexyl, and cycloheptyl are preferable.

  Examples of the lower alkoxycarbonyl referred to in the present invention include methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl and the like, preferably methoxycarbonyl or ethoxycarbonyl.

Examples of the aminocarbonyl referred to in the present invention include -CONR ₂ (R represents a hydrogen atom which may be the same or different, a lower alkyl group exemplified above, and a phenyl group which may have a substituent). It is done.

Examples of the secondary aminocarbonyl group referred to in the present invention include -CONHR (which represents the lower alkyl group exemplified above and a phenyl group which may have a substituent). Examples of the tertiary aminocarbonyl group include -CONR ₂ (R represents the lower alkyl group exemplified above which may be the same or different, or a phenyl group which may have a substituent).

  As a salt of the compound exemplified in the present invention, a pharmaceutically acceptable salt is desirable. For example, hydrochloride, sulfate, phosphate, hydrobromide, acetate, maleate, fumarate, succinate, methanesulfonate, p-toluenesulfonate, citrate, tartaric acid A salt or the like may be formed.

  As the compound names described in the present invention, names converted using IUPAC nomenclature or commercially available software (for example, ChemDraw Ultra, CambridgeSoft, USA) are used.

The compounds of the present invention can be produced by carrying out the following methods and methods analogous thereto, or known methods (for example, Khim. Geterotsikl. Soedin., No. 1, p. 40 (1992)). A method for producing the compound represented by the formula (1) is shown below.

Compound (II) can be produced by refluxing equal amounts of Compound (I) and nitromethane in ethanol.

  A suitable solvent that does not adversely affect the reaction between compound (II) and an equal amount or more of any aniline derivative, and an equal amount of p-toluenesulfonic acid (for example, amides such as dimethylformamide and dimethylacetamide, ethers such as tetrahydrofuran) ) And stirred at room temperature.

  Compound (1) can be produced by dissolving an equal amount or more of compound (III) in any amount of benzoquinone derivative in acetic acid and acetic anhydride and stirring.

A method for producing the compound represented by the formula (2) is shown below.

  A compound in which R4 is hydrogen in the formula (1) can be produced by mixing equal amounts or more of N, N, N ′, N′-tetraalkyldiaminomethane in dioxane and refluxing.

A method for producing the compound represented by the formula (3) is shown below.

  A compound represented by the formula (2) is mixed in an alcohol (for example, methanol, ethanol, isopropyl alcohol) with an equal amount or more of a compound having a primary or secondary amino group having an optional substitution machine, and refluxed. Can be manufactured.

The compound represented by the formula (1) can be derivatized by the following scheme.

  A compound represented by the formula (1) and an equal amount or more of any aldehyde derivative in an appropriate solvent that does not adversely influence the reaction (for example, amides such as dimethylformamide and dimethylacetamide, ethers such as tetrahydrofuran, benzene, Compound (4) can be obtained by stirring with aromatic hydrocarbons such as toluene.

The compound represented by the formula (1) can be derivatized by the following scheme.

  A compound represented by the formula (1) and an equal amount or more of dimethylformamide diethyl acetal in an appropriate solvent that does not adversely influence the reaction (for example, amides such as dimethylformamide and dimethylacetamide, ethers such as tetrahydrofuran, benzene, Compound (5) can be obtained by stirring with aromatic hydrocarbons such as toluene. Furthermore, in an alcohol (for example, methanol, ethanol, isopropyl alcohol), an equal amount or more of a compound having a primary or secondary amino group having an optional substitution machine is mixed and refluxed to obtain compound (6). be able to.

The compound represented by the formula (1) can be derivatized by the following scheme.

  In an appropriate solvent that does not adversely affect the reaction between the compound represented by the formula (1) and an equal amount or more of di-N-bromosuccinimide (for example, amides such as dimethylformamide and dimethylacetamide, ethers such as tetrahydrofuran, benzene Compound (7) can be obtained by stirring with aromatic hydrocarbons such as toluene, halogen solvents such as carbon tetrachloride and chloroform. Furthermore, in an appropriate solvent that does not adversely influence the reaction (for example, amides such as dimethylformamide and dimethylacetamide, ethers such as tetrahydrofuran, aromatic hydrocarbons such as benzene and toluene, halogen solvents such as carbon tetrachloride and chloroform) The compound (8) can be obtained by mixing an equal amount or more of any nucleophilic reagent (for example, primary and secondary amine derivatives, mercapto derivatives, alcohol derivatives) with stirring.

The compound represented by the formula (9) can be derivatized by the following scheme.

  Compound (9) in an appropriate solvent that does not adversely influence the reaction (for example, amides such as dimethylformamide and dimethylacetamide, ethers such as tetrahydrofuran, aromatic hydrocarbons such as benzene and toluene, carbon tetrachloride, chloroform Compound (10) can be obtained by mixing with a halogenated derivative in the presence of a base such as potassium carbonate) or potassium carbonate.

  The thus obtained compound of the present invention or a salt thereof can be isolated and purified by usual separation means such as recrystallization, distillation, chromatography and the like. Thus, when the compound of the present invention is obtained in a free form, it can be converted into a salt by a known method or a method analogous thereto, and conversely when obtained in a salt. It can be converted into a free form or other salt by a known method or a method analogous thereto. The compound or a salt thereof may have an asymmetric carbon, but when it is obtained as an optically active mixture (racemate), it can be separated into each optical activity by ordinary optical resolution means.

  The compound of the present invention has an activity capable of proliferating stem cells, preferably embryonic stem cells, in an undifferentiated state. The compound provided by the present invention proliferates stem cells, preferably embryonic stem cells, more conveniently and safely than before. Cell culture methods comprising the compounds of the invention can also be used to screen for specific differentiation inducers and useful combinations of differentiation inducers. The ability to grow undifferentiated embryonic stem cells using the compounds of the present invention and culture methods has the ability to produce embryonic stem cell lines with single or multiple genetic modifications with important therapeutic applications. Provides important benefits including.

  Cells cultured using the compounds of the present invention include all stem cells and embryonic stem cells that can be obtained using known methods and materials. Examples of stem cells include stem cells that can be obtained by the following known methods. Bone marrow cells ("Bone marrow transplantation guide" HJ Dieg, HG Klingeman, GL Phillips, translated by Shinhei Kasakura), Bone marrow stem cells (Osawa et al., Science, 273, p242-245, 1996, Goodell et al., JEMed.183, p1797 1806, 1996, Verfaillie et al., Nature, 418, p41, 2002), neural stem cells (Reynolds et al., Science, p1707-1710, 1992), tissue stem cells (Goodell et al., JEMed. 183, p1797-1806, 1996) Matsuzaki et al., Experimental Medicine, 19, p345-349, 2001, Blau et al., Cell, 105, p829-841, 2001), Mesenchymal stem cells (Liechty et al., NatureMedicine, 6, p1282-1286, 2000) Pittenger et al., Science, 284, p143-147, 1999). Skin stem cells / epidermal stem cells (Murota Seiji, “Regenerative Medicine / Regenerative Therapy”, Modern Chemistry Extra Number 41, Tokyo Chemical Doujin).

  Further, embryonic stem cells to be cultured can be obtained using known methods and materials shown below. Mouse embryonic stem cells: Evans et al., Nature, 292, p154, 1981, Bovine ES cells: Schellander et al., Theriogenology, 31, p15-17, 1989, Porcine ES cells: Strojek et al., Theriogenology, 33: p901, 1990 , Sheep ES cells: Handyside, Roux's Arch. Dev. Biol., 196: p185, 1987, hamster ES cells: Doetschman et al., Dev. Biol., 127, p224, 1988, monkey ES cells: Thomson et al., Proc. Natl. Acad. Sci. USA, 92, p7844, 1995, Cynomolgus monkey ES cells: Suemori et al., Dev. Dyn. 222, P273, 2001, human ES cells: Thomson et al., Science, 282, p1145, 1998, Reubinoff Nature Biotech, 18, p399, 2000, human EG cells: Gearhart et al., Proc. Natl. Acad. Sci. USA, 95, p13726, 1998. Mouse embryonic stem cells (129SV and C57 / BL6) can be purchased from Dainippon Pharmaceutical.

  The Nanog gene has been identified as one of the molecules that control undifferentiation of embryonic stem cells (Mitsui et al., Cell, 113, p631, 2003, Chambers et al., Cell, 113, p643, 2003). The presence of the Nanog gene has been confirmed in mice and humans, and if the expression is suppressed by gene disruption, the totipotency of the embryonic stem cell is lost. Conversely, if the Nanog gene is strongly expressed to increase the expression level, Undifferentiated sex stem cells can be maintained.

  Therefore, a substance capable of increasing the expression level of the Nanog gene can be used for maintaining undifferentiated embryonic stem cells, that is, for culturing totipotent embryonic stem cells.

  In addition, strong expression of Nanog gene does not induce STAT3 activation, and activation of STAT3 does not induce Nanog gene expression. Therefore, Nanog gene is undifferentiated in embryonic stem cells independent of LIF and STAT3. To maintain. That is, a substance capable of increasing the expression level of Nanog gene can be used for maintenance culture even in embryonic stem cells in which the effect of maintaining undifferentiation by LIF is not observed, such as monkey ES cells and human ES cells. It is suggested.

  The compounds of the present invention include low molecular weight compounds that increase the expression level of the Nanog gene. That is, the compound of the present invention includes a low molecular weight compound that exhibits the ability to maintain undifferentiation of embryonic stem cells through an increase in the expression level of Nanog gene.

  According to a recent report, in bone marrow-derived mesenchymal stem cells, which are a type of pluripotent stem cell, the culture of mouse-derived bone marrow mesenchymal cells is LIF-dependent, whereas human-derived cells are LIF-independent It has been reported that a phenomenon similar to that of embryonic stem cells has been observed (Verfaillie et al., Nature, 418, p41, 2002). This suggests that stem cells exhibiting pluripotency may have the same undifferentiation maintenance mechanism as embryonic stem cells. Therefore, in primate stem cells, as in embryonic stem cells, it is possible that undifferentiation is maintained by a signal different from the LIF-STAT3 pathway. The compound of the present invention includes a low molecular compound having an activity of maintaining an undifferentiated state of a cell without using STAT3.

  The compounds provided by the present invention can be used for all stem cells and embryonic stem cells, but are desirably used for mammalian stem cells and embryonic stem cells, and are preferably used for primate stem cells and embryonic stem cells.

  The degree of undifferentiation of embryonic stem cells cultured using the compound of the present invention can be confirmed by measuring the alkaline phosphatase (ALP) activity present on the cell membrane of the stem cells. It is known that ALP activity is maintained in undifferentiated embryonic stem cells and decreases when differentiated (Williams et al., Nature, 336, p684, 1988. Thomson et al., Science, 282, p1145, 1998). ). Examples thereof include a method of detecting alkaline phosphatase (ALP) activity by a staining method using an insoluble substrate or a spectroscopic measurement method using a water-soluble substrate.

  In one embodiment, ALP activity can be quantified by spectroscopic methods. Add an alkaline solution of paranitrophenyl phosphate (pNPP) to the cells on the culture dish. PNPP is hydrolyzed by ALP present on the cell membrane to produce paranitrophenol. By measuring the absorbance of the resulting solution at 405 nm, alkaline phosphatase activity can be quantified. When the ALP activity of embryonic stem cells cultured with the compound of the present invention added by the method described in Examples was measured, the ALP activity was significantly higher than that of control embryonic stem cells cultured in a medium not containing the compound. Is shown to have. That is, it is confirmed that the compound of the present invention can be proliferated while maintaining the undifferentiated state of embryonic stem cells.

  In another embodiment, ALP activity can also be detected by ALP staining. A reaction solution containing a phosphate ester salt and a diazonium salt as a substrate is added to the cells on the culture dish. Phosphate ester salt is hydrolyzed by alkaline phosphatase present in the form of a cell membrane, and then coupled with a diazonium salt to produce an azo dye, which precipitates at the ALP active site. By measuring the number of stained colonies, the ALP activity of the cells can be quantified, that is, the degree of undifferentiation of the cells can be quantified. When the embryonic stem cells cultured with the differentiation inhibitor of the present invention were ALP stained by the method described in the Examples, it was shown that the ALP activity was significantly higher than that of control cells without the differentiation inhibitor, ie It is confirmed that the differentiation inhibitor of the invention can be grown while maintaining the undifferentiated state of embryonic stem cells.

  In addition, the degree of embryonic stem cell undifferentiation can be confirmed by measuring the expression level of the Oct-3 / 4 gene. The Oct-3 / 4 gene is a transcription factor belonging to the POU family and is specifically expressed in embryonic stem cells and embryonic cancer cells (EC cells) in an undifferentiated state (Okamoto et al., Cell, 60, p461, 1990) In embryogenesis, it is expressed only in the undifferentiated cell lineage (Scholer, Trends Genet, 7, p323, 1991). In addition, homozygous mice with Oct-3 / 4 gene disruption stop development at the blastocyst stage Therefore, it has been clarified that it has an important function for maintaining an undifferentiated state (Nichols et al., Cell, 95, p379, 1998). On the other hand, it has recently been clarified that overexpression of the Oct-3 / 4 gene promotes differentiation of embryonic stem cells (Niwa et al., Nat. Genet. 24, p372, 2000). Maintaining the expression level within a certain range is important for maintaining an undifferentiated state. As one embodiment for measuring the expression level of the Oct-3 / 4 gene, a quantitative PCR (polymerase chain reaction) method can be used.

  In one embodiment, a real-time PCR method is used, which has a wide dynamic range and enables simple and reliable quantitative measurement. Real-time PCR technology includes a method using a Taq Man probe using ABIPRISM7700TM (Applied Biosystems) and a method using LightCyclerTM (Roche Diagnostics). In particular, in the latter case, changes in the amplification amount of DNA synthesized in each cycle can be detected in real time under a high-speed reaction cycle in which the PCR temperature cycle is completed in several tens of minutes. As a DNA detection method of the real-time PCR method, there are four types of methods using a DNA binding dye (intercalator), a hybridization probe (kissing probe), a TaqMan probe, and a Sunrise uniprimer (molecular beacon). In addition, the expression level of the Oct-3 / 4 gene can be analyzed using a DNA binding dye such as SYBR GreenI. SYBR GreenI is a double-strand-specific binding dye for DNA, and its original fluorescence intensity is enhanced by binding to the double-strand. By adding SYBR GreenI during the PCR reaction and measuring the fluorescence intensity at the end of each cycle of the extension reaction, an increase in the PCR product can be detected. In order to detect the Oct-3 / 4 gene, primers are designed using commercially available gene analysis software based on the Oct-3 / 4 gene sequence in the same manner as in normal PCR. Since SYBR GreenI also detects non-specific products, it is necessary to set optimal primers. As design criteria, attention should be paid to the length of the oligomer, the base composition of the sequence, the GC content, and the Tm value.

  In many cases it is the amount of DNA of interest per fixed sample that is intended to be revealed in quantitative PCR. For this purpose, it is necessary to first evaluate the amount of sample added to the reaction system. In this case, another DNA serving as an internal standard reflecting the sample amount can be measured separately from the target DNA, and the sample amount initially added to the reaction system can be corrected. As an internal standard used for the purpose of correcting the sample amount, a housekeeping gene that is considered to have no difference in expression level depending on tissues can be used. Examples include genes such as glyceroaldehyde phosphate dehydrogenase (GAPDH), which is a major glycolytic enzyme, β-actin or γ-actin, which is a constituent component of the cytoskeleton, and S26, which is a constituent protein of ribosome.

  The expression level of the Oct-3 / 4 gene can be determined for cells exposed to the compounds of the present invention. Compared to the expression level of the Oct-3 / 4 gene in control cells that have not been exposed to the compound, that is, induced to differentiate from embryonic stem cells, the activity can significantly maintain the expression level of the Oct-3 / 4 gene. What is possessed is considered to be a compound that maintains the undifferentiation of embryonic stem cells.

  As yet another method for screening for an undifferentiated maintenance compound of an optimized embryonic stem cell, stage-specific embryo antigen (Stage Specific Embryonic Antigen) (hereinafter referred to as SSEA) -1, SSEA-3, SSEA-4, etc. Examples include methods for detecting antigens that are specifically expressed in undifferentiated cells (Smith et al., Nature, 336, p688, 1988, Solter et al., Proc. Natl. Acad. Sci. USA, 75, p5565, 1978, Kannagi et al., EMBO J.2, p2355, 1983).

  In one embodiment, a surface antigen such as SSEA-1 is incubated with a specific antibody that recognizes the same antigen (primary antibody), and further a second antibody (secondary antibody) bound to a reporter such as a fluorescent label. Can be labeled by incubating with. By this operation, cells expressing the target antigen become fluorescent. The labeled cells can then be counted and even sorted using standard methods, such as a flow cytometer. The number of labeled and unlabeled cells can then be compared to determine the effect of the intended culture substrate. Alternatively, after exposure to an unlabeled cell surface marker antibody, in an ELISA (enzyme linked immunosorbent assay) format, the cell is a second antibody specific for an anti-cell surface antigen antibody (eg, an anti-SSEA-1 antibody). From which the number of cells expressing the desired surface antigen can be quantified colorimetrically or by measuring fluorescence. Still other methods for quantifying cells expressing surface antigens are well known to those skilled in the art of cell culture.

  The compound of the present invention can be used by adding to any mammalian cell culture basal medium which is a basal medium for animal cell culture. Examples of animal cell basal medium include, but are not limited to, Dulbecco's modified Eagle medium: DMEM, knockout DMEM, Glasgow MEM: GMEM, RPMI1640, IMDM (GIBCO, USA). In one embodiment, the cell culture medium is Dulbecco's Modified Eagle Medium (DMEM). Furthermore, serum or various growth factors as serum substitutes can be added to these basal media. The serum can be any serum or serum-based solution that provides nutrients that are effective in maintaining the growth and viability of stem cells and embryonic stem cells. Examples of such sera include fetal calf serum (FCS), bovine serum (CS), horse serum (HS), etc., and serum substitutes well known to those skilled in the art, proteins, amino acids, lipids Vitamins can be used alone or in combination. Proteins include insulin, transferrin, albumin, peptone, FGF (Fibroblast Growth Factor), EGF (Epitherial Growth Factor), and amino acids such as arginine, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine Examples of vitamins include, but are not limited to, pantothenic acid, choline, folic acid, inositol, nicotinic acid amide, riboflavin, thiamine, and pyridoxine. In one embodiment, the serum is fetal bovine serum. In a more particular embodiment, fetal calf serum is provided at a concentration between about 25% and about 1%. In an even more specific embodiment, the fetal calf serum concentration in the cell culture medium is 15%. In another embodiment, the serum replacement is knockout serum replacement: KSR (GIBCOBRL, USA).

  The cell culture medium also contains an antioxidant (reducing agent) (for example, β-mercaptoethanol). In certain preferred embodiments, β-mercaptoethanol has a concentration of about 0.1 mM. Other antioxidants such as monothioglycerol or dithiothreitol (DTT) alone or in combination can be used for similar effects. Still other equivalent materials are well known to those skilled in the art of cell culture.

  The concentration of the compound of the present invention when used by adding to any cell culture basic medium is desirably 0.1 ng / ml to 1 mg / ml, preferably 10 ng / ml to 100 μg / ml. More preferably, it is used in the range of 100 ng / ml to 10 μg / ml.

  The compound of the present invention and its active ingredient can be used together with an arbitrary culture substrate or fixed to the culture substrate. A porous material can be used as the culture substrate. The porous body refers to a substrate having a large number of fine pores, and the material, thickness, shape, dimensions and the like are not particularly limited. The material of the porous body may be an organic material, an inorganic material, or a composite material composed of an organic material and an inorganic material. The shape of the porous body may be any of a flat plate shape, a spherical shape, a rod shape, a fiber shape, and a hollow shape, for example, a film, a sheet, a membrane, a plate, a nonwoven fabric, a filter paper, a sponge, a woven fabric, a knitted fabric, a lump, Examples thereof include yarn, hollow fiber, and particle. In culturing cells, a non-woven fabric is more preferable in consideration of the ability to easily control the size of the holes that support the cells so that they can be cultivated three-dimensionally, the ease of manufacturing the substrate, and the cost. The size of the pores of the porous body is not particularly limited, but considering that the cells can be supported three-dimensionally, the average pore diameter is preferably 0.1 μm or more and 100 μm or less, preferably 1 μm or more. More preferably, it is 50 μm or less. The fiber diameter is not particularly limited, but is preferably 0.03 denier or less.

  The porous body may be subjected to a surface coating treatment with a polymer compound in order to improve cell adhesion, differentiation maintenance function, and proliferation ability. The polymer substance means a substance having a molecular weight of several hundreds or more in which monomers of one or more kinds of repeating structural units are linked one-dimensionally, two-dimensionally and three-dimensionally. Polymer materials can be broadly classified into three categories: natural polymer materials, semi-synthetic polymer materials, and synthetic polymer materials, and any polymer material can be used in the present invention.

  For example, natural polymer substances include mica (mica), asbestos (asbestos), graphite (graphite), saccharides such as diamond, starch, cellulose, and alginic acid, and proteins such as gelatin, fibronectin, and fibrinogen. Is mentioned. Examples of the semi-synthetic polymer material include glass, cellulose nitrate, cellulose acetate, hydrochloric acid rubber, carboxymethyl cellulose and the like. Synthetic polymer materials include polyphosphonitrile chloride, polyethylene, polyvinyl chloride, polyamide, polyethylene terephthalate, polysulfone, polyacrylonitrile, polyvinyl alcohol, polymethyl methacrylate, polyhydroxyethyl methacrylate, polydimethylaminoethyl methacrylate and hydroxyethyl methacrylate. Examples thereof include a copolymer composed of two or more kinds of synthetic monomers represented by a copolymer of dimethylaminoethyl methacrylate.

  Considering the ease of coating treatment, organic polymer materials are preferable, and proteins and peptides, and organic synthetic polymer materials are more preferable.

  By using a culture substrate on which the compound of the present invention is immobilized as a cell-capturing material, a method and an apparatus capable of separating and culturing stem cells or embryonic stem cells from a population consisting of a plurality of different cells can be provided. That is, a cell-containing solution containing stem cells or embryonic stem cells and cells to be removed is introduced into a container filled with a cell capturing material made of a culture substrate such as a porous body, to which the compound of the present invention is fixed. A stem cell or embryonic stem cell culturing method characterized in that stem cells or embryonic stem cells are captured by a capturing material, and the cells to be removed are led out of the container and then cultured together with the container, and the compound of the present invention is immobilized A cell culture apparatus in which a container is filled with a cell trapping material comprising a culture substrate, wherein the cell trapping material can be used as a carrier for cell culture, and the container can be used for cell culture. Is a cell culture device characterized by the above. The cells to be removed refer to all cells other than stem cells or embryonic stem cells. This also includes cells that have differentiated from stem cells or embryonic stem cells and have lost pluripotency. The cell-containing solution to be introduced into the cell trapping material may be any cell solution containing stem cells or embryonic stem cells. For example, blood, bone marrow, debris tissue fluid, or stem cell or embryonic stem cell culture solution Etc.

  The compound provided by the present invention is applied as an improved differentiation inhibitor against the proliferation of stem cells or embryonic stem cells, and the method and medium for culturing stem cells and / or embryonic stem cells using the compounds are stem cells Or it is expected to apply to all techniques where embryonic stem cells are useful.

  Cells produced using the compound, culture method and culture solution of the present invention can be used as they are or differentiated for cell transplantation, or used for artificial tissue construction in combination with the use of artificial support tissue. It can be used as an artificial organ. The use of stem cells for cell transplantation treatment and tissue engineering can solve the problems of conventional transplantation treatments including autologous transplantation, such as tissue loss after donor graft removal and donor shortage. Cells and tissues cultured for transplantation may be returned to the same person as the collected person or transplanted to another person for treatment, but the cells of the present invention can be used for both. Furthermore, the compound of the present invention, the culture method using the same, and the cells obtained by the culture medium containing the same are used for screening for substances that improve the monitoring or stem cell collection of stem cells, preferably embryonic stem cells. For example, a putative stem cell or embryonic stem cell differentiation inducer can be added to a cell culture grown using the method described above. A substance capable of inducing differentiation into three germ layers is identified as an embryonic stem cell differentiation inducing factor as compared to a control cell culture lacking a putative stem cell or embryonic stem cell differentiation inducing substance.

  Since the compound of the present invention or a salt thereof has excellent stem cell undifferentiation maintenance and proliferation ability, it can be used as a therapeutic agent for tissues or organs damaged by diseases or trauma. Examples of the target diseases include burns, refractory skin ulcers, acne, hypertrophic scars, nevus, and tattoos for skin-related, fractures and osteoporosis for bone-related, and osteoarthritis for cartilage-related. , Chronic rheumatism, intervertebral disc herniation, osteophysis, sports disorders, etc. Injuries related to periodontal disease and alveolar pyorrhea, alveolar bone injuries due to periodontal diseases, alveolar pyorrhea, male pattern baldness in the hair, congenital abnormalities in the cornea, endothelial cell decompensation, corneal infection Opacity, corneal degeneration, abnormal corneal shape, etc., vascular related hypertension, chronic arterial occlusion, ischemic heart disease, myocardial infarction in myocardium, diabetes in pancreas, liver in liver , Cirrhosis, but like liver failure include, but are not limited to.

  The compound of the present invention can be administered orally or parenterally as a prophylactic and / or therapeutic agent for the above-mentioned diseases, and is usually mixed with a pharmaceutically acceptable carrier. It is orally administered as a solid preparation such as a granule or a powder, or parenterally administered as an injection, a suppository, a sublingual tablet or the like intravenously, subcutaneously or intramuscularly. Alternatively, it may be administered sublingually, subcutaneously or intramuscularly as a sustained release preparation such as a sublingual tablet or a microcapsule. The daily dose varies depending on the degree of symptoms; age, sex, weight, sensitivity difference of administration subject; timing of administration, interval, nature of pharmaceutical preparation, preparation, type; type of active ingredient, etc., but is not particularly limited , Usually used for the treatment of sex hormone-dependent cancer (eg, prostate cancer, uterine cancer, breast cancer, pituitary tumor), prostatic hypertrophy, uterine fibroids, endometriosis, precocious puberty About 0.01 to 100 mg, preferably about 0.02 to 20 mg, more preferably 0.1 to 10 mg, and most preferably 0.5 to 10 mg per kg body weight of a mammal, usually divided into 1 to 4 times a day. To administer. The dosage for use in the field of livestock or fisheries is also the same as above, but about 0.01 to 30 mg, preferably about 0.1 to 10 mg per kg body weight of the subject organism is usually divided into 1 to 3 times a day. Administer. The content of the compound in the pharmaceutical composition of the present invention is about 0.01 to 100% by weight of the whole composition. As the pharmaceutically acceptable carrier, various organic or inorganic carrier substances commonly used as pharmaceutical materials are used. Excipients, lubricants, binders, disintegrants in solid preparations; solvents in liquid preparations, dissolution aids It is formulated as an agent, suspending agent, isotonic agent, buffering agent, soothing agent and the like. Further, if necessary, preparation additives such as preservatives, antioxidants, colorants, sweeteners and the like can be used. Preferable examples of the excipient include lactose, sucrose, D-mannitol, starch, crystalline cellulose, light anhydrous silicic acid and the like. Preferable examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like. Preferable examples of the binder include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone and the like. Preferable examples of the disintegrant include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, carboxymethyl starch sodium and the like. Preferable examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil and the like. Preferable examples of the solubilizer include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like. Suitable examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; And hydrophilic polymers such as polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose. Preferable examples of the isotonic agent include sodium chloride, glycerin, D-mannitol and the like. Preferable examples of the buffer include buffers such as phosphate, acetate, carbonate and citrate. Preferable examples of the soothing agent include benzyl alcohol. Preferable examples of the preservative include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like. Preferable examples of the antioxidant include sulfite and ascorbic acid.

  Suspending agents, solubilizers, stabilizers, tonicity agents, preservatives and the like can be added to the compounds of the present invention to give intravenous, subcutaneous and intramuscular injections by methods known per se. At that time, if necessary, a freeze-dried product can be obtained by a method known per se. When the compound of the present invention is administered to, for example, humans, it can be safely administered orally or parenterally as a pharmaceutical composition by itself or mixed with an appropriate pharmacologically acceptable carrier, excipient or diluent. can do. Examples of the pharmaceutical composition include oral preparations (eg, powders, granules, capsules, tablets), parenteral preparations (eg, injections, 10 infusions, external preparations (eg, nasal preparations, transdermal preparations, etc.) ), Suppositories (eg, rectal suppositories, vaginal suppositories, etc.). These preparations can be produced by a method known per se generally used in the preparation process.

  The compound of the present invention contains a dispersant (eg, Tween 80 (manufactured by Atlas Powder, USA), HCO 60 (manufactured by Nikko Chemicals), polyethylene glycol, carboxymethylcellulose, sodium alginate, etc.), preservative (eg, methylparaben, propylparaben, Benzyl alcohol, etc.), isotonic agents (eg, sodium chloride, mannitol, sorbitol, glucose, etc.), etc. in aqueous injections, or in vegetable oils such as olive oil, sesame oil, cottonseed oil, corn oil, and propylene glycol. Or it can emulsify and shape | mold into an oily injection and can be set as an injection. In order to obtain an oral preparation, according to a method known per se, the compound of the present invention is treated with, for example, an excipient (eg, lactose, sucrose, starch, etc.), a disintegrant (eg, starch, calcium carbonate etc.), a binder (eg, starch). , Gum arabic, carboxymethyl cellulose, polyvinyl pyrrolidone, hydroxypropyl cellulose, etc.) or a lubricant (eg, talc, magnesium stearate, polyethylene glycol 6000, etc.), etc. For enteric or long-lasting purposes, an oral preparation can be obtained by coating by a method known per se. Examples of the coating agent include hydroxypropylmethylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, polyoxyethylene glycol, Tween 80, Pluronic F68, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxymethylcellulose acetate succinate, Eudragit (Rohm) Made in Germany, methacrylic acid / acrylic acid copolymer) and dyes (eg, bengara, titanium dioxide, etc.). In the case of an enteric preparation, an intermediate phase can be provided between the enteric phase and the drug-containing phase by a method known per se for the purpose of separating both phases.

  In order to obtain an external preparation, the compound of the present invention can be made into a solid, semi-solid or liquid external preparation according to a method known per se. For example, as the solid state, the compound of the present invention is used as it is, or an excipient (eg, glycol, mannitol, starch, microcrystalline cellulose, etc.), a thickener (eg, natural gums, cellulose derivatives, acrylic acid) Polymer etc.) is added and mixed to form a powdery composition. The liquid form is an oily or aqueous suspension, almost the same as in the case of injections. In the case of a semi-solid form, an aqueous or oily gel or an ointment is preferred. In addition, these are all pH adjusters (eg, carbonic acid, phosphoric acid, citric acid, hydrochloric acid, sodium hydroxide, etc.), preservatives (eg, paraoxybenzoic acid esters, chlorobutanol, benzalkonium chloride, etc.), etc. May be added. For example, to make a suppository, the compound of the present invention can be converted into an oily or aqueous solid, semisolid or liquid suppository according to a method known per se. Examples of the oily base used in the composition include glycerides of higher fatty acids (eg, cacao butter, witepsols (manufactured by Dynamite Nobel, Germany)), intermediate fatty acids (eg, miglyols (manufactured by Dynamite Nobel, Germany)) Etc.], or vegetable oils (eg, sesame oil, soybean oil, cottonseed oil, etc.). Examples of the aqueous base include polyethylene glycols and propylene glycol. Examples of the aqueous gel base include natural gums, cellulose derivatives, vinyl polymers, and acrylic acid polymers.

EXAMPLES The present invention is illustrated in detail by the following examples and examples, which are merely illustrative and do not limit the invention. Moreover, you may change in the range which does not deviate from the range of this invention. The “room temperature” described in the following examples and examples represents 0 to 30 ° C. “%” Means weight percent unless otherwise specified. Other abbreviations used in the text have the following meanings.

Example 1: Synthesis of dimethyl- (1-methyl-2-nitro-vinyl) -amine

N, N-dimethylacetamide dimethyl acetal (6.3 ml), nitromethane (2.1 ml) and ethanol (10 ml) were added and refluxed. After the reaction proceeded, the solvent was distilled off and the residue was purified by silica gel column chromatography (dichloromethane: methanol = 100: 1 (v / v)) to give dimethyl- (1-methyl-2-nitro-vinyl) -amine (3.55 g ) ¹ H-NMR (DMSO-d6, TMS standard) δ (ppm): 2.54 (s, 3H), 3.05 (s, 6H), 6.72 (s, 1H)

Example 2: Synthesis of 1- (4-methoxy-2-methyl-phenyl) -2-methyl-3-nitro-1H-indole-6-ol

Dimethyl- (1-methyl-2-nitro-vinyl) -amine (515 mg), p-toluenesulfonic acid (750 mg), 4-methoxy-2-methyl-phenylamine (1.63 g), DMF (3 ml) were added, room temperature And stirred. About 100 ml of distilled water was added, and the resulting precipitate was filtered off and used for the next reaction. Acetic acid (2 ml) and acetic anhydride (0.2 ml) were added to the previous precipitate (240 mg) and p-benzoquinone (67 mg). After purging with argon, the mixture was stirred overnight at room temperature. Washing with water gave 1- (4-methoxy-2-methyl-phenyl) -2-methyl-3-nitro-1H-indole-6-ol (34 mg). ¹ H-NMR (DMSO-d6, TMS standard) δ (ppm): 1.88 (s, 3H), 2.50 (s, 3H), 3.86 (s, 3H), 6.19 (d, J = 2.4Hz, 1H), 6.87 (dd, J = 2.0 and 8.4Hz, 1H), 7.03 (dd, J = 2.8Hz and 7.0Hz, 1H), 7.13 (d, J = 2.4Hz, 1H), 7.35 (d, J = 8.8Hz, 1H), 7.96 (d, J = 8.4Hz, 1H), 9.45 (s, 1H)

Example 3: Synthesis of 1- (4-methoxy-phenyl) -2,5-dimethyl-3-nitro-1H-indole-6-ol

Dimethyl- (1-methyl-2-nitro-vinyl) -amine (479 mg), p-toluenesulfonic acid (700 mg), 4-methoxy-phenylamine (1.37 g) and DMF (2 ml) were added, and the mixture was stirred at room temperature. About 100 ml of distilled water was added, and the resulting precipitate was filtered off and used for the next reaction. Acetic acid (2 ml) and acetic anhydride (0.2 ml) were added to the previous precipitate (170 mg) and 2-methyl-p-benzoquinone (68 mg). After purging with argon, the mixture was stirred overnight at room temperature, and the resulting precipitate was filtered off. By washing with acetic acid and distilled water, 1- (4-methoxy-phenyl) -2,5-dimethyl-3-nitro-1H-indole-6-ol (6 mg) was obtained. ¹ H-NMR (DMSO-d6, TMS standard) δ (ppm): 2.43 (s, 3H), 2.56 (s, 3H), 3.87 (s, 3H), 6.45 (s, 1H), 7.21 (d, J = 8.8Hz, 2H), 7.46 (d, J = 8.8Hz, 2H), 7.86 (s, 1H)

Example 4: Synthesis of 4- (6-hydroxy-2-methyl-3-nitro-indol-1-yl) -benzoic acid ethyl ester

Add dimethyl- (1-methyl-2-nitro-vinyl) -amine (0.67 g), p-toluenesulfonic acid (1 g), 4-aminobenzoic acid ethyl ester (2.6 g), DMF (6 ml) and stir at room temperature did. About 100 ml of distilled water was added, and the resulting precipitate was filtered off and used for the next reaction. Acetic acid (2 ml) and acetic anhydride (0.2 ml) were added to the previous precipitate (190 mg) and p-benzoquinone (34 mg). After purging with argon, the mixture was stirred overnight at room temperature. Washing with water gave 4- (6-hydroxy-2-methyl-3-nitro-indol-1-yl) -benzoic acid ethyl ester (19 mg). ¹ H-NMR (DMSO-d6, TMS standard) δ (ppm): 1.37 (t, J = 7.2 Hz, 3H), 2.60 (s, 3H), 4.39 (q, J = 7.2 Hz, 2H), 6.42 ( d, J = 2.0Hz, 1H), 6.90 (dd, J = 8.6 and 2.0Hz, 1H), 7.61 (d, 7.4Hz, 2H), 7.98 (d, J = 8.8,1H), 8.23 (d, J = 8.4Hz, 2H), 9.60 (s, 1H)

Example 5: Synthesis of 1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol

Dimethyl- (1-methyl-2-nitro-vinyl) -amine (217 mg), p-toluenesulfonic acid (317 mg), 4-methoxy-phenylamine (620 mg) and DMF (3 ml) were added, and the mixture was stirred at room temperature. About 100 ml of distilled water was added, and the resulting precipitate was filtered off and used for the next reaction. Acetic acid (1 ml) and acetic anhydride (0.1 ml) were added to the previous precipitate (50 mg) and p-benzoquinone (18 mg). After purging with argon, the mixture was stirred overnight at room temperature. Washing with water gave 1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol (4 mg). ¹ H-NMR (DMSO-d6, TMS standard) δ (ppm): 2.56 (s, 3H), 3.88 (s, 3H), 6.37 (d, J = 2.0Hz, 1H), 6.87 (dd, J = 8.8 And 2.0Hz, 1H), 7.20 (dd, 6.8Hz and 2.0Hz, 2H), 7.47 (dd, J = 7.0 and 1.6Hz, 2H), 7.95 (d, J = 8.4Hz, 1H)

Example 6: Synthesis of 7-dimethylaminomethyl-1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol

1- (4-Methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol (107 mg), dioxane (2 ml) and N, N-tetramethylmethane (62 ul) were added and refluxed. After returning to room temperature and distilling off the solvent, the residue was purified by silica gel column chromatography (dichloromethane: methanol = 10: 1 (v / v)), and 7-dimethylaminomethyl-1- (4-methoxy-phenyl) -2- Methyl-3-nitro-1H-indole-6-ol (86 mg) was obtained. ¹ H-NMR (DMSO-d6, TMS standard) δ (ppm): 1.93 (s, 6H), 2.42 (s, 3H), 2.98 (s, 2H), 3.88 (s, 3H), 6.86 (d, J = 8.8Hz, 1H), 7.18 (d, J = 8.8Hz, 2H), 7.44 (d, J = 8.8Hz, 2H), 7.99 (d, J = 8.8Hz, 1H)

Example 7: Synthesis of 1- (4-methoxy-phenyl) -2-methyl-3-nitro-7-propylaminomethyl-1H-indole-6-ol

7-dimethylaminomethyl-1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol (20 mg), 2-propanol (5 ml), n-propylamine (14 ul) Added and refluxed. After returning to room temperature and distilling off the solvent, the residue was purified by silica gel column chromatography (dichloromethane: methanol = 25: 1 (v / v)) to give 1- (4-methoxy-phenyl) -2-methyl-3-nitro- 7-propylaminomethyl-1H-indole-6-ol (7.2 mg) was obtained. ¹ H-NMR (DMSO-d6, TMS standard) δ (ppm): 0.77 (t, J = 7.4 Hz, 3H), 1.27 (m, 2H), 2.14 (t, J = 7.0 Hz, 2H), 2.42 ( s, 3H), 3.25 (s, 2H), 3.87 (s, 3H), 6.80 (d, J = 8.8Hz, 1H), 7.17 (d, J = 8.8Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.95 (d, J = 8.8Hz, 2H)

Example 8: Synthesis of 1- (3-chloro-4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol

Dimethyl- (1-methyl-2-nitro-vinyl) -amine (0.52 g), p-toluenesulfonic acid (0.76 g), 3-chloro-4-methoxy-phenylamine (0.94 g), DMF (5 ml) added And stirred at room temperature. About 100 ml of distilled water was added, and the resulting precipitate was filtered off and used for the next reaction. Acetic acid (20 ml) and acetic anhydride (2 ml) were added to the previous precipitate (1.39 g) and p-benzoquinone (0.42 g). After purging with argon, the mixture was stirred overnight at room temperature. By washing with distilled water, 1- (3-chloro-4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol (61 mg) was obtained. Identification was performed by ¹ H-NMR (DMSO-d6, TMS standard).

Example 9: Synthesis of 5-chloro-1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol

Add dimethyl- (1-methyl-2-nitro-vinyl) -amine (0.74g), p-toluenesulfonic acid (1.1g), 4-methoxy-phenylamine (1.07g), DMF (5ml) and stir at room temperature did. About 100 ml of distilled water was added, and the resulting precipitate was filtered off and used for the next reaction. Acetic acid (5 ml) and acetic anhydride (0.5 ml) were added to the previous precipitate (0.92 g) and 2-chloro-p-benzoquinone (0.44 g). After purging with argon, the mixture was stirred overnight at room temperature. Separately, it was washed with acetic acid and distilled water to obtain 5-chloro-1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol (138 mg). Identification was performed by ¹ H-NMR (DMSO-d6, TMS standard).

Example 10: Synthesis of 4- (6-hydroxy-2-methyl-3-nitro-indol-1-yl) -benzoic acid

4- (6-Hydroxy-2-methyl-3-nitro-indol-1-yl) -benzoic acid ethyl ester (65 mg), 1N aqueous potassium hydroxide solution (10 ml) and methanol (10 ml) were added and stirred for 3 hours. . After confirming the disappearance of insoluble matter, the solution was gradually acidified with 8N aqueous hydrochloric acid solution, and the resulting precipitate was filtered off, and then 4- (6-hydroxy-2-methyl-3-nitro-indol-1-yl) -benzoic acid The acid (23 mg) was obtained. Identification was performed by ¹ H-NMR (DMSO-d6, TMS standard).

Example 11: Synthesis of 1- (4-hydroxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol

Dimethyl- (1-methyl-2-nitro-vinyl) -amine (0.89 g), p-toluenesulfonic acid (1.3 g) 4-amino-phenol (1.1 g) and DMF (5 ml) were added, and the mixture was stirred at room temperature. About 100 ml of distilled water was added, and the resulting precipitate was filtered off and used for the next reaction. Acetic acid (25 ml) and acetic anhydride (2.5 ml) were added to the previous precipitate (0.98 g) and p-benzoquinone (0.37 g). After purging with argon, the mixture was stirred overnight at 40 ° C., and the resulting precipitate was filtered off. By washing with acetic acid and distilled water, 1- (4-hydroxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol (120 mg) was obtained. Identification was performed by ¹ H-NMR (DMSO-d6, TMS standard).

Example 12: Synthesis of 1- (3-hydroxy-4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol

Dimethyl- (1-methyl-2-nitro-vinyl) -amine (1.40 g), p-toluenesulfonic acid (2.05 g) 5-amino-2-methoxy-phenol (2.25 g), DMF (10 ml) added, room temperature And stirred. About 100 ml of distilled water was added, and the resulting precipitate was filtered off and used for the next reaction. Acetic acid (15 ml) and acetic anhydride (1.5 ml) were added to the previous precipitate (1.17 g) and p-benzoquinone (0.38 g). After purging with argon, the mixture was stirred overnight at 40 ° C., and the resulting precipitate was filtered off. By washing with acetic acid and distilled water, 1- (3-hydroxy-4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol (422 mg) was obtained. Identification was performed by ¹ H-NMR (DMSO-d6, TMS standard).

Example 13: Synthesis of 1- (4-Ethoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol

Dimethyl- (1-methyl-2-nitro-vinyl) -amine (0.64 g), p-toluenesulfonic acid (0.94 g) 4-ethoxy-phenylamine (1.02 g) and DMF (5 ml) were added and stirred at room temperature. . About 100 ml of distilled water was added, and the resulting precipitate was filtered off and used for the next reaction. Acetic acid (10 ml) and acetic anhydride (1 ml) were added to the previous precipitate (0.77 g) and p-benzoquinone (0.25 g). After purging with argon, the mixture was stirred overnight at 40 ° C. And washed with distilled water to give 1- (4-ethoxy-phenyl) -2-methyl-3-nitro-1H-indol-6-ol (319 mg). Identification was performed by ¹ H-NMR (DMSO-d6, TMS standard).

Example 14: Synthesis of 1- (4-methoxy-phenyl) -2-methyl-3-nitro-6-propoxy-1H-indole

1- (4-Methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol (56.66 mg), potassium carbonate (105 mg), potassium iodide (15.8 mg), DMF (10 ml), odor Propyl chloride (21 ul) was added and stirred at room temperature overnight. After removing the solvent, the residue was purified by silica gel column chromatography (dichloromethane: methanol = 100: 1) to give 1- (4-methoxy-phenyl) -2-methyl-3-nitro-6-propoxy-1H-indole (10 mg ) Identification was performed by ¹ H-NMR (DMSO-d6, TMS standard).

Example 15: 5- (2-Oxo-hexahydro-thieno [3,4-d] imidazol-4-yl) -pentenoic acid (2- [2- [2- (2-[[6-hydroxy-1- Synthesis of (4-Methoxy-phenyl) -2-methyl-3-nitro-1H-indol-7-ylmethyl] -amino] -ethoxy) -ethoxy] -ethoxy] -ethyl) -amide

7-dimethylaminomethyl-1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol (28.3 mg), 2-propanol (5 ml), 5- (2-oxo- Hexahydro-thieno [3,4-d] imidazol-4-yl) -pentenoic acid (2- [2- [2- (2-amino-ethoxy) -ethoxy] -ethoxy] -ethyl) -amide (Pierce) ) (50 mg) was added and refluxed. After returning to room temperature and distilling off the solvent, the residue was purified by silica gel column chromatography (dichloromethane: methanol = 10: 1 (v / v)) to give 5- (2-oxo-hexahydro-thieno [3,4-d] imidazole. -4-yl) -pentenoic acid (2- [2- [2- (2-[[6-hydroxy-1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-7- Irmethyl] -amino] -ethoxy) -ethoxy] -ethoxy] -ethyl) -amide (28 mg) was obtained. Identification was performed by ¹ H-NMR (DMSO-d6, TMS standard) and ESI-MS. [M + H] + = 729.3m / z, calc.728.3

Example 16:
"Preparation of ES cell medium"
For the purpose of proliferating ES cells, factors were added to Dulbecco's Modified Eagle Medium (hereinafter DMEM) (GIBCOBRL Cat. No. 11995) at the final concentrations shown below to prepare an ES cell medium. 15% fetal bovine serum (GIBCO BRL) or 15% KSR (Invitrogen), 0.1 mM β-mercaptoethanol (SIGMA), 1 × non-essential amino acid stock (GIBCO BRL)
Cat. No. 11140-050), 2 mM L-G1utamine (Cat. No. 25030-081 manufactured by GIBCO BRL), 103 unit / ml ESGRO (manufactured by CHEMICON International Inc., Inc.).
However, ESGRO contains mouse LIF as an active ingredient. As a medium for ES cell differentiation inhibition assay, an assay medium was prepared by removing ESGRO from the above ES cell medium.

“Culture of ES cells”
Dissolve Gelatin (SIGMA TypeA: from porcinESkin, G2500) in distilled water at a concentration of 0.1% in a cell culture dish with a diameter of 6 cm, add 5 ml of sterile 0.1% gelatin aqueous solution, and continue at 37 ° C for 30 minutes or more Left to stand. Except gelatin aqueous solution, 2x106 mouse embryonic primary cultured cells (Lifetech Oriental Cat. No. YE9284400) treated with mitomycin C (manufactured by Kyowa Hakko) were seeded and 10% fetal bovine serum (GIBCO BRL) Incubated for 5 hours or more in a 5% CO2 incubator (manufactured by Tabay Espec) with 5 ml of DMEM. Mouse embryonic stem cell line D3ES cells (available from Rolf Kemler, Max Planck Institut fur Immunbiologie, Steuheweg51, D-79108 Freiburg, Germany) are seeded on a 6 cm diameter fibroblast feeder layer and in 37 ml of 5 ml ES medium. The cells were grown for 2 days in a 5% CO2 incubator at 0 ° C.

"ES cell differentiation inhibition assay"
The D3 ES cells cultured in the “ES cell culture” are washed twice with PBS. A 0.25% trypsin solution (GIBCOBRL) was added and incubated at 37 ° C. for 5 minutes, and undifferentiated D3ES cell colonies were detached from the feeder. 5 ml of ES cell medium was added, cell colonies were dispersed using a small-diameter pipette, transferred to a 15 ml sterilized tube, and pelleted by centrifugation at 800 rpm for about 5 minutes in a tabletop centrifuge (Tomy Seiko). The supernatant was removed and the cells were resuspended in 5 ml of fresh ES cell medium, seeded in a 10 cm diameter cell culture dish pre-coated with 0.1% gelatin aqueous solution and incubated at 37 ° C. for 20 minutes. After 20 minutes, the medium containing floating cells is collected with a pipette, transferred to a 15 ml sterilized tube, pelleted by centrifugation at 1000 rpm for about 5 minutes in a desktop centrifuge, the supernatant is removed, and 5 ml of ES cell assay medium is obtained. Resuspend. In a 24-well cell culture dish (FALCON Cat. No. 3047, USA) previously coated with 0.1% gelatin aqueous solution, 3 × 10 ² to 1 × 10 ³ cells per well were added per well. ES cell assay medium was seeded to 500 μl. 50 μl of dimethyl sulfoxide (DMSO), compound A to K of the present invention dissolved in water or a mixture thereof, or ESGRO is added to each well so as to be 0.4 to 40 μg / ml, and 37 ° C., 5% CO 2 The cells were cultured for 7 days in an incubator. DMSO was brought into the medium at a final concentration of 0.1% or less. Also, DMSO alone was added to the control well so that the final concentration was 0.1%. The structures of compounds A to K are shown in FIG.

"Quantification of alkaline phosphatase"
The alkaline phosphatase activity of ES cells was determined using p-NITROPHENYL PHOSPHATE SOLUTION (MOSS Inc., PRODUCT NO.NPPD-1000, USA, or SIGMA, A-3469, USA) (hereinafter p-NPP). And quantified. The medium was aspirated and removed from each well of ES cells cultured for 7 days by the method described in Example 3. The cells were washed once with 100 μl of phosphate buffered saline (PBS), and then 100 μl of p-NPP was added to each well. In addition to the wells, they were allowed to stand at room temperature for 10 minutes. 12.5 μl of 8M sodium hydroxide solution was added to each well to stop the reaction. The absorbance at 405 nm (OD405) and the absorbance at 690 nm (OD690) of the solution were measured with an absorptiometer (Molecular Devices, model: SPECTRA MAX190), and the value calculated by OD405-OD690 was defined as alkaline phosphatase activity. A graph of the quantitative results is shown in FIG. The differentiation inhibitors of the present invention, Compounds A to K, significantly amplified alkaline phosphatase activity compared to DMSO (0.1%) as a control. That is, the differentiation inhibitor of the present invention supported the culture of undifferentiated ES cells.

  The indole derivative of the present invention can be suitably used in the field of cell culture and / or regenerative medicine.

FIG. 1 shows the structures of compounds AK of the present invention. In the compounds A to K, R1 to R10 in the formula (1) are represented by groups or elements shown in the following table. FIG. 2 represents the alkaline phosphatase quantification results of the present invention.

Claims (6)

General formula (2)
[Wherein, R ¹ , R ² and R ⁴ represent a hydrogen atom, R ³ represents a hydroxyl group, R ⁵ represents a methyl group, and R ⁶ represents a nitro group. A is
Represents. Here, R ⁷ , R ¹⁰ and R ¹¹ represent a hydrogen atom, R ⁸ represents a hydroxyl group, and R ⁹ represents a methoxy group. Or a salt thereof. A stem cell differentiation inhibitor comprising the compound according to claim 1 or a salt thereof as an active ingredient. A stem cell culturing method comprising culturing stem cells in an undifferentiated state using the compound according to claim 1 or a salt thereof. An embryonic stem cell culturing method comprising culturing embryonic stem cells in an undifferentiated state using the compound according to claim 1 or a salt thereof. A medium for proliferating stem cells in an undifferentiated state, comprising the compound according to claim 1 or a salt thereof as an active ingredient. The medium according to claim 5, wherein the concentration of the active ingredient is 10 ng / ml to 100 μg / ml.